A MODIFIED YEAST ONE-HYBRID SYSTEM TO INVESTIGATE PROTEIN-PROTEIN AND PROTEIN:DNA
INTERACTIONS
by
Gang Chen
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Graduate Department of Chemistry University of Toronto
© Copyright by Gang Chen 2008
ii
A MODIFIED YEAST ONE-HYBRID SYSTEM TO INVESTIGATE
PROTEIN-PROTEIN AND PROTEIN:DNA INTERACTIONS
Gang Chen
Doctor of Philosophy
Graduate Department of Chemistry University of Toronto
2008
Abstract A modified yeast one-hybrid (MY1H) system has been developed for in vivo
investigation of simultaneous protein-protein and protein:DNA interactions. The traditional yeast
one-hybrid assay (Y1H) permits examination of one expressed protein targeting one DNA site,
whereas our MY1H allows coexpression of two different proteins and examination of their
activity at the DNA target. This single-plasmid based MY1H was validated by use of the DNA-
binding protein p53 and its inhibitory partners, large T antigen (LTAg) and 53BP2. The MY1H
system could be used to examine proteins that contribute inhibitory, repressive, coactivational or
bridging functions to the protein under investigation, as well as potential extension toward
library screening for identification of novel accessory proteins.
After development and validation of the MY1H with the p53/LTAg/53BP2 system, we
applied the MY1H system to investigate the DNA binding activities of heterodimeric proteins,
the bHLH/PAS domains of AhR and Arnt that target the xenobiotic response element (XRE).
The AhR/Arnt:XRE interaction, which served as our positive control for heterodimeric protein
binding of the XRE DNA site, showed negative signals in initial MY1H experiments. These false
negative observations were turned into true positives by increasing the number of DNA target
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sites upstream of the reporter genes and increasing the number of activator domains fused to the
two monomers. This methodology may help trouble-shooting false negatives stemming from
unproductive transcription in yeast genetic assays, which can be a common problem.
In the study of XRE-binding proteins, two bHLHZ-like hybrid proteins, AhRJunD and
ArntFos were designed and coexpressed in the MY1H and yeast two-hybrid (Y2H) systems;
these proteins comprise the bHLH domains of AhR and Arnt fused to the leucine zipper (LZ)
elements from bZIP proteins JunD and Fos, respectively. The in vivo assays revealed that in the
absence of the XRE DNA site, heterodimers and homodimers formed, but in the presence of the
nonpalindromic XRE, only heterodimers bound to the XRE and activated reporter transcription.
The present results provide valuable information on DNA-mediated protein heterodimerization
and specific DNA binding, as well as the relationship between protein structure and DNA-
binding function.
iv
Acknowledgments First and Foremost I would like to express my sincere appreciation to Dr. Jumi Shin for
giving me the right amount of freedom and guidance during my graduate studies at UofT. Your
dedication, encouragement and enthusiasm served as a constant source of inspiration.
I would like to express my gratitude to the rest of my Supervisory Committee Dr.
Deborah Zamble and Dr. Ulrich Krull for their involvement in my research and their advice that
helped to shape my research skills. I am very thankful to Dr. Erica Golemis (University of
Pennsylvania) for being my external reviewer, and Dr. Andrew Woolley, Dr. Patrica Harper, and
Dr. Mark Nitz for sitting on my Examination Committee, critical reading of my thesis and
offering useful suggestions.
I would like to thank Dr. Kuniyoshi Iwabuchi (Kanazawa Medical University, Japan) and
Dr. Stanley Fields (University of Washington) for kindly providing human 53BP2 cDNA, and Dr.
Patricia Harper and Dr. Allan Okey (University of Toronto) for kindly providing human AhR
and Arnt cDNA. These gifts greatly helped the progress of my thesis work and eventually made
my thesis possible.
I would like to thank all the past and present members of Shin group for providing
invaluable help, for both experimental work and discussion, in my thesis work. They are Dr.
Adrian Schwartz, Dr. Jing Xu, Ms. Lisa Denboer, Ms. Antonia De Jong, Ms. Joanna Chow, Dr.
Christopher Damaso, Ms. Cassie Ho, Dr. Anna Fedorova, Ms. Alevtina Pavlenco, Ms. I-San
Chan, and Mr. Hesam Shahravan. Special thanks to Dr. Adrian Schwartz, who introduced me to
the magic world of yeast genetic reporter systems, and to Ms. Antonia De Jong, who is now
working on the downstream work of Chapter 5 as well as some interesting ideas I would
otherwise love to try.
v
The thesis work was funded by National Institutes of Health (R01 GM069041), Premier's
Research Excellence Award (PREA), Canadian Foundation for Innovation/Ontario Innovation
Trust (CFI/OIT), Natural Sciences and Engineering Research Council of Canada (NSERC)
Discovery Grant, and the University of Toronto.
I would like to express my earnest gratitude to my parents for their constant support and
love, without which any of my achievements would not have been possible. I am deeply indebted
to my dear wife Ying for her love and understanding, as well as her unconditional support right
behind me. Finally thanks to our son, Bernie, for the joy and the happiness he brings to us all the
time. Sharing my life with them has been a wonderful experience and I look forward to our many
more years to come.
Toronto, August 2008
Gang Chen
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Table of Contents Abstract ........................................................................................................................................... ii
Acknowledgments.......................................................................................................................... iv
vi
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Table of Contents...........................................................................................................................
List of Tables ................................................................................................................................
List of Figures ..............................................................................................................................
List of Abbreviations ...................................................................................................................
Chapter 1 Introduction ....................................................................................................................
1.1 Yeast genetic reporter assays ..............................................................................................
1.1.1 Yeast two- and one-hybrid systems ........................................................................
1.1.2 Modified yeast genetic reporter systems for detection of simultaneous protein-
protein and protein:DNA interactions..................................................................... 3
1.1.3 Advantages and disadvantages of yeast genetic reporter systems ..........................
1.2 Transcription factors ...........................................................................................................
1.2.1 The bZIP family......................................................................................................
1.2.2 The bHLH superfamily ...........................................................................................
1.2.3 Heterodimeric transcription factors increase regulatory diversity........................
1.3 Studies of bHLH superfamily proteins in yeast genetic reporter systems ........................
1.4 Scope.................................................................................................................................
Chapter 2 Design of a single plasmid-based modified yeast one-hybrid system for
investigation of in vivo protein-protein and protein:DNA interactions ...................................
2.1 Abstract .............................................................................................................................
2.2 Introduction.......................................................................................................................
vii
2.3 Materials and Methods...................................................................................................... 23
23
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Chapter 3 AhR/Arnt:XRE interaction: turning false negatives into true positives in the
modified yeast one-hybrid assay.............................................................................................. 38
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2.3.1 Bacterial and yeast strains.....................................................................................
2.3.2 Transformation, DNA preparation, and plasmid rescue .......................................
2.3.3 Plasmid Construction ............................................................................................
2.3.4 3-AT titration analysis ..........................................................................................
2.3.5 X-gal colony-lift filter assay and ONPG liquid assay ..........................................
2.4 Results...............................................................................................................................
2.4.1 Design of protein expression vectors....................................................................
2.4.2 Coexpression of LTAg or 53BP2 decreases the transactivation potential of
GAL4AD-p53 in the MY1H.................................................................................
2.5 Discussion .........................................................................................................................
2.5.1 The DNA-binding activity of p53 and its interaction with LTAg or 53BP2:
comparison of MY1H observations with earlier studies.......................................
2.5.2 Different types of interactions between two proteins and a DNA target can be
examined in our MY1H system ............................................................................
2.6 Supplemental Information ................................................................................................
3.1 Abstract .............................................................................................................................
3.2 Introduction.......................................................................................................................
3.3 Materials and methods ......................................................................................................
3.3.1 Bacterial and yeast strains.....................................................................................
3.3.2 Construction of reporter strains ............................................................................
3.3.3 Transformation, DNA preparation, and plasmid rescue .......................................
viii
3.3.4 Plasmid Construction ............................................................................................ 43
45
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3.3.5 HIS3 reporter assay ...............................................................................................
3.3.6 LacZ reporter assay ...............................................................................................
3.4 Results...............................................................................................................................
3.4.1 Initial trials with pCETT: fusion of the GAL4 AD to NAhR only.......................
3.4.2 Next generation trials with pCETT2: fusion of the GAL4 AD to both NAhR
and NArnt..............................................................................................................
3.5 Discussion .........................................................................................................................
3.5.1 Are false negatives due to unproductive transcription?........................................
3.5.2 Copy number of XRE target sites .........................................................................
3.5.3 Double AD system................................................................................................
3.5.4 Synergistic activation is due to both increase of XRE target sites and doubling
of AD ....................................................................................................................
3.6 Supplemental Information ................................................................................................
Chapter 4 Forced protein heterodimerization and specific DNA binding to a nonpalindromic
DNA sequence in vivo and in vitro: bHLHZ-like hybrid heterodimers of bHLH/PAS
proteins AhR and Arnt and bZIP proteins JunD and Fos as a model ......................................
4.1 Abstract .............................................................................................................................
4.2 Introduction.......................................................................................................................
4.3 Materials and methods ......................................................................................................
4.3.1 Bacterial and yeast strains.....................................................................................
4.3.2 Transformation, DNA preparation, and plasmid rescue .......................................
4.3.3 Plasmid Construction ............................................................................................
4.3.4 HIS3 reporter assay ...............................................................................................
ix
4.3.5 LacZ reporter assay ............................................................................................... 64
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4.3.6 Protein-protein interactions in the Y2H system....................................................
4.4 Results...............................................................................................................................
4.4.1 The AhRJunD/ArntFos and AhR(ΔL)JunD/ArntFos heterodimers bind the
XRE site in the MY1H..........................................................................................
4.4.2 Heterodimerization between two monomers confirmed in the Y2H assay ..........
4.5 Discussion .........................................................................................................................
4.5.1 Fusion of the JunD and Fos LZ domains to the Arnt and AhR bHLH domains
reconstitutes the heterodimeric structure and specific DNA-binding function
of the bHLH/PAS domains of AhR and Arnt .......................................................
4.5.2 The nonpalindromic XRE DNA target mediates the heterodimerized structure
of AhRJunD and ArntFos, resulting in DNA-binding function............................
4.5.3 What causes the negative signal in XRE binding by the bHLH domains of
AhR and Arnt in the MY1H?................................................................................
4.5.4 Deletion of one leucine residue in the JunD LZ negatively affects
heterodimerization but not DNA binding .............................................................
4.5.5 Our domain swapping experiments support the hypothesis of domain shuffling
in the evolutionary pathway of the bHLH superfamily ........................................
4.6 Supplemental Information ................................................................................................
Chapter 5 Summary and future work............................................................................................
5.1 Summary ...........................................................................................................................
5.2 Future work.......................................................................................................................
5.2.1 Downstream work on the AhRJunD/ArntFos:XRE interaction............................
5.2.2 Improvement on the MY1H system......................................................................
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5.2.3 Other DNA-binding hybrid proteins of interest.................................................... 89
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Chapter 6 Materials and Methods .................................................................................................
6.1 General ..............................................................................................................................
6.1.1 Two reporter assays used in the MY1H................................................................
6.1.2 Examination of protein-protein interactions in the Y2H system ..........................
6.2 Modified Y1H for identification of protein-protein/protein:DNA interactions................
6.2.1 Construction of p53 target-reporter strains ...........................................................
6.2.2 Plasmid construction.............................................................................................
6.2.3 3-AT titration analysis ..........................................................................................
6.3 Turning false negatives into true positives in the MY1H.................................................
6.3.1 Construction of XRE target-reporter strains .........................................................
6.3.2 Plasmid construction.............................................................................................
6.4 DNA binding forces heterodimerization of hybrids of bHLH/PAS and bZIP..................
6.4.1 Plasmid construction.............................................................................................
References.....................................................................................................................................
Appendix A Design of a single plasmid-based modified yeast one-hybrid system for
investigation of in vivo protein-protein and protein:DNA interactions .................................
A.1 Western blot analysis ......................................................................................................
A.2 Expression levels of LTAg and 53BP2 in the MY1H system ........................................
A.3 Expression of the AD-fusion protein from MCS I is not affected by expression from
MCS II ............................................................................................................................
Appendix B AhR/Arnt:XRE interaction: turning false negatives into true positives in the
modified yeast one-hybrid assay............................................................................................
B.1 Construction of reporter strains ......................................................................................
xi
B.1.1 Three-copy strains............................................................................................... 112
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B.1.2 Six-copy strains...................................................................................................
B.2 Construction of AhR6-436 (NAhR) fragment ................................................................
B.3 Construction of Arnt82-464 (NArnt) fragment...............................................................
Appendix C Forced protein heterodimerization and specific DNA binding to a
nonpalindromic DNA sequence in vivo and in vitro: bHLHZ-like hybrid heterodimers of
bHLH/PAS proteins AhR and Arnt and bZIP proteins JunD and Fos as a model.................
C.1 Plasmid Construction ......................................................................................................
C.1.1 pCETT2/AhRbHLH/ArntbHLH.........................................................................
C.1.2 pCETT/AhRJunD/ArntFos and pCETT2/AhRJunD/ArntFos ............................
C.1.3 pCETT2/AhR(ΔL)JunD/ArntFos........................................................................
C.1.4 pGBKT7/AhRbHLH and pGADT7/AhRbHLH.................................................
C.1.5 pGBKT7/ArntbHLH and pGADT7/ArntbHLH .................................................
C.1.6 pGBKT7/AhRJunD and pGADT7/AhRJunD.....................................................
C.1.7 pGBKT7/AhR(ΔL)JunD and pGADT7/AhR(ΔL)JunD .....................................
C.1.8 pGBKT7/ArntFos and pGADT7/ArntFos ..........................................................
C.2 Expression of GAL4AD-AhRJunD and ArntFos by use of pCETT in the MY1H ........
Appendix D Table of Oligonucleotides...................................................................................
xii
List of Tables Table 2.1 Oligonucleotides used in this study............................................................................. 25
74
109
112
116
122
Table 4.1 Dimerization of AhR- and Arnt-hybrid proteins.........................................................
Table A.1 Reporter activation of GAL4AD fusions of p53 or Max expressed from different
vectors .........................................................................................................................................
Table B.1 Oligonucleotides used in this study..........................................................................
Table C.1 Oligonucleotides used in this study..........................................................................
Table D.1 Oligonucleotides used in this thesis .........................................................................
xiii
List of Figures Figure 1.1 Schematic representation of Y2H and Y1H systems.................................................... 1
8
11
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Figure 1.2 Crystal structure of the GCN4 bZIP homodimer bound to the AP-1 DNA site ..........
Figure 1.3 Crystal structure of the MyoD:DNA complex (A) and Max:DNA complex (B) ......
Figure 1.4 Ribbon presentation of the Drosophila Period dimer................................................
Figure 2.1 Five different types of interactions between proteins and DNA can be detected with
the MY1H system .........................................................................................................................
Figure 2.2 Plasmids pCETT and pCETF were constructed for coexpression of two proteins in a
yeast model system .......................................................................................................................
Figure 2.3 3-AT titrations reveal that the survival rates of transformants decreases when the
inhibitory proteins are expressed ..................................................................................................
Figure 2.4 Colony-lift filter assay indicates LTAg and 53BP2 inhibit DNA binding of p53 to
different extents ............................................................................................................................
Figure 2.5 Histogram comparing the effects of different expression levels of inhibitory proteins
LTAg and 53BP2 on DNA binding by p53 ..................................................................................
Figure 3.1 Plasmid pCETT2 for coexpression of two AD fusion proteins in the MY1H system
.......................................................................................................................................................
Figure 3.2 HIS3 reporter assay for detection of NAhR/NArnt:XRE interaction from protein
expression vector pCETT or pCETT2 in YM4271[pHISi-1/XRE-3] (A and C) and
YM4271[pHISi-1/XRE-6] (B and D) strains................................................................................
Figure 3.3 Colony-lift filter assay for detection of NAhR/NArnt:XRE interaction from protein
expression vector pCETT or pCETT2 in YM4271[pHISi-1/XRE-3] (A and C) and
YM4271[pHISi-1/XRE-6] (B and D) strains................................................................................
xiv
Figure 3.4 ONPG assay for detection of NAhR/NArnt:XRE interaction from protein expression
vector pCETT or pCETT2 in YM4271[pHISi-1/XRE-3] (A and C) and YM4271[pHISi-1/XRE-6]
(B and D) strains ........................................................................................................................... 48
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105
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Figure 4.1 Primary sequence alignment of the native Max bHLHZ domain and bHLH•LZ
hybrids...........................................................................................................................................
Figure 4.2 HIS3 reporter assay for detection of interactions between heterodimers and the XRE
cognate sequence ..........................................................................................................................
Figure 4.3 HIS3 reporter spot titration assay for comparison of the relative strengths of
AhRJunD/ArntFos:XRE and AhR(ΔL)JunD/ArntFos:XRE interactions.....................................
Figure 4.4 Colony-lift filter assay for the detection of interactions between heterodimers and the
XRE cognate sequence .................................................................................................................
Figure 4.5 ONPG measurements for detection of AhRJunD/ArntFos:XRE and
AhR(ΔL)JunD/ArntFos:XRE interactions by use of protein expression vector pCETT2 in strain
YM4271[pHISi-1/XRE-6] ............................................................................................................
Figure 4.6 Y2H assay of homo- and heterodimerization of AhR and Arnt hybrid proteins.......
Figure A.1 Plasmids pCETT (truncated ADH1 promoter) and pCETF (full-length ADH1
promoter) were constructed for coexpression of two proteins in a yeast model system ............
Figure A.2 Promoter length dictates differential expression levels of inhibitory proteins when
YM4271[p53HIS] cells were transformed with the indicated plasmids and grown to exponential
phase in YPDA media.................................................................................................................
Figure B.1 Colony-lift filter assay for detection of NAhR/NArnt:XRE interaction from protein
expression vector pCETT or pCETT2 in YM4271[pHISi-1/XRE-3] (A and C) and
YM4271[pHISi-1/XRE-6] (B and D) strains..............................................................................
xv
Figure C.1 The HIS3 reporter assay for detection of the AhRJunD/ArntFos:XRE interaction by
use of protein expression vector pCETT in strain YM4271[pHISi-1/XRE-6] ........................... 120
120
121
Figure C.2 The X-gal colony-lift filter assay............................................................................
Figure C.3 ONPG measurements for detection of AhRJunD/ArntFos:XRE interactions by use
of protein expression vector pCETT in strain YM4271[pHISi-1/XRE-6] .................................
xvi
List of Abbreviations Abbreviation Full Name
3-AT 3-amino-1,2,4-triazole
AD activation domain
AhR aryl hydrocarbon receptor
AhRJunD AhRbHLH20-86-JunD296-332 fusion
AhR(ΔL)JunD AhRbHLH20-86-JunD297-332 fusion
Arnt AhR nuclear translocator
ArntFos ArntbHLH82-148-Fos165-201 fusion
ARRE2 antigen receptor response element
bHLH basic helix-loop-helix
bHLH/PAS basic helix-loop-helix/Per-Arnt-Sim
bHLHZ basic helix-loop-helix/leucine zipper
bZIP basic region/leucine zipper
CD circular dichroism
DBD DNA binding domain
DBP DNA-binding protein
DR dioxin receptor
DRE dioxin response element
E-box enhancer box
GAL4AD GAL4 activation domain
GAL4DBD GAL4 DNA-binding domain
GFP green fluorescent protein
HIF hypoxia-inducible factor
Hsp90 heat shock protein 90
HTH helix-turn-helix
Kd dissociation constant
LTAg large T antigen
LZ leucine zipper
mAb monoclonal antibody
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Abbreviation Full Name
Max Myc-associated factor X
MCS multiple cloning site
MRF muscle regulatory factor
MY1H modified yeast one-hybrid
NAhR human AhR6-436 fragment
NArnt human Arnt82-464 fragment
NMR nuclear magnetic resonance
ONPG ortho-nitrophenyl-galactoside
PAS Per-Arnt-Sim
PCB polychlorinated biphenyl
Per Period protein
PMSF phenylmethylsulfonyl fluoride
SD minimal synthetic dropout medium
SEM standard error measurement
Sim single-minded protein
S/N signal-to-noise ratio
SRE serum response element
SRF serum response factor
SURE Stop Unwanted Rearrangement Events
SV40 Simian Virus 40
TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin
VDR vitamin D receptor
XRE xenobiotic response element
Y1H yeast one-hybrid
Y2H yeast two-hybrid
1
Chap 1ter Introduction
1.1 Yeast genetic reporter assays
1.1.1 Yeast two- and one-hybrid systems Yeast two-hybrid (Y2H) systems are genetic assays that allow identification of novel
protein-protein interactions, confirmation of suspected interactions, and definition and mapping
of interacting domains (1-4). Y2H systems, as well as variant yeast one-hybrid (Y1H) systems,
exploit the modular nature of transcription factors, which typically comprise discrete DNA
binding domains (DBD) and activation domains (AD) (1). In Y2H systems, one protein of
interest “X” is expressed as a fusion with the DBD, which serves as the bait, while the other
protein “Y” is expressed as a fusion to the AD serving as the prey. When both chimeric proteins
are coexpressed and localized to the nucleus, interaction between the bait and the prey brings the
DBD and AD into proximity, thereby reconstituting the functional transcription factor, and
reporter gene expression is activated (Figure 1.1A).
Figure 1.1 Schematic representation of Y2H and Y1H systems. A) In Y2H systems, the bait “X” is fused to a DBD, which targets specific DNA binding sites in the promoter region upstream of reporter genes such as HIS3 and lacZ. The prey “Y” is fused to an AD. B) In Y1H systems, the DNA element E placed upstream of reporter genes serves as the bait; the DNA-binding protein (DBP), being the prey, is expressed as a fusion with the AD. Reporter gene expression is activated when the AD-fused DBP interacts with the specific DNA element E.
Y1H systems are genetic assays for isolation of novel genes encoding proteins that bind
to a target, cis-acting regulatory element and for further characterization of known protein:DNA
2
interactions (4-6). In Y1H systems, the bait is not a protein, but a DNA element placed upstream
of reporter genes such as lacZ and HIS3; the protein of interest, expressed as a fusion with an AD,
serves as the prey. Reporter gene expression is activated when the AD-fused protein interacts
with the specific DNA element upstream of the reporter gene (Figure 1.1B).
Over the years of development, yeast genetic reporter systems have undergone
considerable modifications and modernizations to improve the overall performance of their
utilization, therefore allowing greater robustness and flexibility, and/or greatly expanding their
applicable scope. Classic yeast genetic reporter systems commonly utilize a DBD derived from
GAL4 or LexA, and AD from GAL4, B42, or the stronger VP16. The earliest systems developed
were usually based on one reporter gene, lacZ; subsequently, growth selection markers, such as
HIS3, LEU2, and URA3 were introduced. In order to increase the sensitivity of these systems,
multiple reporter genes under the control of different promoters were implemented into one
single system, which has been proven to enable detection of weak interactions and efficiently
eliminate false positives (7,8). In addition to the aforementioned traditional reporter genes,
counter-selectable reporters such as CYH2 or URA3 can also be used to detect de novo
autoactivators to minimize false positive interactions or for functional analysis by dissociation of
protein-protein or protein:DNA interactions (8-10). Other than lacZ, the reporter green
fluorescent protein (GFP) protein was also applied in both the Y2H and Y1H systems (11-13).
The application of GFP in yeast genetic reporter systems simplifies and accelerates the
identification and screening process of putative interactions, as well as increases the detection
sensitivity.
Moreover, several systems have been developed to detect protein-protein and/or
protein:DNA interactions occurring outside the yeast nucleus. For instance, split-GFP systems, in
which the two halves of GFP are fused to the two proteins of interest and upon interaction,
3
reconstitute functional GFP, were applied to Y2H systems to localize interactions in living cells,
which might be used to detect the compartment where the protein-protein interaction occurs in
yeast (14). In order to circumvent problems associated with the analysis of membrane proteins in
classic Y2H systems, split-ubiquitin systems were developed which allow the detection of
interactions of membrane proteins (15,16). In the split-ubiquitin systems, a functional ubiquitin
is reconstituted by the interaction between the two proteins of interest fused to two halves of
ubiquitin, respectively, and then recognized by endogenous ubiquitin-specific proteases, leading
to release of the originally attached artificial transcription factor. The released transcription
factor thereby enters the nucleus and activates reporter transcription.
In addition to straightforward protein-protein and protein:DNA interactions, yeast genetic
reporter systems have been modified to explore much broader interactions such as multiprotein
interactions, multiprotein:DNA interactions, protein:RNA interactions, and interactions between
proteins and small molecules (for a recent comprehensive review on yeast hybrid approaches by
Golemis and coworkers, see ref. (17)). Because of the scope of this thesis, we will only discuss
in detail the study of simultaneous protein-protein and protein:DNA interactions by use of yeast
genetic reporter systems.
1.1.2 Modified yeast genetic reporter systems for detection of simultaneous protein-protein and protein:DNA interactions
Several approaches that combine features of the Y1H and Y2H have been reported for
investigation of simultaneous protein-protein and protein:DNA interactions. These approaches
enable identification of a protein in complex with a known partner, thereby forming a
heterodimeric complex that binds DNA; these complexes may include one partner lacking
intrinsic DNA-binding capability that is only enabled in the presence of an accessory protein. A
modified Y1H system was developed to identify SAP-1, which binds the c-fos serum response
4
element (SRE) only when complexed with serum response factor (SRF) (18). This system was
later used to isolate BETA2, a novel member of the basic helix-loop-helix (bHLH) superfamily
that heterodimerizes with the ubiquitous bHLH protein E47, where the heterodimer binds to the
insulin enhancer box (E-box) sequence with high affinity and is important for the regulation of
insulin genes (19). The same concept was applied to investigate the trimer complex
NFAT/Jun/Fos, which targets the upstream antigen receptor response element (ARRE2) located
in an enhancer region that controls antigen-dependent induction of the interleukin 2 gene (20,21).
