MBB331_Week9-Fall-2011 - Readings for Week 9 Readings...

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Unformatted text preview: Readings for Week 9 Readings Weaver, Molecular Biology 5th ed. Chapter 12, p.315­320 (zinc fingers, Gal4, steroid receptors); p. 323­325 (activators); p. 329­334 and Chapter 9, p. 238­241 and Chapter 25, 805­ 808 (enhancers) Chapter 5, p. 112 and Chapter 25, p.802­ 805(ChIP) Readings for Week 9 Readings Weaver, Molecular Biology 4th ed. Chapter 12, p.322­328 (zinc fingers, Gal4, steroid receptors); p. 330­333 (activators); p. 337­341 Chapter 9, p. 244­247 (enhancers) Chapter 13, p. 384­385 (ChIP) MODELS FOR ENHANCER ACTION MODELS -How can an enhancer function at varying distances and orientations? -Can think of enhancer as serving to bring transcription factors into general vicinity of basal apparatus at promoter -the effect of “tethering” a protein with an enhancer is same as increasing the protein’s local concentration at the promoter -how then, does this “locally concentrated” protein affect the promoter? MODELS FOR ENHANCER ACTION MODELS SUPERCOILING SCANNING LOOPING FACILITATED TRACKING Weaver 2002 Molecular Biology Ed. 2 ENHANCERS CAN INTERACT WITH PROMOTERS ON SEPARATE DNAs PROMOTERS Weaver 2002 Molecular Biology Ed. 2 • Looping is likely involved in enhancer/promoter Looping interaction - evidence: in some cases, enhancer must evidence in be >70bp away from promoter (allowing room for DNA to loop out?), an enhancer & promoter can function even if on separate DNA molecules provided DNA is catenated i.e. supercoiling or sliding not relevant i.e supercoiling not glnA ENHANCER: NtrC LOOPING glnA • DNA looped DNA out between enhancer & promoter may permit interaction between enhancerenhancerbound factors bound & the basal transcription apparatus apparatus NtrC σ 54 glnA NtrC, a rare bacterial enhancer­binding protein, activates RNAP at specific sites by DNA looping as seen by electron microscopy ENHANCER TRAPS ENHANCER CNS & CNS neuronal lacZ lacZ enhancer traps in Drosophila embryos embryos Balakireva et al., (2000) J. Balakireva et ., Neuroscience 20, 3425. 20 • • Enhancer traps: used to identify enhancers that confer tissue or Enhancer temporal gene expression patterns-useful in developmental biology temporal Enhancer trap constructs: TATA-Inr-lacZ integrated randomly in genome - if inserted near active enhancer, lacZ expressed & after XlacZ gal add’n, blue color indicates tissue-specific pattern of transcription gal controlled by enhancer… ie. blue staining = lacZ turned-on by ie blue lacZ enhancer (can also use anti-β -galactosidase antibodies to see enhancer -galactosidase expression pattern) expression ENHANCER TRAPS ENHANCER CNS & CNS neuronal lacZ lacZ enhancer traps in Drosophila embryos embryos Balakireva et al., (2000) J. Balakireva et ., Neuroscience 20, 3425. 20 • • • • enhancer trapping allows one to go after genes that may participate enhancer in the development of a particular tissue in for example, if you are interested in CNS development, go after for enhancers expressed in CNS enhancers clone out genes normally under control of these enhancers presence of reporter gene near enhancer provides “tag” reporter gene A number of approaches can be used to clone gene near enhancer: -make genomic library from transgenic animal and screen with probe from reporter gene or use inverse PCR: -genomic DNA from transgenic animal cut with r.e. that doesn’t cut in reporter gene, but will give some flanking genomic sequence -DNA fragments circularized by ligation and subjected to PCR using primers based on reporter gene that face opposite directions -inverse PCR amplifies flanking genomic sequences joined by r.e. site corresponding to original r.e. reporter gene (may be present with plasmid) -some Drosophila vectors used to insert transgenes also incorporate a complete bacterial plasmid into genome if plasmid present transform bacteria get “plasmid rescue” of flanking sequences -in this case, can