Regulation by changes in histones, nucleosomes and chromatin Opening and activation Movement from heterochromatin to euchromatin Nucleosomes and transcription factors Chromatin remodeling activities Histone acetyl transferases and deacetylases Thanks: Dr. Jerry Workman

Human b-globin gene cluster Domain 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 events Loca- tion, hetero- chrom- atin General histone hyper- Ac’n Human HBB complex wildtype N-MEL DNase sensi- tive H3 hyper Ac’n LCR HSs Txn e g g d b ORGs + + + + away Del. HS2-HS5 + + - - away T-MEL, Hisp. del. - - - - x close Reik et al. (1988) Mol. Cell. Biol. 18:5992-6000. Schübeler et al. (2000) Genes & Devel. 14:940- 950

Chromosome localization in interphase In interphase, chromosomes appear to be localized to a sub-region of the nucleus.

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 heterochromatin

Domain opening is associated with movement to non-hetero-chromatic regions

Proposed 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 transcription

A scenario for transitions from silenced to open to actively transcribed chromatin

From silenced to open chromatin

Movement from hetero- to euchromatin

Nucleosome remodelers and HATs further open chromatin

Assembly of preinitiation complex on open chromatin

Transcription 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 remodeling

Chromatin 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 nucleosome II: form a stably remodeled dimer, altered DNAse digestion pattern III: transfer a histone octamer to a different DNA fragment

Covalent modification of histones in chromatin

Histones are acetylated and deacetylated Histone acetyl transferases Histone deacetylases

Covalent modification of histone tails N-ARTKQTARKSTGGKAPRKQLATKAARKSAP...- H3 4 9 10 14 18 23 27 28 N-SGRGKGGKGLGKGGAKRHRKVLRDNIQGIT...- H4 1 5 8 12 16 20 phosphorylation acetylation methylation

Two 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 chromatin. Acetylate K5 and K12 in histone H4

Acetylation by nuclear HATs is associated with transcriptional activation Highly acetylated histones are associated with actively transcribed chromatin Increasing histone acetylation can turn on some genes. Immunoprecipitation of DNA cross-linked to chromatin with antibodies against Ac-histones enriches for actively transcribed genes. Acetylation of histone N-terminal tails affects the ability of nucleosomes to associate in higher-order structures The acetylated chromatin is more “open” DNase sensitive accessible to transcription factors and polymerases HATs are implicated as co-activators of genes in chromatin, and HDACs (histone deacetylases) are implicated as co-repressors

Nuclear HAT As are coactivators Gcn5p is a transcriptional activator of many genes in yeast. It is also a HAT. PCAF (P300/CBP associated factor) is a HAT and is homologous to yeast Gcn5p. P300 and CBP are similar proteins that interact with many transcription factors (e.g. CREB, AP1 and MyoD). P300/CBP are needed for activation by these factors, and thus are considered coactivators. P300/CBP has intrinsic HAT activity as well as binding to the HAT PCAF.

HAT complexes often contain several trancription regulatory proteins. Example of the SAGA complex components: Gcn5: catalytic subunit, histone acetyl transferase Ada proteins transcription adaptor proteins required for function of some activators in yeast. Spt proteins (TBP-group) regulate function of the TATA-binding protein. TAF proteins associate with TBP and also regulate its function. Tra1 homologue of a human protein involved in cellular transformation. May be direct target of activator proteins.

Yeast SAGA interacting with chromatin

Roles of histone acetylation Increase access of transcription factors to DNA in nucleosomes. Decondense 30nm chromatin fibers Serve as markers for binding of non-histone proteins (e.g. bromodomain proteins).

Histone deacetylases are associated with transcriptional repression A mammalian histone deacetylase: Histone deacetylases: Are recruited by inhibitors of transcription. Are inhibited by trichostatin and butyrate.

Repression by deacetylation of histones

Methylated DNA can recruit HDACs

Connections in eukaryotic transcriptional activation Transcriptional activators Coactivators Nucleosome remodeling Histone modification Interphase nuclear localization

The functions of SWI/SNF and the SAGA complex are genetically linked. Some genes require both complexes for activation. Other genes require one or the other complex. Many genes require neither - presumably utilize different ATP-dependent complexes and/or HATs

The yeast HO endonuclease gene requires both SWI/SNF and SAGA The order of recruitment at the promoter: 1. SWI5 activator: sequence recognition 2. SWI/SNF complex: remodel nucleosomes 3. SAGA: acetylate histones 4. SBF activator (still at specific sequences) 5. general transcription factors Cosma, Tanaka and Nasmyth (1999) Cell 97:299-311. The order is likely to differ at different genes