MBB331_Week10-Fall-2011 - Readings for Week 10 Readings...

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Unformatted text preview: Readings for Week 10 Readings Weaver, Molecular Biology 5th ed. Chapter 12, p.320­323 (helix­turn­helix, leucine zipper and HLH proteins), p. 339­343 (insulators); Chapter 13, p.356­364 (nucleosomes), p.366­372 (GAGA factor, nucleosome positioning, DNase hypersensitivity), p. 372­376 (histone acetylation/deacetylation) Chapter 15, p. 437­456 (capping and polyadenylation of mRNA) Chapter 14, p. 395­405 (mRNA splicing) 1 Readings for Week 10 Readings Weaver, Molecular Biology 4th ed. Chapter 12, p.328­330 (helix­turn­helix, leucine zipper and HLH proteins), p. 343­346 (insulators); Chapter 13, p.360­368 (nucleosomes), p.371­377 (GAGA factor, nucleosome positioning, DNase hypersensitivity), p. 377­381 (histone acetylation/deacetylation) Chapter 15, p. 445­465 (capping and polyadenylation of mRNA) Chapter 14, p. 400­409 (mRNA splicing) 2 TRANSCRIPTION FACTOR FAMILIES: HELIX­TURN­HELIX MOTIFS HELIX­TURN­HELIX MOTIFS • First described helixturn-helix factors: λ turn-helix repressor & Cro repressor • Common eukaryotic Common helix-turn-helix factors: homeodomain developmental regulators regulators Weaver 2002 Molecular Biology Ed. 2 3 TRANSCRIPTION FACTOR FAMILIES: TRANSCRIPTION HOMEODOMAIN MOTIFS • Homeodomain (homeobox): 60 amino acid sequence first seen in the gene products of Drosophila Drosophila homeotic loci, mutations in which cause transformations of 1 body part into another - often involved in developmental regulation-found in many eukaryotes many • Homeodomain sometimes combined Homeodomain with other motifs - eg. Oct proteins which bind to eg Oct octamer motif --> Pou motif + homeodomain homeodomain Antennapedia 4 TRANSCRIPTION FACTOR FAMILIES: TRANSCRIPTION HOMEODOMAIN MOTIFS -homeodomains have 3 helical regions, the best conserved between different homeodomains being helix 3 -homeodomain helix 3 functions like the λ repressor recognition helix but is larger (17 amino acids compared to 9) Weaver 2002 Molecular Biology Ed. 2 5 HYDROGEN BONDS BETWEEN λ REPRESSOR & OPERATOR DNA REPRESSOR OL1 Weaver 2002 Molecular Biology Ed. 2 • in addition to hydrogen bonding, helix-2 undergoes ionic interactions in with phosphate backbone due to fact that its amino end, which has a net positive charge, is pointing directly towards negatively charged DNA backbone DNA 6 TRANSCRIPTION FACTOR FAMILIES: TRANSCRIPTION HOMEODOMAIN MOTIFS • • Homeodomain C-term region: Homeodomain helix-turn-helix motif (like λ cI repressor) repressor) crystal structure of crystal homeodomain of Drosophila Drosophila Engrailed bound to DNA determined determined Lewin Genes VII 2000 Oxford University Press 7 TRANSCRIPTION FACTOR FAMILIES: TRANSCRIPTION HOMEODOMAIN MOTIFS • Helix-3 lies in major groove and Helix-3 makes majority of contacts with DNA, helix-1 projects into minor groove and makes other contacts with DNA contacts Lewin Genes VII 2000 Oxford University Press 8 TRANSCRIPTION FACTOR FAMILIES: HELIX­LOOP­ HELIX­ HELIX (HLH) MOTIFS • • • • • Helix-loop-helix motif: 40-50 amino acids - 2 amphipathic α helices separated by loop HLH amphipathic helices: one HLH helix face hydrophobic, other face charged & hydrophilic HLH proteins form both HLH homodimers and heterodimers through interactions between hydrophobic amino acids in helices helices bHLH proteins have highly basic HLH DNA binding region adjacent to HLH motif HLH a homodimer or heterodimer in homodimer which both subunits contain basic both region can bind DNA like a pair of tongs tongs Weaver 2002 Molecular Biology Ed. 2 9 TRANSCRIPTION FACTOR FAMILIES: HELIX­LOOP­HELIX (HLH) MOTIFS HELIX­ A. B. Lewin Genes VII 2000 Oxford University Press • Heterodimers between Heterodimers bHLH & nonbasic HLH nonbasic proteins block DNA binding binding • ie. MyoD/MyoD homodimer MyoD/MyoD homodimer ---> active active • MyoD/Id heterodimer ---> MyoD/Id heterodimer 10 inactive inactive CLASSES OF HLH