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DATA TO SUPPORT DATA TO SUPPORT STATEMENTS IN STATEMENTS IN PRESENTATIONPRESENTATION

Outline of talkOutline of talk

General backgroundGeneral background

Introduction to the projectIntroduction to the project

Experimental designExperimental design

Experiments and resultsExperiments and results

ConclusionsConclusions

Future experimentsFuture experiments

Proteins Proteins involved in involved in

complexcomplexMethodMethod SummarySummary ReferencReferenc

ee

11 HP1HP1, KAP1, KAP1 Yeast 2 HybridYeast 2 Hybrid HP1HP1 was used as a bait and was used as a bait and TIF1TIF1 (KAP1) was isolated (KAP1) was isolated

Le Douarin et Le Douarin et alal

19961996

22 KOX1, KAP1KOX1, KAP1 Yeast 2 HybridYeast 2 Hybrid KRAB domain of KOX1 was KRAB domain of KOX1 was used as a bait and KAP1 used as a bait and KAP1

was isolatedwas isolated

Moosmann et Moosmann et al.al.

19961996

33NuRD/HDAC, NuRD/HDAC,

KAP1KAP1Yeast 2 HybridYeast 2 Hybrid

Mi2Mi2 (= NuRD/HDAC) was (= NuRD/HDAC) was shown interact with KAP-1-shown interact with KAP-1-PHD/BROMO but not Mi2PHD/BROMO but not Mi2

Schultz et al.Schultz et al.20012001

44 KIP21, KAP1KIP21, KAP1Yeast 2 HybridYeast 2 Hybrid

KIP21 and KIP41 (=SETDB1) KIP21 and KIP41 (=SETDB1) were shown interact with were shown interact with

KAP-1-PHD/BROMOKAP-1-PHD/BROMO

Schultz et al.Schultz et al.2002200255 KIP41, KAP1KIP41, KAP1

66KOX1, KAP1, KOX1, KAP1,

HP1HP1

Gel Electrophoretic Gel Electrophoretic Mobility Shift Mobility Shift

assay (in vitro)assay (in vitro)

DNA+GAL4-KRAB+KAP1 DNA+GAL4-KRAB+KAP1 ternary complex was ternary complex was

shown to complex with shown to complex with HP1HP1

Ryan et al. Ryan et al. 19991999

Prior demonstrations Prior demonstrations of KAP1 complexesof KAP1 complexes

Structure of CSD complexStructure of CSD complex

Epigenetic Gene SilencingEpigenetic Gene Silencing

Epigenetic effects are those changes in Epigenetic effects are those changes in gene function which are heritable gene function which are heritable through mitosis and/or meiosis and are through mitosis and/or meiosis and are not due to changes in DNA sequence.not due to changes in DNA sequence.

Main types of epigenetic information:Main types of epigenetic information:– Cytosine DNA MethylationCytosine DNA Methylation– Genomic impritningGenomic impritning– Histone modificationsHistone modifications

CSD interacting proteinsCSD interacting proteins

Chromatin based Chromatin based epigeneticsepigenetics

Controls chromosome domains and also Controls chromosome domains and also helps in cell differentiation helps in cell differentiation

X –inactivation (XIST locus. Genomic X –inactivation (XIST locus. Genomic imprinting, parent-of origin-specific allele imprinting, parent-of origin-specific allele silencing)silencing)

Developmental reprogramming of cell Developmental reprogramming of cell lineageslineages

Plasticity of stem cellsPlasticity of stem cells

Implications Implications human biology and human biology and disease, including cancer and agingdisease, including cancer and aging

Expression VectorsExpression Vectors

pBridge HP1 KAP19525 bp

HP1a

KAP1

Gal4 DBD

TRP1

MET25

HA/NLS

Gal4DBD

BAIT HP1

pACT2 Hela cDNA Lib10114 bp

Gene Library

GAL4 AD

LEU2

HA

Gal4AD??

PREY

KAP1

(BD-HP1a)-(HA-KAP1)

(AD-?)

HeLa cell cDNA libraryHeLa cell cDNA library

Source of human genes (cDNA).Source of human genes (cDNA).

