Regulation by changes in histones, nucleosomes and chromatinHuman b-globin gene clusterDomain opening and gene activation are separable eventsChromosome localization in interphaseGene activation and location in the nucleusDomain opening is associated with movement to non-hetero-chromatic regionsProposed sequence for activationA scenario for transitions from silenced to open to actively transcribed chromatinFrom silenced to open chromatinMovement from hetero- to euchromatinNucleosome remodelers and HATs further open chromatinAssembly of preinitiation complex on open chromatinTranscription factor binding to DNA is inhibited within nucleosomesStimulate binding of transcription factors to nucleosomesBinding of transcription factors can destabilize nucleosomesNucleosome remodelingChromatin remodeling ATPases are large complexes of multiple proteinsChromatin remodeling ATPases catalyze stable alteration of the nucleosomeCovalent modification of histones in chromatinHistones are acetylated and deacetylatedCovalent modification of histone tailsTwo types of Histone Acetyltransferases (HATs).Acetylation by nuclear HATs is associated with transcriptional activationNuclear HAT As are coactivatorsHAT complexes often contain several trancription regulatory proteins.Yeast SAGA interacting with chromatinRoles of histone acetylationHistone deacetylases are associated with transcriptional repressionRepression by deacetylation of histonesMethylated DNA can recruit HDACsConnections in eukaryotic transcriptional activationThe functions of SWI/SNF and the SAGA complex are genetically linked.The yeast HO endonuclease gene requires both SWI/SNF and SAGARegulation by changes in histones, nucleosomes and chromatinOpening and activationMovement from heterochromatin to euchromatinNucleosomes and transcription factorsChromatin remodeling activitiesHistone acetyl transferases and deacetylasesThanks: Dr. Jerry WorkmanHuman -globin gene clusterεγγψηδβGA020406080kbLCRDNase HSsYesEmbryonicFetal >EmbryonicAdultLocus Control Region is needed to: • open a chromatin domain in erythroid cells • express of linked globin genes at a high level • override position effects in transgenic miceDomain opening?Locus control region:Activate linked globin gene expression in erythroid cells.Overcome position effects at many integration sites in transgenic mice.Role in switching expression?Domain opening and gene activation are separable eventsLCR HSs Human HBB complexORGswildtypeN-MELDNasesensi-tiveGeneralhistonehyper-Ac’nH3 hyperAc’nLoca-tion,hetero-chrom-atin Txn+-Del. HS2-HS5T-MEL, Hisp. del.x x- -close-awayaway--+ ++ + +Reik et al. (1988) Mol. Cell. Biol. 18:5992-6000.Schübeler et al. (2000) Genes & Devel. 14:940- 950Chromosome localization in interphaseIn interphase, chromosomes appearto be localized to a sub-region of thenucleus.Gene activation and location in the nucleus•Condensed chromatin tends to localize close to the centromeres–Pericentromeric heterochromatin•Movement of genes during activation and silencing–High resolution in situ hybridization–Active genes found away from pericentromeric heterochromatin –Silenced genes found associated with pericentromeric heterochromatinDomainopening is associated with movement to non-hetero-chromatic regionsProposed sequence for activation•1. Open a chromatin domain–Relocate away from pericentromeric heterochromatin–Establish a locus-wide open chromatin configuration•General histone hyperacetylation•DNase I sensitivity•2. Activate transcription–Local hyperacetylation of histone H3–Promoter activation to initiate and elongate transcriptionA scenario for transitions from silenced to open to actively transcribed chromatinFrom silenced to open chromatinMovement from hetero- to euchromatinNucleosome remodelers and HATs further open chromatinAssembly of preinitiation complex on open chromatinTranscription factor binding to DNA is inhibited within nucleosomes•Affinity of transcription factor for its binding site on DNA is decreased when the DNA is reconstituted into nucleosomes •Extent of inhibition is dependent on:–Location of the binding site within the nucleosome.•binding sites at the edge are more accessible than the center–The type of DNA binding domain. •Zn fingers bind more easily than bHLH domains.Stimulate binding of transcription factors to nucleosomes•Cooperative binding of multiple factors.•The presence of histone chaperone proteins which can compete H2A/H2B dimers from the octamer.•Acetylation of the N-terminal tails of the core histones•Nucleosome disruption by ATP-dependent remodeling complexes.Binding of transcription factors can destabilize nucleosomes•Destabilize histone/DNA interactions.•Bound transcription factors can thus participate in nucleosome displacement and/or rearrangement.•Provides sequence specificity to the formation of DNAse hypersensitive sites.•DNAse hypersensitive sites may be–nucleosome free regions or –factor bound, remodeled nucleosomes which have an increased accessibility to nucleases.Nucleosome remodelingChromatin remodeling ATPases are large complexes of multiple proteins•Yeast SWI/SNF–10 proteins–Needed for expression of genes involved in mating-type switching and sucrose metabolism (sucrose non-fermenting).–Some suppressors of swi or snf mutants are mutations in genes encoding histones.–SWI/SNF complex interacts with chromatin to activate a subset of yeast genes.–Is an ATPase•Mammalian homologs: hSWI/SNF–ATPase is BRG1, related to Drosophila Brahma•Other remodeling ATPase have been discovered.Chromatin remodeling ATPases catalyze stable alteration of the nucleosomeII: form a stably remodeled dimer, altered DNAse digestion patternIII: transfer a histone octamer to a different DNA fragmentCovalent modification of histones in chromatinHistones are acetylated and deacetylatedAcCoACOCHNHCH2CONH......CH2CH2CH2CH2NH3+COCHNHCH2CONH......CH2CH2CH2CH2NHGlyLysCCH3OCoAAcPositive charge on amino groupNo charge on amide groupHistone acetyl transferasesHistone deacetylasesCovalent modification of histone tailsN-ARTKQTARKSTGGKAPRKQLATKAARKSAP...- H34 9 10 141823 27 28N-SGRGKGGKGLGKGGAKRHRKVLRDNIQGIT...- H45 8 12 16 201acetylationphosphorylation methylationTwo types of Histone Acetyltransferases (HATs).•Type A nuclear HATs: acetylate histones in chromatin.•Type B cytoplasmic HATs: acetylate free histones prior to their assembly into