1. BioSci 145A lecture 14 page 1 © copyright Bruce Blumberg 2000. All rights reserved BioSci 145A Lecture 14 - Transcription factors I Principles of gene regulation Identification of regulatory element binding proteins Functional analysis Transcription factors - introduction Additional reading –nuclear transport Nakielny and Dreyfuss (1999) Cell 99, 677-690. –Nuclear pore structure Daneholt (1997) Cell 88, 585- 588. –Nuclear receptors Evans (1988) Science 240, 889-895 Orphan receptors - 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

2. BioSci 145A lecture 14 page 2 © copyright Bruce Blumberg 2000. All rights reserved Principles of gene regulation Why does gene expression need to be controlled anyway? –Primary purpose in multicellular organisms is to execute precise developmental decisions so that proper genes are expressed at –appropriate time –correct place –at the required levels so that development, growth and differentiation proceed correctly –maintenance of homeostasis produce required substances in appropriate amounts –nutrients, cofactors, etc. degrade undesired substances from –diet –metabolism –injury inter and intracellular signaling processes Where are the control points? –Activation of gene structure –initiation of transcription –processing of the transcript to mRNA –transport of mRNA to cytoplasm –translation of mRNA –processing and stability of protein

3. BioSci 145A lecture 14 page 3 © copyright Bruce Blumberg 2000. All rights reserved Principles of gene regulation (contd) Activation of gene structure –genes are active only in cells where they are expressed –structure of gene determines whether it is can be transcribed or not –activation of an active structure may be one of the first steps in gene regulation modification of DNA –methylation of DNA inactivates genes –active genes are hypomethylated modification of histones –methylation and acetylation of histones activates gene expression »acetylase activates active genes are in an open, hypomethylated conformation. –associated histones are hyper-acetylated –one of the primary responsibilities of cell-type specific transcription factors is to facilitate the formation of an active chromatin conformation majority of alleged co-activator and co-repressor proteins are relatively non-specific modifiers of chromatin conformation that interact with specific factors targeting chromatin remodeling

4. BioSci 145A lecture 14 page 4 © copyright Bruce Blumberg 2000. All rights reserved Principles of gene regulation (contd)

5. BioSci 145A lecture 14 page 5 © copyright Bruce Blumberg 2000. All rights reserved Principles of gene regulation (contd) Initiation of transcription –Once the DNA template is accessible, the next requirement is to form the initiation complex although other forms of regulation are important, the majority of regulatory events occur at the initiation of transcription –genes under common control share response elements (aka cis-acting elements, enhancers) these sequences are presumed to be recognized by specific protein(s) the protein(s) functions as a transcription factor needed for RNA polymerase to initiate the active protein is only available when the gene is to be expressed –response elements are often cell-type or tissue- specific because binding proteins are cell-type specific but this is a tautology –each gene has multiple response elements each regulatory event depends on the binding of a protein to a particular response element any one of these can independently activate the gene combinatorial regulation by multiple elements and proteins is a central mechanism by which levels of gene expression are modulated

6. BioSci 145A lecture 14 page 6 © copyright Bruce Blumberg 2000. All rights reserved Principles of gene regulation (contd) –cis-acting control elements can be located many kilobases away from the transcriptional start site in intergenic regions in introns some elements may be quite close to TATA box or other intitiator elements –cis-acting elements are responsible for allowing the recruitment of TBP and assembly of the initiation complex.

7. BioSci 145A lecture 14 page 7 © copyright Bruce Blumberg 2000. All rights reserved Transcription factors and the preinitiation complex cooperative assembly of an activated transcription initiation complex at the TTR promoter in hepatocytes. –Four activators enriched in hepatocytes plus ubiquitous AP-1 factors bind to sites in the hepatocyte-specific enhancer and promoter- proximal region of the TTR gene –Activation domains of the bound activators interact extensively with co-activators, TAF subunits of TFIID,Srb/mediator proteins and general transcription factors. This causes looping of DNA and formation of stable initiation complex –Highly cooperative nature of complex assembly prevents initiation complex from forming in other cells that lack all four of the hepatocyte-enriched transcription factors.

8. BioSci 145A lecture 14 page 8 © copyright Bruce Blumberg 2000. All rights reserved Principles of gene regulation (contd) processing of the transcript to mRNA –RNA is synthesized as an exact copy of DNA heterogeneous nuclear RNA (hnRNA) –hnRNA gets capped and polyadenylated –introns are spliced out by the spliceosome, a large complex of RNA and proteins. exons can also be spliced out as well. Alternative splicing may produce proteins with new functions. –Molecular mechanisms underlying alternative splicing are still only poorly understood –regulation of alternative splicing is important in the CNS and for sex determination –splice junctions are read in pairs spliceosome binds to a 5’ splice donor and scans for a lariat sequence followed by a 3’ splice acceptor mutations in either site can lead to exon skipping –principle underlying gene trapping –mRNA is now ready for transport to cytoplasm –some organisms perform trans splicing between mRNAs another way to generate mRNA diversity

