BioSci 145A lecture 14 page 1 ©copyright Bruce Blumberg 2000. All rights reserved
BioSci 145A Lecture 14 - 2/21/2002Transcription factors III
• Finish up with identifying regulatory regions
• Major families of transcription factors and their functions
– zinc finger genes
• nuclear hormone receptors
– helix-turn-helix
• homeobox genes
– helix-loop-helix
• myogenic genes
– bZIP proteins
• Additional reading
– Evans (1988) Science 240, 889-895
– Blumberg and Evans (1998) Genes and Development 12, 3149-3155
• Last year’s final exam is now posted.
– I will post answers in a couple of weeks after you have had time to work through the questions
BioSci 145A lecture 14 page 2 ©copyright Bruce Blumberg 2000. All rights reserved
Identification of regulatory elements
• Given a gene of interest, how does one go about studying its regulation?
– First step is to isolate cDNA and genomic clones.
– Map cDNA to genomic sequence
• identify introns, exons
• locate approximate transcriptional start
– recognizing elements, e.g. TATA box
– 5’ primer extension or nuclease mapping
• get as much 5’ and 3’ flanking sequence as is possible
– fuse largest chunk of putative promoter you can get to a suitable reporter gene.
– Test whether this sequence is necessary and sufficient for correct regulation
• how much sequence is required for correct regulation?
– what is correct regulation?
» In cultured cells
» in animals?
– typical result is the more you look, the more you find.
• questions are usually asked specifically. That is, what part of the putative promoter is required for activity in cultured liver cells?
– doesn’t always hold in vivo.
BioSci 145A lecture 14 page 3 ©copyright Bruce Blumberg 2000. All rights reserved
Identification of regulatory elements (contd)
• Promoter mapping
– nuclease footprinting of promoter to identify regions that bind proteins
– make various deletion constructs
• Previously made by ExoIII deletions or insertion of linkers (linker scanning)
• typical method today is to PCR parts of the promoter and clone into a promoterless reporter
– map activity of promoter related to deletions
• incremental changes in activity indicate regions important for activity
– test elements for activity
BioSci 145A lecture 14 page 4 ©copyright Bruce Blumberg 2000. All rights reserved
Identification of binding proteins
• How to identify what factors bind to putative elements?
– examine the sequence
• does it contain known binding sites?
• if yes, do such proteins bind to the isolated element in gel-shift experiments?
– do the elements bind proteins from nuclear extracts?
• gel shift (EMSA) experiments
– clone the elements into reporters with minimal promoters.
• do these constructs recapitulate activity?
• Biochemical purification of binding proteins
– tedious, considerable biochemical skill required
– two basic approaches
• fractionate nuclear extracts chromatographically and test fractions for ability to bind the element in EMSA
• DNA-affinity chromatography
– multimerize the element and bind to a resin
– pass nuclear extracts across column and purify specific binding proteins
– protein microsequencing
– predict DNA sequence from amino acid sequence
• look in GENBANK database
• prepare oligonucleotides and screen library
BioSci 145A lecture 14 page 5 ©copyright Bruce Blumberg 2000. All rights reserved
Identification of binding proteins (contd)
• Biochemical purification of binding proteins (contd)
– advantages
• gold standard
• if you can purify proteins, this will always work
– disadvantages
• slow, tedious
• need good protein sequencing facility
• biochemical expertise required
• expense of preparing preparative quantities of nuclear extracts
• Molecular biological approaches
– oligonucleotide screening of expression libraries (Singh screening)