Another approach was developed to simultaneously identify proteins that bind to the ftz proximal
enhancer element and cofactors that directly interact with the Ftz protein (22). This approach was
also used to isolate BAF60a, a mammalian protein capable of interacting with the Vitamin D
receptor (VDR) heterodimer complex while driving expression from a repressor VDR element
(23).
1.1.3 Advantages and disadvantages of yeast genetic reporter systems There are several advantages of using yeast genetic reporter systems to investigate
protein-protein or protein:DNA interactions. First, an obvious advantage of using yeast genetic
reporter systems over classic biochemical or other genetic methods is that they are in vivo
techniques that use the yeast cell as a living test tube. The eukaryotic yeast host bears a greater
resemblance to higher eukaryotic systems than does bacteria, while less likely to have
endogenous interference issues than higher eukaryotic hosts. Second, the yeast machinery can
provide post-translational modifications required by eukaryotic protein-protein or protein:DNA
interactions. Third, yeast genetic reporter systems can be used to detect weak or transient
interactions important in signaling pathways. In some cases, transient interactions not stable
under the in vitro conditions of immunoprecipitation are able to display a transcriptional
response in vivo in yeast genetic reporter systems (24). Fourth, yeast assays do not require the
5
purification of the proteins under investigation as only the cDNA of the gene of interest is
needed, which is in contrast to classic biochemical approaches that require either high quantities
of purified proteins and/or good quality antibodies. Last, although these yeast genetic assays do
not provide direct, quantitative measurement of the strength of interactions under investigation,
the transcriptional readouts of reporter activation generally correlate with the strength of the
interactions as determined in vitro, therefore permitting discrimination of interactions with
different affinities (25).
However, various factors lead to the observation of false positives and false negatives,
which are commonly observed in yeast genetic reporter assays. Interference from endogenous
proteins may cause either false positives or false negatives. Autoactivator and some “sticky” or
“promiscuous” proteins are common sources of false positives. In addition, as the proteins under
investigation must be fused to the DBD or AD in the reporter systems, some physically
interactive, yet physiologically irrelevant, interactions might also be detected (26).
Recent explorations of protein-protein and protein:DNA interactions at genome- or
pathway-wide scale by use of yeast reporter systems have shown the frequent occurrence of false
negatives that had otherwise been severely underestimated (26,27). Factors that lead to false
negatives generally include weak interactions beyond detection limitations, proteins unstably
expressed and/or improperly folded in the host cell and/or unable to localize to the nucleus,
toxicity caused by heterologous expression of some hybrid proteins, and structural hindrance by
fused domains or epitope tags (26,28-30). Such issues with in vivo systems necessitate
confirmation of results by other independent methods.
Although there are certain disadvantages involved in the use of yeast genetic reporter
systems, the relative ease of manipulation, high efficiency and superior efficacy of these systems
have already made their use widespread. These genetic systems will surely continue to be useful
6
methods for investigation of the assembly and specificity of a variety of protein-protein and
protein:DNA interactions that provide the basis for further understanding of cell function.
1.2 Transcription factors Transcription factors are a large and diverse class of DNA-binding proteins that
specifically target DNA for transcriptional regulation of gene expression. They play a critical
role in the control of important physiological functions including cell development, growth, and
differentiation. Thus, aberrant expression or incorrect processing of transcription factors
contributes to the progression of a variety of diseases, including developmental abnormalities
and cancer (31).
Transcription factors are typically composed of an autonomous DBD responsible for
directing the protein to a specific DNA target site, and an effector domain mediating activation
or repression of targeted genes. In general, transcription factors function by binding to specific
DNA sequences located upstream of the gene promoter region in chromatin and induce or
repress gene expression by recruitment of appropriate global regulatory complexes including
RNA polymerase II, basal transcription factors, and other cofactors bound at the gene promoter
(31,32).
Because the DBD of transcription factors determines which promoter sequences are
bound and therefore, which genes are regulated, much attention has been focused on deciphering
the basis of protein:DNA recognition at the molecular level. Tremendously valuable information
on how different transcription factors bind their cognate DNA targets has been revealed with
advances in genetic and biochemical techniques. Particularly, the structures of protein:DNA
complexes determined by both X-ray and nuclear magnetic resonance (NMR) provide clues for
understanding the mechanism of protein:DNA recognition. However, analysis of the available
7
functional and structural data on DNA binding by transcription factors has shown that there is no
clear “recognition code” that underlies protein:DNA interactions (33-35).
Although no general rules exist in protein:DNA recognition, transcription factors can still
be grouped into families based on sequence and structural homologies within their DBDs. These
families include helix-turn-helix (HTH), zinc finger, basic region/leucine zipper (bZIP), bHLH,
and homeodomain (36,37). Of all these DNA binding proteins, the simple α-helix scaffold is
usually the common secondary structure untilized to bind to the DNA major groove (38). Several
excellent reviews summarize the general scaffolds that proteins use to recognize DNA and
discuss in detail the diversity of ways in which proteins can contact DNA (32,37-39).
Three classes of transcription factors are the focus of our interest: the bZIP family and
two families within the bHLH superfamily: the basic helix-loop-helix/leucine zipper (bHLHZ)
and basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS). All these motifs utilize a dimer of α-
helices to bind specific DNA sequences in the major groove. The following is a brief
introduction to these three classes of transcription factors.
1.2.1 The bZIP family The bZIP transcription factors comprise a large family of DNA binding proteins involved
in the regulation of DNA transcription. Fifty-three and sixty-seven bZIP proteins were identified
in the genomes of Homo sapiens (40) and Arabidopsis thaliana (41), respectively. All of these
factors bind to specific DNA sequences as homo- or heterodimers with the DNA interaction
surface, which is achieved by a region of basic amino acids that immediately precedes the
leucine zipper domain in the primary protein sequence. Members of the bZIP family are
exclusively eukaryotic nucleoproteins that are widely expressed in diverse cell types and tissues
and responsible for regulation of a variety of cellular processes including cell development,
proliferation, differentiation, apoptosis, and oncogenesis (42).
8
The α-helical bZIP motif, which forms hetero- and/or homodimers of α-helices of 60-80
residues, is the smallest and simplest protein structure that recognizes specific DNA sites with
high affinity (44,45). The classic domain swapping experiments between GCN4 and C/EBP
confirmed that sequence-specific DNA-binding activity resides in the basic region, and that
dimerization specificity is determined by the leucine zipper (46). The crystal structures of several
bZIP domains bound to their cognate sequences, including GCN4 bound either to the AP-1 (43)
or CRE sites (47,48), the Jun/Fos heterodimer (49), CREB (50), and PAP1 (51), provide clearer
images of DNA recognition by bZIP proteins.
Figure 1.2 Crystal structure of the GCN4 bZIP homodimer bound to the AP-1 DNA site (43). The double-stranded DNA is in light gray and the bZIP α-helices are in dark gray with the leucines in the fourth position of the heptads shown in the gray ball model. The basic region and the leucine zipper are labeled. The carboxyl-terminal leucine zippers of the two monomers pack together as a coiled coil, which gradually diverges to allow the amino-terminal basic region to follow the major groove of either DNA half site. (PDB ID: 1YSA)
Figure 1.2 presents the structure of the GCN4 homodimer bound to the AP-1 DNA site,
5'-TGACTCA (43). The entire structure is a dimer of continuous bipartite α-helices. In each
monomer, the N-terminal basic region, rich in positively charged residues, interacts with the
DNA site in a sequence-specific manner. Comparisons of DNA-binding basic regions show a
9
high degree of sequence similarity (51). Each basic region of the two monomers contacts a half-
site in the DNA major groove (45,52). A unique aspect of the basic region of bZIP proteins is
that it undergoes a coil-to-helix transition to form an α-helical extension of the leucine zipper
upon sequence-specific DNA binding (53-55); hence, bZIP transcription factors are not stable
and fully folded until binding to DNA.
The C-terminal leucine zipper region, which typically contains a leucine residue (or
occasionally other hydrophobic residues such as Met, Ile or Val) every seven amino acids, is
responsible for regulating dimerization stability and specificity. The two leucine zippers, one
from each monomer, dimerize to generate a parallel amphipathic coiled-coil to position the basic
region for specific DNA binding. The leucine zippers contain various heptad repeats, each of
which is composed of two α-helical turns or seven amino acids (56). The residues at the
dimerization interface, occupying the first and fourth positions of the heptad, are typically
hydrophobic, and the remaining positions are mostly polar or charged. The residues at the
dimerization interface dictate partner specificity together with neighboring residues near the
leucine zipper interface (40).
1.2.2 The bHLH superfamily The bHLH proteins form an important and versatile superfamily of eukaryotic
transcription factors found in organisms from yeast to human and are involved in diverse
fundamental biological processes, including cell cycle and developmental regulation, apoptosis
and homeostasis, and stress response pathways (57-60).
Compared to bZIP proteins, bHLH proteins utilize a similar mode of DNA binding: a
highly conserved structural motif organized into a DNA-binding basic region and a dimerization
domain. Same as for bZIP proteins, the N-terminal basic region of bHLH proteins, adjacent to
the dimerization domain, contacts the DNA recognition site. In contrast to the leucine zipper in
10
bZIP proteins, the dimerization domain of bHLH proteins is composed of two amphipathic
helices separated by a nonconserved loop typically 5-12 residues in length, forming a compact
hydrophobic four-helix bundle that positions the contiguous basic regions for DNA binding. The
loop separating the two helices leads to more flexibility in positioning the basic region on the
DNA site (61,62). In addition to the basic region that determines specificity of DNA recognition,
the loop and Helix 2 have been observed to make DNA contacts in some bHLH:DNA complexes
(57,61,63). Both the basic region and HLH dimerization domain are required for formation of
functional DNA binding complexes.
In addition to those bHLH proteins containing the bHLH domain only, there are two
other families of bHLH proteins: bHLHZ and bHLH/PAS proteins. Members of these two
families contain an additional structural motif contiguous with the bHLH domain: a leucine
zipper or PAS domain, which additionally regulates dimerization (65,66).
The bHLH proteins typically associate as homo- or heterodimers that recognize the
hexameric E-box DNA site (5'-CANNTG) (58,65). Figure 1.3 shows the crystal structure of
bHLH protein MyoD and bHLHZ protein Max (Myc-associated factor X) bound to their
corresponding DNA target sites, respectively. MyoD is a mammalian protein involved in
myogenesis (67), whereas Max is a member of the Myc/Max/Mad network of transcription
factors involved in cell proliferation, differentiation, and death (65,68). In the MyoD:DNA
complex, Helices 1 and 2 from each monomer are connected by an eight-residue loop and
participate in forming a parallel, left-handed, four-helix bundle, which allows the basic region
contiguous with Helix 1 to contact the DNA major groove, and the N-terminal basic region and
Helix 1 form a long uninterrupted α-helix (64). In contrast, in addition to the bHLH domain,
bHLHZ protein Max contains an additional secondary leucine zipper dimerization domain that is
contiguous with the C-terminus of Helix 2 in each monomer, forming a long seamless α-helix.
11
The two leucine zippers form a parallel, left-handed coiled coil in a similar manner to the leucine
zipper in the bZIP motif (61).
Figure 1.3 Crystal structure of the MyoD:DNA complex (64) (A) and Max:DNA complex (61) (B). Both MyoD and Max form a four-helix bundle with Helix 1 and Helix 2 from each monomer, allowing the basic region contiguous with Helix 1 to contact both sides of the DNA major groove. Max contains a secondary dimerization domain, the leucine zipper, which forms a long continuous α-helix with the preceding Helix 2 in each monomer. (A: PDB ID: 1MDY and B: PDB ID: 1AN2)
Similar to the basic region of the bZIP motif, the disordered basic regions of both bHLH
and bHLHZ proteins display an induced α-helical structure upon DNA binding. Furthermore, in
addition to contributing to the overall stability of bHLHZ proteins by adding substantial buried
surface area to the dimerization interface, the leucine zipper, not the HLH domain of bHLHZ
proteins, acts as the determinant of dimerization specificity (69,70), which is different from
bHLH proteins where the HLH domain determines dimerization preferences. This also indicates
12
the intrinsic subtle structural differences between the HLH domains of bHLH and bHLHZ
proteins (71).
Figure 1.4 Ribbon presentation of the Drosophila Period dimer. Each monomer comprises two tandemly organized PAS domains (PAS A and PAS B) and two additional C-terminal α helices (74). The two monomers are shown in dark gray and light gray, respectively. The disordered regions are depicted as dotted lines. (PDB ID: 1WA9)
In comparison, bHLH/PAS proteins contain structurally and functionally more
complicated PAS domain, which typically consists of 250–350 amino acids in conjunction with
the HLH domain and contains two adjacent, highly degenerate 50 amino-acid subdomains
termed PAS A and PAS B. The PAS domain is a well conserved signaling module that responds
to environmental and developmental stimulus. In addition, the PAS domain is also involved in
protein dimerization and specification of heterodimerization partner (72,73). Figure 1.4 presents
the crystal structure of an N-terminal Drosophila Period (Per) fragment comprising PAS A and
PAS B domains as well as two additional C-terminal α helices. Structural Analysis revealed a
noncrystallographic dimer mediated by intermolecular interactions of PAS A with PAS B and C-
terminal α helix (74). Unlike bHLH and bHLHZ families, in which quite a few high resolution
structures of protein:DNA complexes have been solved, including MyoD (64), E47 (75), USF
(76), Max homodimers (61,77) and heterodimers with Myc (78) and Mad (70), the structural data
13
of the complete DNA-binding and dimerization regions of any bHLH/PAS family member is still
unavailable. However, the bHLH domains of bHLH/PAS proteins are predicted to be structurally
similar and adopt a similar DNA-binding mode as does the closely related bHLH and bHLHZ
proteins based on their homologous primary sequences and DNA binding specificities (59).
Typical members of the bHLH/PAS family include the aromatic hydrocarbon receptor
(AhR, also know as dioxin receptor, DR), a mammalian protein that regulates xenobiotic
metabolizing enzymes in response to environmental contaminants such as prototypical 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD); hypoxia-inducible factor (HIF) 1α, a protein involved in
mediating cellular responses to hypoxia; the AhR nuclear translocator (Arnt), a central regulator
that functions by associating with AhR or HIF; and single-minded (Sim) and Period proteins,
Drosophila proteins that are involved in central nervous system midline development and
circadian rhythms, respectively (59,65).
In addition to their significant signal transduction activity under physiological conditions,
there are several unique characteristics involved in DNA recognition by the AhR/Arnt
heterodimer. First, biochemical and genetics studies have determined that the AhR/Arnt
heterodimer functions by binding the asymmetric xenobiotic response element (XRE, also
known as dioxin response element, DRE) site, 5'-TNGCGTG (79,80), which is in contrast to
bHLH and bHLHZ proteins that usually target the consensus hexameric E-box DNA site. AhR
targets the 5'-TNGC half site, and Arnt the 5'-GTG half site. Arnt is able to homodimerize and
target the symmetric E-box site; the amino acids critical for E-box recognition by Max and USF
are conserved in Arnt (81). However, AhR is unable to form homodimers and can only function
through heterodimerization with Arnt. Given the fact that the basic region of AhR is proline rich,
AhR probably does not undergo the characteristic coil-to-helix transition upon DNA binding (63).
14
Therefore, AhR and Arnt provide interesting targets for exploration of heterodimeric recognition
of DNA.
1.2.3 Heterodimeric transcription factors increase regulatory diversity All three types of DNA binding proteins discussed above function by forming homo- or
heterodimers. As a matter of fact, many prokaryotic and eukaryotic transcriptional factors are
dimeric DNA-binding proteins. This fact, from the perspective of evolution, probably indicates
that dimerization capability of transcriptional factors might lead to the regulation of biological
processes more specific and efficient while genomically economical.
Heterodimeric formation provides several potential advantages over monomer and
homodimer counterparts. First, heterodimeric formation of two proteins that have distinct DNA-
binding specificities in their homodimeric form could create a complex with a novel binding
specificity, therefore expanding the repertoire of potential DNA sequences that a family of
factors can bind (82). Second, heterodimeric formation may also form more subtle interactions
within gene regulatory regions, thereby generating diverse transcriptional control from a limited
number of transcription factors (82). For instance, protein dimers within one family can bind to
DNA sites containing common half-site sequences that differ in polarity and inter-half-site
separation (32). This provides a fine tuning of gene expression by competition of different
complexes able to bind the same or similar DNA target sequences. Third, heterodimeric
formation can increase specificity of gene regulation. Some tissue-specific DNA-binding
proteins are incapable of forming homodimers and preferentially heterodimerize to exert
function with a constitutively expressed DNA-binding protein capable of forming either homo-
or heterodimers. In this way, the transcriptional regulation is tightly and specifically controlled.
Finally, different combinations of activation and/or repression domains brought together by
heterodimeric formation at regulatory DNA sequences also allows the change of regulatory
15
properties of the specific protein bound at that fixed DNA site (36). This change of regulation
would not be possible if the DNA-binding proteins functioned only as monomers or homodimers.
Therefore, the mechanisms that control the formation of heterodimerzation of DNA-
binding proteins at the molecular level may help us further understand the relationship between
protein structure and its resultant functional activity and may potentially guide the development
of useful tools to promote or repress the corresponding function.
1.3 Studies of bHLH superfamily proteins in yeast genetic reporter systems Yeast genetic reporter systems were utilized to study bZIP and bHLH family proteins
shortly after the development of the Y2H and Y1H. Because transcriptional activation studies of
human oncoproteins and other transcription factors in mammalian cells have often been hindered
by the presence of endogenous proteins, the study of these proteins in yeast provides an obvious
advantage, as the chance of endogenous interference by their homologous counterparts in yeast is
greatly decreased.
Moreover, a more open chromatin structure in yeast than that in higher eukaryotes means
a higher level of DNA accessibility, a critical point as gene activation relies on the binding of
transcription factors to their accessible consensus sites (83,84). Analysis of the crystal structure
of the yeast nucleosome core particle reveals that the overall principles of DNA organization in
the nucleosome are conserved between lower and higher eukaryotes. However, yeast
nucleosomes are likely to be subtly destabilized as compared with nucleosomes from higher
eukaryotes. As a result, much of the yeast genome is constitutively open for transcription, as
opposed to the small percentage of actively transcribed genes at any given time in the cells of
higher eukaryotes (83). In addition, the activation of gene expression in yeast genetic reporter
systems is achieved through the use of activation domains such as GAL4 or VP16 AD. These
16
domains have been shown to interact with factors implicated in the perturbation of chromatin
structure, leading to transcriptional activation (85). Therefore, yeast genetic reporter systems
provide a simplified model for the study of DNA-binding function of mammalian transcription
factors, as complications stemming from the contribution of chromatin structure to gene
regulation is not a major concern in yeast.
In addition to the studies mentioned in Section 1.1.2 of this Chapter, there are also a
number of studies on mammalian transcription factors using yeast genetic assays. For instance,
the activities of mammalian bHLH proteins MyoD and muscle regulatory factor (MRF) were
examined in yeast, demonstrating that in vivo yeast systems can be a useful approach to facilitate
functional studies of bHLH transcription factor regulation (86). This work was followed by the
study of regulation of bHLHZ proteins Myc/Max-mediated transactivation in yeast (87). Another
elegant use of the yeast genetic system was to decipher the interplay between two mammalian
bHLHZ proteins, Mad1 and Max, and the yeast protein Sin3. The study revealed that Mad1 and
Max form a complex with inhibitory Sin3 and therefore, are unable to activate reporter gene
expression in yeast, as the expression of both proteins in a SIN3-knockout yeast strain activated
reporter gene expression. These experiments provided direct evidence that yeast protein Sin3 can
repress transcription through interaction with DNA-binding proteins by the same repression
mechanism as its mammalian counterpart (88).
Several yeast genetic reporter systems have been developed to study signal transduction
by bHLH/PAS transcription factors AhR and Arnt. A study of transcription factor AhR in a yeast
model system provided the first genetic evidence that heat shock protein 90 (Hsp90) is critical to
AhR signaling (89), which was confirmed by Poellinger et al. in a similar yeast reporter system
(90). A cDNA library screening study revealed that Arnt was able to interact with the AhR
bHLH/PAS in yeast (91). This work was followed by two similar systems developed to
17
investigate the AhR/Arnt heterodimer's (full-length or bHLH/PAS domain) response to different
AhR ligands in the Y1H system (92,93).
1.4 Scope The thesis is organized as follows. Chapters 2-4 are entire manuscripts that comprise
three different projects. With the intention of maintaining the integrity of each manuscript as a
published entity, only necessary changes for each chapter, such as bibliographic referencing and
figure formatting, have been made for thesis organization purposes. Therefore, some unavoidable
redundancy will appear in the text. In Chapter 2, a modified yeast one-hybrid (MY1H) system is
described that was developed for in vivo investigation of protein-protein and protein:DNA
interactions. This single-plasmid expression system was validated by use of the well-
characterized DNA-binding protein p53 and its inhibitory partners, large T antigen (LTAg) and
53BP2 (in press in BioTechniques). Chapter 3 focuses on turning a false negative protein
heterodimer:DNA interaction into a true positive control in the MY1H system. Negative signals
were observed when we initially coexpressed the heterodimeric bHLH/PAS domains of AhR and
Arnt that target the cognate XRE sequence as a positive control in the study of XRE-binding
proteins in the MY1H system. By increasing the number of DNA target sites upstream of the
reporter genes and increasing the number of activator domains fused to the proteins of interest,
false negative results were salvaged into true positives (in press in Analytical Biochemistry).
Analysis of how a pair of bHLHZ-like hybrid proteins, AhRJunD and ArntFos, undergoes
target sequence-mediated heterodimerization upon specific DNA binding in MY1H is described
in Chapter 4. This current manuscript focuses on the in vivo yeast assays, and the final
manuscript will be submitted upon completion of in vitro fluorescence anisotropy titrations and
western blotting assays; these bacterially expressed proteins are currently being expressed and
purified. Although this work is not yet completed, Chapter 4 maintains the format of the previous
18
two chapters that are in press. Chapter 5 summarizes the thesis results and recommends future
directions for research. Chapter 6 comprehensively discusses all the materials and methods that
are used in this thesis. Appendices A-C comprise the supplemental materials supplied with the
manuscripts presented in Chapters 2-4. Appendix D lists and describes all the oligonucleotides
used in the thesis.
It is noted that the MY1H system developed in this thesis could be particularly useful for
testing the effects of a new protein, or mutant versions of a protein, on the DNA-binding activity
of a transcription factor. Our lab applied this system to explore the repression of the Max:E-box
DNA site interaction with several competitor mutants of Max. The results revealed different
repression capabilities of these designed Max mutants on the Max:E-box interaction in vivo. This
manuscript is in preparation, and I am the second author.
19
Chap 2ter
Design of a single plasmid-based modified yeast one-hybrid system for investigation of in vivo protein-protein and protein:DNA
interactions Gang Chen, Lisa M. DenBoer, and Jumi A. Shin
(Short running title: Modified Y1H for identification of protein-protein/protein:DNA interactions)
Contributions:
I initiated this project. I performed part of the plasmid construction, part of the 3-AT
titration assays, and all of the X-gal colony-lift assays and ONPG assays. Lisa DenBoer
performed most of the plasmid construction and 3-AT titration assays. I completed the data
interpretation. Lisa DenBoer provided intellectual input and editing to the manuscript. Jumi Shin
provided supervision and intellectual input to the project and manuscript.
This chapter is adapted from the original manuscript accepted by BioTechniques, with
necessary changes for thesis organization purposes.
Authors retain copyright of manuscript published in BioTechniques, as per publisher's
agreement.
20
2.1 Abstract We have developed a modified yeast one-hybrid system (MY1H) useful for in vivo
investigation of protein-protein and protein:DNA interactions. Our single-plasmid expression
system is capable of differential protein expression levels; in addition to a GAL4 activation
domain (AD) fusion protein, a second protein can be coexpressed at either comparable or higher
transcriptional levels from expression vectors pCETT or pCETF, respectively. This second
protein can play a structural, modifying, or inhibitory role that restores or blocks reporter gene
expression. Our MY1H was validated by use of the well-characterized DNA-binding protein p53
and its inhibitory partners, large T antigen (LTAg) and 53BP2. By coexpressing LTAg or 53BP2
at comparable or higher levels than the GAL4AD-p53 fusion in the MY1H, we show that DNA
binding of p53 decreases by different, measurable extents dependent on the expression level of
inhibitory partner. As with the traditional Y1H, our system could also be used to investigate
proteins that provide coactivational or bridging functions and to identify novel protein- or DNA-
binding partners through library screening. Our MY1H provides a system for investigation of
simultaneous protein-protein and protein:DNA interactions, and thus, is a useful addition to
current methods for in vivo investigation of such interactions.
21
2.2 Introduction
Figure 2.1 Five different types of interactions between proteins and DNA can be detected with the MY1H system. Top: If P1 is able to target the binding element E, the second protein P2 can be expressed to block the DNA binding region of P1, directly bind to binding element E, or recruit repressors to P1, thereby inhibiting reporter gene expression. Bottom: If P1 is unable to target the binding element E, P2 can be expressed as a bridging protein or coregulatory accessory protein, or it can modify the structure of P1 to enable DNA binding, thereby restoring reporter gene expression. As a coregulatory protein, P2 may function with or without making direct contact with P1.
Gene expression is a sophisticated, finely tuned process that involves the regulated
interactions of multiple proteins with promoter and enhancer elements. A variety of approaches
are currently employed in the study of these interactions, including phage display and yeast-
based assays, as well as other biophysical and biochemical methods (94). The yeast one-hybrid
22
system (Y1H), a variant of the yeast two-hybrid system (Y2H) (1), is a powerful and commonly
used in vivo genetic assay for identification of protein:DNA interactions. The Y1H is useful for
isolation of genes encoding proteins that bind to cis-acting regulatory elements and for further
characterization of known protein:DNA interactions, whereas the Y2H allows detection of
protein-protein interactions (1-3).