Human cervical carcinoma cell lineHuman cervical carcinoma cell line

Estimated number of Independent Estimated number of Independent Clones: Clones: 3.5 x 103.5 x 1066

– Average insert (cDNA) size: 2.0 kbAverage insert (cDNA) size: 2.0 kb– cDNA size range: 0.5 – 4.0 kbcDNA size range: 0.5 – 4.0 kb

HeLa cancer cells

Hybrid proteins used in this Hybrid proteins used in this studystudy

BAITSBAITS– BD-HP1aBD-HP1a– (BD-HP1A)-(HA-(BD-HP1A)-(HA-

KAP1)KAP1)– BD-KAP1BD-KAP1– (BD-KAP1)-(HA-HP1)(BD-KAP1)-(HA-HP1)– (BD-HP1A)-(HA-(BD-HP1A)-(HA-

CAF1p150)CAF1p150)

PREYSPREYS– AD-?AD-? (HeLa cell cDNA (HeLa cell cDNA

library; Clontech)library; Clontech)

– AD-KIP21 (SETDB1)AD-KIP21 (SETDB1)– AD-KIP41 (SETDB1)AD-KIP41 (SETDB1)– AD-Mi2bAD-Mi2b– AD-KOX1AD-KOX1– AD-Y2H6.2 (AD-Y2H6.2 (POGZPOGZ))

Proteins Proteins involved in involved in

complexcomplexMethodMethod SummarySummary ReferencReferenc

ee

11 HP1HP1, KAP1, KAP1 Yeast 2 HybridYeast 2 Hybrid HP1HP1 was used as a bait and was used as a bait and TIF1TIF1 (KAP1) was isolated (KAP1) was isolated

Le Douarin et Le Douarin et alal

19961996

22 KOX1, KAP1KOX1, KAP1 Yeast 2 HybridYeast 2 Hybrid KRAB domain of KOX1 was KRAB domain of KOX1 was used as a bait and KAP1 used as a bait and KAP1

was isolatedwas isolated

Moosmann et Moosmann et al.al.

19961996

33NuRD/HDAC, NuRD/HDAC,

KAP1KAP1Yeast 2 HybridYeast 2 Hybrid

Mi2Mi2 (= NuRD/HDAC) was (= NuRD/HDAC) was shown interact with KAP-1-shown interact with KAP-1-PHD/BROMO but not Mi2PHD/BROMO but not Mi2

Schultz et al.Schultz et al.20012001

44 KIP21, KAP1KIP21, KAP1Yeast 2 HybridYeast 2 Hybrid

KIP21 and KIP41 (=SETDB1) KIP21 and KIP41 (=SETDB1) were shown interact with were shown interact with

KAP-1-PHD/BROMOKAP-1-PHD/BROMO

Schultz et al.Schultz et al.2002200255 KIP41, KAP1KIP41, KAP1

66KOX1, KAP1, KOX1, KAP1,

HP1HP1

Gel Electrophoretic Gel Electrophoretic Mobility Shift Mobility Shift

assay (in vitro)assay (in vitro)

DNA+GAL4-KRAB+KAP1 DNA+GAL4-KRAB+KAP1 ternary complex was ternary complex was

shown to complex with shown to complex with HP1HP1

Ryan et al. Ryan et al. 19991999

77HP1HP1, POGZ , POGZ

(Y2H6.2)(Y2H6.2)

Co-Co-immunoprecipitatimmunoprecipitat

ion and Yeast ion and Yeast Two Hybrid Two Hybrid

genetic screengenetic screen

Novel prey (POGZ / Y2H6.2) Novel prey (POGZ / Y2H6.2) shown to interact with shown to interact with

HP1HP1

Lechner et al,Lechner et al,UnpublishedUnpublished

Prior demonstrations Prior demonstrations of KAP1 & HP1 complexesof KAP1 & HP1 complexes

Histone CodeHistone Code

                                                                                          

Strahl et al. Nature 2000

HP1 gene silencing

Long term GoalsLong term Goals

Build complete HP1 network with all Build complete HP1 network with all its partner proteins and to build a its partner proteins and to build a chromatin network database !chromatin network database !

Map both the temporal and Spatial Map both the temporal and Spatial formation of these complexesformation of these complexes

Get in depth and complete Get in depth and complete understanding of gene regulation in understanding of gene regulation in cellscells

Statement of ProblemStatement of Problem

Studies show that HP1Studies show that HP1 binds with binds with several proteins and presumably several proteins and presumably performs different activities (mainly performs different activities (mainly in regulating chromatin structure and in regulating chromatin structure and function).function).