PROTEINS CLASSES OF HLH PROTEINS • bHLH proteins: E12 & E47 (Ig enhancer-binding bHLH factors); MyoD, myogenin, & Myf-5 (myogenesis/muscle formation); Myc proto-oncogene (growth reg); da & AC-S (CNS development in Drosophila) Drosophila • Class A: ubiquitously expressed - E12/E47 & da • Class B: tissue-specific - MyoD & AC-S • Combinatorial associations between HLH proteins Combinatorial enable complex regulations ie. various combinations of ie various MyoD/E12/E47/Id regulate muscle cell formation MyoD/E12/E47/Id 11 TRANSCRIPTION FACTOR FAMILIES: HELIX­LOOP­HELIX (HLH) MOTIFS HELIX­ -dimers of bHLH proteins vary in their ability to bind DNA and dimer formation introduces a degree of regulation eg. E12/E47 heterodimer binds DNA strongly but E12 homodimer binds poorly -in Drosophila, the gene emc is involved in establishing pattern of adult sensory organs, and functions by repressing activity of genes such as da (daughterless) and achaete-scute complex (AC-S) 12 Lewin Genes VII 2000 Oxford University Press TRANSCRIPTION FACTOR FAMILIES: HELIX­LOOP­HELIX (HLH) MOTIFS HELIX­ -if da and AC-S not suppressed too many cells adopt sensory organ fate -AC-S and da are genes of the bHLH type, while the suppressor codes for an HLH protein that lacks the basic region -in the absence of emc function, da and AC-S proteins form dimers that activate transcription of target genes; when emc protein is available it forms heterodimers with da and AC-S that can’t bind DNA 13 Lewin Genes VII 2000 Oxford University Press TRANSCRIPTION FACTOR FAMILIES: LEUCINE ZIPPER MOTIFS LEUCINE ZIPPER MOTIFS • • • • DNA binding protein dimerization DNA often important- as we have seen in bacteria, phage, eukaryotes bacteria, leucine zipper proteins well studied leucine example of how dimer formation positions DNA-binding regions of ecah subunit ecah each subunit contains half the each “zipper”: an α -helix with leucines -helix spaced 7 amino acids apart such that they all protrude from one face of helix of the zipper is a coiled coil formed the when leucines of two subunits interdigitate interdigitate Weaver 2002 Molecular Biology Ed. 2 14 TRANSCRIPTION FACTOR FAMILIES: LEUCINE ZIPPER MOTIFS LEUCINE ZIPPER MOTIFS • • • • adjacent to leucine dimerization adjacent zipper, DNA binding basic region basic formation of the zipper by 2 formation proteins forms a tong-like structure called bZIP which “grasps” DNA bZIP target sequences which are inverted repeats without separating nucleotides nucleotides bZIP is similar to DNA-binding bZIP structure of dimerized bHLH proteins proteins some proteins, such as Myc have some hybrid bHLH-ZIP domains where hybrid leu zipper stabilizes bHLH dimer leu Weaver 2002 Molecular Biology Ed. 2 15 LEUCINE ZIPPER TRANSCRIPTION FACTORS LEUCINE ZIPPER TRANSCRIPTION FACTORS • • • • Leucine zippers form homo- or heterodimers eg. Fos & Jun dimerization to form AP1 transcription factor, Fos orginally identified as SV40 enhancer binding protein orginally Fos and Jun are encoded by the cellular counterparts, c-fos & cFos c-fos cjun, of the oncogenes v-fos & v-jun found in murine and avian jun, v-fos sarcoma viruses, respectively sarcoma Jun/Jun binds AP1 site 10X lower affinity than Jun/Fos (Fos cannot Jun/Jun form homodimers) form 16 CHROMATIN & GENE EXPRESSION CHROMATIN & GENE EXPRESSION • Transcription factors do not interact with naked DNA Transcription chromosome organization of eukaryotic DNA has direct effects on transcription regulation: effects • (1) DNA packaging - transcription can be repressed (1) when DNA coiled into nucleosomes when • (2) Chromosomal domains - large regions of DNA (2) organized into regulatory domain organized • (3) DNA methylation & imprinting - methylated DNA is (3) generally not expressed generally 17 (1) DNA PACKAGING & TRANSCRIPTION: HISTONES (1) DNA PACKAGING & TRANSCRIPTION: HISTONES • Eukaryotes: DNA packaged Eukaryotes: into chromatin: DNA wound around histone octamer - the nucleosome core core • Histone octamer core: (2X