In many cases, protein-protein and protein:DNA interactions are intertwined in vivo:
DNA-binding proteins are often modulated by the recruitment of accessory proteins that cannot
bind DNA directly but rather serve to repress or coactivate transcription through the formation of
transcriptional complexes (95,96). Most bZIP and bHLH families, such as Jun-Fos (97), Myc-
Max (98), and the classic bacteriophage λ repressor and Cro proteins, belong to this class of
transcription factors. A number of yeast genetic approaches have been reported for investigation
of a protein in complex with a known partner, thereby forming a heterodimeric complex that
binds DNA; these complexes may include one partner lacking intrinsic DNA-binding capability
enabled by dimerization with an accessory protein (18-23). These studies have traditionally used
two separate plasmids for expression of the two different proteins.
We have developed a single plasmid-based modified Y1H system (MY1H) useful for
examination of both protein-protein and protein:DNA interactions in vivo. In addition to an AD
fusion protein, a second protein is coexpressed at either comparable or excess levels. The
interaction of this second protein with the AD fusion via cooperative oligomerization, structural
modification, or inhibition, can restore or block reporter gene expression (Figure 2.1). We chose
to validate our MY1H using the extensively studied interactions of DNA-binding protein p53
and its inhibitory partners, Simian Virus 40 (SV40) LTAg and 53BP2 (99-103). Both LTAg and
53BP2 inhibit wild-type p53 function through a protein-protein interaction at the DNA-binding
domain of p53, thereby preventing p53 from binding to its consensus DNA target site (also
23
known as the p53 cis-acting DNA target element) (102-106). It was reported that the p53-53BP2
complex forms with a dissociation constant (Kd) of about 30 nM as determined by surface
plasmon resonance (106). However, isothermal titration calorimetry revealed that p53 interacted
with 53BP2 with a Kd of 2.2 μM (107), so there is significant discrepancy in these quantitative
measurements. No quantitative data is available regarding the relative affinity of the LTAg-p53
interaction, to the best of our knowledge. The well-characterized p53-LTAg and p53-53BP2
interactions—protein-protein interactions that modulate DNA-binding ability—provide an ideal
system for validation of our MY1H.
Our MY1H combines the features of the Y1H and Y2H systems, and also extends their
scopes, such that simultaneous protein-protein and protein:DNA interactions can be investigated;
hence, this MY1H is speculated to have broad utility (Figure 2.1) and may provide a widely
applicable approach for investigation of various types of interactions, including heterodimer-
DNA interactions or the effects of different protein modifiers on the DNA-binding capability of a
transcription factor.
2.3 Materials and Methods Reagents were purchased from BioShop Canada (Burlington, ON), enzymes were
purchased from New England Biolabs (Pickering, ON), and oligonucleotides were synthesized
by Operon Biotechnologies (Huntsville, AL) unless otherwise stated.
2.3.1 Bacterial and yeast strains Escherichia coli DH5α (Stratagene, La Jolla, CA) or dam-/dcm- C2925H (New England
Biolabs) was used for standard cloning and for rescue of plasmids from yeast cells.
Saccharomyces cerevisiae YM4271 [MATa, ura3-52, his3-200, ade2-101, lys2-801, leu2-3, 112,
trp1-901, tyr1-501, gal4-∆512, gal80-∆538, ade5::hisG] was used for plasmid construction via
homologous recombination and reporter strain construction. Two yeast reporter strains,
24
YM4271[p53HIS] and YM4271[p53BLUE], were created according to the Matchmaker™ One-
hybrid System User Manual (Clontech, Palo Alto, CA) for reporter assay analysis in the MY1H.
These two strains contain three tandem copies of the consensus p53 binding site upstream of the
HIS3 and lacZ reporter genes, respectively.
2.3.2 Transformation, DNA preparation, and plasmid rescue Recombinant plasmids were transformed into E. coli by the standard TSS procedure
(108). Plasmids were isolated from bacteria using the Wizard® Plus SV Miniprep DNA
Purification System (Promega, Madison, WI). Yeast transformations were performed using
either the standard lithium acetate method (Yeast Protocols Handbook, Clontech) or the Frozen-
EZ Yeast Transformation II™ Kit (Zymo Research, Orange, CA). Transformants were selected
by leucine prototrophy. Isolation of yeast plasmids was performed using the Zymoprep™ II
Yeast Plasmid Miniprep Kit (Zymo Research). PCR reactions were performed using Phusion™
high-fidelity DNA polymerase (New England Biolabs). PCR products and DNA fragments for
cloning were purified using the QIAquick Spin Kits or MinElute Kits (Qiagen, Mississauga, ON).
2.3.3 Plasmid Construction All new constructs were confirmed by dideoxynucleotide DNA sequencing on an ABI
(Applied Biosystems) 3730XL 96 capillary sequencer at the DNA Sequencing Facility in the
Centre for Applied Genomics, Hospital for Sick Children (Toronto, ON).
2.3.3.1 pGAD424-MCS I and pGAD424-MCS II pGAD424-MCS I and pGAD424-MCS II were constructed by homologous
recombination (109) in YM4271 to replace the original multiple cloning site (MCS) in
pGAD424 (110) using the 6.6 kb EcoR I/Pst I pGAD424 fragment along with the CE4MCS
fragment and 679 bp BstZ17 I/Mlu I pGADT7 fragment, respectively. The CE4MCS fragment
25
was assembled by self-priming PCR (111) using oligonucleotides 2-1 to 2-6 (Table 2.1; all
oligonucleotides discussed in Chapter 2 are listed in Table 2.1 and also described in Table D.1 of
Appendix D); the fragment contains a T7 promoter, a c-Myc epitope tag, and a multiple cloning
site (MCS I) with recognition sequences for five restriction enzymes (Sac II, Sal I, BssH II, Xba I,
and Bcl I). Similarly, the BstZ17 I/Mlu I pGADT7 fragment contains a T7 promoter, a HA
epitope tag, and a multiple cloning site (MCS II) with recognition sequences for six restriction
enzymes (EcoR I, Sma I, BamH I, Sac I, Xho I, and Pst I).
Table 2.1 Oligonucleotides used in this study No Sequence
2-1 ACTATCTATTCGATGATGAAGATACCCCACCAAACCCAAA
2-2 GGCGCTCGCCCTATAGTGAGTCGTATTAAAGATCTCTTTTTTTGGGTTTGGTGGGGTATC
2-3 CTCACTATAGGGCGAGCGCCGCCATCATGGAGGAGCAGAAGCTGATCTCAGAGGAGGACC
2-4 GCGCGCACCTTGTCGACCGCGGCCTCCATGGCCATATGCAGGTCCTCCTCTGAGATCAGC
2-5 GCGGTCGACAAGGTGCGCGCTCTAGATGATCATGAATCGTAGATACTGAAAAACCCCGCA
2-6 ATGCACAGTTGAAGTGAACTTGCGGGGTTTTTCAGTATCT
2-7 AGAAAGGTCGAATTGGGTACCGCCGCCAATAAAGAGATCTTTAAT
2-8 CTCGCCCTATAGTGAGTCGTATTAAAGATCTCTTTATTGGCGGCG
2-9 AAAGACGTCGCATGCAACTTCTTTTCTTT
2-10 ATTGACGTCAAGCTTGCATGCCGGTAGAGGT
2-11 AAAGACGTCCCTGCAGGTCGAGATCCGGGA
2-12 AAAAGTCGACCCTGTCACCGAGACCCCTGG
2-13 ACGCTCTAGATCAGTCTGAGTCAGGCCCCA
2-14 AAAGAATTCGGAACTGATGAATGGGAGCAG
2-15 AAAGGATCCTTATGTTTCAGGTTCAGGGGGAG
2-16 AAAGAATTCCCGCCTGAAATCACCGGGCAG
2-17 AAAGGATCCTCAGGCCAAGCTCCTTTGTCTT
*Oligonucleotide sequences are shown in 5’ to 3’ direction. Restriction sites used for cloning are in bold.
26
2.3.3.2 pGAD424-MCS IIΔAD and pGADT7ΔAD In pGAD424-MCS IIΔAD and pGADT7ΔAD, the GAL4AD was deleted while the open
reading frame was maintained. To create these two recombinant plasmids, the FINALREC
fragment was assembled by mutually primed synthesis (112) using oligonucleotides 2-7 and 2-8.
The 5’ and 3’ ends of the FINALREC fragment contain 30 and 35 bp homology, respectively, to
both Bgl II-linearized pGAD424-MCS II and Bgl II-linearized pGADT7. YM4271 was
cotransformed with either Bgl II-linearized pGAD424-MCS II or Bgl II-linearized pGADT7 and
the FINALREC fragment to give rise to pGAD424-MCS IIΔAD and pGADT7ΔAD, respectively.
2.3.3.3 pCETT and pCETF The T2 fragment was amplified with oligonucleotides 2-9 and 2-10 from pGAD424-MCS
IIΔAD. The F2 fragment was amplified with oligonucleotides 2-10 and 2-11 from pGADT7ΔAD.
The amplified fragments T2 and F2 were digested with Aat II, treated with alkaline phosphatase,
and then ligated into the Aat II site of pGAD424-MCS I to generate pCETT and pCETF (Figure
2.2), respectively.
2.3.3.4 pCETT/53 and pCETF/53 The sequence encoding amino acids 72-390 of the murine p53 gene was amplified from
pGAD53m (Clontech) using oligonucleotides 2-12 and 2-13. This fragment was inserted
between the Sal I and Xba I sites of pCETT and pCETF to construct pCETT/53 and pCETF/53,
respectively.
2.3.3.5 pCETT/53/T and pCETF/53/T The fragment encoding amino acids 87-708 of SV40 LTAg was amplified from
pGADT7/T (Clontech) with oligonucleotides 2-14 and 2-15. This fragment was inserted into the
27
EcoR I and BamH I sites of pCETT/53 and pCETF/53 to construct pCETT/53/T and
pCETF/53/T, respectively.
2.3.3.6 pCETT/53/BP2 and pCETF/53/BP2 The cDNA plasmid pGBT9, which contains the human 53BP2 sequence, was kindly
given to us by Dr. Kuniyoshi Iwabuchi (Kanazawa Medical University, Japan) (103). The
sequence encoding amino acids 768-1005 of 53BP2 was amplified with oligonucleotides 2-16
and 2-17 and inserted between the EcoR I and BamH I sites of pCETT/53 and pCETF/53 to
generate pCETT/53/BP2 and pCETF/53/BP2, respectively.
2.3.4 3-AT titration analysis HIS3 gene expression was measured by growing transformed yeast cells on selective
media lacking leucine, uracil, and histidine. Activity of the HIS3 reporter was quantified as
survival rates of yeast transformants on plates containing increasing amounts of 3-aminotriazole
(3-AT), a competitive inhibitor of the His3 protein.
Transformed yeast cells were initially grown at 30 °C with shaking in SD/-L media for 2
days or until OD600 >1.5 was reached, and then used to inoculate a fresh culture of SD/-L media.
This secondary culture was grown overnight until OD600 1.0-1.3 was reached. An aliquot of the
secondary culture was resuspended in YPDA to give a starting OD600 ~0.2. The YPDA culture
was then grown for 3-5 hrs until OD600 0.60-0.65 was reached. The culture was then diluted by a
factor of 4000 and 100 μl of the diluent was plated on SD/-H/-L/-U plates containing increasing
3-AT concentrations ranging from 0 to 80 mM. Individual colonies were counted after 5 days
growth at 30 °C. The survival rate of a specific transformant was calculated as the number of
colonies on the SD/-H/-L/-U plate containing a specific 3-AT concentration divided by the
28
number of the colonies on the control SD/-L/-U plate. This assay was performed in triplicate and
independently repeated at least 3 times in order to ensure reproducibility.
2.3.5 X-gal colony-lift filter assay and ONPG liquid assay The X-gal colony-lift filter assay and ortho-nitrophenyl-galactoside (ONPG) liquid assay
were performed according to the protocols provided in the Yeast Protocols Handbook (Clontech)
with the following modifications: in the X-gal assay, the cells were subjected to two cycles of
freeze/thaw in order to lyse the cells. In the ONPG assay, yeast cells were grown as described
above for the 3-AT titration assay before harvesting for lysis. Results are presented as mean
values ± S.E.M. of 3-4 independent experiments, each performed in triplicate.
2.4 Results
2.4.1 Design of protein expression vectors In order to examine protein-protein and protein:DNA interactions simultaneously in a
single yeast genetic system, two GAL4AD fusion vectors, pCETT and pCETF, were constructed
based on pGAD424, the protein expression plasmid provided in the Matchmaker™ One-Hybrid
System (Clontech). In both pCETT and pCETF, the gene encoding the AD fusion protein is
inserted into MCS I where transcription is under the control of a truncated ADH1 promoter,
leading to low protein expression levels (113,114). A second gene inserted into the second
multiple cloning site, MCS II, can also be expressed from the same plasmid under the control of
the truncated ADH1 promoter identical to that in MCS I. In pCETF, MCS II is under the control
of the full-length ADH1 promoter, leading to higher transcription levels (114,115).
Our plasmid design provides the option of allowing the second protein to be expressed at
a comparable level with the AD fusion protein (pCETT; truncated ADH1 promoters in MCS I
and II) or in excess (pCETF; truncated ADH1 promoter in MCS I, full-length ADH1 in MCS II).
In addition, these plasmids were designed with different epitope tag-coding sequences upstream
29
from each multiple cloning site, and a T7 promoter upstream of both multiple cloning sites to
allow in vitro transcription and translation.
Figure 2.2 Plasmids pCETT and pCETF were constructed for coexpression of two proteins in a yeast model system. The two vectors have unique restriction sites located in both the MCS I and MCS II regions. MCS I is at the 3'-end of the open reading frame for the GAL4 AD sequence allowing a fusion protein combining amino acids 768–881 of the GAL4 AD and the cloned protein of interest to be expressed at low levels from a truncated constitutive ADH1 promoter. The expression of genes inserted into MCS II is controlled by either the truncated ADH1 promoter (pCETT) or full-length ADH1 promoter (pCETF). Therefore, a second protein can be coexpressed at either low levels (pCETT) or high levels (pCETF). Both vectors also contain a T7 promoter at both MCS regions, a c-Myc epitope tag at MCS I, and an HA epitope tag at MCS II.
2.4.2 Coexpression of LTAg or 53BP2 decreases the transactivation potential of GAL4AD-p53 in the MY1H
In our MY1H, we used three assays to measure the inhibitory strengths of LTAg and
53BP2 on binding of the p53 DNA consensus site by GAL4AD-p53, including titration on
inhibitory 3-AT-containing media (HIS3 assay) and two colorimetric lacZ reporter-based assays:
qualitative X-gal colony-lift filter assay and quantitative ONPG liquid assay. All three assays
showed that the ability of GAL4AD-p53 to activate transcription of reporter genes was adversely
affected when coexpressed with LTAg or 53BP2 in the MY1H.
30
Figure 2.3 3-AT titrations reveal that the survival rates of transformants decreases when the inhibitory proteins are expressed. YM4271[p53HIS] cells were transformed with A) pCETT (□), pCETT/53 (◊), pCETT/53/T (○) or pCETT/53/BP2 (Δ) or B) pCETF (□), pCETF/53 (◊), pCETF/53/T (○) or pCETF/53/BP2 (Δ). Key for labels as follows. pCETT and pCETF: negative controls, no protein expression. pCETT/53 and pCETF/53: positive controls, expression of GAL4AD-p53 from MCS I only. pCETT/53/T and pCETF/53/T: low-level expression of GAL4AD-p53 from MCS I, low- or high-level expression of LTAg from MCS II. pCETT/53/BP2 and pCETF/53/BP2: low-level expression of GAL4AD-p53 from MCS I, low- or high-level expression of 53BP2 from MCS II. Cells were grown to exponential phase in YPDA media and plated at equal densities on selective media containing increasing concentrations of 3-AT. Individual colonies were counted after 5 days growth at 30 °C. The survival rate of a specific transformant was calculated as the number of colonies on the SD/-H/-L/-U plate containing a specific 3-AT concentration divided by the number of the colonies on the control SD/-L/-U plate. The assay was conducted in triplicate and independently repeated at least 3 times. Data is presented as average values ± standard error measurement.
We titrated the yeast transformants on SD/-H/-L plates containing 0 to 80 mM 3-AT (3-
amino-1,2,4-triazole) and plotted the survival rates of each transformant at increasing 3-AT
concentrations (Figure 2.3). Survival rates in the presence of 3-AT correlate with transcriptional
activity (116). Therefore, lower survival of transformants on 3-AT-containing plates indicates
stronger inhibitory strengths of LTAg or 53BP2 on the ability of GAL4AD-p53 to bind the p53
consensus DNA site. As expected, the survival rates of yeast cells transformed with pCETT or
pCETF decrease sharply as 3-AT concentration increases and reach zero at 20 mM 3-AT, while
cells transformed with pCETT/p53 or pCETF/p53 only begin to show decreased survival at 3-AT
concentrations over 70 mM. In contrast, when LTAg or 53BP2 is coexpressed with GAL4AD-
p53, an immediate decrease in cell survival is observed at 10-20 mM 3-AT and continues to
31
decline at higher 3-AT concentrations, consistent with the inhibitory role both LTAg and 53BP2
play in the DNA-binding ability of p53.
The sensitive X-gal colony-lift filter assay corroborates the 3-AT titration results in the
HIS3 assay (Figure 2.4). In the colony-lift assay, reporter gene activation is visualized by the
blue chromophore released by the action of β-galactosidase encoded by the lacZ gene.
Transformants expressing only GAL4AD-p53 become blue very quickly (within 10 min, Figure
2.4B and F), and continue to increase in intensity, becoming vivid blue after 45 minutes. When
LTAg or 53BP2 is coexpressed at low expression levels from pCETT (Figure 2.4C and D), blue
color only begins to appear after 15 minutes and is considerably less intense after 45 minutes,
indicating discernible inhibition of reporter gene expression. Higher expression of either LTAg
or 53BP2 from pCETF (Figure 2.4G and H) inhibits lacZ transcription to a much greater extent,
as only a faint blue color is achieved after 45 minutes.
Figure 2.4 Colony-lift filter assay indicates LTAg and 53BP2 inhibit DNA binding of p53 to different extents. YM4271[p53BLUE] cells were transformed with A) pCETT; B) pCETT/53; C) pCETT/53/T; D) pCETT/53/BP2; E) pCETF; F) pCETF/53; G) pCETF/53/T; or H) pCETF/53/BP2 and plated on SD/-L/-U plates. The X-gal colony-lift assay was performed after 4 days growth at 30 °C. Photos were taken after 45 min incubation at 30 °C. See caption for Figure 2.3 for the key to labels.
32
The quantitative ONPG assay further corroborates the qualitative results obtained from
both the 3-AT titration and the colony-lift assays. Expression of GAL4AD-p53 from either
pCETT or pCETF leads to comparable β-galactosidase activities (Figure 2.5), demonstrating that
expression from MCS I is not affected by the different ADH1 promoters (truncated vs. full-length)
that control the transcription of genes cloned into MCS II. When no gene is cloned into MCS II,
a nonsense protein (76 residues) from the cloning vector is expressed. We further tested the
effect of this nonsense protein by assay of β-galactosidase activity and compared these results to
those obtained when using singly transformed yeast expressing GAL4AD-p53 from the original
pGAD53m plasmid supplied by Clontech. The results of this assay are not statistically different,
suggesting that this nonsense protein expressed from pCETT or pCETF, regardless of its high or
low expression levels, does not affect GAL4AD-p53 in either an enhancing or inhibitory fashion,
nor does its expression adversely affect normal yeast growth (data not shown).
Figure 2.5 Histogram comparing the effects of different expression levels of inhibitory proteins LTAg and 53BP2 on DNA binding by p53. Quantitative assessment of β-galactosidase activity of the GAL4AD-p53 fusion in yeast strain YM4271[p53BLUE] in the presence/absence of high/low expression of 53BP2 or LTAg. Vertical axis indicates the mean values of β-galactosidase units. Error bars represent standard error from at least three independent trials conducted in triplicate. See caption for Figure 2.3 for the key to histogram labels.
33
Low level coexpression from pCETT of GAL4AD-p53 and LTAg (pCETT/53/T, Figure
2.5) gives a ~3.5-fold decrease in β-galactosidase activity compared with the same cells
expressing GAL4AD-p53 alone (pCETT/53, Figure 2.5), compared to a ~26-fold decrease when
LTAg is expressed from the full-length ADH1 promoter in pCETF (pCETF/53/T, Figure 2.5).
Similar results were observed for the coexpression of GAL4AD-p53 and 53BP2: a minimal
decrease in β-galactosidase activity was observed when 53BP2 was expressed from pCETT
(pCETT/53/BP2, Figure 2.5), while a 36-fold decrease was observed when using pCETF
(pCETF/53/BP2, Figure 2.5). As expected, expression of either LTAg or 53BP2 alone does not
result in reporter gene activation (data not shown).
2.5 Discussion
2.5.1 The DNA-binding activity of p53 and its interaction with LTAg or 53BP2: comparison of MY1H observations with earlier studies
In our MY1H system, the decrease in positive signal from transactivation of GAL4AD-
p53 requires the interaction of LTAg or 53BP2 with GAL4AD-p53: LTAg and 53BP2 prevent
the DNA binding of p53 in vivo, thereby leading to decreased activation potential. This
conclusion is supported both by our experimental data and by evidence gained from previously
published studies. Firstly, as discussed above, previous studies have already proven that LTAg
and 53BP2 inhibit DNA binding of p53 by binding to and masking p53's DNA-binding domain
(102-106). Second, we observed by the HIS3 reporter assay that neither LTAg nor 53BP2
interacts with the p53 consensus DNA site (data not shown), which excludes the possibility that
LTAg or 53BP2 inhibits transcription of GAL4AD-p53 through occupying the p53 DNA
consensus target. Third, there is no evidence that LTAg or 53BP2 can interact with GAL4AD,
indicating the unlikelihood of LTAg or 53BP2 interference with GAL4AD function. Fourth, no
abnormal growth of yeast upon expression of either LTAg or 53BP2 was observed in our
34
experiments, and none was reported by other labs in their experiments on p53 interactions with
either LTAg or 53BP2 (103,104,117). In our MY1H, all three assays provide convincing in vivo
evidence that either LTAg or 53BP2 excludes p53 from binding to its DNA consensus sequence.
Interestingly, all three assays show that at low expression levels, the inhibitory activity of
53BP2 is not vastly different from the positive control expressing only GAL4AD-p53, whereas
LTAg shows strong inhibition of reporter gene activation (Figures 2.3-2.5). At high expression
levels, both 53BP2 and LTAg efficiently inhibit transcription potential of the reporter gene,
leading to comparable minimal levels of reporter gene activity; the 3-AT titration assay shows,
however, that LTAg still appears to be a more effective inhibitor at low and high expression
levels (Figure 2.3). Although these observations suggest that LTAg may be a more capable
inhibitor of transcription potency than 53BP2, they do not necessarily prove that LTAg inhibits
the binding of p53 to its consensus site more efficiently than does 53BP2 due to the complexity
of the in vivo system and the inherent differences between LTAg and 53BP2, including size,
ability to permeate the yeast nucleus, and potential differences in cellular concentrations. Given
such differences between dissimilar proteins in the in vivo environment and that truly
quantitative comparisons of data obtained from in vivo genetic systems is impractical (25), a
more appropriate and reliable use of our MY1H would be, for example, the in vivo comparison
of the effect of several mutant versions of a targeted protein on the DNA-binding ability of a
transcription factor, with potential for correlation of in vivo data with in vitro measurements.
2.5.2 Different types of interactions between two proteins and a DNA target can be examined in our MY1H system
By expressing a second protein in our modified system, different interactions can be
investigated (Figure 2.1). If P1, which is expressed as a fusion to GAL4AD, is able to target
DNA element E, the second protein P2 can interact with the DNA-binding domain of P1
35
(protein-protein interaction) or directly bind to element E (protein:DNA interaction), or P2 can
recruit repressors to P1: either of these scenarios can result in blockage or decrease of reporter
gene activation. If P1 itself is unable to target element E, the second protein can serve to rescue
or restore reporter gene expression by serving as a bridge between P1 and element E, by
dimerizing with P1 to enable binding at element E, or by modifying the structure of P1 to allow
DNA binding.
As demonstrated here, our MY1H system might be particularly useful for testing the
effects of a new protein, or mutant versions of a protein, on the DNA-binding activity of a
transcription factor. As we study the effects of a particular protein on the DNA-binding activity
of a transcription factor, this protein can be transcribed at low or high levels from MCS II, which
is a considerable advantage during the study of inhibitors of a DNA-binding protein or DNA-
binding competition assays. The activities of strong inhibitors can be evaluated with pCETT,
while the activities of weaker inhibitors can be distinguished with pCETF. Such a combined use
of the two plasmids allows reliable qualitative assessment of the strength of the protein:DNA or
protein-protein interaction of interest. For example, we have successfully applied this MY1H
system to examine two other protein:DNA systems (unpublished results). In the first case, the
MY1H was used to test the repression of Max-DNA interactions with several mutants of Max
such that the competitive binding of two proteins vying for the same DNA target was examined:
hence, a protein:DNA interaction was assayed, as shown in Figure 2.1 where P2 serves as a
repressor. In the second case, we utilized the MY1H for examination of mutants of AhR and
Arnt that must heterodimerize in order to bind to a specific DNA target site: hence, a protein
heterodimer-DNA interaction was assayed, as shown in Figure 2.1 where P2 acts as a
coregulatory protein. In the p53/LTAg/53BP2 system presented here, a protein-protein
interaction was assayed, and hence, P2 serves as a blocker.