begin as with inverse PCR technique, but after circularizing DNA, simply transform it into bacteria -this approach is called plasmid rescue and net result is isolation of a plasmid with flanking genomic sequences cloned into it ENHANCER TRAPS ENHANCER -as an example of enhancer trapping, it has been used to clone genes expressed at high levels in the CNS of developing mouse embryos DIRECTED GENE EXPRESSION IN Drosophila Drosophila Gene X = lacZ when checking enhancers • • • Drosophila Gal4p-Enhancer Trap: GAL4 gene inserted randomly into GAL4 genome (by P-element) and then crossed with flies carrying integrated UAS-lacZ promoter fusions integrated lacZ promoter Interesting cell specific lacZ expression patterns seen by lacZ Interesting lacZ lacZ staining identifies tissue-specific enhancers These enhancer GAL4 lines used to drive expression of UAS These regulated transgene (UAS-Gene X) regulated TRANSCRIPTION FACTORS: INDEPENDENT DNA BINDING & ACTIVATION DOMAINS DNA Lewin Genes VII 2000 Oxford University Press • Transcription factors: often one DNA binding domain Transcription & one activating domain contacting basal apparatus one • eg. Gal4p - Zn binding transcription factor (but not Gal4p Zn not Zn-finger transcription factor) contains separable Zn DNA binding & transcription activation domains DNA Gal4p TRANSCRIPTION FACTOR: MODULAR DOMAINS MODULAR Lewin Genes VII 2000 Oxford University Press • Gal4p: binds to DNA at UAS; activates transcription; & Gal4p: binds Gal80p regulatory protein binds • When galactose levels low, Gal80p blocks Gal4p When transcriptional activation transcriptional • Gal4p activation domain attached to another DNA Gal4p binding domain still activates transcription - ie. Gal4p ie Gal4p DNA binding and activation domains are separable Gal4p ACTIVATION DOMAIN INDEPENDENT OF BINDING SPECIFICITY OF Activated by Activated endogenous Gal4p endogenous Weaver 2002 Molecular Biology Ed. 2 • Gal4p activation domain joined to LexA DNA binding Gal4p domain activates gene expression from LexA binding site (LexA operator) (remember LexA is a bacterial repressor) (LexA • Gal4p modular transcription factor: if Gal4p in vicinity of Gal4p promoter, regardless of how it got there, it activates transcription (doesn’t “care” about how it binds DNA) transcription HIV tat ACTIVATION REQUIRES NONHIV SPECIFIC TETHERING Lewin Genes VII 2000 Oxford University Press • HIV tat stimulates HIV transcription without even binding DNA - tat binds tar mRNA & tar while attached, tat activation domain acts at promoter acts • If tat RNA binding If domain exchanged with DNA binding domain, tat still works as activator works UPSTREAM TRANSCRIPTION FACTORS: INTERACTIONS WITH BASAL APPARATUS INTERACTIONS Lewin Genes VII 2000 Oxford University Press • Eukaryotic transcription factors do not directly contact RNA Eukaryotic polymerase (unlike prokaryotes and phage) polymerase • Although not generally called a transcription factor, CAP-cAMP Although activates transcription by direct interaction with RNA pol activates • Activation domains of eukaryotic transcription factors interact with Activation basal apparatus through contact with general (TFIIX) factors - often TFIID, TFIIB, or TFIIA which are involved in relatively early stages of TFIID TFIIB, assembly assembly UPSTREAM TRANSCRIPTION FACTORS: INTERACTIONS WITH BASAL APPARATUS INTERACTIONS Lewin Genes VII 2000 Oxford University Press • given that contact is made with basal apparatus during assembly, it given is likely that upstream transcription factors enhance transcription by increasing a rate-limiting step of basal apparatus formation rather than influencing its activity than • TFIID most common target for upstream factors, where contact can TFIID be made with a TAF; a major role of TAFs is to enable such links be • different TAFs contact different upstream factors, and this different interaction can assist either binding of TFIID to TATA box, or assist binding of other factors around TFIID-TATA