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BioSci 145A Lecture 14 - 2/21/2002 Transcription 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 1 ©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 2 ©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 3 ©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 4 ©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 5 ©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 His Bait elements BioSci 145A lecture 14 page 6 ©copyright lacZ Reporter(s) 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 7 ©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 8 ©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 9 ©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 10 ©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 TRANSFAC http://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 11 ©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 12 ©copyright Bruce Blumberg 2000. All rights reserved Regulating transcription factor activity (contd) -catenin/ armadillo BioSci 145A lecture 14 page 13 ©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 14 ©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-X5-leu-X2-his-X3-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-X13-cys-X2-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 15 ©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 16 ©copyright Bruce Blumberg 2000. All rights reserved Zinc finger genes (contd) • • BioSci 145A lecture 14 page 17 ©copyright 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 ecdysoneinducible system 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 18 ©copyright Bruce Blumberg 2000. All rights reserved Nuclear hormone receptors A/B • • • • • C D D 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 BioSci 145A lecture 14 page 19 ©copyright Bruce Blumberg 2000. All rights reserved F Nuclear hormone receptors (contd) • • • • bind to specific target DNA sequences activate transcription of target genes upon ligand binding -9 function at very low levels of ligand (~10 M or ~ ppb) bind to small (~300d) lipophilic molecules – steroids – retinoids – thyroid hormone – vitamin D3 BioSci 145A lecture 14 page 20 ©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 • CH2OH O OH HO OH 1,25 dihydroxy D3 cortisol O HO OH I NH2 HO COOH O all-trans retinoic acid COOH I I tri-iodo thyronine OH OH testosterone O BioSci 145A lecture 14 estradiol HO page 21 ©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 22 ©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 23 ©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 24 ©copyright Bruce Blumberg 2000. All rights reserved Nuclear hormone receptors (contd) Known ligands RAR,, TR , VDR EcR all-trans RA thyroid hormone 1,25-(OH)2-VD3 ecdysone Recent EX-orphans PPAR ,,, FXR BXR LXR , fatty acids, eicosanoids bile acids benzoates oxysterols Activatable orphans SXR/PXR CAR , • steroids, xenobiotics androstans, xenobiotics 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 BioSci 145A lecture 14 page 25 ©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 26 ©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 27 ©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 28 ©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 BioSci 145A lecture 14 page 29 ©copyright Bruce Blumberg 2000. All rights reserved