Bio Sci 145 A Lecture 13 2192002 Transcription

Bio. Sci 145 A Lecture 13 - 2/19/2002 Transcription factors II • Topics we will cover today – Hybrid tetracycline regulated system – implications of transgenic technology – Principles of gene regulation – Identification of regulatory elements – Identification of regulatory element binding proteins – Functional analysis – Transcription factors - introduction – Modulation of transcription factor activity • transcription factor resources – http: //transfac. gbf-braunschweig. de/TRANSFAC/ – http: //bioinformatics. weizmann. ac. il/transfac/ • detailed transcription factor database – http: //copan. bioz. unibas. ch/homeo. html • collected information about homeobox genes – http: //biochem 1. basic-ci. georgetown. edu/nrr. html • nuclear receptor resource • references – nuclear transport Nakielny and Dreyfuss (1999) Cell 99, 677 -690. – Nuclear pore structure Daneholt (1997) Cell 88, 585588. Bio. Sci 145 A lecture 13 1 ©copyright Bruce Blumberg 2000. All rights reserved

Regulated expression - hybrid tetracycline systems • • Background – references • Kringstein et al (1998) PNAS 95, 13670 -75 • Baron et al (1999) PNAS 96, 1013 -1018 • Blau and Rossi (1999) PNAS 96, 797 -799 – utilizes highly engineered tet and reverse tet proteins to get specific effects properties of the system – what happens when one puts proteins into the cell that respond differently to the same effector compound? • If they can dimerize with each other • can not dimerize with each other • or if they do not dimerize and bind to different and non-overlapping operator sequences – different function, can heterodimerize • in this case, a fair number will make unproductive heterodimers and interfere with desired effect • this will also be problematic even if the two dimers have been engineered to bind different targets • so for maximum effect, we must prevent heterodimerization between effectors that – can bind to different sequences – have different functions (activator vs repressor Bio. Sci 145 A lecture 13 2 ©copyright Bruce Blumberg 2000. All rights reserved

Regulated expression - hybrid tetracycline (contd) Heterodimers possible same DNA targets different DNA targets Heterodimers not possible functional discrimination works – Opposite function - same DNA target • use pure tet. R and VP 16 -rtet. R • at low dox, the repressor will dominate • as dox increases, the repressor will dissociate and VP 16 -rtet. R will activate • increases dynamic range of activation ~105 fold – increases sensitivity as well Bio. Sci 145 A lecture 13 3 ©copyright Bruce Blumberg 2000. All rights reserved

Regulated expression - hybrid tetracycline (contd) – Opposite function - different DNA target • use std tet with one type of tet. O to regulate gene A • and rev tet with another type of tet. O to regulate gene B • in the absence of dox, gene A will be activated while B will be silent • in the presence of high dox, gene A will be inactivated and gene B will be activated Bio. Sci 145 A lecture 13 4 ©copyright Bruce Blumberg 2000. All rights reserved

Regulated expression - hybrid tetracycline (contd) • Applications – activator and repressor • very sensitive regulation of responsive gene expression • tightly regulated expression over 5 logs • can readily measure effects of subtle changes in gene expression – how much change in expression is required to get effects? – Do effects differ at different levels of expression? – Activator/repressor two targets • can create “conditional mutants” that have one activity at low levels of effector substance and another at high levels • can regulate two different genes or two alleles of a single gene – mutually exclusive expression – or expression of neither • can repeatedly switch between two states and observe effects at high resolution • possible to perturb intracellular equilibria in small increments and follow the effects on phenotype Bio. Sci 145 A lecture 13 5 ©copyright Bruce Blumberg 2000. All rights reserved

Regulated expression - hybrid tetracycline (contd) • • • Caveats and pitfalls – best done with stable cell lines advantages – much more versatile than ecdysone – possible to fine tune expression of a single gene, or two genes with unprecendented resolution – can make conditional mutants without genetics • eg in model systems not amenable to genetics such as Xenopus or chicken – can make conditional replacements in vivo • knock the repressible tet. O into an endogenous gene • introduce a transgene under the control of the activatable tet. O • breed these mice with a line expressing the dual tet repressors • increasing dox will inactivate the endogenous gene and activate the transgene disadvantages – technically demanding – multiple steps required – may not work as well as predicted due to complexity – virus-based systems may not work in ES cells – Clontech’s TRE-effector plasmids have high background - need to be remade Bio. Sci 145 A lecture 13 6 ©copyright Bruce Blumberg 2000. All rights reserved