9. BioSci 145A lecture 14 page 9 © copyright Bruce Blumberg 2000. All rights reserved Principles of gene regulation (contd) transport of mRNA to cytoplasm –capping, polyadenylation and splicing of mRNA are prerequisites to transport –macromolecules are specifically transported bidirectionally though nuclear pores direction controlled by nuclear import and export signals in macromolecules –fully processed mRNAs are packaged into ribonucleoprotein particles, mRNPs hnRNP proteins contain nuclear export sequences –These are transported through the pore complex, unwinding as they do so –On the cytoplasmic side of the pore, the mRNA is stripped from the RNP by binding to ribosomes –those with signal sequences are paused and subsequently associate with ER –those without are translated directly

10. BioSci 145A lecture 14 page 10 © copyright Bruce Blumberg 2000. All rights reserved Principles of gene regulation (contd) translation of mRNA –by default, mRNAs are all translated –efficiency is important for protein levels. regulatory genes tend to be poorly translated –two primary mediators of efficiency consensus around the ATG –optimum is ACCACCATGG –most important factor is a G following ATG (A gives about 40% of protein –underlined sequence will give very high levels of translation - NcoI site stability of mRNA in the cytoplasm –many short lived mRNAs have multiple copies of the sequence AUUUA in 3’ UTR

11. BioSci 145A lecture 14 page 11 © copyright Bruce Blumberg 2000. All rights reserved Principles of gene regulation (contd) stability of mRNA (contd) –others mRNAs are specifically degraded, e.g. transferrin –in the absence of iron, a specific protein (IRE-BP) binds to a region of the transferring mRNA containing AUUUA sequences –this protects the mRNA from degradation, transferrin is synthesized and iron accumulates –iron binds to IRE-BP and dissociates it from mRNA »AUUUA mediates degradation

12. BioSci 145A lecture 14 page 12 © copyright Bruce Blumberg 2000. All rights reserved Principles of gene regulation (contd) Recap – control of gene expression –Activation of gene structure Physical structure of chromatin –initiation of transcription Ability of preinitiation complex to bind Cooperative binding of activators –processing of the transcript to mRNA Alternative splicing –transport of mRNA to cytoplasm Only capped, polyadenylated, spliced mRNAs –translation of mRNA Cap? Kozak sequence? Stability of mRNA –processing and stability of protein Many transcription factors very unstable

13. BioSci 145A lecture 14 page 13 © 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.

14. BioSci 145A lecture 14 page 14 © 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

15. BioSci 145A lecture 14 page 15 © 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

16. BioSci 145A lecture 14 page 16 © 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 32 P 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

17. BioSci 145A lecture 14 page 17 © 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

18. BioSci 145A lecture 14 page 18 © 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

19. BioSci 145A lecture 14 page 19 © 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

20. BioSci 145A lecture 14 page 20 © 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

21. BioSci 145A lecture 14 page 21 © copyright Bruce Blumberg 2000. All rights reserved Identification of binding proteins (contd) 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

22. BioSci 145A lecture 14 page 22 © 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

23. BioSci 145A lecture 14 page 23 © copyright Bruce Blumberg 2000. All rights reserved Identification of binding proteins (contd) 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

24. BioSci 145A lecture 14 page 24 © 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. –many types Usually classified by DNA-binding domains and intermolecular interaction domains Features of transcription factors –typically have multiple functional domains can frequently be rearranged or transferred –DNA-binding domains many forms see also the list in TRANSFAC http://transfac.gbf.de/TRANSFAC/ –Activation domains polypeptide sequences that activate transcription when fused to a DNA-binding domain diverse in sequence, 1% of random sequences fused to GAL4 can activate many 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

25. BioSci 145A lecture 14 page 25 © 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 (HDACs) –stabilize nucleosomes and condense 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

26. BioSci 145A lecture 14 page 26 © copyright Bruce Blumberg 2000. All rights reserved Regulating transcription factor activity (contd)  -catenin/ armadillo

27. BioSci 145A lecture 14 page 27 © 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

28. BioSci 145A lecture 14 page 28 © 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-X 2-4 -cys-X 3 -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-X 2 -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

29. BioSci 145A lecture 14 page 29 © 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

30. BioSci 145A lecture 14 page 30 © 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

31. BioSci 145A lecture 14 page 31 © 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

32. BioSci 145A lecture 14 page 32 © 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/BC D D F

33. BioSci 145A lecture 14 page 33 © 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 -9 M or ~ ppb) bind to small (~300d) lipophilic molecules –steroids –retinoids –thyroid hormone –vitamin D3

34. BioSci 145A lecture 14 page 34 © 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

35. BioSci 145A lecture 14 page 35 © 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

36. BioSci 145A lecture 14 page 36 © 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

37. BioSci 145A lecture 14 page 37 © 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.

38. BioSci 145A lecture 14 page 38 © 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 ligands RAR , ,  all-trans RA TR ,  thyroid hormone VDR1,25-(OH) 2 -VD3 EcRecdysone Recent EX-orphans PPAR , , ,  fatty acids, eicosanoids FXRbile acids BXRbenzoates LXR ,  oxysterols Activatable orphans SXR/PXRsteroids, xenobiotics CAR ,  androstans, xenobiotics

39. BioSci 145A lecture 14 page 39 © 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 VDRESXRE –DR-4 TRESXRE, LXRE, FXRE, BXRE –DR-5 RARESXRE, CARRE –DR-1 RXRE PPRE –DR-2 RARE selectivity is not absolute but these provide a good model for determining response elements

40. BioSci 145A lecture 14 page 40 © 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

41. BioSci 145A lecture 14 page 41 © 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

42. BioSci 145A lecture 14 page 42 © 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