36
Moreover, investigation of cooperative heterodimer-DNA interactions benefits from
expression of both proteins from the same plasmid. The dimeric complex comprises two
different monomers at equimolar ratio, and such expression is more controllable when plasmid
copy number is not a complicating factor. In the MY1H system, the two genes inserted into
pCETT are transcribed at the same level, resulting in comparable protein concentrations within
the cell. (We note that although both proteins encoded in each plasmid should be expressed
comparably, their actual concentrations in the cell can depend on other factors including protein
size, stability, and post-translational processing.) In contrast, previously published yeast genetic
approaches for investigation of multiprotein:DNA interactions utilize two separate plasmids to
express two different proteins, commonly controlled by different promoters (18-23). These two-
plasmid systems inherently lack the ability to control the expression of both proteins at
comparable levels, as fluctuations in plasmid copy numbers and differential promoter strengths
can lead to variable and unpredictable expression levels of the two different proteins. Our one-
plasmid system, therefore, eliminates the issue of variable protein expression levels stemming
from differential copy numbers between plasmids within the same cell.
Although not shown in this report, our system could also be used to examine proteins that
contribute coactivational or bridging functions to the protein under investigation (Figure 2.1).
Furthermore, with some prior knowledge of possible target proteins, this system could
potentially be extended toward library screening for identification of novel accessory proteins
that rescue DNA-binding capability of a target protein that is otherwise incapable of specific
DNA binding.
In summary, we have developed a modified Y1H system that can be used to detect both
protein-protein and protein:DNA interactions in vivo. The system was validated by use of DNA
binding protein p53 and inhibitors LTAg and 53BP2. This MY1H system should be particularly
37
useful in the investigation of the effects of a regulatory protein on the target transcription factor-
DNA interaction, and should complement current methods available for identifying and
investigating novel protein-protein or protein:DNA interactions in yeast Saccharomyces
cerevisiae.
2.6 Supplemental Information Supplemental Information associated with this manuscript is provided in Appendix A.
38
Chap 3ter
AhR/Arnt:XRE interaction: turning false negatives into true positives in the modified yeast one-hybrid assay
Gang Chen and Jumi A. Shin
(Short running title: Turning false negatives into true positives in modified Y1H)
Contributions:
I performed all the experiments and completed the data interpretation. Jumi Shin
provided supervision and intellectual input to the project and manuscript.
This chapter is adapted from the original manuscript accepted by Analytical Biochemistry,
with necessary changes for thesis organization purposes.
Reproduced with permission from Analytical Biochemistry. Copyright (2008) Elsevier
Inc.
39
3.1 Abstract Given the frequent occurrence of false negatives in yeast genetic assays, it is both
interesting and practical to address the possible mechanisms of false negatives, and more
importantly, turn false negatives to true positives. We recently developed a modified yeast one-
hybrid system (MY1H) useful for investigation of simultaneous protein-protein and protein:DNA
interactions in vivo. We coexpressed the bHLH/PAS domains of AhR and Arnt—namely NAhR
and NArnt, respetively—which are known to form heterodimers and bind the cognate XRE
sequence both in vitro and in vivo, as a positive control in the study of XRE-binding proteins in
the MY1H system. However, we observed negative results, i.e. no positive signal detected from
binding of the NAhR/NArnt heterodimer and XRE site. We demonstrate that by increasing the
copy number of XRE sites integrated into the yeast genome and using double GAL4 activation
domains, the NAhR/NArnt heterodimer forms and specifically binds the cognate XRE sequence,
an interaction that is now clearly detectable in the MY1H. This methodology may be helpful in
trouble-shooting and correcting false negatives that arise from unproductive transcription in
yeast genetic assays.
40
3.2 Introduction Since their introduction almost two decades ago, the yeast two-hybrid (Y2H) (1) and
yeast one-hybrid (Y1H) (4) in vivo genetic assays have been widely utilized for identification
and characterization of protein-protein and protein:DNA interactions. With the arrival of the
post-genome era, these two systems, together with their mammalian and bacterial counterparts
(118,119), are being extensively applied to search networks of interactions in pathways and
genomes (28,120,121).
A general pitfall of genetic assays is the all-too-common observation of false positives
and false negatives in yeast reporter assays (122). Endogenous proteins can affect the protein-
protein or protein:DNA interactions under investigation either positively (false positive) or
negatively (false negative). Additionally, some activation domain (AD) fusions auto-activate
transcription without protein- or DNA-binding as a prerequisite, and some AD fusions can bind
or regulate promoter sequences by themselves without interaction with the DNA-binding domain
(DBD) fusion in the Y2H or designated DNA target element in the Y1H; in the case of the Y2H,
the DBD fusion may also activate transcription independent of the AD fusion (10,29,110). In
addition, there exist a relatively small number of “sticky” or “promiscuous” proteins frequently
detected using multiple baits. Moreover, some physically true, yet physiologically irrelevant,
interactions may also be categorized as one class of false positives (26).
Compared with false positives, false negatives have received much less attention.
However, as researchers shift their focus from detecting any protein that interacts with their
protein or DNA targets of interest toward mapping all of the protein-protein and protein:DNA
interactions on a genome- or pathway-wide scale, the issue of false negatives becomes more
important (26). False negatives stem from various factors, including weak interactions beyond
the detection limitations of a yeast-based genetic system, proteins not stably expressed or folded
41
improperly in the host cell, proteins not localizing to the nucleus, and post-translational
modifications not provided by the host cell's machinery. Additionally, high expression levels of
some hybrid proteins can be toxic to the host cell, and eukaryotic regulatory proteins may
interfere with the function of their yeast homologs. Also, fused domains or epitope tags can have
unintended effects by perturbing protein structure or occluding the site of interaction.
Furthermore, false negatives can simply be due to unproductive transcription of the reporter gene,
caused by an inappropriately positioned DNA target in the promoter and/or insufficient AD
potency (26,28,30,110). In some cases, inappropriate intersite spacing between multiple binding
sites can also lead to unproductive gene transcription, especially for a weak activator (123).
We examined the human aryl hydrocarbon receptor (AhR) and its partner aryl
hydrocarbon receptor nuclear translocator (Arnt), which are bHLH/PAS (basic helix-loop-
helix/Per-Arnt-Sim) proteins that regulate genes involved in the metabolism of carcinogens, such
as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls (PCBs). As AhR
alone is incapable of DNA binding, Arnt regulates the transcriptional activity of AhR by
heterodimerization; thus, the AhR/Arnt heterodimer targets the cognate xenobiotic response
element (XRE), 5'-TNGCGTG, also known as the dioxin response element (DRE) (91-93,124-
126).
In this paper, we demonstrate that a false negative interaction between the AhR/Arnt
heterodimer and cognate XRE DNA site can be turned into a positive control in our modified
Y1H system (MY1H) developed to investigate simultaneous protein-protein and protein:DNA
interactions in vivo (127). The methodology presented may assist in trouble-shooting and
correcting false negatives that arise from unproductive transcription in yeast genetic assays.
42
3.3 Materials and methods Reagents were purchased from BioShop Canada (Burlington, ON), enzymes were
purchased from New England Biolabs (Pickering, ON), and oligonucleotides were synthesized
by Operon Biotechnologies (Huntsville, AL) unless otherwise stated.
3.3.1 Bacterial and yeast strains Escherichia coli SURE® strain (Stop Unwanted Rearrangement Events, Stratagene, La
Jolla, CA) or dam-/dcm- C2925 (New England Biolabs) was used for standard cloning and rescue
of plasmids from yeast cells. SURE cells were used for routine cloning of DNA with secondary
structures likely to be rearranged or deleted in conventional strains. C2925 is a
methyltransferase-deficient E. coli strain used for growth and purification of plasmids free of
dam and dcm methylation; this allows cloning to be performed with dam- or dcm-sensitive
restriction sites. Saccharomyces cerevisiae YM4271 [MATa, ura3-52, his3-200, ade2-101, lys2-
801, leu2-3, 112, trp1-901, tyr1-501, gal4-∆512, gal80-∆538, ade5::hisG] was purchased from
Clontech (Palo Alto, CA) and used for plasmid construction via homologous recombination and
reporter-strain construction.
3.3.2 Construction of reporter strains Four yeast reporter strains were classified into two sets: three-copy strains and six-copy
strains were created according to the Matchmaker™ One-hybrid System User Manual (Clontech)
for reporter assay analysis in the MY1H. Three-copy strains YM4271[pHISi-1/XRE-3] and
YM4271[pLacZi/XRE-3] contain three tandem copies of the consensus XRE (5’-TTGCGTG)
(128) upstream of the HIS3 and lacZ reporter genes, respectively. Similarly, the six-copy strains
YM4271[pHISi-1/XRE-6] and YM4271[pLacZi/XRE-6] contain six tandem copies of the
consensus XRE upstream of the HIS3 and lacZ reporters, respectively. 3-amino-1,2,4-triazole (3-
AT) titration assay revealed that 30 mM 3-AT, necessary for inhibition of background HIS3
43
expression, was sufficient to suppress background growth from both YM4271[pHISi-1/XRE-3]
and YM4271[pHISi-1/XRE-6] on SD/–His medium (minimal synthetic dropout medium lacking
histidine). Details of reporter strain construction are provided in the Supplemental Information.
3.3.3 Transformation, DNA preparation, and plasmid rescue Recombinant plasmids were transformed into E. coli by the standard TSS procedure
(108). Plasmids were isolated using the Wizard® Plus SV Minipreps DNA Purification System
(Promega, Madison, WI). Yeast transformations were performed using either the standard
lithium acetate method (Yeast Protocols Handbook, Clontech) or the transformation procedure
developed by Dohmen et al. (129). Transformants were selected by leucine prototrophy.
Isolation of yeast plasmids was performed using the Zymoprep™ II Yeast Plasmid Miniprep Kit
(ZymoResearch). PCR reactions were performed with Phusion™ high-fidelity DNA polymerase
(New England Biolabs). PCR products and DNA fragments for cloning were purified using
QIAquick Spin Kit, MinElute Kit, or QIAEX II Gel Extraction Kit (Qiagen, Mississauga, ON).
3.3.4 Plasmid Construction All new constructs were confirmed by DNA sequencing on an ABI (Applied Biosystems)
3730XL 96 capillary sequencer at the DNA Sequencing Facility in the Centre for Applied
Genomics, Hospital for Sick Children (Toronto, ON).
3.3.4.1 pCETT2 Plasmid pCETT2 (Figure 3.1) was constructed by homologous recombination in
YM4271 by insertion of a GAL4 AD gene upsteam of the MCS II site (multiple cloning site) in
pCETT (127). This was achieved by use of Sma I-linearized pCETT and the T2AD fragment
amplified by mutually primed synthesis (112) from pGAD424-MCS II (127) with
oligonucleotides 2-9 and 2-10 (Table 2.1 in Chapter 2 and Table D.1 in Appendix D). The
44
homology between the T2AD fragment and both ends of Sma I-linearized pCETT allows
generation of pCETT2. In pCETT2, the gene encoding amino acids 768–881 of the GAL4 AD is
inserted between PADH1(T) and PT7 upstream of both MCS I and MCS II (in pCETT, this gene is
inserted between PADH1(T) and PT7 upstream of MCS I only). Therefore, both monomers
expressed from pCETT2 are expressed as GAL4 AD fusion proteins.
Figure 3.1 Plasmid pCETT2 for coexpression of two AD fusion proteins in the MY1H system. The plasmid contains unique restriction sites located in the MCS I and MCS II regions. Both MCS I and MCS II are at the 3'-end of the open reading frame of the GAL4 AD sequence, allowing two fusion proteins, each of which combines the GAL4 AD and cloned protein of interest, to be expressed at low levels from a truncated constitutive ADH1 promoter.
3.3.4.2 pCETT/NAhR/NArnt and pCETT2/NAhR/NArnt Both human AhR (pRc-CMV/AhR) and human Arnt variant 3 (pRc-CMV/Arnt) cDNA
(130) were generously provided by Patricia Harper and Allan Okey (Department of
Pharmacology and Toxicology, University of Toronto). Details regarding the construction of the
human AhR6-436 fragment (NAhR) and the human Arnt82-464 fragment (NArnt) are provided
in the Supplemental Information. The NAhR and NArnt fragments contain cDNA fragments
45
encoding the bHLH/PAS domains of each protein, respectively. The NAhR fragment was
amplified and inserted into the Sal I and Xba I sites of MCS I in pCETT and pCETT2 to generate
pCETT/NAhR and pCETT2/NAhR, respectively. In order to be consistent with a previously
reported in vitro study where the dissociation constant (Kd) of the XRE DNA complex of the
NAhR/NArnt heterodimer was 2.0 nM as determined by electrophoretic mobility shift assay
(126), the NArnt fragment was based on the sequence of human Arnt variant 1, which we
generated from the human Arnt variant 3 template provided in pRc-CMV/Arnt. The NArnt
fragment was then inserted into the Bam HI and Xho I sites of MCS II in pCETT and pCETT2 to
give pCETT//NArnt and pCETT2//NArnt, respectively. (We note that the recombinant plasmids
pCETT and pCETT2 are named in the format "plasmid/MCS I/MCS II." When a gene is inserted
into MCS I only, the format is "plasmid/MCS I"; when a gene is inserted into MCS II only, the
format is "plasmid//MCS II.") Similarly, the NAhR fragment was inserted into the Sal I and Xba
I sites of pCETT//NArnt and pCETT2//NArnt to generate pCETT/NAhR/NArnt and
pCETT2/NAhR/NArnt, respectively.
3.3.5 HIS3 reporter assay The integrated HIS3 reporter strain was transformed with an AD-fusion plasmid. The
transformants were plated on SD/-H/-L plates. Interactions between the proteins under
investigation and corresponding DNA target element were determined by activation of the HIS3
reporter gene, which was measured by spotting 15 μL diluted fresh cell resuspensions at OD600
~0.01 on SD/-H/-L control plates and SD/-H/-L testing plates that contained 30 mM 3-AT. A
positive interaction was indicated by growth of colonies on both types of plates.
3.3.6 LacZ reporter assay Two commonly used β-galactosidase assays, qualitative X-gal colony-lift filter assay and
quantitative ortho-nitrophenyl-galactoside (ONPG) liquid assay, were performed as described
46
previously (127). In the ONPG assay, results are presented as mean values ± standard error
measurement (SEM) from 3-4 independent experiments, each performed in triplicate.
3.4 Results
3.4.1 Initial trials with pCETT: fusion of the GAL4 AD to NAhR only
Figure 3.2 HIS3 reporter assay for detection of NAhR/NArnt:XRE interaction from protein expression vector pCETT or pCETT2 in YM4271[pHISi-1/XRE-3] (A and C) and YM4271[pHISi-1/XRE-6] (B and D) strains. The transformants that coexpress both GAL4AD-NAhR and NArnt (or GAL4AD-NAhR and GAL4AD-NArnt in the cases of B and D) as well as their corresponding controls were grown as patches on SD/-H/-L control plates (left in each subfigure) that select only for the presence of plasmid; these were replica-plated on SD/-H/-L/30 mM 3-AT testing plates (right in each subfigure) that select for HIS3 reporter activity (30 °C, 5 days). The transformants on the same plate were identified according to the following order. A and B: upper left, pCETT/NAhR/NArnt; upper right, pCETT/NAhR; lower left, pCETT//NArnt; lower right, pCETT. C and D: upper left, pCETT2/NAhR/NArnt; upper right, pCETT2/NAhR; lower left, pCETT2//NArnt; lower right, pCETT2.
In order to construct a positive control in the study of XRE-binding proteins in our
MY1H system, we first tried to coexpress both NAhR and NArnt from pCETT in yeast strain
YM4271[pHISi-1/XRE-3], in which three copies of the XRE site were integrated into the yeast
genome upstream of the HIS3 reporter gene. Arnt can homodimerize, although it preferentially
heterodimerizes (124). In order to minimize the effect of Arnt homodimerization on reporter
activation, NAhR is expressed as the fusion to the GAL4 AD (GAL4AD-NAhR, AD at N
terminus), and NArnt is expressed as the independent protein with no AD fusion. The
transformant expressing GAL4AD-NAhR and NArnt did not grow on SD/-H/-L plates
containing 30 mM 3-AT, nor did the corresponding controls (Figure 3.2A). Because increasing
47
the copy number of XRE sites integrated upstream of the reporter gene may increase the
probability of the NAhR/NArnt heterodimer binding at the promoter and/or optimize the distance
between the promoter and site of transcription initiation, we constructed the six-copy strain
YM4271[pHISi-1/XRE-6], wherein six copies of the XRE were integrated upstream of the
reporter. However, coexpression of GAL4AD-NAhR and NArnt from pCETT in the
YM4271[pHISi-1/XRE-6] strain did not show positive signal by HIS3 assay as well (Figure
3.2B).
Figure 3.3 Colony-lift filter assay for detection of NAhR/NArnt:XRE interaction from protein expression vector pCETT or pCETT2 in YM4271[pHISi-1/XRE-3] (A and C) and YM4271[pHISi-1/XRE-6] (B and D) strains. The X-gal colony-lift assay was performed after 4 days growth of transformants that were transformed with pCETT/NAhR/NArnt (A and B) or pCETT2/NAhR/NArnt (C and D) and plated on SD/-L/-U plates at 30 °C. Photos were taken after 60 min incubation at 30 °C. The colony-lift filter assay results of their corresponding controls are shown in Figure B.1 in the Appendix B.
The X-gal colony-lift filter assay shows similar results to that of the HIS3 assay. After 60
minutes incubation, both three-copy and six-copy transformants that coexpress GAL4AD-NAhR
48
and NArnt proteins developed a faint blue color indistinguishable from their corresponding
controls (Figure 3.3A and B).
Figure 3.4 ONPG assay for detection of NAhR/NArnt:XRE interaction from protein expression vector pCETT or pCETT2 in YM4271[pHISi-1/XRE-3] (A and C) and YM4271[pHISi-1/XRE-6] (B and D) strains. Vertical axis indicates the mean values of β-galactosidase units. Error bars represent standard error measurements (SEM) from at least three independent trials conducted in triplicate.
The quantitative ONPG assay provides more information than the HIS3 and colony-lift
assays (Figure 3.4A and B). When pCETT is utilized as the coexpression vector, the control
transformant that contains vector only with no inserted genes gives background expression at
1.38±0.06 in the three-copy strain strain; in contrast, in the six-copy strain, the background is
4.83±0.30, and therefore, increasing the copy number of DNA target elements increases
49
background expression. When GAL4AD-NAhR and NArnt are coexpressed, the ONPG values
are 3.16±0.37 and 11.31±0.81 in the three- and six-copy strains, respectively. Although the
absolute value increases in the latter case, the actual signal-to-noise ratios (S/N) in both cases are
not statistically different (2.29 vs. 2.34).
3.4.2 Next generation trials with pCETT2: fusion of the GAL4 AD to both NAhR and NArnt
Plasmid pCETT2 is a double AD vector based on pCETT for coexpression of two AD
fusion proteins in the MY1H system (Figure 3.1). The difference between pCETT2 and pCETT
lies in the expression cassette of the second protein; in pCETT, the GAL4 AD is only fused to
the protein expressed from MCS I, while in pCETT2, the GAL4 AD is fused to proteins
expressed from both MCS I and II. In addition, the transcription of both genes is under the
control of the same truncated ADH1 promoter, leading to comparable low levels of protein
expression (113).
From pCETT2, we coexpressed GAL4AD-NAhR and GAL4AD-NArnt in yeast strain
YM4271[pHISi-1/XRE-3]. This time, transformants clearly grew normally on SD/-H/-L test
plates containing 30 mM 3-AT, while the corresponding controls, which expressed either
GAL4AD-NAhR or GAL4AD-NArnt or neither, were still clear on test plates (Figure 3.2C). The
same test was performed in YM4271[pHISi-1/XRE-6] with the same results (Figure 3.2D).
Because the HIS3 reporter assay we performed is qualititative and only shows growth or death,
this assay did not indicate a difference in signal from the three- or six-copy strain.
Results from the X-gal colony-lift filter assay were consistent with the HIS3 reporter
assay. The cells transformed with pCETT2/NAhR/NArnt developed vivid blue color in the filter
assay, regardless of whether the plasmid was transformed into the three- or six-copy strain
(Figure 3.3C and D). The corresponding controls that express only one of the two proteins or
50
neither showed no blue color development even after 60 minutes incubation (Figure B.1C and D
in the Appendix B). Furthermore, when GAL4AD-NAhR and GAL4AD-NArnt were
coexpressed in the six-copy strain, blue color began to show after twenty minutes, while in the
three-copy strain, color started to develop after forty minutes; after one hour incubation, the
former transformant showed blue color much more intense than the latter.
The ONPG assay quantitatively confirmed the results obtained from both HIS3 and
colony-lift assays. When GAL4AD-NAhR and GAL4AD-NArnt were coexpressed in
YM4271[pLacZi/XRE-3], the ONPG value was 3.82±0.13; its corresponding negative control
transformed with pCETT2 gave ONPG value 1.59±0.13 (Figure 3.4C) corresponding to S/N 2.40.
In contrast, when GAL4AD-NAhR and GAL4AD-NArnt were coexpressed in
YM4271[pLacZi/XRE-6], the ONPG value was 62.3±5.9; its corresponding negative control
transformed with pCETT2 gave ONPG value 5.67±0.52 (Figure 3.4D) corresponding to S/N
10.98. In addition, ONPG values from these two negative controls in yeast strains with different
copy numbers of XRE sites indicate again that increasing the copy number of DNA target
elements also increases the background expression (1.59±0.13 for three-copy strain vs. 5.67±0.52
for six-copy strain).
As expected, the controls that express either GAL4AD-NAhR or GAL4AD-NArnt only
give ONPG values close to their corresponding negative controls that express GAL4 AD only.
Although Arnt can homodimerize and target the symmetric sequence 5'-CACGTG (131), we did
not anticipate that the Arnt homodimer would target the asymmetric XRE sequence, 5'-
TTGCGTG, and our data show that GAL4AD-NArnt displays activity indistinguishable from
backgound.
51
3.5 Discussion
3.5.1 Are false negatives due to unproductive transcription? Yeast model systems have already been used to study the protein-protein and
protein:DNA interactions of AhR and Arnt. In 1995, successful isolation of a cDNA encoding
Arnt was reported by using the recombinant AhR bHLH/PAS domain as a probe in cDNA
library screening in the Y2H (91); this work was followed by two similar systems developed to
investigate the AhR/Arnt heterodimer's (full-length or bHLH/PAS domain only) response to
different AhR ligands in the Y1H system (92,93). We therefore believe the factors leading to the
false negative observations in our initial experiments with pCETT may include unproductive
transcription of the reporter gene from inappropriate positioning of XRE sites in the promoter
region, masking effects of endogenous proteins that interact with DNA sites in the promoter
region, steric hindrance from the NAhR/NArnt heterodimer that adversely affect transactivation
of the GAL4 AD, and/or insufficient transactivation potency of a single AD on the heterodimer.
The above possibilities can be classified into two categories: target DNA element and AD.
There are numerous factors that affect levels of gene transcription. From the standpoint of
artificial transcription factor design, by using a DBD that binds its cognate response element
with higher affinity or by using an AD that possesses stronger activation potency, levels of gene
transcription can be increased (132). Alternatively, increasing the potential for the DBD to find
its specific target element by increasing the number of cognate DNA sequences in the promoter
(132,133) or multimerizing the AD on a single transcriptional factor (134) can also produce
similar effects. Although intersite spacing between multiple binding sites can also be a critical
factor for effective gene transcription (123), given that high background was obtained when a
spacer of 4 bp or 6 bp was inserted between adjacent XRE binding sites, tandem copies of
52
multiple XRE binding sites without intersite spacing sequences eventually became our desired
option.
The NAhR/NArnt:XRE interaction in our MY1H initially showed a false negative. In
order to turn this false negative into a true positive, the tactics mentioned above were combined
to increase the levels of reporter gene transcription.
3.5.2 Copy number of XRE target sites Transcription factor binding sites can function as regulated enhancer elements when
multimerized and ligated to a promoter (132,135). Therefore, increasing the copy number of
XRE sites will likely increase the number of transcription factors bound in the promoter region,
thereby synergistically increasing the activation potency of the reporter gene. As a matter of fact,
it is not uncommon in nature for multiple DNA response elements to exist in a given gene's
promoter region. For instance, yeast transcriptional activator GAL4 binds to multiple sites on
DNA to activate transcription synergistically; the presence of two such sites can more than
double the level of transcription from a single site (136). In the case of XRE, at least six XRE
sites have been identified in the upstream region of both rat and human CYP1A1 genes (137),
and eight XREs are located within 2.3 kb of the 5'-flanking region of human CYP1B1 gene (138).
Although it is unclear as to the synergistic activation roles of these different XREs on
gene regulation, in vitro experiments revealed that the AhR/Arnt heterodimer has different
affinities for these XRE variants sharing the 5'-GCGTG core sequence (137,138). These different
XRE sites in the same promoter may be used to perform complicated regulation of downstream
gene expression, as in the case of cI repressor and Cro proteins in phage λ or GAL4 in yeast
(136,139). Alternatively, it is also possible that these XRE sites are just evolutionary footprints
from the different positions where XRE sites were placed to find a favorable spatial
conformation for optimal, synergistic gene regulation. These trials eventually led to a fraction of
53
the total sites being physiologically relevant. Therefore, the presence of more XRE sites may
simply enable transcription factors to perform their regulatory functions more advantageously.
However, the presence of more target elements in the promoter region also risks
increased nonspecific interactions from endogenous biomolecules. Background expression will
be more “leaky” as the number of DNA targets increases. Indeed, background expression
increases by 3.6-fold in our studies when the number of XRE sites integrated into the yeast
genome increases from three to six. The increase in background expression can become
overwhelming when too many binding sites are present in the promoter. We constructed a yeast
strain with twenty-three integrated copies of XRE sites in a pilot experiment and found that
reporter gene activation from the negative control was so high that in the X-gal colony-lift filter
assay, the negative controls developed blue color within five minutes and intense blue after 15
minutes of incubation.