box complex binding • specific example of specific transcription factor contributing to assembly of basal apparatus-studies on GAL4 and VP16 GAL4 • GAL4 & VP16 (herpes GAL4 simplex virus protein) are acidic activators acidic that recruit TFIIB that AFFINITY CHROMATOGRAPHY ASSAYS FOR TRANSCRIPTION FACTORS TRANSCRIPTION Factor TFIIB binds an affinity column containing the acidic activation domain of VP16: omitted -pass crude nuclear extract thorugh column, collect what binds (eluate) and test its ability to transcribe in presence of various components of apparatus -eluate can cause transcription even when TFIIB not added, indicating that TFIIB present in eluate -conversely, only thing missing from flowthrough (ie stuff that didn’t bind column) is TFIIB, as only TFIIB addition can restore transcription Weaver 1999 Molecular Biology Ed. 1 + Nuclear extract TFIIB VP16 VP16 TFIIB TFIIB VP16 TFIIB VP16 Eluate contains only TFIIB TFIIB VP16 VP16 TFIIB VP16 VP16 + TFIIB TFIIB Flowthrough is everything but TFIIB Transcription TFIIB is only factor that can excluded from added back factors and still get get transcription (note that TFIIA is not needed for in vitro transcription) Transcription Transcription will only work when TFIIB is added back AFFINITY CHROMATOGRAPHY ASSAYS FOR TRANSCRIPTION FACTORS TRANSCRIPTION • Transcription factor effects can be Transcription assayed by affinity chromatography based on factor binding to target site and general RNAP factors and • 2-step assay: DNA containing TATA 2-step box & UASGAL4 site coupled to box chromatography beads • Step 1: Gal4p & TFIID added & Step washed ---> Step 2: RNAP & all other TFIIs… Active transcription Active • Step 1: TFIIB & TFIID added & Step washed ---> Step 2: Gal4p + RNAP & other TFIIs… No transcription No Step 1 Step 2 – THUS, Gal4p recruits TFIIB to complex Weaver 2002 Molecular Biology Ed. 2 ACIDIC ACTIVATORS FACILITATE TFIIB BINDING TO PREINITIATION COMPLEX BINDING • (a) For transcription, Gal4p (a) must be present with TFIIB during preinitiation complex assembly - TFIID binds with or without Gal4p, TFIIB becomes limiting without Gal4p limiting • (b) In order to associate (with (b) Gal4p), TFIIB requires TFIID to be bound in the first step be Weaver 2002 Molecular Biology Ed. 2 Step 1 Step 2 Step 1 Step 2 • specific example of specific transcription factor contributing to assembly of basal apparatus-studies on GAL4 and VP16 GAL4 • GAL4 & VP16 (herpes GAL4 simplex virus protein) are acidic activators acidic that recruit TFIIB that COACTIVATORS COACTIVATORS Lewin Genes VII 2000 Oxford University Press • Transcription factors can contact basal Transcription apparatus indirectly through coactivator coactivator intermediates intermediates • DNA binding and transcriptional activation are in DNA essence encoded by two separate proteins as opposed to one protein with two domains opposed REPRESSORS BLOCK BASAL APPARATUS ASSEMBLY APPARATUS • Transcription activators --> assemble basal Transcription activators assemble transcription apparatus transcription • Transcription repressor --> blocks basal Transcription repressor blocks apparatus assembly apparatus • eg. DR1 repressor prevents TFIIB from joining DR1 TFIID complex --> assembly blocked --> assembly blocked transcription initiation blocked initiation • Eukaryotic repressors block assembly whereas Eukaryotic bacterial or phage repressors interfere with RNAP activity (eg λ repressor or lac repressor) OVERVIEW OF “UPSTREAM” FACTORS OVERVIEW • Upstream transcription factors: flexible activators of Upstream transcription - for instance… transcription • Upstream enhancers: DNA binding sites can be moved Upstream all around a gene all • Transcription factors: DNA binding domains can be Transcription swapped between factors swapped • Conserved mechanism: transcription factor from one Conserved eukaryote functions in distant eukaryotic species (eg. eukaryote yeast GAL4 in Drosophila) Drosophila – Eukaryotic promoter activation - conserved