H2a) + (2X H2b) + (2X H3) + (2X H4) (2X • Histone H1: can be Histone removed without affecting nucleosome structure suggesting H1 external to core particle core Acid-urea Acid-urea PAGE for histones histones 18 Weaver 2002 Molecular Biology Ed. 2 CHROMATIN: BEADS ON A STRING CHROMATIN: 30nm fiber Partially unwound 10nm fiber - beads on a string • • lyse nuclei & chromatin fiber released… tightly packed material lyse (30nm fiber) & DNA thread running thru a series of particles (nucleosomes) (nucleosomes) 1 nucleosome = 200bp DNA (includes DNA on & between particles) 19 CHROMATIN: CORE PARTICLE • Micrococcal nuclease releases Micrococcal individual nucleosomes individual • Micrococcal endonuclease Micrococcal cuts DNA between nucleosomes & then digests away linker DNA between particles particles • H1 extracted with moderately H1 low salt low • Nucleosome disk removed Nucleosome from DNA with high salt 20 CHROMATIN: CORE PARTICLE Weaver 2002 Molecular Biology Ed. 2 • Nuclease digestion produces Nuclease core particle of 146bp DNA core wrapped around 4 core histones (H2a, H2b, H3, & H4) histones H3 H4 • DNA follows “tracks” around DNA histone octamer histone 21 CHROMATIN: NUCLEOSOME Weaver 2002 Molecular Biology Ed. 2 • • • • H2A (yellow), H2B (red), H3 (blue), H4 (green) Core histones: histone fold ­ 3 α helices linked by 2 loops DNA wraps twice around core H4 basic tail exposed on side of core, H4 tail interacts with H2A & B acidic regions of neighboring particle ­ mediates nucleosome X­linking 22 CHROMATIN PACKAGING CHROMATIN • Increasing ionic strength Increasing results in increased chromatin condensation chromatin • Nucleosome string Nucleosome eventually condenses to 30nm fiber 30nm • DNA condensed ~7 fold in DNA nucleosome ---> add’nal ~7 fold condensation in 30nm fiber fiber • 30nm fiber: solenoid or 30nm irregular zig-zag? 23 Weaver 2002 Molecular Biology Ed. 2 CHROMATIN: 30 nm SOLENOID? CHROMATIN: • 30nm fiber solenoid 30nm model: Nucleosome string coiled into hollow tube (solenoid) tube • Model consistent with Model X-ray diffraction data X-ray • Inconsistent with Inconsistent irregular structure observed by electron microscopy Weaver 2002 Molecular Biology Ed. 2 24 25 CHROMATIN: 30nm FIBER • 30nm zigzag 30nm ribbon model: alternating reg & irregular zigzag string of nucleosomes nucleosomes • Regardless of Regardless model, 30nm fiber further looped into chromatin domains domains Transcription factors must act on DNA in chromatin 26 CHROMATIN & TRANSCRIPTION: CHROMATIN PRE-EMPTIVE MODEL PRE-EMPTIVE • Pre-emptive model: histones & Pre-emptive transcription factors battle for DNA possession possession • Histones displaced at replication, Histones permits transcription factors to gain access gain • eg. in vitro adenovirus promoter transcription: transcription: • (1) TFIID + RNAP II (& other general (1) factors) + histones --> transcribed transcribed • (2) histones + TFIID + RNAP II (& (2) other general factors) --> NOT transcribed Lewin Genes VII 2000 Oxford University Press 27 CHROMATIN & TRANSCRIPTION: CHROMATIN DYNAMIC MODEL DYNAMIC • Dynamic model: Dynamic transcription factor actively displaces nucleosomes using ATP hydrolysis hydrolysis • eg. Drosophila hsp70 Drosophila promoter: promoter: – GAGA transcription factor GAGA displaces nucleosomes in ATP-dependent manner ATP-dependent Lewin Genes VII 2000 Oxford University Press 28 CHROMATIN ASSEMBLY: TRANSCRIPTION • RNAP displaces DNA from RNAP octamer during transcription octamer • DNA behind RNAP loops forward DNA and octamer slides from DNA ahead of RNAP to DNA behind ahead • Octamer displaced by +ve DNA Octamer supercoiling ahead of moving RNAP RNAP • Octamer is displaced as intact Octamer unit - evidence: crosslinking evidence crosslinking histones together does not inhibit transcription suggesting octamer dissociation not necessary dissociation • add’n of H1 reduces transcription add’n suggesting H1 must be modified or removed from active chromatin or Lewin Genes VII 2000 Oxford University