biosci 145a lecture 14 page 1 © copyright bruce blumberg 2000. all rights reserved biosci 145a...

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


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.


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


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


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



• 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



• 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



– 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



– 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



– 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


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

http://transfac.gbf.de/TRANSFAC/


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


Regulating transcription factor activity (contd)

-catenin/armadillo


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


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


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


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


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


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


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



• 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



• 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



• 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



• 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.


• 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


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



• 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



• 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



• 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



• 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

biosci 145a lecture 14 page 1 © copyright bruce blumberg 2000. all rights reserved biosci 145a...

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