But, little is known about what But, little is known about what complexes exist complexes exist in vivoin vivo and what and what controls their formationcontrols their formation

AssumptionsAssumptions

The main assumption in this experiment is The main assumption in this experiment is that there are regulatory factors that help that there are regulatory factors that help in the formation of HP1 complexes. in the formation of HP1 complexes.

Yeast system does not interfere with any Yeast system does not interfere with any of the interactions. (relatively ‘safe’; most of the interactions. (relatively ‘safe’; most of the initial HP1 interactions were done in of the initial HP1 interactions were done in yeast screens.)yeast screens.)

Feasibility of this screen can be Feasibility of this screen can be demonstrated by showing a ternary demonstrated by showing a ternary complex formation with positive controls.complex formation with positive controls.

LimitationsLimitations

Yeast might inactivate or destroy the foreign Yeast might inactivate or destroy the foreign (human) proteins. (human) proteins. Some of these human proteins might be toxic to Some of these human proteins might be toxic to yeast. yeast. Functional homology might be present between Functional homology might be present between yeast proteins and the human proteins.yeast proteins and the human proteins.Presence of false positives or false negatives.Presence of false positives or false negatives.Bait proteins that auto-activate cannot be used.Bait proteins that auto-activate cannot be used.Cannot directly extrapolate the results to higher Cannot directly extrapolate the results to higher eukaryotes without further verification.eukaryotes without further verification.

Y : TyrosineW: TryptophanE : GlutamineT : ThreonineK : LysineV : ValineN : AsparagineS : SerineA : Alanine

Histone H3 tail bound to Histone H3 tail bound to Chromo Domain of Chromo Domain of DrosophilaDrosophila HP1 HP1

Yelow: Me2k9Red/Orange: Me3k9

Jacobs and Khorasanizadeh (2002), Science express

Mock transformationMock transformation

Number of Transformants Number of Transformants = = ~ ~ 2 x 102 x 1066 per ml; per ml;

For a 5ml final volume of For a 5ml final volume of transformed cells number transformed cells number of library colonies of library colonies screened is around screened is around ~~101077 ! !

Large enough to cover all Large enough to cover all clones in the library. clones in the library. ((Number of independent Number of independent clones in the library: clones in the library: 3.5 3.5 x 10x 1066; clontech; clontech ) )

HIS3 expression seems to be HIS3 expression seems to be Leaky on –L/T/H platesLeaky on –L/T/H plates

S.No

PARENTPLATE PREY TO COLONIES

1 

SD - LT 

pACT-Mi2b 

SD - LT 10 -20

SD -LTH 0

2 

SD - LT 

pACT-KIP21 

SD - LT 100-150

SD -LTH 20 -30

3 

SD - LT 

pACT-KIP41 

SD - LT 100-150

SD -LTH 1

4 

SD - LT 

pACT 

SD - LT Lawn

SD -LTH 20 -30

All colonies have BAIT in them (pBRIDGE-Gal4HP1a-HA-All colonies have BAIT in them (pBRIDGE-Gal4HP1a-HA-KAP1).KAP1).

BAIT and empty pACT seem to grow on –L/T/H but not BAIT and empty pACT seem to grow on –L/T/H but not –L/T/H/A (observation from mock transformation).–L/T/H/A (observation from mock transformation).

HIS3HIS3 titration with competitive titration with competitive inhibitor 3-ATinhibitor 3-AT

S.no

plasmid

-- L/T/H with 3-AT @ different concentrations --

L/T/H/A0mM 1mM 3mM

5mM

10mM

15mM

1 pACT  ~60 ~24 3 No colonies

2 KIP21  TNC ~100 10-20 No colonies

3 KIP41  TNC ~100 0 No colonies

positivecontrol

SV40T + p53

 Lawn

 notplated

 Lawn

 Lawn Lawn

Lawn

 Lawn

All colonies have BAIT in them (pBRIDGE-Gal4HP1a-HA-KAP1).All colonies have BAIT in them (pBRIDGE-Gal4HP1a-HA-KAP1).

NOTE : 3-AT is 3-AMINO-1,2,4-TRIAZOLE, a competitive inhibiotorof HIS3 gene product.