Press 29 SV40 NUCLEOSOME­FREE SV40 NUCLEOSOME­FREE ZONES • Some regulatory DNA Some regions too stiff for nucleosome formation nucleosome • Nucleosome-free zones Nucleosome-free identified in control regions of active genes regions • SV40 minichromosome SV40 visible nucleosome-free gap that corresponds to nuclease-sensitive region…. region…. Lewin Genes VII 2000 Oxford University Press 30 DNase HYPERSENSITIVE SITES DNase • Chromatin exposed by Chromatin transcription or structural changes susceptible to digestion by low DNase I concentration concentration • DNase I hypersensitive DNase sites: 100X more sensitive to DNase I to • Chromatin DNase I treated Chromatin --> DNA isolated --> distance from known restriction site determines hypersensitive site 31 Lewin Genes VII 2000 Oxford University Press TRANSCRIPTION AIDED BY NUCLEOSOME TRANSCRIPTION POSITIONING • Nucleosomes not necessarily Nucleosomes hindrance to transcription - ie. ie positioning nucleosomes with respect to regulatory sites respect • Mouse mammary tumor virus Mouse (MMTV) promoter: 3 sites for dimeric hormone receptors, and NF1 & OTF-1 sites NF1 • Naked DNA ---> NF1 excluded • Nucleosomal DNA ---> all Nucleosomal factors bind factors • HR may bind on nucleosome HR surface and make alterations that allow binding of other factors to build transcription activation complex activation Lewin Genes VII 2000 Oxford University Press 32 HISTONE DEACETYLATION & TRANSCRIPTIONAL REPRESSION • • • Lysine on histone tails are targets of acetylation by histone acetyltransferases (HATs) Histone acetylation “loosens” bound DNA in nucleosomes ---> gene activation Histone deacetylase “tightens” DNA binding in nucleosomes ---> gene repression Weaver 2002 Molecular Biology Ed. 2 33 HISTONE DEACETYLATION & TRANSCRIPTIONAL REPRESSION • • a variety of co-activators of transcription have histone acetyltransferase (HAT) activity-may loosen association of nucleosomes with gene control region known repressors of transcription can interact with co-repressors wich interact with histone deactylases to tighten grip of histones on DNA Weaver 2002 Molecular Biology Ed. 2 eg. in absence of retinoic acid, retinoic acid receptor (RAR) & retinoic acid receptor X (RXR) heterodimer binds sites that repress transcription N-CoR/SMRT (nuclear receptor co-repressor/silencing mediator for retinoid & thyroid hormone receptors) bind histone deacetylase HDAC1 34 35 ChIP can be used to elucidate “histone code” of single genes or whole genomes • ChIP using antibodies against methylated and acetylated histones can reveal the histone code of single genes or whole genomes under particular conditions • For example, ChIP was used to look at the histone code of upstream control region of the var gene of the malaria parasite, P. falciparum, during its life cycle • var facilitates long-term chronic infection of the host 36 37 38 REGULATING TRANSCRIPTION BY AFFECTING CHROMATIN STRUCTURE • Global changes in chromatin structure can regulate transcription: • eg. yeast SWI/SNF “chromatin remodeling” favours transcription by altering nucleosome density/structure • Drosophila Polycomb proteins repress homeotic genes by affecting chromatin structure - polycomb null mutants cause homeotic gene expression in incorrect locations 39 40 (2) LONG RANGE TRANCRIPTION REGULATION: (2) β -GLOBIN GENE CLUSTER Lewin Genes VII 2000 Oxford University Lewin Press Press • • • • • Transcriptional regulation = promoters + enhancers + locus control Transcriptional regions (LCRs) for some genes regions β -globin gene cluster: distant DNase I hypersensitive sites ---> 5 ´cluster of sites is LCR required for expression of all β -globin genes ´cluster -globin in cluster; chromosomal domain showing sensitivity to DNase I chromosomal Erythroid cells: DNA packaging of globin chromatin domain DNA “opened” by LCRs, permitting transcription initiation “opened” In cells with no globin expression, no DNase sensitivity In no no 41 Delete LCRs in erythroid cells, no DNase