When we increased the number of XRE sites integrated into the yeast genome, we still
observed false negatives in our first trials with pCETT. Thus, we re-evaluated the role of
increased the number of target elements, which may be only one of many factors responsible for
false negatives. As insufficient transactivation potency from the single AD may also contribute
to the false negative (140), the double AD expression vector pCETT2 was additionally employed
to increase gene transcription levels.
3.5.3 Double AD system In addition to increasing the number of transcription factors bound to DNA to trigger
synergistic activation (136), oligomerization of the AD on a single transcriptional factor is
another strategy for enhancement of gene transcription. For example, a transcription factor
containing two copies of the VP16 AD upregulated transcription 5-fold over the single copy AD
activator (134).
54
Our strategy for AD oligomerization differs from that discussed above due to the
heterodimeric nature of the NAhR/NArnt complex. By use of the double AD coexpression
plasmid pCETT2, heterodimerization of GAL4AD-NAhR and GAL4AD-NArnt intrinsically
leads to doubling the local concentration of ADs present at the promoter. This double AD
heterodimer is somewhat different in structure from the multimerized AD on a single
transcriptional factor, but we expected our strategy to yield a similar effect.
When both NAhR and NArnt proteins were coexpressed as GAL4 AD fusion proteins in
strain YM4271[pLacZi/XRE-3], the sensitive HIS3 reporter selection assay showed positive
growth, demonstrating that doubling the AD concentration in the promoter region lead to
detectable transcriptional activation in the MY1H. However, the ONPG assay showed the
synergistic activation of this double AD in the three-copy strain to be insignificant (S/N 2.40).
Once the two fusion proteins were coexpressed in strain YM4271[pLacZi/XRE-6], the
synergistic activation of this double AD was so considerable that S/N increased over 4-fold
compared with the S/N ratios for the single AD heterodimer in the six-copy strain or the double
AD heterodimer in the three-copy stain. This result proves that both increasing the number of
DNA binding sites and increasing the number of activation domains contribute to the
considerable augmentation of transcription potency of the reporter gene through synergistic
activation, and that implementation of both strategies was critical for avoiding the false negative
result.
3.5.4 Synergistic activation is due to both increase of XRE target sites and doubling of AD
The data from trials with pCETT and pCETT2 reveal three important points. First,
increase of the number of XRE sites in the yeast genome from three to six raises background
expression. Thus, it is feasible to “amplify” the signal from a specific protein:DNA interaction
55
by increasing the copy number of DNA elements. However, the presence of too many DNA
target elements can adversely affect the differentiation of true positive and negative signals.
Second, only when both NAhR and NArnt are expressed as GAL4 AD fusion proteins can the
reporter be activated in the MY1H system. This result indicates that the double AD system leads
to more efficient reporter transcription. Third and most significantly, the considerable increase in
S/N requires both increase of the copy number of XRE sites and attachment of ADs to both
NAhR and NArnt. Overall, the data indicate that the combination of increasing target element
number and increasing the concentration of activation domains can make a false negative truly
positive.
Another potential bonus of introducing synergistic activation into our MY1H system is
that it increases the chances for detection of only weakly active proteins that would be
considered inactive, and therefore missed, during testing of large libraries by genetic assays.
In summary, by increasing the copy number of XRE sites integrated into the yeast
genome and doubling the GAL4 activation domains, we successfully turned a false negative
NAhR/NArnt:XRE interaction into a true positive control that paves the way to further study of
XRE-binding proteins in our MY1H system. Given that there is no general means for addressing
all possible false positives and false negatives, the methodology we present here provides an
effective, productive way to pinpoint false negatives that arise from unproductive transcription.
3.6 Supplemental Information Supplemental Information associated with this manuscript is provided in Appendix B.
56
Chap 4ter
Forced protein heterodimerization and specific DNA binding to a nonpalindromic DNA sequence in vivo and in vitro: bHLHZ-like hybrid heterodimers of bHLH/PAS proteins AhR and Arnt and
bZIP proteins JunD and Fos as a model Gang Chen, Antonia T. De Jong, S. Hesam Shahravan, and Jumi A. Shin
(Short running title: DNA binding forces heterodimerization of hybrids of bHLH/PAS and bZIP)
Contributions:
I initiated this project and performed all the in vivo experiments. Antonia De Jong and
Hesam Shahravan are now working on protein expression and purification and the in vitro
quantitative fluorescent anisotropy analysis. I completed the in vivo data interpretation and wrote
the manuscript. Jumi Shin provided supervision and intellectual input to the project and
manuscript.
57
4.1 Abstract The molecular basis of specific partner selection and DNA binding of the basic helix-
loop-helix (bHLH) superfamily of dimeric transcription factors is of great interest to both
biologists and chemists. The bHLH proteins use the helix-loop-helix dimerization domain to
form a four-helix bundle, which positions the contiguous basic regions for targeting the DNA
major groove. In bHLHZ and bHLH/PAS proteins, dimerization is further controlled by an
adjacent leucine zipper (LZ) or Per-Arnt-Sim homology (PAS) domain, respectively. Domain
swapping was employed to probe the relationship between the bHLH and PAS domains of the
bHLH/PAS proteins AhR and Arnt as well as their roles in specific DNA binding. Two hybrid
proteins, AhRJunD and ArntFos, were designed wherein the leucine zippers of JunD and Fos
were fused to the bHLH domains of AhR and Arnt, respectively. In the modified yeast one-
hybrid (MY1H) system, the HIS3 assay and two lacZ-based assays, X-gal colony-lift assay and
quantitative ONPG liquid assay, demonstrate that these two bHLH•LZ hybrids specifically
bound to the XRE cognate sequence with enhanced DNA-binding affinity relative to the
heterodimer of the truncated AhR and Arnt bHLH domains, with no PAS or LZ domains. The
JunD or Fos LZ domains may serve to induce properly folded, α-helical structure that stabilizes
the hydrophobic HLH dimerization interface as a bundle of four parallel α-helices.
Although each monomer is able to homodimerize, as demonstrated in the Y2H, we
demonstrate that the presence of the nonpalindromic XRE cognate sequence forces
heterodimerization of the two hybrid monomers for specific DNA binding in vivo. These hybrids
provide a scaffold for design of dimeric helical proteins forced to heterodimerize by a specific
DNA ligand and might be informative for further exploration of the relationship between protein
structure and DNA-binding function. Moreover, they provide additional evidence that supports
the hypothesis of domain shuffling in the evolutionary pathway of the bHLH superfamily.
58
4.2 Introduction The basic helix-loop-helix (bHLH) proteins are a large and diverse class of DNA-binding
proteins that target specific DNA sites for transcriptional regulation of genes involved in
fundamental biological processes, such as cell cycle and developmental regulation, apoptosis and
homeostasis, and stress response pathways (57-60). The bHLH proteins typically associate in
homo- or heterodimeric complexes that recognize the hexameric E-box DNA site, 5'-CANNTG
(58,65). Members of the bHLH superfamily share a highly conserved structural motif comprising
a DNA-binding basic region and a dimerization domain. Similar to that in bZIP proteins, the
basic region in bHLH proteins targets the DNA major groove. The dimerization domain
comprises two amphipathic helices separated by a nonconserved loop typically 5-12 residues in
length; upon dimerization, a compact hydrophobic four-helix bundle forms that positions the
contiguous basic regions for DNA binding (61,70, Kewley, 2004 #206,141).
Two subclasses of bHLH proteins, namely the bHLHZ and bHLH/PAS families, contain
an additional leucine zipper (LZ) or Per-Arnt-Sim (PAS) domain contiguous with the bHLH
domain, respectively, which further regulates protein dimerization (72,142). The LZ region in
bHLHZ proteins is believed to contribute to the overall stability of the proteins as well as to
determine partner selection (69,70).
In comparison, the PAS domain in bHLH/PAS proteins functions as a conserved
signaling module in response to environmental stimulus (128,143-146). Structurally, the PAS
domain is known to be involved in protein dimerization and specification of heterodimerization
partner and may also directly interact with the DNA target element as well as the adjacent bHLH
domain (72,74). However, the function of the PAS domain in protein dimerization and DNA
binding is far from being well understood. Although high-resolution structural data of the bHLH
domain of bHLH/PAS proteins is not available, the bHLH domains of bHLH/PAS proteins are
59
predicted to adopt a similar DNA-binding mode and to be structurally similar to the closely
related bHLH and bHLHZ proteins, based on their homologous primary sequences and DNA-
binding specificities (59).
AhR and Arnt are bHLH/PAS proteins that heterodimerize in the presence of
environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and
polychlorinated biphenyls (PCBs). The activated complex binds specific xenobiotic response
elements (XREs, also known as the dioxin response element, DRE), 5'-TNGCGTG, and activates
genes encoding xenobiotic metabolizing enzymes (128,143-145). Biochemical and genetics
studies have concluded that the AhR/Arnt heterodimer functions by binding the atypical XRE
cognate sequence (79,80): AhR targets the 5'-TNGC half site, and Arnt the 5'-GTG half site.
Arnt is able to homodimerize and target the E-box site (124,126,131,147). However, AhR is
unable to form homodimers and can only function through heterodimerization with Arnt.
In order to gain more insight into the role the PAS domains of AhR and Arnt play in
protein dimerization and DNA binding, and to further understand DNA recognition by
heterodimeric proteins, we constructed a pair of bHLHZ-like hybrid proteins, in which the bHLH
domains of AhR and Arnt are fused to the LZ regions of bZIP proteins JunD and Fos,
respectively (AhRJunD and ArntFos). We used in vivo yeast genetic assays and are currently
conducting in vitro quantitative fluorescence anisotropy titrations to explore their dimerization
and specific DNA-binding activities. In vivo results reveal that the presence of the
nonpalindromic XRE cognate sequence forces heterodimerization of the two hybrids for specific
DNA binding.
This chapter describes the results of the dimerization and specific DNA binding of
AhRJunD and ArntFos obtained from MY1H and Y2H assays. The in vitro quantitative
fluorescence anisotropy assays to assess the free energies of protein:DNA binding are ongoing,
60
as well as western blotting assays to examine the expression of the hybrid proteins examined in
the Y2H. The data presented here is not complete, and work remains to confirm these
interactions observed in vivo and accordingly determine the affinities of the corresponding
interactions in vitro.
4.3 Materials and methods Reagents were purchased from BioShop Canada (Burlington, ON), enzymes were
purchased from New England Biolabs (Pickering, ON), and oligonucleotides were synthesized
by Operon Biotechnologies (Huntsville, AL) or Integrated DNA Technologies (Coralville, IA)
unless otherwise stated.
4.3.1 Bacterial and yeast strains Escherichia coli DH5α (Stratagene, La Jolla, CA), SURE® strain (Stop Unwanted
Rearrangement Events, Stratagene), or dam-/dcm- C2925 (New England Biolabs) was used for
standard cloning and rescue of plasmids from yeast cells. SURE cells were used for routine
cloning of DNA with secondary structures likely to be rearranged or deleted in conventional
strains. C2925 is a methyltransferase-deficient E. coli strain used for growth and purification of
plasmids free of dam and dcm methylation; this allows cloning to be performed with dam- or
dcm-sensitive restriction sites.
Saccharomyces cerevisiae YM4271 [MATa, ura3-52, his3-200, ade2-101, lys2-801, leu2-
3, 112, trp1-901, tyr1-501, gal4-∆512, gal80-∆538, ade5::hisG] was used for reporter strain
construction. Two yeast reporter strains, YM4271[pHISi-1/XRE-6] and YM4271[pLacZi/XRE-
6], for reporter assay analysis in the MY1H were created as reported previously (148). These two
strains contain six tandem copies of the consensus xenobiotic response element (XRE), 5'-
TTGCGTG, upstream of the HIS3 and lacZ reporter genes, respectively. 3-amino-1,2,4-triazole
(3-AT) titration assay revealed that 30 mM 3-AT, a competitive inhibitor of the HIS3 gene
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product leading to background expression, was sufficient to suppress YM4271[pHISi-1/XRE-6]
background growth on SD/-H medium. AH109 [MATa, trp1-901, leu2-3, 112, ura3-52, his3-200,
gal4∆, gal80∆, LYS2 : : GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2, URA3 : :
MEL1UAS-MEL1 TATA-lacZ, MEL1] was used for assessing protein-protein interactions in the Y2H
system. Both YM4271 and AH109 were purchased from Clontech (Palo Alto, CA).
4.3.2 Transformation, DNA preparation, and plasmid rescue The procedures for molecular cloning, plasmid harvesting, and yeast transformation were
performed as described previously (127,148). The following is a brief summary. PCR reactions
were performed with Phusion™ high-fidelity DNA polymerase (New England Biolabs). PCR
products and DNA fragments for cloning were purified using the corresponding Qiagen DNA
purification kits (Qiagen, Mississauga, ON). Recombinant plasmids were transformed into E.
coli by the standard TSS procedure (108) and harvested from bacteria using the Wizard® Plus
SV Minipreps DNA Purification System (Promega, Madison, WI). Yeast transformations were
performed using either the standard lithium acetate method (Yeast Protocols Handbook,
Clontech) or the transformation procedure developed by Dohmen et al. (129).
4.3.3 Plasmid Construction Detailed information of plasmid construction is provided in the Supplemental
Information. All new constructs were confirmed by dideoxynucleotide DNA sequencing
performed at The Centre for Applied Genomics, The Hospital for Sick Children (Toronto, ON).
4.3.3.1 pCETT2/AhRbHLH/ArntbHLH All AhR and Arnt fragments used in this study were based on the human AhR and Arnt
cDNA (130) generously provided by Patricia Harper and Allan Okey (Department of
Pharmacology and Toxicology, University of Toronto). The AhRbHLH sequence encoding
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amino acids 20-90 of human AhR was amplified from AhR cDNA and cloned into MCS I of
pCETT2 to generate pCETT2/AhRbHLH. The ArntbHLH sequence encoding amino acids 82-
149 of human Arnt isoform 1 was amplified from the Arnt82-464 cDNA fragment (148) and
cloned into MCS II of pCETT2 and pCETT2/AhRbHLH to generate pCETT2//ArntbHLH and
pCETT2/AhRbHLH/ArntbHLH, respectively.
4.3.3.2 pCETT/AhRJunD/ArntFos and pCETT2/AhRJunD/ArntFos The AhRJunD fragment encoding a hybrid protein of human AhR20-86 fused to human
JunD296-332 was ligated into MCS I of both pCETT and pCETT2 to generate pCETT/AhRJunD
and pCETT2/AhRJunD, respectively. The ArntFos fragment encoding a hybrid protein of human
Arnt82-148 fused to human Fos165-201 was similarly ligated into MCS II of both
pCETT/AhRJunD and pCETT2/AhRJunD to generate pCETT/AhRJunD/ArntFos and
pCETT2/AhRJunD/ArntFos, respectively. The ArntFos fragment was also inserted into MCS II
of pCETT and pCETT2 to generate pCETT//ArntFos and pCETT2//ArntFos, respectively.
We note that AhRJunD contains AhR20-86 and ArntFos contains Arnt82-148, both of
which encompass the full-length bHLH domains of AhR and Arnt, respectively. This is in
comparison to AhR20-90 in the AhRbHLH construct and Arnt82-149 in the ArntbHLH construct
above that contain four and one additional amino acid(s) C-terminal to Helix 2 of AhR and Arnt,
respectively (Figure 4.1).
4.3.3.3 pCETT2/AhR(ΔL)JunD/ArntFos The AhR(ΔL)JunD fragment was constructed similarly to AhRJunD and ligated into
MCS I of both pCETT2 and pCETT2//ArntFos to generate pCETT2/AhR(ΔL)JunD and
pCETT2/AhR(ΔL)JunD/ArntFos, respectively. Compared with the AhRJunD fragment, the
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codon encoding Leu296 of JunD in AhR(ΔL)JunD was deleted. Therefore, this fragment encodes
a hybrid protein of human AhR20-86 fused to human JunD297-332 (Figure 4.1).
4.3.3.4 Plasmids constructed for the Y2H assay The aforementioned five fragments (AhRbHLH, ArntbHLH, AhRJunD, AhR(ΔL)JunD,
and ArntFos) were amplified with appropriate primers, and each was ligated into both GAL4
DNA-bidning domain (DBD) fusion vector pGBKT7 and GAL4 activation domain (AD) fusion
vector pGADT7 using the Matchmaker™ Two-hybrid System 3 (Clontech).
4.3.4 HIS3 reporter assay The HIS3 reporter assays were performed as described previously (148). A brief
summary of this procedure follows: fresh colonies of YM4271[pHISi-1/XRE-6] transformed
with an appropriate AD-fusion plasmid were resuspended in sterile water and then diluted in 10-
fold steps to ~0.01. 15 μL of each diluted resuspension were spotted on SD/-H/-L control plates
and on SD/-H/-L/ test plates containing 30 mM 3-AT. A positive interaction was indicated by the
growth of cultures on both types of plates.
4.3.4.1 Spot titration assay The HIS3 reporter gene was also used to compare the transactivation potency of different
GAL4 AD fusion proteins. Ten-fold serial dilutions of fresh colony resuspensions were
generated such that the diluted cultures of each transformant gave OD600 from 10-1 to 10-4. 15 µL
of each dilution were spotted on SD/-H/-L plates and SD/-H/-L test plates containing 30 mM 3-
AT. The relative strengths of reporter gene activation were estimated by comparing the growth
status of different transformants with the same cell densities on the test plates.
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4.3.5 LacZ reporter assay Two commonly used β-galactosidase assays, qualitative X-gal colony-lift filter assay and
quantitative ortho-nitrophenyl-galactoside (ONPG) liquid assay, were performed as described
before (127). In the ONPG assay, results are presented as mean values ± standard error of the
mean (SEM) of 3-4 independent experiments, each performed in triplicate.
4.3.6 Protein-protein interactions in the Y2H system The Matchmaker™ GAL4 Two-hybrid System 3 (Clontech) was utilized to examine
homo- and heterodimerization of the proteins under investigation. The recombinant plasmids of
DBD fusion vector pGBKT7 and AD fusion vector pGADT7 (or their respective parental vectors
in the controls) were cotransformed into strain AH109. Transformed cells were selected on SD/-
L/-W plates at 30 °C, 4 days. Interactions between the two proteins under investigation were
determined by simultaneous activation of the HIS3, ADE2, and MEL1 reporter genes. Activation
of the three reporter genes was measured by spotting 15 μL diluted fresh cell resuspensions at
OD600 ~0.01 on SD/-L/-W plates and on SD/-A/-H/-L/-W/X-α-Gal plates. A positive interaction
was indicated by the growth of cultures on both types of plates, with blue colony color showing
on the latter plates. The X-α-Gal indicator plates were prepared as per the Yeast Protocol
Handbook, Clontech.
4.3.6.1 Spot titration assay Activation strength of two-hybrid activity was measured by spotting 15 μL of 10-fold
serial dilutions (OD600: 10-1-10-5) of fresh colony resuspensions at OD600 0.1 on both SD/-H/-L
control plates and SD/-A/-H/-L/X-α-Gal plates.
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4.4 Results The original goal of our design was to generate a protein heterodimer that targets a
specific cognate DNA element. Several considerations were made in the process of protein
design. First, we surmised that compared with palindromic or pseudopalindromic sites,
nonpalindromic sites are more suitable for heterodimer:DNA interactions in that these sites are
normally not targeted by homodimers, therefore minimizing the possibility of homodimeric
interference with DNA binding. So the atypical nonpalindromic XRE site targeted by the native
AhR/Arnt heterodimer became our ideal candidate; as a positive control, the interaction between
the bHLH/PAS domains of the AhR and Arnt proteins and XRE DNA site showed positive
signal in the MY1H (148). Second, in order for the hybrid proteins to target the specific DNA
site, the ideal scenario would be that neither of the two monomers be able to form homodimers,
and that heterodimerization of the two monomers must be of high affinity and stability, as it is
unlikely that monomeric proteins containing disordered basic regions strongly target the specific
DNA site without partner participation.
Arnt is able to homodimerize and bind to the 5'-CACGTG sequence (124,126,131,147),
and it serves as a common heterodimerization partner within the bHLH/PAS family to regulate a
number of proteins such as AhR, HIF-1α and SIM proteins (149). AhR alone, in contrast, is
unable to bind DNA. Jun and Fos, members of the bZIP superfamily, act in parallel fashion to
AhR and Arnt, in that Jun is able to form homodimers and is a common heterodimerization
partner within the Fos subfamily to regulate a number of proteins including c-Fos, whereas c-Fos
is restricted to pairing with Jun family members only (42). Additionally, Jun and Fos
preferentially heterodimerize rather than homodimerize, again similar to AhR and Arnt (150).
Members of the Jun subfamily have been shown to homodimerize with different affinities, and
JunD homodimerizes with lower affinity than does c-Jun (151).
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Our aim was to generate small bHLHZ-like hybrid protein structures that strongly
preferred to heterodimerize and bind a desired DNA site, in comparison with the much larger
native bHLH/PAS domains: the typical LZ is approximately 30 aa, whereas the PAS domain
exceeds 300 aa. These heterodimeric hybrid proteins of nonnative structure were also designed to
maintain the high DNA-binding affinity and sequence specificity of the full-length native
proteins.
MaxbHLHZ
basic region helix 1 loop helix 2 :
AhRJunD: AhR(ΔL)JunD
:
ArntFos:
MaxbHLHZ: AhRJunD:
AhR(ΔL)JunD
: ArntFos: AhR(20-90): Arnt(82-149):
ADKRAHHNALERKRR-DHIKDSFHSLRDSVP-SLQGEKAS -RAQILDKATEYIQYM- QKTVKPIPAEGIKSNPSKRHR-DRLNTELDRLASLLP-FPQDVINKLD-KLSVLRLSVSYLRAK- QKTVKPIPAEGIKSNPSKRHR-DRLNTELDRLASLLP-FPQDVINKLD-KLSVLRLSVSYLRAK- SSADKERLARENHSEIERRRR-NKMTAYITELSDMVP-TCSALARKPD-KLTILRMAVSHMKSL- ucine zipper leRRKNDT QQDIDD KRQNAL EQQVRA H L L LSFFDVA EEKVKT KSQNTE ASTASL REQVAQ KQKVLSHV L L L L LSFFDVA EEKVKT KSQNTE ASTASL REQVAQ KQKVLSHV- L L L L RGTGNTLQAETDQLEDEKSALQTEIANLLKEKEKLEFILAAHR basic region helix 1 loop helix 2
QKTVKPIPAEGIKSNPSKRHR-DRLNTELDRLASLLP-FPQDVINKLD-KLSVLRLSVSYLRAKSFF-DVALKSS SSADKERLARENHSEIERRRR-NKMTAYITELSDMVP-TCSALARKPD-KLTILRMAVSHMKSLRGT-GNTS
Figure 4.1 Primary sequence alignment of the native Max bHLHZ domain and bHLH•LZ hybrids. The sequence alignment is based on refs. (142) and (61). The bHLHZ region of reference sequence Max was aligned with the putative bHLH regions of AhR and Arnt, as well as the LZ of JunD and Fos. The bHLH domains of AhR or Arnt were fused to the LZ domains of JunD or Fos, respectively; the hydrophobic heptad repeat register of Helix 2 and the LZ is not maintained in AhR(ΔL)JunD, as the first leucine in the JunD LZ is deleted. The leucines/hydrophobic residues that define the leucine zipper motif are highlighted in bold, and the highly conserved residues in the basic regions of Max and Arnt are underlined.
We therefore decided to fuse the JunD LZ to the bHLH domain of the AhR protein, and
the c-Fos leucine zipper to the bHLH domain of the Arnt protein; the LZ is directly fused to
Helix 2 of the bHLH domain in order to maintain the register of the hydrophobic heptad repeat
that forms the protein-protein interface. Although the groups of Brennan and Whitelaw showed
that just the Arnt bHLH domain is sufficient for binding of the palindromic E-box DNA site (5'-
CACGTG) under certain conditions in in vitro studies (142,152), we found the Arnt bHLH
domain incapable of targeting the E-box in in vivo yeast studies (153). Therefore, we added the
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LZ not only to provide additional dimerization interface and promote heterodimerization, but
also to provide stability and nucleate the α-helical structure necessary for proper folding and
solubility. Although there are a few endogenous bZIP proteins present in yeas such as GCN4 and
Yap family proteins, it is unlikely that they interfere with the JunD/Fos LZ pair, as no interaction
between JunD or Fos zipper and either of the yeast bZIP proteins has been observed (151).
In order to better understand the role the leucine zipper plays in the dimerization structure
and DNA-binding function of the hybrid proteins, we also constructed the mutant AhR(ΔL)JunD,
in which the first leucine residue of the JunD LZ immediately after Helix 2 of AhR bHLH is
deleted—hence, disruption of the heptad repeat and hydrophobic interface (Figure 4.1).
4.4.1 The AhRJunD/ArntFos and AhR(ΔL)JunD/ArntFos heterodimers bind the XRE site in the MY1H
Two reporter assays, HIS3 and lacZ, were utilized to examine our protein design in the
MY1H system. These reporter assays provide indirect, qualititative (or semi-quantitative)
measurement of the DNA-binding affinities of the hybrid proteins under investigation. Because
transcriptional potency of reporter activation generally correlates with the strength of the
interaction under investigation (25), these in vivo measurements provide a reasonable guide
regarding the DNA-binding affinities of the hybrid proteins.
