MCB 252 Topic 11 Chromosome Organization and Gene Regulation III Co Activators and Co Repressors Prof David Rivier MCB 252 Spring 2015 New set of questions 1 How is the expression of a gene regulated at the transcriptional level 2 How is the expression of a gene regulated at the posttranscriptional level 3 How are mRNAs exported to the cytoplasm A mammalian organism needs to regulate the expression of 30 000 40 000 genes in the proper spatial and temporal pattern In eukaryotes DNA is assembled into chromatin which regulates access of the transcriptional machinery to genes Genes and the general transcriptional machinery Transcription factors activators and repressors Chromatin dynamics Histone H3 phosphorylated on Serine 10 is only detected in cells undergoing mitosis CH3 NH3 CH2 CO Histone Acetyl Transferases HATs NH CH2 CH2 CH2 CH2 CH2 N H C CH2 Histone Deacetylases HDACs C O Side chain of a lysine CH2 N H C C O Histone Acetyl Transferases HATs Histone Deacetylases HDACs Hypo acetylated histones Hyper acetylated histones Swi2 HP1 Closed state chromatin Open state chromatin A typical gene transcribed by RNA polymerase II RNAPII Start site BE Enhancer 4000 RP 500 CP Core Promoter RP Regulatory Promoter BE Boundary Element CP Gene 40 50 Pre mRNA BE Mediator RNAPII TF TF TF TAFs IIB TBP Pre initiation complex Holoenzyme Genetic links between gene activation and chromatin 1 Nucleosome depletion in vivo was found to result in transcriptional activation in a general manner This result suggested that removal of nucleosomes is coupled to gene activation Actual result is that the original in vitro txn reactions on naked DNA only needed GTFs for txn 2 Evidence for how the coupling might occur was derived from mutations of a gene called SWI2 or SNF2 Mutation in Swi2 Swi2 Snf2 gene Reduced Transcription of 2 unrelated genes The HO gene DNA endonuclease that is required for mating type switching The SUC2 gene Invertase for using sucrose as a source of carbohydrates Remarkably the mutations in SWI2 SNF2 could be suppressed by mutations in Histone H4 Hypothesis SWI2 SNF2 played a role in gene expression by modulating chromatin structure It was later found that the SWI2 SNF2 protein is a component of a large multi protein complex that could disrupt or reconfigure nucleosomes and stabilize the binding of TFs to nucleosomal DNA This complex was named the SWI SNF complex Suppression of Snf2 Defects by Reduced Histone H4 Function WT HO Suc2 is transcribed Snf2 LOF HO Suc2 not transcribed Snf2 LOF reduced histone function HO Suc2 transcribed Snf2 Histone H4 HO Suc2 Txn Suppressor Mutation A suppressor mutation suppresses the phenotypic effect of another mutation so that the double mutant seems normal Suppressor mutations alleviate the phenotypic abnormality caused by another mutation Logic Snf2 normally required for txn of SUC2 and HO genes Snf2 is not needed for txn of SUC2 or HO genes when chromatin structure is disrupted by a mutation in the gene that encodes Histone H4 Hypothesis Suggestion The role of Snf2 is to alter or disrupt normal chromatin structure and that disruption of chromatin structure is required to activate transcription of SUC2 and HO in normal wildtype cells Swi1 Swp82 Snf11 SWP73 Swi2 Snf2 Swi3 Arp9 Swi3 Arp 7 Snf5 Snf6 Swp29 Swi Snf complex Swi2 Snf2 subunit is an ATPase that is stimulated by both DNA and nucleosomes ATP dependent chromatin remodeling complexes Organism Name of subunits ATPase Yeast Swi Snf RSC 11 15 Swi2 Snf2 STH1 Human BAF NuRD NRD NURD 9 12 7 6 18 BRG1 Mi 2 CHD3 4 CHD4 Drosophila RSF NURF CHRAC ACF Brahma 2 4 5 4 ND hSnf2h ISWI ISWI ISWI BRM Frog Mi 2 complex 6 Mi 2 The current three central issues regarding the chromatin remodeling complexes are 1 What is the mechanism of chromatin remodeling 2 Are the complexes functionally equivalent or do they play different roles in vivo 3 How are these complexes targeted to DNA during activation or repression ATP dependent chromatin remodeling complexes Bind both DNA and nucleosomes in the nM range High affinity Generate nucleosome free regions in vitro promote unwinding of the DNA around the nucleosome slide nucleosomes along DNA Nucleosome Promoter Off Octamer sliding or Octamer transfer Promoter On Octamer sliding Octamer Transfer TAFs IIB TBP Octamer sliding TAFs IIB TBP Octamer Transfer Closed state chromatin Which factors are recruited When are these factors recruited Open state chromatin Experimental evidences suggest Promoter Off 1 An activator must find access to its binding site on chromatin 2 This activator recruits remodeling complexes and other factors 3 The holoenzyme is then recruited to form the pre initiation complex Promoter On TAFs IIB TBP TF DNA Binding site on naked DNA is readily accessible Binding site is no longer accessible after nucleosome deposition and chromatin folding Sometime the binding site is located in the linker DNA and is partially accessible Sometime the binding site is displayed properly even when wrapped around the histone octamer and is partially accessible A good example of the sequence of events associated with the activation of a promoter is provided by the HO gene 1 Swi5 finds access to its binding sites Swi5 SAGA complex Swi Snf 2 Swi 5 recruits the Swi Snf complex 3 Chromatin remodeling by Swi Snf leads to the recruitment of the SAGA complex HAT A good example of the sequence of events associated with the activation of a promoter is provided by the HO gene 4 Remodeling by Swi Snf and hyper acetylation of histone tails by SAGAresult in opening chromatin and the recruitment of the transcription factor SBF Promoter On TAFs SBF IIB TBP 5 In turn SBF is implicated in the recruitment of GTFs Swi2 Snf2 can interact directly with acetylated histones which causes the retention of the Swi Snf complex NH2 ATPase domain Bromo domain COOH Acetylated histones GCN5 containing complex SAGA a Co Activator Two Types of Co Activators Chromatin Remodeling Complexes Histone Acetyl Transferases HATs HATs are similar to Chromatin Remodeling Complexes in that each are complexes of proteins that are conserved from yeast to Humans Neither binds DNA directly Both can bind modified histones Examples of Human HATs p300 P300 CBP CREB Binding Protein CREB Activator AD SAGA complex TBP DBD Activator UAS TATA TBP can bind to the SAGA complex When TBP interacts with SAGA it cannot bind to TATA box Upon induction the activator binds the UAS and directly recruits the SAGA complex to the promoter region