TNC means too numerous to count

This assay is to determine the amount of 3-AT needed to reduce This assay is to determine the amount of 3-AT needed to reduce noise (growth when there is no interaction) on selection plates.noise (growth when there is no interaction) on selection plates.

Cell Lysate prepared for Cell Lysate prepared for Western blot used for Co-IPsWestern blot used for Co-IPs

Harsh conditions used to obtain cell lysateHarsh conditions used to obtain cell lysateBlot demonstrates that the Anti-HA antibody is not as sensitive as expected. Blot demonstrates that the Anti-HA antibody is not as sensitive as expected.

Anti-HA used for IMMUNOPRECIPITATION

Who's calling the shots?Who's calling the shots?Some transcription factors have, or recruit proteins that have, Some transcription factors have, or recruit proteins that have, histone modification and remodeling activities (histone modification and remodeling activities (Fig. 1Fig. 1). ). Presumably, gene activation requires at least one such factor that Presumably, gene activation requires at least one such factor that can bind its recognition sequence within 'inactive' chromatin and can bind its recognition sequence within 'inactive' chromatin and recruit other factors that collaborate in altering local chromatin recruit other factors that collaborate in altering local chromatin structure. These altered regions of chromatin would then expose structure. These altered regions of chromatin would then expose binding sites for other factors, including the basal transcription binding sites for other factors, including the basal transcription machinerymachinery33. Histone modifications may also be necessary to allow . Histone modifications may also be necessary to allow RNA polymerase to transit across nucleosomal DNA sequencesRNA polymerase to transit across nucleosomal DNA sequences77..Also, whereas acetylation can be reversed by various histone Also, whereas acetylation can be reversed by various histone deacetylases, there are no known histone demethylases. deacetylases, there are no known histone demethylases. Therefore, once a genomic region is methylated, modified Therefore, once a genomic region is methylated, modified nucleosomes must be replaced rather than altered to remove this nucleosomes must be replaced rather than altered to remove this epigenetic mark. Further work will be required to understand the epigenetic mark. Further work will be required to understand the mechanisms responsible for the spread of local histone mechanisms responsible for the spread of local histone modifications and the impact of these modifications on chromatin modifications and the impact of these modifications on chromatin structure and transcriptional regulation.structure and transcriptional regulation.

Figure 1. Transcription factors recruit chromatin modification enzymes, which in turn regulate chromatin structure in the vicinity of the promoter.Dashed lines indicate spread of chromatin changes outward from the promoter region. In this model, changes are reversible until nucleosomes are modified by histone methylation. Ac, acetylated; Me, methylated.

Nature GeneticsNature Genetics  3636, 438 - 440 (2004) , 438 - 440 (2004)

Trends Genet. 2002 May;18(5):252-8. Trends Genet. 2002 May;18(5):252-8.

Fig. 3. Model to explain the role of positive and negative factors in heterochromatin and euchromatin. Methylated amino acids in the histone H3 tail are indicated by red lettering, and acetylated residues are shown in blue. The underlying sequence of the satellite repeats promotes the formation of a regular array of stable nucleosomes, which are favoured substrates for methylation at H3 lysine 9 (K9) by SUVAR39H1. Binding of HP1 to long arrays of nucleosomes containing H3 methylated at K9 promotes the formation of the higher-order heterochromatin structure. Transcriptionally active euchromatin is generated by transcription factors binding to clustered recognition sequences resulting in the formation of DNase I hypersensitive sites (HS). The HS generate and maintain the open structure of the euchromatin by promoting H3 K9 acetylation and K4 methylation of neighbouring nucleosomes. They can also act as barriers preventing the spread of heterochromatin into neighbouring euchromatin.

How to identify different How to identify different classes of interaction?classes of interaction?

My InterestsMy Interests

How do cells in different tissues have How do cells in different tissues have different functions when they have different functions when they have the same genome? the same genome? Is entire human genome expressed?Is entire human genome expressed?– NONO

There is approximately one gene every There is approximately one gene every

~~75,000 base pairs.75,000 base pairs.

And only a fraction (And only a fraction (~~2%)of this part codes 2%)of this part codes for polypeptides. for polypeptides.

Global gene expression patterns?Global gene expression patterns?