• multimerize oligonucleotide and label with 32P
• screen expression library to identify binding proteins
• advantages
– straightforward
– much less biochemical expertise required
– relatively fast
• disadvantages
– can’t detect binding if multiple partners are required
– fair amount of “touch” required
BioSci 145A lecture 14 page 6 ©copyright Bruce Blumberg 2000. All rights reserved
Identification of binding proteins (contd)
• Molecular biological approaches (contd)
– yeast one-hybrid assay
• clone element of interest into a reporter construct (e.g. -gal) and make stable yeast strain
• transfect in aliquots of cDNA expression libraries that have fragments of DNA fused to yeast activator
• if the fusion protein binds to your element then the reporter gene will be activated
• advantages
– somewhat more of a functional approach
– eukaryotic milieu allows some protein modification
• disadvantages
– slow, tedious purification of positives
– can’t detect dimeric proteins
– sensitivity is not so great
AD
Bait elements Reporter(s)His lacZ
BioSci 145A lecture 14 page 7 ©copyright Bruce Blumberg 2000. All rights reserved
Identification of binding proteins (contd)
• Molecular biological approaches (contd)
– expression cloning (sib screening)
• clone element of interest (or promoter) into a suitable reporter construct (e.g. luciferase)
• transfect (or inject, or infect, etc) pools (~10,000 cDNAs each) of cDNA expression libraries and assay for reporter gene
• retest positive pools in smaller aliquots (~1000)
• repeat until a pure cDNA is found
– advantages
– functional approach
– presumably using the appropriate cell type so modifications occur
– possibility to detect dimers with endogenous proteins
– disadvantages
• VERY TEDIOUS
• very slow, much duplication in pools, extensive rescreening is required
• could be expensive
BioSci 145A lecture 14 page 8 ©copyright Bruce Blumberg 2000. All rights reserved
Identification of binding proteins (contd)
– in vitro expression cloning (IVEC)
• Make small pools of cDNAs (~100)
• transcribe and translate cDNA libraries in vitro into protein pools
• EMSA to test protein pools for element binding
• unpool cDNAs and retest
• advantages
– functional approach
– smaller pools increase sensitivity
• disadvantages
– can’t detect dimers
– very expensive (TNT lysate)
– considerable rescreening still required
– tedious, countless DNA minipreps required
BioSci 145A lecture 14 page 9 ©copyright Bruce Blumberg 2000. All rights reserved
Identification of binding proteins (contd)
– hybrid screening system 1
• begin with cDNA libraries in 384-well plates, 1 cDNA per well
• pool cDNAs using robotic workstation
• prepare DNA with robotic workstation
• transcribe and translate protein in vitro
• test for ability to bind DNA element using sensitive, high-throughput assay
– fluorescence
– radioactive assay
• retest components of positive pools
• advantages
– very fast, only two steps required, ~ 2 weeks
– little work required
• disadvantages
– expense of robotics
– won’t detect dimers (unless 1 partner known)
– expense of reagents (TNT, radionuclides, fluorescent labels
BioSci 145A lecture 14 page 10 ©copyright Bruce Blumberg 2000. All rights reserved
Identification of binding proteins (contd)
– hybrid screening system 2
• prepare reporter cell line with element or promoter driving reporter gene (e.g. luciferase)
• prepare cDNA pools as in system 1
• use robotic workstation to transfect cDNA libraries into reporter cells
• assay for reporter gene
• advantages
– very fast
– truly functional approach
– use of cells allows modifications
– can detect dimers if one partner is already present in cell
• disadvantages
– expense of equipment
• OK, you have your element and binding protein, now what?