Eukaryotic mechanism and general in nature mechanism – Despite use of general mechanisms, an incredible Despite range of specifities of transcriptional activation can be built up using “mix and match” combinations of upstream factor binding sites in promoter and enhancers enhancers COOPERATIVE ASSEMBLY: TTR TTR PROMOTER IN HEPATOCYTES PROMOTER • Complexity of Complexity eukaryotic gene expression exemplified by TTR TTR transcription transcription • TTR promoter: model for cooperative assembly of basal transcription apparatus in hepatocytes hepatocytes COOPERATIVE ASSEMBLY: TTR TTR PROMOTER IN HEPATOCYTES PROMOTER • Complexity of Complexity eukaryotic gene expression exemplified by TTR TTR transcription transcription • TTR promoter: model for cooperative assembly of basal transcription apparatus in hepatocytes hepatocytes RESPONSE ELEMENTS: COMMON REGULATORY CONTROL REGULATORY • Groups of genes under Groups • common regulation often share specific factorshare binding sequences (1) • RESPONSE ELEMENTS: RESPONSE bound by inducible transcription factors that (2) respond to signals respond • Often found in promoter & Often enhancer regions enhancer • Not found at fixed Not positions but in promoters usually <200bp from start site site eg. COMMON COMMON RESPONSE ELEMENTS: ELEMENTS: HSE: heat shock HSE: response element - in promoters & bound by HSTF HSTF GRE: glucocorticoid GRE: response element enhancer recognized by steroid receptor by RESPONSE ELEMENTS: COMMON REGULATORY CONTROL REGULATORY -heat shock response that is controlled thorugh HSE underscores differences between prokaryotic and eukaryotic gene expression -heat shock response in bacteria mediated by production of new σ factor that enables RNAP to recognize special promoters of heat shock genes -eukaryotic heat shock genes are activated by binding of HSTF to HSE sequences in their promoters -heat shock apparatus well conserved among eukaryotes, for example, Drosophila heat shock genes can be activated in mammals and sea urchins METALLOTHIONEIN GENE: MULTIPLE RESPONSE ELEMENTS Weaver 2002 Molecular Biology Ed. 2 • • • Complex interactions of enhancers permits regulation of eukaryotic gene Complex expression in response to wide-range of conditions expression eg. Metallothionein gene regulation: constitutive factors (Sp1/GC boxes) + Metallothionein response elements (GRE - gluccocorticoid, MRE - inductive response to heavy metal ions, TRE (inside one BLE)- AP1 binding triggered by phorbol esters) esters) metallothionein protein protects cells from excess concentrations of heavy metallothionein metals by binding and removing them from the cell metals USE OF CHROMATIN IMMUNOPRECIPITATION (ChIP) TO DETERMINE TRANSCRIPTION FACTOR BINDING SITES DETERMINE -if you have an antibody against a transcription factor, ChIP can be used to determine where in a gene of interest that transcription factor binds -PCR primers targeting segments of the gene of interest are used to determine which sequences are present in the immunoprecipitation USE OF CHROMATIN IMMUNOPRECIPITATION (ChIP) TO DETERMINE TRANSCRIPTION FACTOR BINDING SITES FACTOR -genome-wide analysis of transcription factor binding sites can be done using microarrays (ChIP-chip) or high-throughput sequencing (ChIP-seq) -sample results of ChIP-seq reveal variations in transcription factor binding patterns YEAST ONE-HYBRID CAN BE USED TO FIND TRANSCRIPTION FACTOR(S) BINDING GIVEN SEQUENCE FACTOR(S) -in reverse expt to ChIP, this approach can find transcription factors binding to given DNA sequence -the DNA sequence of interest (“bait”) is inserted upstream of the minimal promoter of a reporter gene as multiple tandem copies -cDNA library is expressed as fusion to a transcriptional activation domain (AD) -if expressed protein binds to bait DNA, this will result in functional transcription factor and reporter gene expression -one then sequences cDNA to determine identity of binding protein CONTROL OF REGULATORY TRANSCRIPTION FACTORS • Lewin