We first coexpressed both GAL4AD-AhRJunD and GAL4AD-ArntFos hybrid proteins,
which are fused to the GAL4 activation domain (GAL4AD), by use of pCETT2 in yeast strain
YM4271[pHISi-1/XRE-6] (Figure 4.2A), in which six tandem copies of the XRE site were
integrated into the yeast genome upstream of the HIS3 reporter gene. The transformant grew on
SD/-H/-L/30 mM 3-AT test plates, whereas its corresponding controls, in which only one of the
two hybrid proteins or none was expressed, did not grow on test plates; therefore, neither
AhRJunD nor ArntFos is capable of binding to the XRE site without a heterodimerization partner.
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Thus, the transcriptional activation of the HIS3 reporter gene in the XRE-integrated strain
requires the presence of both GAL4AD-AhRJunD and GAL4AD-ArntFos hybrid proteins. These
results indicate that in yeast, AhRJunD and ArntFos must heterodimerize and bind to the XRE
site, as the basic regions determine the DNA-binding specificity.
Figure 4.2 HIS3 reporter assay for detection of interactions between heterodimers and the XRE cognate sequence. The heterodimeric pairs of (A) AhRJunD/ArntFos, (B) AhR(ΔL)JunD/ArntFos, and (C) AhRbHLH/ArntbHLH were coexpressed by use of protein expression vector pCETT2 in strain YM4271[pHISi-1/XRE-6]. The transformants that coexpress both GAL4AD-fused monomers, as well as their corresponding controls, were grown as patches on SD/-H/-L control plates (left in each subfigure) that select only for the presence of the plasmid and replica-plated on SD/-H/-L/30mM 3-AT test plates (right in each subfigure) that select for HIS3 reporter activity (incubated at 30 °C, 5 days). The transformants on the same plate are identified according to the following order. (A) upper left: pCETT2/AhRJunD/ArntFos, upper right: pCETT2/AhRJunD, lower left: pCETT2//ArntFos, and lower right: pCETT2; (B) upper left: pCETT2/AhR(ΔL)JunD/ArntFos, upper right: pCETT2/AhR(ΔL)JunD, lower left: pCETT2//ArntFos, and lower right: pCETT2; (C) upper left: pCETT2/AhRbHLH/ArntbHLH, upper right: pCETT2/AhRbHLH, lower left: pCETT2//ArntbHLH, and lower right: pCETT2.
The coexpression of GAL4AD-AhR(ΔL)JunD and GAL4AD-ArntFos hybrids in the
same HIS3 reporter strain (Figure 4.2B) displayed similar results to that of GAL4AD-AhRJunD
and GAL4AD-ArntFos. Again, only the transformant that coexpresses both hybrid proteins was
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able to grow on SD/-H/-L/30 mM 3-AT plates. The cells expressing only GAL4AD-
AhR(ΔL)JunD did not grow on the test plates. These data reveal that AhR(ΔL)JunD is also able
to heterodimerize with ArntFos, and this heterodimer targets the XRE as does the
AhRJunD/ArntFos heterodimer.
In contrast, the cells that coexpress GAL4AD-AhRbHLH and GAL4AD-ArntbHLH did
not grow on 3-AT containing test plates (Figure 4.2C), indicating that coexpression of just the
bHLH domains of AhR and Arnt is insufficient to trigger reporter gene activation.
Figure 4.3 HIS3 reporter spot titration assay for comparison of the relative strengths of AhRJunD/ArntFos:XRE and AhR(ΔL)JunD/ArntFos:XRE interactions. YM4271[pHISi-1/XRE-6] cells were transformed with either pCETT2/AhRJunD/ArntFos or pCETT2/AhR(ΔL)JunD/ArntFos and plated on SD/-H/-L plates. Fresh colonies were first resuspended in 1 mL H2O and diluted to OD600 0.1. 15 μL of 10-fold serial dilutions (OD600: 10-1 to 10-4) of the resuspensions at OD600 0.1 were spotted on SD/-H/-L plates and on SD/-H/-L plates containing 30 mM 3-AT.
Although the HIS3 reporter assay is qualititative and only shows growth or death, the
survival rate of a particular transformant in the presence of 3-AT generally correlates with
transcriptional activity (116). Therefore, lower survival of transformants on 3-AT-containing
plates indicates weaker transcriptional potency of the GAL4 AD fusion, probably caused by
weaker affinity of the protein of interest to its response element (25). As both AhRJunD and
AhR(ΔL)JunD are able to heterodimerize with ArntFos and target the XRE site, we performed a
spot titration assay to differentiate the transcriptional potencies of the transformants that
coexpress GAL4AD-ArntFos with either GAL4AD-AhRJunD or GAL4AD-AhR(ΔL)JunD
(Figure 4.3). Fresh colony dilutions of both transformants at a gradient of cell densities from
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OD600 10-1 to 10-4 were spotted on both SD/-H/-L control plates and on SD/-H/-L test plates
containing 30 mM 3-AT.
Figure 4.4 Colony-lift filter assay for the detection of interactions between heterodimers and the XRE cognate sequence. YM4271[pLacZi/XRE-6] cells were transformed with different pCETT2 recombinant plasmids for coexpression of heterodimeric pairs of (A) AhRJunD/ArntFos, (B) AhR(ΔL)JunD/ArntFos, (C) AhRbHLH/ArntbHLH, as well as their corresponding controls, and were plated on SD/-L/-U plates. X-gal colony-lift assay was performed after 4 days growth, 30 °C. Photos were taken after 60 min incubation at 30 °C. The transformants are identified from left to right: (A) pCETT2/AhRJunD/ArntFos, pCETT2/AhRJunD, pCETT2//ArntFos, and pCETT2; (B) pCETT2/AhR(ΔL)JunD/ArntFos, pCETT2/AhR(ΔL)JunD, pCETT2//ArntFos, and pCETT2; (C) pCETT2/AhRbHLH/ArntbHLH, pCETT2/AhRbHLH, pCETT2//ArntbHLH, and pCETT2.
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As the hydrophobic heptad repeat continuing through AhR Helix 2 and the JunD leucine
zipper of AhR(ΔL)JunD is out of register due to the deletion of a leucine at the junction between
Helix 2 and the LZ, we expected that the AhR(ΔL)JunD/ArntFos heterodimer would display a
weaker signal from transcriptional activation as compared with AhRJunD/ArntFos in the MY1H.
Surprisingly, the growth of both transformants on the test plates was quite similar. Both were
capable of growth to OD600 10-4, indicating that the transcriptional potencies of both
heterodimers are comparable, thereby implying that both heterodimers are likely able to bind the
XRE site with comparable affinities.
Two lacZ-based β-galactosidase reporter assays, the X-gal colony-lift filter assay and
ONPG assay, further confirmed the results from the HIS3 reporter assays above. In the
qualititative colony-lift filter assay, the transformants coexpressing GAL4AD-ArntFos and either
GAL4AD-AhRJunD or GAL4AD-AhR(ΔL)JunD showed similar transcriptional activation of the
lacZ reporter gene. Both started to show blue color within 20 minutes and became bright blue
after 60 minutes (Figure 4.4A and B). None of their controls, which expressed only one of the
two proteins or neither, produced detectable blue color after 60 minutes. Similar to the negative
controls, the transformant expressing GAL4AD-AhRbHLH and GAL4AD-ArntbHLH (Figure
4.4C), as well as transformants expressing only one of the two proteins, showed color
development indiscernible from the background control transformed with pCETT2.
The ONPG assay further quantified these results. Expression of GAL4AD-ArntFos with
either GAL4AD-AhRJunD or GAL4AD-AhR(ΔL)JunD in the lacZ reporter strain led to the
same level of β-galactosidase activity, 95.7±3.1 and 95.1±9.0, respectively (Figure 4.5;
pCETT2/AhRJunD/ArntFos and pCETT2/AhR(ΔL)JunD/ArntFos), demonstrating that both
AhRJunD and AhR(ΔL)JunD confer similar XRE-binding capability upon heterodimerization
with ArntFos. These quantitative results corroborate those gained from the HIS3 reporter spot
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titration assay. As expected, expression of only one of the two proteins displayed β-galactosidase
activities comparable to background (Figure 4.5; pCETT2/AhRJunD, pCETT2/AhR(ΔL)JunD,
pCETT2//ArntFos, and pCETT2). Because expression of AhRbHLH and ArntbHLH displayed
negative signals in both the HIS3 reporter assay and colony-lift filter assay, the ONPG assay was
not performed.
Figure 4.5 ONPG measurements for detection of AhRJunD/ArntFos:XRE and AhR(ΔL)JunD/ArntFos:XRE interactions by use of protein expression vector pCETT2 in strain YM4271[pHISi-1/XRE-6]. Vertical axis indicates the mean values in β-galactosidase units. Error bars represent standard error from at least three independent trials conducted in triplicate.
4.4.2 Heterodimerization between two monomers confirmed in the Y2H assay
The homo- and heterodimerization activity of our hybrid proteins was examined in the
GAL4 yeast two-hybrid system. To exclude the possibility that the transformants show Ade+His+
and α-galactosidase-positive phenotypes simply because they encode a polypeptide that interacts
73
with either the GAL4 DBD of the fusion protein or the GAL4 AD of the fusion protein and not
the protein(s) under investigation, we cotransformed yeast strain AH109 with either the
corresponding GAL4 AD fusion plasmids (pGADT7-based) and the control containing only the
GAL4 DBD (pGBKT7), or the corresponding GAL4 DBD fusion plasmids (pGBKT7-based) and
the control containing only the GAL4 AD (pGADT7), and tested for adenine and histidine
prototrophy and α-galactosidase activity. Also, in order to exclude the possibility that some
proteins under investigation are transcriptionally active, a reverse test was performed that
exchanges the two proteins that are fused to the DBD and AD (17).
Figure 4.6 Y2H assay of homo- and heterodimerization of AhR and Arnt hybrid proteins. Each transformant is identified by a single letter and number, indicated in the figure and described in Table 4.1. The transformants were grown as patches on SD/-L/-W medium (A) that selects only for the presence of the plasmids and replica-plated on SD/-A/-H/-L/-W/X-α-Gal medium (B) that selects for two-hybrid activity.
None of these controls exhibited two-hybrid activity in the test except one (Figure 4.6,
Table 4.1). The GAL4AD-ArntFos fusion gave a weak positive signal when expressed with the
GAL4 DBD, indicating that there is no dependence on any hybrid proteins, i.e. ArntFos,
AhRJunD, or AhR(ΔL)JunD, that are fused to the GAL4 DBD to activate reporter transcription.
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Although this transformant is able to grow on the test plate, its blue color is much less intense
than the other true positives, which indicates this transactivation is relatively weak.
Table 4.1 Dimerization of AhR- and Arnt-hybrid proteins.1 pGBKT7/DBD fusion pGADT7/AD fusion X-α-Gal plate
A1 AhRJunD ArntFos + A2 AhRJunD vector only − A3 vector only ArntFos −2 A4 ArntFos AhRJunD + A5 ArntFos vector only − A6 vector only AhRJunD − B1 AhRbHLH ArntbHLH − B2 AhRbHLH vector only − B3 vector only ArntbHLH − B4 ArntbHLH AhRbHLH − B5 ArntbHLH vector only − B6 vector only AhRbHLH − C1 AhR(ΔL)JunD ArntFos + C2 AhR(ΔL)JunD vector only − C3 ArntFos AhR(ΔL)JunD + C4 vector only AhR(ΔL)JunD − C5 AhRJunD AhRJunD + C6 ArntFos ArntFos + D1 AhRbHLH AhRbHLH − D2 ArntbHLH ArntbHLH − D3 AhR(ΔL)JunD AhR(ΔL)JunD + D4 vector only vector only −
1 The pGBKT7- and pGADT7-encoded fusions were coexpressed in yeast AH109 cells and tested for adenine and histidine prototrophy, as well as expression of X-α-galactosidase; + : blue color detected; − : no detectable growth and/or no color development. 2 False positive.
The most straightforward interpretation of this result is that ArntFos binds to the GAL4
DBD or a DNA sequence in the promoter of the reporter gene. According to the intensity of the
blue color, the GAL4AD-ArntFos fusion displayed measurably stronger transcriptional activity
in the presence of the GAL4 DBD fusions with AhRJunD, AhR(ΔL)JunD, or ArntFos than when
expressed with the GAL4DBD alone; thus, the ArntFos protein interacts with AhRJunD,
AhR(ΔL)JunD, and ArntFos. In addition, since the GAL4DBD-ArntFos fusion gave a negative
signal when expressed with the GAL4AD, but displayed a positive signal when expressed with
the GAL4AD fusions with AhRJunD, AhR(ΔL)JunD, or ArntFos, a conclusion can be drawn
with confidence that ArntFos can homodimerize and also heterodimerize with AhRJunD or
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AhR(ΔL)JunD. Additionally, both AhRJunD and AhR(ΔL)JunD can homodimerize, while
neither AhRbHLH nor ArntbHLH can homodimerize or heterodimerize with each other in yeast.
As transcriptional potency of reporter activation usually reflects the strength of the
assayed interaction in the Y2H (25), a similar spot assay to that in the MY1H assay was
performed in order to further examine the relative strengths of homo- or heterodimerization of
the hybrid proteins, as they possess relatively similar primary sequences and structures. Fresh
colony dilutions of transformants at a gradient of cell densities from OD600 10-1 to 10-5 were
spotted on both SD/-L/-W control plates and SD/-A/-H/-L/-W/X-α-Gal test plates. The survival
status and color development are dependent on the relative strength of the interaction between
the two proteins coexpressed in the transformant: the stronger the interaction, the lower the
density of the spot at which the transformant is able to survive and the more blue color the
transformant develops on the test plate.
All five transformants showed different dimerization affinities, as measured by
transcription potencies in the Y2H. The transformant expressing GAL4DBD-ArntFos and
GAL4AD-AhRJunD showed similar dimerization strength to the transformant expressing
GAL4DBD-AhRJunD and GAL4AD-AhRJunD. These two transformants displayed the
strongest transcriptional potencies among the five. The transformant expressing GAL4DBD-
ArntFos and GAL4AD-AhR(ΔL)JunD displayed similar dimerization affinity to the transformant
expressing GAL4DBD-AhR(ΔL)JunD and GAL4AD-AhR(ΔL)JunD.
Therefore, both AhRJunD and AhR(ΔL)JunD are able to homodimerize and
heterodimerize with ArntFos, and the homo- and heterodimers that contain the AhRJunD
monomer are of stronger dimerization affinity than those containing AhR(ΔL)JunD. In addition,
ArntFos was also titrated to test the strength of dimerization. As GAL4AD-ArntFos gave false
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positive results in the yeast reporter assays, no reliable conclusion can be made as to the relative
strength of the ArntFos homodimer without in vitro measurement.
4.5 Discussion
4.5.1 Fusion of the JunD and Fos LZ domains to the Arnt and AhR bHLH domains reconstitutes the heterodimeric structure and specific DNA-binding function of the bHLH/PAS domains of AhR and Arnt
Our results clearly reveal that in yeast, the GAL4 AD fusions of the bHLH domains of
AhR and Arnt are unable to activate the transcription of reporter genes. However, when the
leucine zippers of bZIP proteins JunD and Fos are fused to the C-termini of the bHLH domains
of AhR and Arnt, respectively, these two hybrid proteins are able to heterodimerize and
specifically target the cognate XRE sequence in yeast, with concomitant reporter gene activation.
The results suggest two points: first, although the bHLH domains of the bHLH, bHLHZ,
and bHLH/PAS proteins are likely to be structurally similar and are proposed to have a similar
mode of DNA binding (59), there are still fine structural differences among these bHLH domains.
Structural analysis of bHLH and bHLHZ proteins demonstrates that the HLH domains from the
two monomers dimerize and form a compact four-helix bundle, which positions the basic regions
for specific DNA binding. For bHLH proteins, such as E-proteins, the HLH domains intrinsically
possess the capability of forming stable homo- or heterodimers with partner bHLH proteins
without the aid of a secondary dimerization region. In contrast, for bHLHZ proteins, the leucine
zipper domain, which is fused to the C- terminus of Helix 2 of the bHLH domain, is necessary
for achieving stable dimeric structure.
The LZ enhances DNA binding by elongating the dimerization interface in a long rigid α-
helical coiled coil (61,70,76,141). We observed that in the Y1H system, deletion of the LZ from
the Max bHLHZ abolished homodimerization and DNA binding of Max, indicating that the LZ
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is critical for Max dimerization in addition to determination of partner specificity (J. Xu, J. A.
Shin, unpublished results). These observations show that unlike bHLH proteins, the isolated
bHLH domains of bHLHZ proteins are incapable of forming stable DNA-binding dimers without
the additional "inducer" element LZ necessary for generating the dimeric structure capable of
specific DNA binding. In addition, analysis of HLH sequences from bHLH and bHLHZ proteins
indicates that more potential attractive pairs of residues exist between the monomers of bHLH
proteins than bHLHZ proteins (71), further proving the requirement of the additional LZ in
bHLHZ proteins for efficient dimerization and DNA-binding function.
Similar to the LZ in the bHLHZ family, the PAS domain in bHLH/PAS proteins also
serves as a secondary dimerization interface to initiate and/or stabilize folding and dimerization
of the bHLH domains (72,142,152). Although the isolated bHLH domains of AhR and Arnt
proteins can homo- and heterodimerize and weakly bind to specific DNA sites in vitro, the
inclusion of the PAS A domains considerably increases the binding affinity and specificity of the
heterodimeric AhR/Arnt:DNA interaction (152,154), indicating that PAS domains serve as the
additional inducer for high affinity DNA binding of the bHLH domains of AhR and Arnt. In this
regard, LZ regions in bHLHZ proteins and PAS domains in bHLH/PAS proteins are the primary
driving force dictating partner specificity leading to heterodimerization and DNA-binding
activity.
The second point suggested by our results is that the leucine zippers of JunD and Fos may
replace the dimerization and protein stabilization function of the PAS domains in AhR and Arnt.
Attachment of the JunD or Fos LZ to the bHLH domains of AhR or Arnt restored protein
heterodimerization and DNA binding at the XRE in vivo; similar chimeric proteins in which the
LZ of Myc and Max were fused to bHLH domains of AhR and Arnt, respectively, were reported
to be capable of heterodimerization and binding to the XRE in vitro (72). We emphasize that we
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do not directly measure protein dimerization in the MY1H, but that we infer dimerization with
positive DNA-binding signal, as we have shown above that protein constructs capable of only
monomeric or homodimeric DNA binding showed no detectable binding to the XRE in in vivo
yeast assays.
Compared with the LZ domains in bHLHZ proteins, the function of the PAS domain in
protein dimerization and DNA binding is quite diverse and not fully understood. The PAS
domain clearly forms a secondary dimerization interface and stabilizes dimer formation (74). In
the absence of the bHLH domain, the interaction between the isolated PAS domains of the AhR
and Arnt can be detected using the mammalian two-hybrid assay (155), and deletion of the entire
PAS region of Arnt markedly reduces dimerization with the AhR (156). The PAS domain of
Arnt may have a role in stabilizing the bHLH domain in the absence of DNA (152). In addition
to providing additional protein dimerization interface and contributing to dimerization strength,
the PAS domains also confer the specificity of partner choice within bHLH/PAS family. The
PAS A domain restricts AhR to heterodimerization with Arnt, as the isolated AhR bHLH domain
is capable of homodimerization (154). Moreover, the PAS domains of bHLH/PAS proteins can
contribute directly to DNA-binding activity, and interdomain communication between the bHLH
and PAS domains of bHLH/PAS proteins probably exists (72,74).
For hybrid proteins AhRJunD and ArntFos, we believe the LZ pair mimics the
dimerization and stabilization function of the PAS domains in bHLH/PAS proteins. Although it
is unlikely that the LZ coiled coil makes direct contact with the XRE DNA site, we suspect the
attachment of the JunD or Fos leucine zippers to the AhR or Arnt bHLH domains likely
increases the stability and/or helicity of the AhR and Arnt bHLH domains, with attendant
increase in DNA-binding affinity. For example, the DNA binding affinity of a MyoD hybrid was
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increased ~4-fold by introducing a disulfide-stabilized helix at the N-terminus of the basic
domain of MyoD (157).
Our results show that the effect of the PAS domains on specific DNA binding by
bHLH/PAS proteins is not unique, as the replacement of PAS domains with the LZ region can
also reconstitute the heterodimeric structure and DNA-binding function of the bHLH domains of
AhR and Arnt. Although our results do not conclusively prove that dimerization and DNA
binding by bHLH/PAS proteins, and similarly for bHLHZ proteins, occurs via an additive
process of dimerization between discrete protein modules, they do provide additional evidence of
the critical role that PAS domains play in dimerization of bHLH/PAS proteins.
4.5.2 The nonpalindromic XRE DNA target mediates the heterodimerized structure of AhRJunD and ArntFos, resulting in DNA-binding function
The LZ regions in bHLHZ proteins are believed to be responsible for dimerization
specificity, as well as providing an additional dimerization interface. The presence of two
distinct dimerization domains was proposed to ensure a high level of discrimination with respect
to dimer partner (82). Our original design also followed this plausible rule. However, our Y2H
results reveal that both AhRJunD and ArntFos are able to homodimerize, as well as
heterodimerize with each other; furthermore, spot assays in the Y2H indicate that AhRJunD
shows almost no preference for homodimerization over heterodimerization with ArntFos (Figure
4.6). Thus, the discrimination regarding dimerization partner selection is at least compromised, if
not totally lost, in our hybrid proteins.
As hypothesized, in the presence of the nonpalindromic XRE target site, neither the
AhRJunD homodimer nor ArntFos homodimer is able to bind the XRE site and activate reporter
gene expression in our MY1H system. Only when AhRJunD and ArntFos are coexpressed do
they heterodimerize and trigger reporter gene activation by specific XRE binding. Apparently,
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assembly of the AhRJunD/ArntFos:XRE complex is driven largely by the strength of each
monomer’s interaction with the DNA: i. e. the heterodimerization of bHLHZ-like AhRJunD and
ArntFos is mediated by the nonpalindromic XRE site and insignificantly, or not at all, by the
differential protein-partner interactions at the dimer interface. This result is in contrast to
dimerization-dependent DNA binding of AP-1 by bZIP proteins Jun and Fos. The Jun/Fos
heterodimer targets the AP-1 binding site in strong preference to the Jun/Jun homodimer.
However, this preference is due to increased dimer stability specified by several amino acids in
the LZ regions of both Jun and Fos, rather than any difference in how the two monomers contact
DNA (82). This mode of DNA-mediated heterodimerization was also found in the C/EBP-ATF
chimeric site targeted by ATF4/IGEBP1 and E-box site targeted by the MyoD-E47 heterodimer
(158,159). Thus, Nature may often utilize DNA-mediated heterodimerization as a common
strategy for specifying protein-protein partnering and protein:DNA recognition in the bZIP and
bHLH superfamilies, as well as using specific interactions at the protein dimer interface.
Therefore, DNA-binding specificity can serve as a feedback mechanism to ensure a
desired protein dimerization preference: a nonpalindromic DNA target site could force the
formation of heterodimer (kinetically) instead of formation of two homodimers that are also
stable in the absence of the DNA target site.
4.5.3 What causes the negative signal in XRE binding by the bHLH domains of AhR and Arnt in the MY1H?
There are several possibilities that may lead to the negative signal observed with
targeting the XRE site by the AhR bHLH and Arnt bHLH in yeast. First, the AhR bHLH and/or
Arnt bHLH may not be stably expressed or present in yeast. Under physiological conditions, the
full-length AhR either depends on chaperones, including Hsp90 and other proteins, or it
heterodimerizes with Arnt once it dissociates from chaperones (160); in both cases properly
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folded structure is most likely maintained. There is evidence that Hsp90 also interacts with the
bHLH domain of the AhR protein (161). In yeast cells, the lack of folding partner may lead to
protein degradation or aggregation. In addition, the expression of the AhR bHLH and Arnt
bHLH domains in E. coli has been shown to be problematic; even coexpression of the AhR
bHLH and Arnt bHLH domains in E. coli failed to recover any DNA binding activity, while
coexpression of the corresponding bHLH/PAS domains dramatically increased recovery of
functional proteins (152).
Second, the AhR bHLH may not be able to heterodimerize with the Arnt bHLH and
target XRE in yeast. The Arnt bHLH homodimer forms a less hydrophobic four-helix bundle
than other bHLH and bHLHZ proteins (142), such as E47 (75) and Max (61,141), both of which
form stable homodimers that target the E-box site. The AhR and Arnt bHLH domains may not be
stable or properly folded in yeast, as they lack the PAS domains to help stabilize the more
hydrophilic bHLH structure. PAS domains of bHLH/PAS proteins have been proposed to initiate
folding and/or stabilize dimerization of the bHLH region in a mode similar to that served by the
LZ in the bHLHZ Myc/Max heterodimer (152). Therefore, the LZ fused to the C-termini of the
AhR and Arnt bHLH domains serves in a manner similar to the LZ in bHLHZ proteins: a
nucleation device encouraging folding of the HLH domain from less ordered structure to
organized four-helix bundle. This may explain why we observed positive signals from the XRE
binding by bHLHZ-like AhRJunD and ArntFos in the MY1H, but not from just the bHLH
domains of AhR and Arnt.
4.5.4 Deletion of one leucine residue in the JunD LZ negatively affects heterodimerization but not DNA binding
The Y2H assay revealed that AhRJunD heterodimerizes with ArntFos more tightly than
does AhR(∆L)JunD. This must be due to the deletion of a Leu residue at the N-terminus of the
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JunD LZ in AhR(∆L)JunD; this deletion disrupts the hydrophobic heptad repeat register between
the AhR bHLH and JunD LZ. In the MY1H, however, the transformant coexpressing
AhR(∆L)JunD and ArntFos surprisingly displayed almost identical transcriptional activation of
the reporter gene to the one coexpressing AhRJunD and ArntFos.