hypersensitivity no Detection of DNase­Hypersensitive Sites Detection of DNase­Hypersensitive Sites (a) Inactive gene (a) (b) Active gene ---> DNase (b) ---> no DNase hyper-sensitive regions not hyper-sensitivity hyper-sensitivity protected ---> nucleosomeprotected free areas digested & free fragments smaller fragments 42 HYPERSENSITIVE SITES IN ACTIVE GENES HYPERSENSITIVE 43 Weaver 2002 Molecular Biology Ed. 2 HYPERSENSITIVE SITES IN ACTIVE GENES HYPERSENSITIVE • • • • DNase hypersensitive sites in DNase (chick) RBC α -globin genes 2 α -2 globin genes, α D & α A, and -2 A, flanking DNA, all DNase digested in RBCs but not in MSB (lymphoid) cells [no DNase control = 0] control Control: ovalbumin gene not Control: DNase I hypersensitive in RBCs or lymphocytes or α -2 globin genes & flanking -2 sequences nucleosome-free, and expressed, in RBCs expressed, 44 Weaver 2002 Molecular Biology Ed. 2 INSULATORS INSULATORS • • • Insulator placed between Insulator enhancer & promoter - insulator blocks enhancer action on promoter; likely explanation for limitation of enhancer activity to particular promoter to Position effect variegation: Position active genes repressed when placed next to heterochromatin (condensed chromatin) placing insulator between heterochromatin & gene blocks spreading inactive condensed chromatin chromatin Models: insulators loop & Models: restrict DNA domains or block factor sliding factor Weaver 2002 Molecular Biology Ed. 2 45 Drosophila SPECIALIZED CHROMATIN STRUCTURES STRUCTURES Lewin Genes VII 2000 Oxford University Press flanking scs & scs´ flanking scs sequences insulate Drosophila hsp70 genes from effects of surrounding genes genes scs sequences resistant to DNase I resistant degradation, flanked by adjacent hypersensitive site hypersensitive -scs placed on either side of Drosophila white gene (for red eyes) ensure gene white function anywhere in genome, even when inserted into heterochromatin function 46 scs BINDING PROTEINS scs Lewin Genes VII 2000 Oxford University Press • Red = DNA, Green = BEAF32, Yellow = overlap Green Yellow • BEAF32 binds at scs insulator sites located at scs ~50% of Drosophila polytene chromosome ‘interbands’ ‘interbands’ • the bands of polytene chromosomes may the represent chromosomal domains separated by insulators in the interbands insulators 47 INSULATORS, MARs, & LCRs INSULATORS, MARs, & LCRs Lewin Genes VII 2000 Oxford University Press • LCR = locus control region, MAR = matrix associated region associated • Chromosomal domains defined by MARs that direct Chromosomal domain attachment to particular locations on inside of nuclear periphery of • 3 types of domain sites: (1) insulators ---> prevent types spreading between domains; (2) MARs ---> attach spreading domain to nuclear matrix; (3) LCRs ---> transcription domain initiation initiation 48 (3) EUKARYOTIC DNA METHYLATION (3) • • • • • • • Eukaryotes: methylation Eukaryotes: important for transcriptional control control 2-7% of cytosines methylated 2-7% most in CG doublets most Distribution of methylation can be Distribution checked using restriction enzymes that cleave target site with CG e.g. HpaII vs. MspI HpaII Msp isoschizomer (cleave same target sequence) digests sequence) MspI ---> cuts all CCGG HpaII ---> cannot cut methylated CCGG CCGG MspI finds all CCGG, HpaII finds HpaII indicates methylation state of these these use to assess CCGG methylation use 49 in various genes... in Lewin Genes VII 2000 Oxford University Press TRANSCRIPTIONAL INACTIVATION BY TRANSCRIPTIONAL INACTIVATION BY METHYLATION General finding: • Methylated genes ---> inactive inactive • Non-methylated/undermethylated genes ---> active active • Undermethylated genes can correspond to Undermethylated chromosomal domains eg. Chick α -globin gene cluster eg Chick -globin - underm...
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  • Spring '11
  • DAVIDSON
  • DNA, Oxford University, Molecular Biology Ed., Lewin Genes

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