Regulated gene expression - summary • • • what system do you need to use? – Transgenic animals? – Gene therapy? – Cell culture? How much tolerance is there for modulation of non target genes? – Eg glucocorticoids, estrogens, progestins and thyroid hormones are very important physiologically and cannot be modulated without collateral effects in adults • however, these are very good in the early embryo of model organisms How stringent must the regulation be? – The more stringent the requirements for control the greater the likelihood that complex techniques will be required • eg hybrid tetracycline Is there a need for regulating multiple genes? – Hybrid tet is the only way to go What are the commercial implications? – Licenses may be required for various technologies • tet is controlled by BASF • ecdysone by the Salk Institute – depending on the license agreement, one technique might be preferred (eg ecdysone) Bio. Sci 145 A lecture 13 7 ©copyright Bruce Blumberg 2000. All rights reserved

Gene transfer technology - implications • • Genetics and reverse genetics – gene transfer and selection technology speeds up genetic analysis by orders of magnitude – virtually all conceivable experiments are now possible • all questions are askable – much more straightforward to understand gene function using knockouts and transgenics • gene sequences are coming at an unprecedented rate from the genome projects • Knockouts and transgenics remain very expensive to practice – other yet undiscovered technologies will be required to understand gene function. Clinical genetics – Molecular diagnostics are becoming very widespread as genes are matched with diseases • huge growth area for the future • big pharma is dumping billions into diagnostics – room for great benefit and widespread abuse • diagnostics will enable early identification and treatment of diseases • but insurance companies will want access to these data to maximize profits Bio. Sci 145 A lecture 13 8 ©copyright Bruce Blumberg 2000. All rights reserved

Gene transfer technology - implications (contd) • • • gene therapy – new viral vector technology is making this a reality • efficient transfer and reasonable regulation possible – long lag time from laboratory to clinic, still working with old technology in many cases – The Biotech Death of Jesse Gelsinger. Sheryl Gay Stolberg, NY Times, Sunday Magazine, 28 Nov 99 • http: //www. frenchanderson. org/history/biotech. pdf protein engineering – not as widely appreciated as more glamorous techniques such as gene therapy and transgenic crops – better drugs, eg more stable insulin, TPA for heart attacks and strokes, etc. – more efficient enzymes (e. g. subtilisin in detergents) – safe and effective vaccines • just produce antigenic proteins rather than using inactivated or attenuated organisms to reduce undesirable side effects metabolite engineering – enhanced microbial synthesis of valuable products • eg indigo (jeans) • vitamin C – generation of entirely new small molecules • transfer of antibiotic producing genes to related species yields new antibiotics (badly needed) – reduction of undesirable side reactions • faster more efficient production of beer Bio. Sci 145 A lecture 13 9 ©copyright Bruce Blumberg 2000. All rights reserved

Gene transfer technology - implications (contd) • • transgenic food – gene transfer techniques have allowed the creation of desirable mutations into animals and crops of commercial value • disease resistance (various viruses) • pest resistance (Bt cotton) • pesticide resistance • herbicide and fungicide resistance • growth hormone and milk production – effective but necessary? – negative implications • pesticide and herbicide resistance lead to much higher use of toxic compounds • results are not predictable due to small datasets • at least one herbicide (bromoxynil) for which resistance was engineered has since been banned plants as producers of specialty chemicals – still very underutilized since plant technology yet lags behind techniques in animals – great interest in using plants as factories to produce materials more cheaply and efficiently • especially replacements for petrochemicals – plants and herbs are the original source of many pharmaceutical products hence it remains possible to engineer them to overproduce desirable substances Bio. Sci 145 A lecture 13 10 ©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 m. RNA – transport of m. RNA to cytoplasm – translation of m. RNA – processing and stability of protein Bio. Sci 145 A lecture 13 11 ©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 coformation. associated histones are hyperracetylated – 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 Bio. Sci 145 A lecture 13 12 ©copyright Bruce Blumberg 2000. All rights reserved