– functional analysis depends on type of protein you are dealing with
– goal will be to prove that this protein is necessary and sufficient to confer regulation onto the promoter, in vivo
• many just stop at works on the element
BioSci 145A lecture 14 page 11 ©copyright Bruce Blumberg 2000. All rights reserved
Transcription factors bind to regulatory elements
• The response element binding proteins you have carefully identified are transcription factors.– There are many types. The primary mode of
classification is via the type of DNA-binding domains and intermolecular interactions
• Features of transcription factors– typically these proteins have multiple functional
domains• can frequently be rearranged or transferred
– DNA-binding domains• these domains take many forms that will be
discussed next time• see also the list in TRANSFAChttp://transfac.gbf.de/TRANSFAC/
– Activation domains• these are polypeptide sequences that activate
transcription when fused to a DNA-binding domain
• these are diverse in sequence, 1% of random sequences fused to GAL4 can activate
• many activation domains are rich in acidic residues and assume an amphipathic -helix conformation when associated with coactivator proteins
• interact with histone acetylases that destabilize nucleosomes and open chromatin
BioSci 145A lecture 14 page 12 ©copyright Bruce Blumberg 2000. All rights reserved
Transcription factors bind to regulatory elements (contd)
• Features of transcription factors (contd)
– repression domains
• functional opposite of activation domains
• short and diverse in amino acid sequence
– some are rich in hydrophobic aa
– others are rich in basic aa
• some interact with proteins having histone deacetylase activity, stabilizes nucleosomes and condenses chromatin
• others compete with activators for the same sequence and contacts with the transcription machinery
– protein:protein interaction domains
• these are diverse in sequence but do contain structural motifs
• leucine zipper
• helix-loop-helix
BioSci 145A lecture 14 page 13 ©copyright Bruce Blumberg 2000. All rights reserved
Regulating transcription factor activity (contd)
-catenin/armadillo
BioSci 145A lecture 14 page 14 ©copyright Bruce Blumberg 2000. All rights reserved
Regulating transcription factor activity (contd)
• How can the activity of a transcription factor be restricted to a particular cell type or time?
– Factor is not generally present but synthesized only where it is needed
• some developmental regulators
– The factor is present but must be modified to be active
• heat shock factors - phosphorylated -catenin/armadillo - dephosphorylated
– A ligand is required for activity (or inactivity)
• nuclear hormone receptors
– The factor is localized to an inactive compartment (e.g. cell membrane) and required cleavage for activity
• sterol response factors (primarily cholesterol)
– The factor may be bound to an inhibitory factor in the cytoplasm
• NF-B and I-B
– A dimeric factor can have multiple partners. Which partner is present determines activity
• some dimers are active
• others are inactive
• eg bHLH and bZip proteins
BioSci 145A lecture 14 page 15 ©copyright Bruce Blumberg 2000. All rights reserved
Zinc finger genes
• Zinc fingers are found in a variety of transcription factors
– two basic types
• Cys-His, consensus sequence is
cys-X2-4
-cys-X3-phe-X
5-leu-X
2-his-X
3-his
– typical gene has 3 or more fingers
– found in factors for Pol II and Pol III
• Cys-Cys, consensus sequence is
cys-X2-cys-X
13-cys-X
2-cys
– typical gene has only 2 fingers
– found in steroid hormone receptor superfamily members
– may be involved in both DNA and RNA binding, presence of finger does not indicate which
• eg TFIIIA binds DNA and RNA product
• eIF2 recognizes translational initiation sites
BioSci 145A lecture 14 page 16 ©copyright Bruce Blumberg 2000. All rights reserved
Zinc finger genes (contd)
• purpose of fingers is to arrange residues such that zn ions can be coordinated
– fingers may form -helical structures that fit into the major groove of the DNA helix
– multiple fingers may act cooperatively to bind nucleic acids
BioSci 145A lecture 14 page 17 ©copyright Bruce Blumberg 2000. All rights reserved
Zinc finger genes (contd)