Apparently, the register of the JunD LZ only affects the dimerization strength of the
heterodimer, not its DNA binding activity. This finding is probably due to the role that the LZ
plays in these hybrid proteins. The leucine zippers serve to stabilize the proper folding of the
contiguous bHLH domains of AhR and Arnt by nucleating α-helix formation and proper protein
folding; they do not contribute positively to the binding affinity between the heterodimer and the
target XRE site. Instead, there is evidence showing that the LZ can negatively affect the DNA-
binding affinity of Arnt bHLH in vitro. Quantitative fluorescence anisotropy analysis revealed
that fusion of the LZ from bZIP protein C/EBP with the bHLH of Arnt yields a protein hybrid
that binds the E-box DNA site with 4-fold reduced affinity compared with E-box binding by the
isolated Arnt bHLH domain (153). In the absence of DNA, however, the JunD/Fos LZ pair,
which is a secondary dimerization element, plays a more significant role with increased
contribution to the protein dimer interface.
In the presence of DNA, the four-helix bundle formed between the bHLH domains of
AhR and Arnt is the dominant interaction stabilizing the protein heterodimer, and ultimately,
contributes to the DNA-binding affinity. Furthermore, we note that the boundaries of each region
of these of bHLH domains were predicted based on sequence similarity with bHLHZ protein
Max; such alignment may not reflect the entire bHLH domain of bHLH/PAS proteins. The
fusion proteins were generated by direct attachment of the LZ to the C-termini of the bHLH
domains of bHLH/PAS proteins; no structural optimization was performed at the junction
between Helix 2 of the bHLH and the LZ, except that the hydrophobic heptad repeat was
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maintained in AhRJunD and ArntFos to give a structure hypothesized to mimic that of Max
bHLHZ. More definitive analysis of the structural differences between AhRJunD and
AhR(∆L)JunD, and conclusions about protein dimerization structure and DNA-binding function,
is possible once high-resolution structural information of the AhR bHLH domain is available and
in vitro measurements of the hybrid proteins are completed.
4.5.5 Our domain swapping experiments support the hypothesis of domain shuffling in the evolutionary pathway of the bHLH superfamily
In addition to the highly conserved HLH domain, members of the bHLH superfamily
may also contain LZ or PAS domains. In some family members, the basic region is even missing,
such as with Id, an HLH negative regulator of bHLH proteins (162). Moreover, the relative
position of the bHLH domain in different family members varies considerably (98). From an
evolutionary perspective, these conserved domains in bHLH proteins are usually independent
and separately evolved. Based on a study of the bHLH domains in 242 bHLH superfamily
members, Atchley and Fitch produced a phylogenetic tree to classify family members according
to evolutionary relationships, and found their analyses could not rigorously support the
hypothesis of a single evolutionary origin for the bHLH domain (98). However, further intensive
investigation of the non-bHLH components of 122 bHLH superfamily members provided strong
evidence that supports the hypothesis that bHLH proteins have undergone modular evolution by
domain shuffling, a process that involves domain insertion and rearrangement (163).
Our domain swapping experiment provides additional, complementary evidence for the
hypothesis of modular evolution by domain shuffling. We found that the PAS domains in the
bHLH/PAS AhR/Arnt heterodimer can be replaced by the LZ domain from the bZIP, a different
transcription factor family from the bHLH, while maintaining dimerized protein structure and
specific DNA-binding function. Taken together, our results provide further evidence that domain
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shuffling is likely the evolutionary pathway that Nature utilized to gain diverse structural
patterns and variable functions in the bHLH superfamily.
4.6 Supplemental Information Supplemental Information associated with this manuscript is provided in Appendix C.
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Chapter 5 Summary and future work
This chapter presents a summary and interpretation of this body of work and recommends
directions for future research.
5.1 Summary Due to the critical role that DNA-binding proteins play in the regulation of diverse
physiological functions including cell development, growth, and differentiation, quite a few
approaches have been employed to investigate the molecular basis of protein:DNA recognition
as well as the cooperative, enhancing (coactivating), and inhibitory effects of cofactors on
particular protein:DNA interactions. Owing to their unique physiologically relevant environment,
high efficiency and sensitivity, and relatively easy manipulation, yeast genetic systems have been
successfully applied to study these interactions.
This thesis is devoted to the development of a modified Y1H system useful for
examination of both protein-protein and protein:DNA interactions in vivo and to applying this
MY1H system to the study of heterodimeric protein:DNA interaction. Hence, we developed a
single plasmid-based MY1H capable of differential protein expression levels. In addition to a
GAL4 AD fusion protein, a second protein can be coexpressed at either comparable or higher
transcriptional levels from expression vectors pCETT or pCETF, respectively. This second
protein can play a structural, modifying, or inhibitory role that restores or blocks reporter gene
expression.
This system was validated by use of the well-characterized DNA-binding protein p53 and
its inhibitory partners, large T antigen (LTAg) and 53BP2. In the MY1H, the DNA binding of
p53 decreases by different extents dependent on different inhibitory partners and their expression
levels. As demonstrated, our system is particularly useful for examining the effects of a
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regulatory protein on the DNA-binding activity of a transcription factor. Moreover, in the study
of inhibitors of a DNA-binding protein or in DNA-binding competition assays, the regulatory
protein can be expressed at comparable or excess levels from MCS II compared with the
GAL4AD-fused protein from MCS I, therefore allowing qualitative assessment of the strength of
the protein:DNA or protein-protein interaction of interest by combined use of pCETT and
pCETF. This feature distinguishes our system from other reported systems and was fully
exemplified in the study of the repression of the Max:E-box DNA site interaction with several
competitor mutants of Max (J. Xu, J. A. Shin, unpublished results), as well as in the
p53/LTAg/53BP2 system (Chapter 2). In addition, this system can also be potentially applied to
investigate coregulatory or bridging proteins and to identify novel protein- or DNA-binding
partners through library screening under certain circumstances.
We then applied this MY1H system to investigate the DNA-binding activities of
heterodimeric proteins from the bHLH/PAS family. A positive control for the study of XRE-
binding proteins was first performed in the MY1H system in which the reporter genes were
driven by the XRE cognate sequence in the promoter. This positive control utilizes the
bHLH/PAS domains of AhR and Arnt (NAhR and NArnt) that are known to form heterodimers
and bind the cognate XRE sequence both in vitro and in vivo. However, negative signals were
observed when these two proteins were coexpressed from pCETT in the MY1H. Several
strategies were implemented together to turn this false negative into a true positive. By
increasing the copy number of XRE sites integrated into the yeast genome from three to six, and
at the same time fusing to each monomer a GAL4 activation domain, the heterodimer formed by
NAhR and NArnt specifically bound the cognate XRE sequence, which displays clear positive
binding signals with high signal-to-noise ratios in the MY1H system. Turning this false negative
interaction into a true positive control made possible the further study of XRE-binding proteins
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in the MY1H system. Moreover, this methodology provides an effective way for trouble-
shooting the false negatives that arise from unproductive transcription in yeast genetic assays.
In order to gain more insight into the role the PAS domains of AhR and Arnt play in
protein dimerization and DNA binding, and to further understand DNA recognition by
heterodimeric proteins, a pair of bHLHZ-like proteins, AhRJunD and ArntFos, were designed
and their dimerization and specific DNA-binding activities were explored in the MY1H and Y2H
systems and are now undergoing in vitro analysis. In vivo results reveal that both AhRJunD and
ArntFos are able to form homodimers, and that the presence of the nonpalindromic XRE cognate
sequence forces heterodimerization of the two hybrids for specific DNA binding.
Therefore, of the five scenarios that can be examined by use of the MY1H system (Figure
2.1), three have been successfully validated: the system investigating the inhibition of
LTAg/53BP2 on DNA binding of p53, where a protein-protein interaction was assayed and the
second protein serves as a blocker; the system investigating the repression of Max mutants on
DNA binding of Max bHLHZ protein, where a protein-DNA interaction was assayed and the
second protein functions as a repressor of DNA binding; the NAhR/NArnt:XRE or
AhRJunD/ArntFos:XRE system where protein heterodimer-DNA interaction was assayed. These
systems clearly demonstrate the wide applicability of this MY1H system.
5.2 Future work In this thesis, emphasis has been placed on validation of the MY1H system useful for
examination of simultaneous protein-protein and protein:DNA interactions and application of the
MY1H system to DNA-binding studies of the bHLH/PAS family of proteins. The following are
some suggestions for future improvements and new research directions.
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5.2.1 Downstream work on the AhRJunD/ArntFos:XRE interaction In addition to in vivo examination, in vitro quantitative assessment of the interaction of
AhRJunD/ArntFos heterodimer with its XRE target element by fluorescence anisotropy titration
is to be performed. The bacterially expressed proteins are currently being expressed and purified
by Antonia De Jong. Circular dichroism (CD) titration assays will also be applied to measure the
dissociation constants of the homodimers formed by these hybrid proteins. As the four-helix
bundle forms upon dimerization in bHLH proteins, an increase in helicity should be detected
with CD spectroscopy. Moreover, western blotting assays are also currently being utilized to
examine the expression of the hybrid proteins examined in the Y2H, for the Y2H is needed to
confirm that all proteins are present in the system as expected and any negative signals are not
due to false negatives.
5.2.2 Improvement on the MY1H system Although great success was achieved in the DNA binding studies of the heterodimer
between bHLHZ-like hybrids AhRJunD and ArntFos by fusing an activation domain to each
hybrid protein, we observed reduced signals when the single AD expression vector pCETT was
utilized to coexpress the two proteins in the MY1H system. For example, the NAhR/NArnt:XRE
interaction is positive only in double AD system, whereas the AhRJunD/ArntFos:XRE
interaction is positive in both the double AD system (ONPG: ~90) and single AD system (ONPG:
~40). Such observations may reflect the differences between strong and weak function; more
activation domains may amplify a weak signal, for instance. However, in some cases, the single
AD system is preferred, as heterodimeric proteins in the same family, usually containing a
monomer capable of both homo- and heterodimerization with a partner incapable of
homodimerization, are able to bind the same or similar DNA target sequences, commonly
palindromic or pseduopalindromic sequences. The double AD system will complicate the
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interactions under question as both homodimer and heterodimer occupy the same DNA binding
site, thereby activating the reporter gene. In contrast, in the single AD system, the problem can
be avoided by fusing the AD to the monomer that is incapable of forming homodimers. Although
the GAL4 AD possesses reasonable activation strength, we may utilize a stronger AD, such as
VP16, in order to facilitate the examination of the aforementioned heterodimeric protein:DNA
interactions in the MY1H system.
A second straightforward experiment would be to introduce a third protein into the
system to study the effects of this protein on the DNA-binding activity of heterodimeric proteins.
Given the fact that unregulated expression of quite a few transcription factors, for instance, the
oncogenic Myc/Max heterodimer, often leads to human diseases such as cancer, dominant
negative inhibitors could be a strategy for suppression of the aberrant function of such
transcription factors (164,165). If a third protein can be expressed in the MY1H system, the
dominant negative function of a protein on a heterodimeric transcription factor can be examined
in the MY1H system. This can be achieved by introducing a protein expression vector similar to
pGAD424-MCS IIΔAD (127) with a different nutritional marker other than LEU2.
5.2.3 Other DNA-binding hybrid proteins of interest As the basic region dictates specific DNA binding, we are now trying to evolve a
nonnative Myc/Max monomer to target the nonnative site 5'-CACCAC. This Myc-Max
monomer contains the basic regions of Myc and Max connected by a short linker. The basic idea
is that rather than using a dimer to target DNA, we fuse the basic regions of the two proteins,
each of which specifically targets a 5'-CAC half site; such covalent fusion of both basic regions
eliminates any need for dimerization and effectively increases the concentration of functional
DNA-binding structural modules at the protein:DNA interface; i.e. analogous to enzyme
catalysis, a similar concept of bringing reactants into close proximity to increase the rate of
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reaction. We anticipate that a protein conformation that optimally positions the two basic regions
of Myc and Max into the major groove of the target element can be found by mutagenizing the
short linker and performing library screening in the Y1H system. Furthermore, once the linker of
the Myc/Max monomer is optimized, one of the monomers can also be mutated to target another
DNA site as in 5'-CACNNN or 5'-NNNCAC; a library can be generated in which one of the two
basic regions is mutated and the other acts as the docking region. Eventually, this monomer
system, similar to zinc finger proteins, can potentially be used to evolve proteins that bind to any
hexameric DNA sequences.
Another hybrid protein of interest is a Max monomer, similar to the preceding concept,
that contains the basic region contiguous with a degenerate short peptide intended to target the
other half site of the E-box. We are trying to evolve a Max monomer to target the E-box cognate
sequence, which is naturally targeted by the Max bHLHZ dimer. As discussed above, this Max
monomer can protentially serve as a dominant negative inhibitor to suppress the expression of E-
box responsive genes.
In the present GAL4-based Y1H system, numerous false positives can appear during
library screening, which limits its use for effectively selecting nonnative DNA-binding protein
precursors that may bind to a DNA target with low affinity. In addition, multiple plasmids can be
transformed into a single yeast cell during library transformation, as high concentrations of DNA
is present, and such aberrant transformation of multiple plasmids makes any positive results
difficult to characterize: which gene is responsible for the protein that produced the positive
signal? We are now introducing the concept of directed evolution into the screening of the Max
monomer library in the Y1H system in an attempt to uncover any weak DNA-binding proteins
that may serve as progenitors of more active progeny and segregation of the multiple plasmids
that are often transformed into a single cell during library transformation.
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Chap 6ter Materials and Methods
6.1 General Details about reagent sources, bacterial and yeast strains, transformation, DNA
preparation, plasmid rescue, and plasmid sequencing are detailed in the Materials and Methods
sections of Chapters 2-4.
6.1.1 Two reporter assays used in the MY1H In the MY1H systems, HIS3 and lacZ are used as reporter genes. The HIS3 reporter gene
is used to test whether the transformants are auxotrophic for histidine. The lacZ reporter gene is
used to perform the β-galactosidase assay that enable colorimetric visualization of protein:DNA
binding. Two commonly used assays based on the lacZ reporter are the qualitative X-gal colony-
lift filter assay and quantitative ONPG liquid assay.
6.1.1.1 HIS3 reporter assay The detailed procedure was described in Chapter 3.
6.1.1.1.1 Spot titration assay The detailed procedure was described in Chapter 4.
6.1.1.2 X-gal colony-lift filter assay The detailed procedure was described in Chapter 2.
6.1.1.3 ONPG liquid assay The ONPG liquid assay was performed according to the protocol provided in the Yeast
Protocols Handbook (Clontech) with the following modifications. In the ONPG liquid assay,
yeast cells were grown as described below before harvesting for lysis. Transformed yeast cells
were initially grown at 30 °C with shaking in SD/-L media for 2 days or until OD600 >1.5 was
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reached, and then used to inoculate a fresh culture of SD/-L media. This secondary culture was
grown overnight until OD600 1.0-1.3 was reached. An aliquot of the secondary culture was
resuspended in YPDA to give starting OD600 ~0.2. The YPDA culture was then grown for 3-5
hrs until OD600 0.60-0.65 was reached. The units of β-galactosidase were calculated according to
β-galactosidase units = 1000 × A420 / (t × v × A600), where t = time (min) required for the reaction
and v = 0.1 × concentration factor. One unit of β-galactosidase is defined as the amount which
hydrolyzes 1 μmol of ONPG to o-nitrophenol and D-galactose per min per cell (114). Results are
presented as mean values ± S.E.M. of 3-4 independent experiments, each performed in triplicate.
6.1.2 Examination of protein-protein interactions in the Y2H system The Matchmaker™ GAL4 Two-hybrid System 3 (Clontech) was utilized to examine
homo- and heterodimerization of the proteins under investigation. The detailed procedure was
described in Chapter 4.
6.2 Modified Y1H for identification of protein-protein/protein:DNA interactions
6.2.1 Construction of p53 target-reporter strains Two yeast reporter strains, YM4271[p53HIS] and YM4271[p53BLUE], were created for
expression analysis in the MY1H from the integrating reporter vectors, p53HIS and p53BLUE
(Clontech), respectively. The p53HIS plasmid contains the minimal promoter of the HIS3 locus
(PminHIS), in which three tandem copies of the consensus p53 binding site, 5'-
AGGCATGCCTAGGCATGCCT, were inserted upstream of the HIS3 reporter gene. Likewise,
p53BLUE contains the minimal promoter of the yeast iso-1-cytochrome C gene (PCYC1), in
which three tandem copies of the consensus p53 binding site were inserted upstream of the lacZ
reporter gene. The p53HIS plasmid was linearized at the Xho I site and integrated into YM4271
93
by recombination to create YM4271[p53HIS]. Similarly, p53BLUE was linearized at the Nco I
site and integrated to generate YM4271[p53BLUE].
To assess background HIS3 expression, 3-AT was used as a competitive inhibitor of the
yeast HIS3 protein. 45 mM 3-AT is sufficient to completely suppress background growth due to
leaky HIS3 expression in the YM4271[p53HIS] reporter strain as per the Matchmaker One-
Hybrid System User Manual (Clontech).
6.2.2 Plasmid construction Detailed information was provided in Chapter 2.
6.2.3 3-AT titration analysis The detailed procedure of the 3-AT titration analysis was described in Chapter 2.
6.3 Turning false negatives into true positives in the MY1H
6.3.1 Construction of XRE target-reporter strains The details of reporter strain construction were described in Appendix B.
6.3.2 Plasmid construction The details of plasmid construction were described in Chapter 3 as well as Appendix B.
6.4 DNA binding forces heterodimerization of hybrids of bHLH/PAS and bZIP
6.4.1 Plasmid construction The details of plasmid construction were described in Chapter 4 as well as Appendix C.
94
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Appendix A
Design of a single plasmid-based modified yeast one-hybrid system for investigation of in vivo protein-protein and protein:DNA
interactions Gang Chen, Lisa M. DenBoer, and Jumi A. Shin
Appendix A contains the original Supplemental Information for the paper accepted by
BioTechniques (Chapter 2 in this thesis), with necessary changes for thesis organization purposes,
as well as western blot data confirming expression of the proteins examined in the MY1H system.
MCS I
CTA TTC GAT GAT GAA GAT ACC CCA CCA AAC CCA AAA AAA GAG ATC TTT AAT ACG ACT
CAC TAT AGG GCG AGC GCC GCC ATC ATG GAG GAG CAG AAG CTG ATC TCA GAG GAG GAC
CTG CAT ATG GCC ATG GAG GCC GCG GTC GAC AAG GTG CGC GCT CTA GAT GAT CAT GAA
TCG TAG ATA CTG AAA AAC CCC GCAAGTTCACTTCAACTGTGCATCGTGCAC
T7 Promoter
c-Myc Epitope Tag
STARTin vitro
GAL4 Activation Domain
BssH II
Xba I
STOP
STOP
Bcl I
STOP
Sac II
Sal I
STOP
STOP
MCS II
CCT CCA AAA AAG AAG AGA AAG GTC GAA TTG GGT ACC GCC GCC AAT AAA GAG ATC TTT
AAT ACG ACT CAC TAT AGG GCG AGC GCC GCC ATG GAG TAC CCA TAC GAC GTA CCA GAT
TAC GCT CAT ATG GCC ATG GAG GCC AGT GAA TTC CAC CCG GGT GGG CAT CGA TAC GGG
ATC CAT CGA GCT CGA GCT GCA GAT GAA TCG TAG ATA CTG AAA AAC CCC GCA AGT TCA
CTTCAACTGTGCATC
T7 Promoter
SV40 Nuclear Localization Sequence
STARTin vitro
HA Epitope Tag
Sma Xma I I/STOP
Nde I
Sfi I STOP
Xho I
Sac I
Pst I
EcoR I STOP
Cla I
BamH I
Figure A.1 Plasmids pCETT (truncated ADH1 promoter) and pCETF (full-length ADH1 promoter) were constructed for coexpression of two proteins in a yeast model system. The two vectors have unique restriction sites located in both the MCS I and MCS II regions. MCS I is at the 3'-end of the open reading frame for the GAL4 AD sequence allowing a fusion protein combining amino acids 768–881 of the GAL4 AD and the cloned protein of interest to be expressed at low levels from a truncated constitutive ADH1 promoter. The expression of genes inserted into MCS II is controlled by either the truncated ADH1 promoter (pCETT) or full length ADH1 promoter (pCETF). Therefore, a second protein can be coexpressed at either low levels (pCETT) or high levels (pCETF). Both vectors also contain a T7 promoter at both MCS regions, a c-Myc epitope tag at MCS I, and an HA epitope tag at MCS II.
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A.1 Western blot analysis Yeast cells were grown as described for the 3-AT titration assay. Cells were then
harvested by centrifugation for 5 min at 3300 rpm and resuspended in 100 µL cold lysis buffer
(50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1%
SDS, and 0.02% sodium azide) containing freshly added protease inhibitors [100 µg/ml
phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml
pepstatin]. The suspensions were incubated on ice for 5 min, and then subjected to 3 rounds of
sonication for 15 sec each. Lysates were again incubated on ice for 5 min, and then centrifuged
for 10 min at 4 °C. The pellet was resuspended in 100 µL 3M urea and incubated at room
temperature for 3 min. An equal volume of SDS-PAGE sample buffer (50 mM Tris-HCl, pH 6.8,
100 mM DTT, 2% SDS, 10% glycerol, and 0.1% bromophenol blue) was added and the samples
were boiled for 10 min. Samples were stored at -80 °C until use.
20 µL of samples were loaded on a 12% SDS-PAGE (Tris-Glycine) gel and run at 100V
for 90 min in SDS-PAGE Running Buffer (25 mM Tris-HCl, 192 mM glycine, 0.5% w/v SDS),
then transferred by tank electroblotting to a nitrocellulose membrane at 100V for 75 min in cold
transfer buffer (25 mM glycine, 192 mM Tris-HCl, 20% v/v methanol). The membrane was
washed in TBS (10 mM Tris-HCl, pH 7.5, 150 mM NaCl) for 10 min and washed twice in TBS-
T (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.05% v/v Tween-20) for 10 min each. The
membrane was blocked in TBS-T containing 5% non-fat skim milk powder at room temperature
with shaking overnight. The membrane was washed twice in TBS-T for 10 min each, then once
in TBS for 10 min. The primary antibody (HA.11 or 9E10(c-Myc), Covance Inc., Princeton, NJ)
was diluted 1000-fold in TBS and the membrane was incubated for 2 hrs at room temperature,
followed by washing as before, and incubation with the secondary antibody (goat anti-mouse
107
IgG-HRP conjugate, Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hr at room temperature.
The membrance was washed again, as before, and the protein bands were visualized using the
ECL Plus Western Blotting Detection System (Amersham Biosciences, Piscataway, NJ) on a
Molecular Dynamics Storm 840 PhosphorImager System.
The gel was washed in water, post-transfer, for 10 min, then stained with Coomassie
Brillant Blue (0.25% w/v in 50% v/v methanol) for 30 min, and de-stained in 10% v/v acetic acid
until the desired color was reached.
A.2 Expression levels of LTAg and 53BP2 in the MY1H system
Figure A.2 Promoter length dictates differential expression levels of inhibitory proteins when YM4271[p53HIS] cells were transformed with the indicated plasmids and grown to exponential phase in YPDA media. Cells were lysed by sonication and 20 µl aliquots were separated by SDS-PAGE and subjected to Western blotting. Proteins were immuno-detected using an anti-HA antibody and visualized by fluorescence detection on a Molecular Dynamics Storm 840 Phosphorimager.
Western blot analysis was performed to examine the expression levels of GAL4AD-p53
and the two inhibitory proteins LTAg and 53BP2 in the MY1H system. We obtained positive
signal from MCS II, but not from MCS I. As stated by Clontech, the truncated ADH1 promoter
in vector pGAD424, the protein expression plasmid provided in the Matchmaker™ One-Hybrid
System, leads to very low levels of fusion protein expression in yeast, such that the signal is not
108
detectable in western blot analysis (114). As our coexpression plasmids, pCETT and pCETF, are
based on pGAD424, they share the same expression cassette in MSC I. Although we also tried
different primary monoclonal antibodies (c-Myc mAb and p53 mAb), none displayed positive
signal.
In contrast, HA-tagged LTAg and 53BP2 fusion proteins expressed from both pCETT
and pCETF in the MY1H system are clearly detectable by using an anti-HA antibody in the
western blot analysis. The LTAg and 53BP2 fusion proteins are approximately 78 kDa and 33
kDa, respectively; the nonsense protein encoded by the cloning vector, which includes the HA
tag, is approximately 8.6 kDa. All HA-tagged proteins of expected molecular weights are
visualized in Figure A.2. Comparison of protein expression from pCETT and pCETF
demonstrates that genes inserted into MCS II of pCETF are expressed at higher levels than those
inserted into MCS II of pCETT, which is due to the different lengths of the ADH1 promoters that
control gene expression in MCS II: the full-length ADH1 promoter leads to higher expression
levels than the truncated promoter.
Although in pCETT, both MCS I and MCS II are under the control of the truncated
ADH1 promoter, western blot results revealed that only protein expressed from the MCS II, not
from the MCS I, is detectable. This observation may indicate the difference in levels of protein
expression between MCS I and MCS II in pCETT. An additional consideration that may affect
protein detection during western blot analysis is that different epitope tags (c-Myc vs. HA) are
used in MCS I and MCS II, with different positioning that may affect accessibility of the tags
during incubation with the respective mAb (c-Myc is in the middile of the fusion protein vs. HA
at the N-terminus)
109
A.3 Expression of the AD-fusion protein from MCS I is not affected by expression from MCS II Although the expression of the GAL4AD-p53 fusion could not be detected in the western
blot analysis, we believe our conclusion that expression of the AD-fusion protein from MCS I is
not affected by expression from MCS II is still valid. The reasons are given briefly in the text, as
well as explicitly described below.
First, 3-AT titration analysis shows that expression of only GAL4AD-p53 from either
pCETT or pCETF leads to comparable reporter gene activation curves (Figure 2.3). The colony
lift assay shows no difference in blue color intensity for these two controls (Figure 2.4).
