BioSci 145A Lecture 13 - 2/20/2001 Transcription factors II Topics we will cover today 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 Tour of molecular interaction screening facility 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://biochem1.basic-ci.georgetown.edu/nrr/nrr.html nuclear receptor resource references nuclear transport Nakielny and Dreyfuss (1999) Cell 99, 677-690. Nuclear pore structure Daneholt (1997) Cell 88, 585588. BioSci 145A lecture 13
1 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 BioSci 145A lecture 13 2 copyright Bruce Blumberg 2000. All rights reserved Gene transfer technology - implications (contd) gene therapy new viral vector technology is making this a reality now possible to get efficient transfer and reasonable regulation long lag time from laboratory to clinic, still working with old technology in many cases 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 BioSci 145A lecture 13 3 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 BioSci 145A lecture 13 4 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 BioSci 145A lecture 13 5 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 BioSci 145A lecture 13 6 copyright Bruce Blumberg 2000. All rights reserved
Principles of gene regulation (contd) BioSci 145A lecture 13 7 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 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 BioSci 145A lecture 13 8 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. BioSci 145A lecture 13 9 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. BioSci 145A lecture 13 10 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 BioSci 145A lecture 13 11 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 BioSci 145A lecture 13 12 copyright Bruce Blumberg 2000. All rights reserved Principles of gene regulation (contd) translation of mRNA
by default, mRNAs 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 - NcoI site stability of mRNA in the cytoplasm varies many short lived mRNAs have multiple copies of the sequence AUUUA in 3 UTR BioSci 145A lecture 13 13 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 BioSci 145A lecture 13 14 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? doesnt always hold in vivo. BioSci 145A lecture 13 15 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 13 16 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 BioSci 145A lecture 13 17 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 cant detect binding if multiple partners are required fair amount of touch required BioSci 145A lecture 13 18 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 cant detect dimeric proteins sensitivity is not so great AD His Bait elements BioSci 145A lecture 13 19 lacZ Reporter(s) 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 13 20 copyright Bruce Blumberg 2000. All rights reserved Identification of binding proteins (contd) in vitro expression cloning (IVEC) transcribe and translate cDNA libraries in vitro into small pools of proteins (~100) EMSA to test protein pools for element binding unpool cDNAs and retest advantages functional approach smaller pools increase sensitivity disadvantages cant detect dimers very expensive (TNT lysate) considerable rescreening still required
tedious, countless DNA minipreps required BioSci 145A lecture 13 21 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 wont detect dimers (unless 1 partner known) expense of reagents (TNT, radionuclides, fluorescent labels
BioSci 145A lecture 13 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 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 13 23 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 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 13 24 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 BioSci 145A lecture 13 25 copyright Bruce Blumberg 2000. All rights reserved
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