• cys-cys fingers in nuclear receptors
– only 1st finger binds to DNA
– second finger is responsible for protein:protein interactions
– spacing between fingers can vary quite a bit
• finger 1 contains a regions that determines target specificity - P-box
– CGSCKA - AGAACA
– CEGCKG - AGTTCA
– these can be swapped and change specificity of the receptor
• used in ecdysone-inducible system
BioSci 145A lecture 14 page 18 ©copyright Bruce Blumberg 2000. All rights reserved
Hormonal signaling pathways
• Hormones are chemical messengers that coordinate cellular activity
• Can act in different ways
– endocrine - on distant cells
– paracrine - on neighboring cells
– autocrine - on cells which secrete them
• Active at very low concentrations - typically less than 1 ppb (1 ppb ~= 3 nM)
• Involved in numerous biological processes - many hundreds of hormones
– reproduction - estrogen, testosterone, progesterone, FSH, LH, activin
– metabolic rate - thyroid hormone, TSH, GH
– stress - glucocorticoids, ACTH, CRF
– blood pressure - aldosterone, renin, angiotensin, vasopressin
– calcium homeostasis - vitamin D3, calcitonin, PH
• Some vitamins or vitamin derivatives are hormones
– Vitamin A
• all-trans-retinoic acid
• 9-cis-retinoic acid
• 14-OH-retroretinol
– Vitamin D3
BioSci 145A lecture 14 page 19 ©copyright Bruce Blumberg 2000. All rights reserved
Nuclear hormone receptors
• Domains are assortable and transferable
• DNA-binding domain (DBD)
– responsible for direct binding to DNA
– discriminates half site sequence
– determines spacing between half sites
– contains an important dimerization motif
• Ligand binding domain (LBD)
– responsible for ligand binding
– has a general dimerization motif
– contains an important transactivation domain
– may interact with amino terminus to modulate activation
• amino terminal region (A/B domain)
– contains an activation domain in many receptors
– may interact with other components of the transcriptional machinery
– many receptors have alternative splicing or promoter usage to yield different A/B domains
• linker region (D) may influence activation, repression, nuclear translocation or DNA-binding
A/B C D D F
BioSci 145A lecture 14 page 20 ©copyright Bruce Blumberg 2000. All rights reserved
Nuclear hormone receptors (contd)
• bind to specific target DNA sequences
• activate transcription of target genes upon ligand binding
• function at very low levels of ligand (~10-9M or ~ ppb)
• bind to small (~300d) lipophilic molecules
– steroids
– retinoids
– thyroid hormone
– vitamin D3
BioSci 145A lecture 14 page 21 ©copyright Bruce Blumberg 2000. All rights reserved
Nuclear hormone receptors (contd)
• Many receptor ligands are related to cholesterol– steroids– bile acids– oxysterols– Vitamin D3– ecdysone
• can move freely through tissues– penetrate to a target– diffuse from a source
OH
I
O
I
INH2
COOH
COOH
O
OH OH
O
CH2OH
all-trans retinoic acid
tri-iodo thyronine
1,25 dihydroxy D3
cortisol
OH
OH OH
O
OH
testosterone
OH
OH
estradiol
BioSci 145A lecture 14 page 22 ©copyright Bruce Blumberg 2000. All rights reserved
Nuclear hormone receptors (contd)
• more orphan than known receptors
• why study orphan receptors (not particularly easy)
– novel signaling pathways
– new developmental hormones
– target gene networks
– potential teratogens
– roles in adult physiology and endocrinology
– cancer treatment
BioSci 145A lecture 14 page 23 ©copyright Bruce Blumberg 2000. All rights reserved
Nuclear hormone receptors (contd)
• Payoff from orphan receptor research so far (4 biotechs)
– LXR and FXR regulate cholesterol metabolism
• LXR diverts cholesterol into bile acid pathway
• FXR negatively regulates uptake of bile acids
– PPARs regulate fat metabolism
• PPAR is insulin sensitizer
– SXR and PXR regulate metabolism of steroids, xenobiotics and environmental compounds
– CAR also mediates drug breakdown
BioSci 145A lecture 14 page 24 ©copyright Bruce Blumberg 2000. All rights reserved
Nuclear hormone receptors (contd)
• Nuclear receptors interact with each other and DNA
– can form homodimers, heterodimers and monomers
– four possible modes of DNA binding
• IR, DR, ER, monomer
– steroid receptors very closely related, others not so much
• bind to HSPs and stay in cytoplasm until ligand bound.