Additionally, in the ONPG assay, the β-galactosidase values are 492±11 and 465±34 when
GAL4AD-p53 only is expressed from vectors pCETT and pCETF, respectively (Figure 2.5 and
Table A.1). These statistically comparable data from ONPG analysis, together with 3-AT titation
and colony lift assay results, reveal that the different ADH1 promoters (truncated vs. full-length)
that control the transcription of genes cloned into MCS II do not affect the expression from MCS
I.
Table A.1 Reporter activation of GAL4AD fusions of p53 or Max expressed from different vectors Strain Plasmid β-galactosidase units
pGAD53m 457* pCETT/53 492±11 YM4271[p53BLUE] pCETF/53 465±34
pGAD424/Max 147±7** pCETT/Max 155±20** YM4271[pLacZi/E-box]pCETF/Max 137±13**
*: One trial performed in triplicate. **: J. Xu, J. A. Shin, unpublished results
Second, the expression of GAL4AD-p53 is not affected by the low or high levels of
nonsense protein (76 aa) expressed from cloning vectors pCETT and pCETF when no gene is
110
cloned into MCS II. We tested the effect of expression of this nonsense protein from MCS II on
transcription activation of GAL4AD-p53 expressed from MCS I by ONPG assay. We then
compared these results to those obtained when using singly transformed yeast expressing
GAL4AD-p53 from the original pGAD53m plasmid supplied by Clontech. The β-galactosidase
value from the tranformant with pGAD53m is 457 (Table A.1), which is comparable to 492±11
and 465±34, the values from transformants with pCETT/53 and pCETF/53, respectively, in
which p53 is expressed from MCS I described above. In addition, we did not observe any
retarding or enhancing growth in cells transformed with pCETT/53 or pCETF/53. These results
suggest that the nonsense protein expressed from MCS II in pCETT or pCETF, regardless of low
or high expression levels, is neutral to the expression of GAL4AD-p53 from MCS I.
A separate project supports the conclusion that the nonsense protein expressed from MCS
II in pCETT or pCETF does not affect the expression of the GAL4AD fusion from MCS I. In
this project, the E-box binding protein Max was expressed from MCS I from different protein
expression vectors including pGAD424, pCETT, and pCETF in the E-box integrated yeast strain
(J. Xu, J. A. Shin, unpublished results). The ONPG assay was performed to compare the reporter
gene activation of the GAL4AD-Max fusion expressed from these different vectors (Table A.1).
The β-galactosidase values are 147±7, 155±20, and 137±13 for pGAD424/Max, pCETT/Max,
and pCETF/Max, respectively: again, quantitative ONPG values that are virtually identical for
protein expressed from MCS I, regardless of no, low or high expression of nonsense protein from
MCS II. Therefore, these experiments further validate the conclusion that the nonsense protein
expressed from MCS II of pCETT or pCETF does not affect the expression of the GAL4AD
fusion expressed from MCS I.
111
Therefore, although we could not produce a western blot for the GAL4AD fusion protein
expressed from the MCS I, we believe that with the evidence from different systems described
both in the text and above, our MY1H system is validated.
112
Appendix B
AhR/Arnt:XRE interaction: turning false negatives into true positives in the modified yeast one-hybrid assay
Gang Chen and Jumi A. Shin
Appendix B is the Supplemental Information for the paper accepted by Analytical
Biochemistry (Chapter 3 in this thesis), with necessary changes for thesis organization purposes.
Table B.1 Oligonucleotides used in this study* No Sequence 3-1 AAAGAATTCTTGCGTGTTGCGTGTTGCGTG
3-2 AAATCTAGACACGCAACACGCAACACGCAA
3-3 AAAGTCGACCACGCAACACGCAACACGCAA
3-4 AAAGAATTCTTGCGTGTTGCGTGTTGCGTGTTGCGTGTTGCGTGTTGCGTG
3-5 AAATCTAGACACGCAACACGCAACACGCAACACGCAACACGCAACACGCAA
3-6 AAAGTCGACCACGCAACACGCAACACGCAACACGCAACACGCAACACGCAA
3-7 ATGCGTCGACGCCAACATCACCTACGCCAG
3-8 ATGCTCTAGATCAACTAGTGCCATTTTTAGTC
3-9 ATGCGGATCCATAGCTCTGCGGATAAAGAGAG
3-10 ATTTTCCCTGGCAAGTCTCTCTTTATCCGCAGAG
3-11 AGAGAGACTTGCCAGGGAAAATCACAGTGA
3-12 ATGCCTCGAGTCACACATTGGTGTTGGTACAGA
*Oligonucleotide sequences are shown in 5′ to 3′ direction. Restriction sites used for cloning are in bold.
B.1 Construction of reporter strains
B.1.1 Three-copy strains The integrated strains YM4271[pHISi-1/XRE-3] and YM4271[pLacZi/XRE-3] contain
three copies of the XRE site upstream of the HIS3 and lacZ reporter genes, respectively, in the
yeast genome.
113
A DNA fragment containing three tandem copies of the XRE target sequence (5'-
TTGCGTG-3') was assembled by mutually primed synthesis (112) using oligonucleotides 3-1
and 3-2 (Table B.1; all oligonucleotides discussed in Appendix B are listed in Table B.1 and also
described in Table D.1 of Appendix D). This fragment was inserted between EcoR I and Xba I
sites of the yeast integration and reporter vector pHISi-1 to create pHISi-1/XRE-3. pHISi-1
contains the yeast HIS3 gene downstream of the MCS and the minimal promoter of the his3
locus. The recombinant vector pHISi-1/XRE-3 was then linearized at the Xho I site in the 3’-
untranslated region immediately following the HIS3 marker and integrated into the mutated his3
locus of YM4271 to produce the reporter strain YM4271[pHISi-1/XRE-3]. The integration
confers a His+ phenotype on the transformants. The 3-AT titration assay was performed to
determine the concentration of 3-AT sufficient for suppression of background growth of
YM4271[pHISi-1/XRE-3]. It was found that 30 mM 3-AT was sufficient to suppress
YM4271[pHISi-1/XRE-3] background growth on SD/–His medium.
Similarly, another DNA fragment containing three tandem copies of the XRE target
sequence was assembled by mutually primed synthesis (112) using oligonucleotides 3-1 and 3-3.
This fragment was inserted between EcoR I and Sal I sites of the yeast integration and reporter
vector pLacZi to create pLacZi/XRE-3. This recombinant reporter plasmid was linearized at the
Nco I site and integrated into the mutated ura3 locus of YM4271. The integration confers a Ura+
phenotype on the transformants.
B.1.2 Six-copy strains YM4271[pHISi-1/XRE-6] and YM4271[pLacZi/XRE-6] contain six copies of the XRE
site upstream of the HIS3 and lacZ reporter genes, respectively, in the yeast genome.
The construction of YM4271[pHISi-1/XRE-6] and YM4271[pLacZi/XRE-6] strains was
similar to that of their corresponding counterparts described above, respectively. The
114
oligonucleotides used for construction of YM4271[pHISi-1/XRE-6] were oligos 3-4 and 3-5; the
oligonucleotides used for construction of YM4271[pLacZi/XRE-6] were oligos 3-4 and 3-6.
3-AT titration assay revealed that 30 mM 3-AT was also sufficient to suppress
YM4271[pHISi-1/XRE-6] background growth on SD/–His medium.
B.2 Construction of AhR6-436 (NAhR) fragment NAhR fragment was amplified from original human AhR cDNA (130) using
oligonucleotides 3-7 and 3-8 as the 5′ and 3′ primers, respectively.
B.3 Construction of Arnt82-464 (NArnt) fragment ArntP1 fragment was assembled by mutually primed synthesis (112) using
oligonucleotides 3-9 and 3-10; ArntP2 fragment was amplified from original human Arnt variant
3 cDNA (130) using oligonucleotides 3-11 and 3-12 as the 5′ and 3′ primers, respectively. The 3’
end of the ArntP1 fragment contains 22 bp homology with the 5′ end of the ArntP2 fragment.
The final NArnt fragment was then amplified with ArntP1 and ArntP2 as templates using
oligonucleotides 3-9 and 3-12 as the 5′ and 3′ primers, respectively.
115
Figure B.1 Colony-lift filter assay for detection of NAhR/NArnt:XRE interaction from protein expression vector pCETT or pCETT2 in YM4271[pHISi-1/XRE-3] (A and C) and YM4271[pHISi-1/XRE-6] (B and D) strains. The X-gal colony-lift assay was performed after 4 days growth of transformants on SD/-L/-U plates at 30 °C. Photos were taken after 60 min incubation at 30 °C. The transformants were identified according to the following order. A and B: upper left, pCETT/NAhR/NArnt; upper right, pCETT/NAhR; lower left, pCETT//NArnt; lower right, pCETT. C and D: upper left, pCETT2/NAhR/NArnt; upper right, pCETT2/NAhR; lower left, pCETT2//NArnt; lower right, pCETT2.
116
Appendix C
Forced protein heterodimerization and specific DNA binding to a nonpalindromic DNA sequence in vivo and in vitro: bHLHZ-like hybrid heterodimers of bHLH/PAS proteins AhR and Arnt and
bZIP proteins JunD and Fos as a model Gang Chen, Antonia T. De Jong, S. Hesam Shahravan, and Jumi A. Shin
Appendix C is the Supplemental Information for Chapter 4.
Table C.1 Oligonucleotides used in this study* No Sequence 4-1 TGCGTCGACCAGAAAACAGTAAAGCCAAT
4-2 TATTCTAGAGGAGGATTTTAATGCAACA
4-3 ACTGAATTCAGCTCTGCGGATAAAGAGAG
4-4 ATACTCGAGGGATGTGTTGCCAGTTCCCC
4-5 AGCTTCTTTGATGTTGCATTAGAAGAAAAGGTTAAGACC
4-6 CAAAGAAGCGGTAGAAGCCAATTCGGTGTTTTGAGACTTCAAGGTCTTAACCTTTTCTTC
4-7 GCTTCTACCGCTTCTTTGTTGAGAGAACAAGTTGCTCAATTGAAGCAAAAGGTTTTGTCT
4-8 GTCCTCTAGAAACGTGAGACAAAACCTTTTGCTT
4-9 TGCGGGGAACTGGCAACACACAAGCTGAAACTGACCAA
4-10 CAACAAGTTAGCAATTTCGGTTTGCAAAGCAGACTTTTCGTCTTCCAATTGGTCAGTTTC
4-11 GAAATTGCTAACTTGTTGAAGGAAAAGGAAAAGTTGGAGTTTATCTTGGCTGCTCACAGA
4-12 AAAGGATCCTGGTCTGTGAGCAGCCAAGAT
4-13 AGCTTCTTTGATGTTGCAGAAGAAAAGGTTAAGACC
4-14 TGCGAATTCCAGAAAACAGTAAAGCCAAT
4-15 TATGGATCCGGAGGATTTTAATGCAACA
4-16 ATACTGCAGGGATGTGTTGCCAGTTCCCC
4-17 GTCCGGATCCAACGTGAGACAAAACCTTTTGCTT
*Oligonucleotide sequences are shown in 5' to 3' direction. Restriction sites used for cloning are in bold.
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C.1 Plasmid Construction
C.1.1 pCETT2/AhRbHLH/ArntbHLH The AhRbHLH sequence encoding amino acids 20-90 of human AhR (GeneID: 196) was
amplified from AhR cDNA (130) with oligonucleotides 4-1 and 4-2 (Table C.1; all
oligonucleotides discussed in Appendix C are listed in Table C.1 and also described in Table D.1
of Appendix D) and inserted between the Sal I and Xba I sites of pCETT2 to construct
pCETT2/AhRbHLH. The ArntbHLH sequence encoding amino acids 82-149 of human Arnt
(GeneID: 405) isoform 1 was amplified from the Arnt82-464 cDNA fragment (148) with
oligonucleotides 4-3 and 4-4 and inserted between the EcoR I and Xho I sites of pCETT2 and
pCETT2/AhRbHLH to generate pCETT2//ArntbHLH and pCETT2/AhRbHLH/ArntbHLH,
respectively.
C.1.2 pCETT/AhRJunD/ArntFos and pCETT2/AhRJunD/ArntFos The RJunD fragment containing the sequence encoding amino acids 81-86 of human
AhR followed by amino acids 296-332 of human JunD (GeneID: 3727) was assembled by self-
priming PCR (111) using oligonucleotides 4-5 to 4-8. The final AhRJunD fragment was then
amplified with RJunD and AhRbHLH described above as templates using oligonucleotides 4-1
and 4-8 as the 5' and 3' primers, respectively. This amplified fragment was digested with Sal I
and Xba I, and then ligated into these restriction sites in pCETT and pCETT2 to construct
pCETT//AhRJunD and pCETT2/AhRJunD, respectively. We note that the AhRJunD hybrid
contains amino acids 20-86 of human AhR, which is in contrast to the AhRbHLH fragment
constructed above that encodes amino acids 20-90 of human AhR.
The ArntFos fragment was constructed in a similar manner to that of AhRJunD: the TFos
fragment containing the sequence encoding amino acids 143-148 of human Arnt followed by
amino acids 165-201 of human c-Fos (GeneID: 2353) was assembled by self-priming PCR (111)
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using oligonucleotides 4-9 to 4-12; the final ArntFos fragment was then amplified with TFos and
ArntbHLH described above as templates using oligonucleotides 4-3 and 4-12 as the 5' and 3'
primers, respectively. This fragment was digested with EcoR I and BamH I and inserted into
these restriction sites in pCETT/AhRJunD and pCETT2/AhRJunD to generate
pCETT/AhRJunD/ArntFos and pCETT2/AhRJunD/ArntFos, respectively. The EcoR I/BamH I-
digested ArntFos fragment was also inserted between these restriction sites in pCETT and
pCETT2 to generate pCETT//ArntFos and pCETT2//ArntFos, respectively. We also note that the
ArntFos hybrid contains amino acids 82-148 of human Arnt, which is in contrast to the
ArntbHLH fragment constructed above that encodes amino acids 82-149 of human Arnt.
C.1.3 pCETT2/AhR(ΔL)JunD/ArntFos The AhR(ΔL)JunD fragment was constructed in a similar fashion to that of AhRJunD.
The R(ΔL)JunD fragment was assembled by self-priming PCR (111) using oligonucleotides 4-13
and 4-6 to 4-8. The final AhR(ΔL)JunD fragment was then amplified with R(ΔL)JunD and
AhRbHLH described above as templates using oligonucleotides 4-1 and 4-8 as the 5' and 3'
primers, respectively. This amplified fragment was digested with Sal I and Xba I, and then
ligated into these restriction sites of pCETT2 and pCETT2//ArntFos to generate
pCETT2/AhR(ΔL)JunD and pCETT2/AhR(ΔL)JunD/ArntFos, respectively.
C.1.4 pGBKT7/AhRbHLH and pGADT7/AhRbHLH The AhRbHLH fragment was amplified from AhR cDNA (130) with oligonucleotides 4-
14 and 4-15 and inserted between the EcoR I and BamH I sites of pGBKT7 and pGADT7 to
generate pGBKT7/AhRbHLH and pGADT7/AhRbHLH, respectively.
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C.1.5 pGBKT7/ArntbHLH and pGADT7/ArntbHLH The ArntbHLH fragment was amplified from NArnt cDNA (148) with oligonucleotides
4-3 and 4-16 and inserted between the EcoR I and Pst I sites of pGBKT7 to generate
pGBKT7/ArntbHLH; the EcoR I/Xho I-digested ArntbHLH fragment described above was
inserted into these restriction sites in pGADT7 to generate pGADT7/ArntbHLH.
C.1.6 pGBKT7/AhRJunD and pGADT7/AhRJunD The AhRJunD fragment was amplified from the AhRJunD fragment with
oligonucleotides 4-14 and 4-17 and inserted between the EcoR I and BamH I sites of pGBKT7
and pGADT7 to generate pGBKT7/AhRJunD and pGADT7/AhRJunD, respectively.
C.1.7 pGBKT7/AhR(ΔL)JunD and pGADT7/AhR(ΔL)JunD The AhR(ΔL)JunD fragment was amplified from the AhR(ΔL)JunD fragment with
oligonucleotides 4-14 and 4-17 and inserted between the EcoR I and BamH I sites of pGBKT7
and pGADT7 to generate pGBKT7/AhR(ΔL)JunD and pGADT7/AhR(ΔL)JunD, respectively.
C.1.8 pGBKT7/ArntFos and pGADT7/ArntFos The EcoR I/BamH I-digested ArntFos fragment was inserted into these restriction sites in
pGBKT7 and pGADT7 to generate pGBKT7/ArntFos and pGADT7/ArntFos, respectively.
C.2 Expression of GAL4AD-AhRJunD and ArntFos by use of pCETT in the MY1H We also tried to express the GAL4AD-AhRJunD and ArntFos proteins by use of pCETT
in both YM4271[pHISi-1/XRE-6] and YM4271[pLacZi/XRE-6]. As expected, both HIS3
(Figure C.1) and lacZ (Figure C.2 and C.3) reporter assays showed the transformant displayed
positive signals while its corresponding controls, in which only one of the two hybrid proteins or
none was expressed, displayed negative signals. In addition, when GAL4AD-AhRJunD and
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ArntFos were coexpressed in the lacZ reporter strain, the ONPG value was 40.2±5.4, which is in
contrast to ONPG value 95.7±3.1 when GAL4AD-AhRJunD and GAL4AD-ArntFos were
coexpressed in the same reporter strain. This indicates that attachment of an AD to both
monomers increases the transcription potency of the reporter gene through synergistic activation,
thereby potentially increasing the sensitivity of the system.
Figure C.1 The HIS3 reporter assay for detection of the AhRJunD/ArntFos:XRE interaction by use of protein expression vector pCETT in strain YM4271[pHISi-1/XRE-6]. The transformants that coexpress both GAL4AD-AhRJunD and ArntFos proteins, as well as their corresponding controls, were plated on both SD/-H/-L control plates (left) and SD/-H/-L/30 mM 3-AT test plates (right) and incubated at 30 °C, 5 days. The transformants on the same plate are labeled according to the following order: upper left: pCETT/AhRJunD/ArntFos; upper right: pCETT/AhRJunD; lower left: pCETT//ArntFos; lower right: pCETT.
Figure C.2 The X-gal colony-lift filter assay. YM4271[pLacZi/XRE-6] cells were transformed with A) pCETT/AhRJunD/ArntFos; B) pCETT/AhRJunD; C) pCETT//ArntFos; D) pCETT and plated on SD/-L/-U plates. The colony-lift assay was performed after 4 days growth, 30 °C. Photos were taken after 60 min incubation at 30 °C.
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Figure C.3 ONPG measurements for detection of AhRJunD/ArntFos:XRE interactions by use of protein expression vector pCETT in strain YM4271[pHISi-1/XRE-6]. Vertical axis indicates the mean values in β-galactosidase units. Error bars represent standard error measurement from at least three independent trials conducted in triplicate.
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Appendix D Table of Oligonucleotides
Table D.1 Oligonucleotides used in this thesis* No. SEQUENCE APPLICATION
2-1 ACTATCTATTCGATGATGAAGATACCCCACCAAACCCAAA
2-2 GGCGCTCGCCCTATAGTGAGTCGTATTAAAGATCTCTTTTTTTGGGTTTGGTGGGGTATC
2-3 CTCACTATAGGGCGAGCGCCGCCATCATGGAGGAGCAGAAGCTGATCTCAGAGGAGGACC
2-4 GCGCGCACCTTGTCGACCGCGGCCTCCATGGCCATATGCAGGTCCTCCTCTGAGATCAGC
2-5 GCGGTCGACAAGGTGCGCGCTCTAGATGATCATGAATCGTAGATACTGAAAAACCCCGCA
2-6 ATGCACAGTTGAAGTGAACTTGCGGGGTTTTTCAGTATCT
Assembly of CE4MCS fragment to construct pGAD424-MCS II by homologous recombination.
2-7 AGAAAGGTCGAATTGGGTACCGCCGCCAATAAAGAGATCTTTAAT
2-8 CTCGCCCTATAGTGAGTCGTATTAAAGATCTCTTTATTGGCGGCG
Assembly of FINALREC fragment to construct pGAD424-MCS IIΔAD and pGADT7ΔAD by homologous recombination.
2-9 AAAGACGTCGCATGCAACTTCTTTTCTTT Forward primer for amplification of T2 fragment.
2-10 ATTGACGTCAAGCTTGCATGCCGGTAGAGGT Reverse primer for amplification of T2 and F2 fragments.
2-11 AAAGACGTCCCTGCAGGTCGAGATCCGGGA Forward primer for amplification of F2 fragment.
2-12 AAAAGTCGACCCTGTCACCGAGACCCCTGG
2-13 ACGCTCTAGATCAGTCTGAGTCAGGCCCCA
Forward and reverse primers for amplification of p53 fragment.
2-14 AAAGAATTCGGAACTGATGAATGGGAGCAG
2-15 AAAGGATCCTTATGTTTCAGGTTCAGGGGGAG
Forward and reverse primers for amplification of LTAg fragment.
2-16 AAAGAATTCCCGCCTGAAATCACCGGGCAG
2-17 AAAGGATCCTCAGGCCAAGCTCCTTTGTCTT
Forward and reverse primers for amplification of 53BP2 fragment.
3-1 AAAGAATTCTTGCGTGTTGCGTGTTGCGTG Forward primer for three-copy XRE target sequence assembly.
3-2 AAATCTAGACACGCAACACGCAACACGCAA Reverse primer for three-copy XRE target sequence assembly (Xba I).
3-3 AAAGTCGACCACGCAACACGCAACACGCAA Reverse primer for three-copy XRE target sequence assembly (Sal I).
3-4 AAAGAATTCTTGCGTGTTGCGTGTTGCGTGTTGCGTGTTGCGTGTTGCGTG Forward primer for six-copy XRE target sequence assembly.
3-5 AAATCTAGACACGCAACACGCAACACGCAACACGCAACACGCAACACGCAA Reverse primer for six-copy XRE target sequence assembly (Xba I).
3-6 AAAGTCGACCACGCAACACGCAACACGCAACACGCAACACGCAACACGCAA Reverse primer for six-copy XRE target sequence assembly (Sal I).
3-7 ATGCGTCGACGCCAACATCACCTACGCCAG
3-8 ATGCTCTAGATCAACTAGTGCCATTTTTAGTC
Forward and reverse primers for amplification of NAhR fragment (Sal I and Xba I).
3-9 ATGCGGATCCATAGCTCTGCGGATAAAGAGAG
3-10 ATTTTCCCTGGCAAGTCTCTCTTTATCCGCAGAG
3-11 AGAGAGACTTGCCAGGGAAAATCACAGTGA
3-12 ATGCCTCGAGTCACACATTGGTGTTGGTACAGA
26 and 29: forward and reverse primers for amplification of NArnt fragment. 27and 28: intermediate primers for the construction of NArnt fragment.
4-1 TGCGTCGACCAGAAAACAGTAAAGCCAAT Forward primer for amplification of AhRbHLH, AhRJunD and AhR(ΔL)JunD fragments (Sal I).
4-2 TATTCTAGAGGAGGATTTTAATGCAACA Reverse primer for amplification of AhRbHLH fragment (Xba I).
4-3 ACTGAATTCAGCTCTGCGGATAAAGAGAG Forward primer for amplification of ArntbHLH and ArntFos fragments.
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No. SEQUENCE APPLICATION
4-4 ATACTCGAGGGATGTGTTGCCAGTTCCCC Reverse primer for amplification of ArntbHLH fragment (Xho I).
4-5 AGCTTCTTTGATGTTGCATTAGAAGAAAAGGTTAAGACC Intermediate primer for the construction of AhRJunD fragment.
4-6 CAAAGAAGCGGTAGAAGCCAATTCGGTGTTTTGAGACTTCAAGGTCTTAACCTTTTCTTC
4-7 GCTTCTACCGCTTCTTTGTTGAGAGAACAAGTTGCTCAATTGAAGCAAAAGGTTTTGTCT
Intermediate primers for the construction of AhRJunD and AhR(ΔL)JunD fragments.
4-8 GTCCTCTAGAAACGTGAGACAAAACCTTTTGCTT Reverse primer for amplification of AhRJunD and AhR(ΔL)JunD fragments (Xba I).
4-9 TGCGGGGAACTGGCAACACACAAGCTGAAACTGACCAA
4-10 CAACAAGTTAGCAATTTCGGTTTGCAAAGCAGACTTTTCGTCTTCCAATTGGTCAGTTTC
4-11 GAAATTGCTAACTTGTTGAAGGAAAAGGAAAAGTTGGAGTTTATCTTGGCTGCTCACAGA
Intermediate primers for the construction of ArntFos fragments.
4-12 AAAGGATCCTGGTCTGTGAGCAGCCAAGAT Reverse primer for amplification of ArntFos fragment.
4-13 AGCTTCTTTGATGTTGCAGAAGAAAAGGTTAAGACC Intermediate primer for the construction of AhR(ΔL)JunD fragment.
4-14 TGCGAATTCCAGAAAACAGTAAAGCCAAT Forward primer for amplification of AhRbHLH, AhRJunD and AhR(ΔL)JunD fragments (EcoR I).
4-15 TATGGATCCGGAGGATTTTAATGCAACA Reverse primers for amplification of AhRbHLH fragment (BamH I).
4-16 ATACTGCAGGGATGTGTTGCCAGTTCCCC Reverse primer for amplification of ArntbHLH fragment (Pst I).
4-17 GTCCGGATCCAACGTGAGACAAAACCTTTTGCTT Reverse primers for amplification of AhRJunD and AhR(ΔL)JunD fragments (BamH I).
*Oligonucleotide sequences are shown in 5′ to 3′ direction. Restriction sites used for cloning are in bold.