Principles of gene regulation (contd) Bio. Sci 145 A lecture 13 13 ©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-cting 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 tissuespecific • 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 Bio. Sci 145 A lecture 13 14 ©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. Bio. Sci 145 A lecture 13 15 ©copyright Bruce Blumberg 2000. All rights reserved

Transcription factors and the preinitiation complex • Model for 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 promoterproximal 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. Bio. Sci 145 A lecture 13 16 ©copyright Bruce Blumberg 2000. All rights reserved

Principles of gene regulation (contd) • processing of the transcript to m. RNA – RNA is synthesized as an exact copy of DNA • heterogeneous nuclear RNA (hn. RNA) – hn. RNA 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 – m. RNA is now ready for transport to cytoplasm – some organisms perform trans splicing between m. RNAs • another way to generate m. RNA diversity Bio. Sci 145 A lecture 13 17 ©copyright Bruce Blumberg 2000. All rights reserved

Principles of gene regulation (contd) • transport of m. RNA to cytoplasm – capping, polyadenylation and splicing of m. RNA are prerequisites to transport – macromolecules are specifically transported bidirectionally though nuclear pores • direction controlled by nuclear import and export signals in macromolecules – fully processed m. RNAs are packaged into ribonucleoprotein particles, m. RNPs • hn. RNP 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 m. RNA 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 Bio. Sci 145 A lecture 13 18 ©copyright Bruce Blumberg 2000. All rights reserved

Principles of gene regulation (contd) • translation of m. RNA – by default, m. RNAs are all translated – efficiency of translation 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 - Nco. I site • stability of m. RNA in the cytoplasm varies – many short lived m. RNAs have multiple copies of the sequence AUUUA in 3’ UTR Bio. Sci 145 A lecture 13 19 ©copyright Bruce Blumberg 2000. All rights reserved

Principles of gene regulation (contd) • stability of m. RNA (contd) – others m. RNAs are specifically degraded, e. g. transferrin – in the absence of iron, a specific protein (IRE-BP) binds to a region of the transferring m. RNA containing AUUUA sequences – this protects the m. RNA from degradation, transferrin is synthesized and iron accumulates – iron binds to IRE-BP and dissociates it from m. RNA » AUUUA mediates degradation Bio. Sci 145 A lecture 13 20 ©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 c. DNA and genomic clones. – Map c. DNA 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. Bio. Sci 145 A lecture 13 21 ©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 Exo. III 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 Bio. Sci 145 A lecture 13 22 ©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 the database • prepare oligonucleotides and screen library Bio. Sci 145 A lecture 13 23 ©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 Bio. Sci 145 A lecture 13 24 ©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 c. DNA 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 Bio. Sci 145 A lecture 13 25 ©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 c. DNAs each) of c. DNA expression libraries and assay for reporter gene • retest positive pools in smaller aliquots (~1000) • repeat until a pure c. DNA 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 Bio. Sci 145 A lecture 13 26 ©copyright Bruce Blumberg 2000. All rights reserved

Identification of binding proteins (contd) – in vitro expression cloning (IVEC) • transcribe and translate c. DNA libraries in vitro into small pools of proteins (~100) • EMSA to test protein pools for element binding • unpool c. DNAs 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 Bio. Sci 145 A lecture 13 27 ©copyright Bruce Blumberg 2000. All rights reserved

Identification of binding proteins (contd) – hybrid screening system 1 • begin with c. DNA libraries in 384 -well plates, 1 c. DNA per well • pool c. DNAs 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 Bio. Sci 145 A lecture 13 28 ©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 c. DNA pools as in system 1 • use robotic workstation to transfect c. DNA 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 Bio. Sci 145 A lecture 13 29 ©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 (next time) 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-braunschweig. 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 GAL 4 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 Bio. Sci 145 A lecture 13 30 ©copyright Bruce Blumberg 2000. All rights reserved

Transcription factors bind to regulatory elements (contd) • Features of transcription factors (contd) – repression domains • functional converse 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 Bio. Sci 145 A lecture 13 31 ©copyright Bruce Blumberg 2000. All rights reserved