Genes VII 2000 Oxford University Press Activation of inducible Activation transcription factors: (i) (i) tissue specific expression eg. homeoproteins; (ii) eg homeoproteins; (ii) modification controlled - e.g. HSTF phosphorylation leads to activation; (iii) ligand (iii) binding - steroid receptors move from cytoplasm to nucleus; (iv) cleavage - eg. (iv) eg absence of sterol causes cleavage of factor bound to nuclear envelope and ER, releasing it to nucleus where it is active it CONTROL OF REGULATORY TRANSCRIPTION FACTORS • • Lewin Genes VII 2000 Oxford University Press (v) inhibitor release - e.g. NFκ B sequestered in sequestered cytoplasm by I-κ B, in cytoplasm I- B, response to signal, NF-κ B response released from I-κ B and released and moves to nucleus to activate transcription transcription (vi) homo- or heterodimerization -eg. HLH dimerization proteins- binding partners that activate or inactivate a factor factor TRANSCRIPTION FACTOR FAMILIES: Zinc FINGER MOTIFS Zinc FINGER MOTIFS DNA binding β -sheet • Zn-finger: first described in Zn-finger: TFIIIA (Aaron Klug 1985), required by RNAP III for 5S rRNA transcription rRNA • Repeated unit in TFIIIA: 9 X 30-residues always with 2 30-residues cysteines & 2 histidines cysteines • 1 Zn per repeat bound by Cys & Zn His in tetrahedral structure His • Zn fingers usually tandem Zn repeats (but can be more than one group of fingers with ~2-9 fingers in total) fingers Weaver 2002 Molecular Biology Ed. 2 TRANSCRIPTION FACTOR FAMILIES: Zinc FINGER MOTIFS Zinc FINGER MOTIFS -Zinc fingers take their names from the finger like structures that result from binding of zinc ions -the “classic” zinc finger proteins have a series of fingers: Cys2-His2 Zn-finger consensus: Cys Cys-X2 to 4-Cys-X3-Phe-X5-Leu-X2-His-X3-His -each finger ~23 amino acids, with linker between fingers usually 7-8 amino acids TRANSCRIPTION FACTOR FAMILIES: Zinc FINGER MOTIFS Zinc FINGER MOTIFS -C-terminal part of each finger forms α -helices that bind DNA, whereas N-terminal part forms a β -sheet Zif268 Zinc FINGER PROTEIN Zif268 Zinc FINGER PROTEIN e.g. -Zif268 TFIIIA Zinc finger protein finger -3 α -helices fit into DNA -3 -helices major groove major -each of 3 fingers has 2 -each amino acids (all Arg except 1 His) that contact Gs in major groove groove -other residues contact -other DNA phosphate backbone Weaver 2002 Molecular Biology Ed. 2 HYDROGEN BONDS BETWEEN λ REPRESSOR & OPERATOR DNA REPRESSOR OL1 OL1 Weaver 2002 Molecular Biology Ed. 2 • • • Cl & Cro are both helix-turn-helix repressors In both CI & Cro, 2 helix-3 amino acids are conserved (neighbouring In helix-3 Gln & Ser) that bond to specific bp in DNA Gln Helix-2 mediates H-bonding with phosphate backbone (for example Gln 33 in λ repressor) but doesn’t control specificity of target Gln TRANSCRIPTION FACTOR FAMILIES: Zinc FINGER MOTIFS Zinc FINGER MOTIFS • Multiple types of Zn fingers Multiple not all involved in DNA binding not • Distinct type (Cys2/Cys2) of Znfinger transcription factor in finger steroid receptors, consensus: steroid Cys-X2-Cys-X13-Cys-X2-Cys Cys-X • binds short palindromes • Cys2/Cys2 fingers are often Cys nonrepetitive nonrepetitive Weaver 2002 Molecular Biology Ed. 2 TRANSCRIPTION FACTOR FAMILIES: STEROID RECEPTORS STEROID RECEPTORS • Steroid receptors: factor family Steroid activated by steroid hormones & share similar response mechanism mechanism • eg. glucocorticoid receptor glucocorticoid binds glucocorticoids & then translocates into nucleus where it binds GRE enhancers associated with inducible genes associated • Unlike other transcription Unlike factors, DNA-binding & activation not independent not though DNA-binding & hormone binding separable binding Weaver 2002 Molecular Biology Ed. 2 • • • STEROIDS STEROIDS Hormones affect growth, tissue Hormones development & body homeostasis homeostasis Steroid hormones enter cell by Steroid simple diffusion & bind receptor in cytoplasm in...
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