• GR, MR, PR and AR all bind each others response elements. Significant crossover between pathways at pharmacological levels of ligands (eg. anabolic steroid use)
– RXR heterodimers is the largest and most diverse family
– monomeric orphan receptors may also dimerize and/or interact with RXR depending on the response element.
BioSci 145A lecture 14 page 25 ©copyright Bruce Blumberg 2000. All rights reserved
• RXR is a common partner in >10 different pathways
– can be silent (non-permissive)
– can be active (permissive)
– offers another means to regulate dimeric receptors
• rexinoids (RXR selective compounds) are being used clinically in treatment of diabetes, breast cancer and other diseases.
– at least part of the reason that vitamin A levels are tightly regulated in vivo
• too much or too little both very harmful, particularly during development
Nuclear hormone receptors (contd)
Known ligandsRAR,, all-trans RA
TR , thyroid hormone
VDR 1,25-(OH)2-VD3
EcR ecdysone
Recent EX-orphansPPAR ,,, fatty acids, eicosanoids
FXR bile acids
BXR benzoates
LXR , oxysterols
Activatable orphansSXR/PXR steroids, xenobiotics
CAR , androstans, xenobiotics
BioSci 145A lecture 14 page 26 ©copyright Bruce Blumberg 2000. All rights reserved
Nuclear hormone receptors (contd)
• P-box determines half-site specificity
– CEGCKGFF in many receptors
– therefore they all bind to the same or similar half sites
– where does specificity come from?
• spacing between half-sites encodes specificity
– 3-4-5 rule of Kaz Umesono
– DR-3 VDRE SXRE
– DR-4 TRE SXRE, LXRE, FXRE, BXRE
– DR-5 RARE SXRE, CARRE
– DR-1 RXRE PPRE
– DR-2 RARE
• selectivity is not absolute but these provide a good model for determining response elements
BioSci 145A lecture 14 page 27 ©copyright Bruce Blumberg 2000. All rights reserved
Nuclear hormone receptors (contd)
• Transcriptional regulation by RXR heterodimers
– most, or all of these bind DNA in the absence of ligand
• unliganded receptor is a repressor
– effect of activators and repressors together ?
• ligand causes a conformational change that kicks off corepressor
• liganded receptor can now recruit coactivators and activate transcription
– coactivators and corepressors alter chromatin conformation by modulating histone modification
– offers possible ways to specifically disrupt complex
BioSci 145A lecture 14 page 28 ©copyright Bruce Blumberg 2000. All rights reserved
Nuclear hormone receptors (contd)
• How does one go about identifying orphan receptor ligands?
– requirements
• receptor expression construct
• response element to make reporter
• cofactors (use tissue where receptor is active)
– good news!
• assay is very sensitive sub parts per billion
– analytical bad news
• chemistry requires parts per thousand-ppm
BioSci 145A lecture 14 page 29 ©copyright Bruce Blumberg 2000. All rights reserved
Nuclear hormone receptors (contd)
• What are some effects of mutations in nuclear receptors?
– steroid receptors
• knockout of SF-1 removes adrenal/gonad axis
• human mutations in DAX-1 similar
• overproduction of adrenal steroids -Cushing’s syndrome
• underproduction - Addison’s disease
• nonfunctional AR - testicular feminization
– genotypic males develop as females externally but male internally (rumor about well-known actress)
• nonfunctional ER
– male, osteoporosis, coronary artery disease, continuous growth
– female lethal in utero, osteoporosis later
• nonfunctional MR- hypotension
• nonfunctional GR- hypertension and low renin
– other receptors
• thyroid hormone receptor - mutations can lead to alterations in metabolism and ADHD
• vitamin D receptor - vitamin D resistant rickets
• retinoic acid receptor
– several types of leukemia result from fusion of RAR to other transcription factors
– some are treatable with RA, others not