Impact of Nucleosomal DNA Sequences on SWR Remodeler Activity and Histone H2A.Z Targeting
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ABSTRACT: The variant histone H2A.Z is deposited into nucleosomes immediately downstream of promoters, where it plays a critical role in transcription. The site-specific deposition of H2A.Z is catalyzed by the SWR complex, a conserved chromatin remodeler with affinity for promoter-proximal nucleosome-depleted regions (NDRs) and histone acetylation. By comparing the genomic distribution of H2A.Z in wild-type and SWR-deficient cells, we found that SWR is also responsible for depositing H2A.Z at thousands of non-canonical sites not directly linked to NDRs or histone acetylation. To understand the targeting mechanism of H2A.Z, we presented SWR to a library of canonical nucleosomes isolated from yeast and analyzed the preferred substrates. Our results revealed that SWR preferentially deposited H2A.Z into a subset of endogenous H2A.Z sites, which are overrepresented by polyadenine tracks on the top strands of the DNA duplex at the nucleosomal entry-exit sites. Insertion of polyadenine sequences into recombinant nucleosomes near the outgoing H2A-H2B dimer enhanced SWR’s affinity for the nucleosomal substrate and increased its H2A.Z insertion activity. These findings suggest that the genome encodes sequence-based information that facilitates remodeler-mediated targeting of H2A.Z.
Project description:The histone variant H2A.Z plays key roles in gene expression, DNA repair, and centromere function. H2A.Z deposition is controlled by SWR-C chromatin remodeling enzymes that catalyze the nucleosomal exchange of canonical H2A with H2A.Z. Here we report that acetylation of histone H3 lysine 56 (H3-K56Ac) alters the substrate specificity of SWR-C, leading to promiscuous dimer exchange where either H2A.Z or H2A can be exchanged from nucleosomes. This result is confirmed in vivo, where genome-wide analysis demonstrates widespread decreases in H2A.Z levels in yeast mutants with hyperacetylated H3K56. Our work also suggests that a conserved SWR-C subunit may function as a M-bM-^@M-^\lockM-bM-^@M-^] that prevents removal of H2A.Z from nucleosomes. Our study identifies a histone modification that regulates a chromatin remodeling reaction and provides insights into how histone variants and nucleosome turnover can be controlled by chromatin regulators. H2A.Z ChIP seq experiments in mutants with constitutive H3K56ac
Project description:The histone variant H2A.Z marking permissive chromatin is preferentially deposited at promoter-proximal +1 nucleosomes by the multicomponent SWR1 chromatin remodeler. Although SWR1 targeting to nucleosome-depleted regions (NDRs) is directed by its free DNA length sensing module, how the remodeler is guided preferentially to flanking +1 nucleosomes has been enigmatic. Here, we show by live-cell, single-molecule tracking (SMT) that SWR1 subunits Bdf1 and Yaf9 harboring bromo and YEATS acetyl-histone reader domains are required for quantitative chromatin binding via distinct kinetic mechanisms: Bdf1 increases SWR1 association, Yaf9-YEATS reduces disassociation. Notably, SMT and genome-wide ChIP-exo reveals Bdf1 and Yaf9 contributions to SWR1 targeting globally and histone exchange at +1 nucleosomes. Our findings highlight the native biochemistry of histone readers and suggest a broadly applicable, two-stage mechanism wherein acetyl-histone interactions initially constrain 3D diffusion of SWR1 to increase local concentration, followed by stochastic 1D diffusion at NDRs with directional capture by acetylated +1 nucleosomes.
Project description:The histone variant H2A.Z plays key roles in gene expression, DNA repair, and centromere function. H2A.Z deposition is controlled by SWR-C chromatin remodeling enzymes that catalyze the nucleosomal exchange of canonical H2A with H2A.Z. Here we report that acetylation of histone H3 lysine 56 (H3-K56Ac) alters the substrate specificity of SWR-C, leading to promiscuous dimer exchange where either H2A.Z or H2A can be exchanged from nucleosomes. This result is confirmed in vivo, where genome-wide analysis demonstrates widespread decreases in H2A.Z levels in yeast mutants with hyperacetylated H3K56. Our work also suggests that a conserved SWR-C subunit may function as a “lock” that prevents removal of H2A.Z from nucleosomes. Our study identifies a histone modification that regulates a chromatin remodeling reaction and provides insights into how histone variants and nucleosome turnover can be controlled by chromatin regulators.
Project description:Positioned nucleosomes limit the access of proteins to DNA and implement regulatory features encoded in eukaryotic genomes. Here we generated the first genome-wide nucleosome positioning map for Schizosaccharomyces pombe and annotated transcription start and termination sites genome-wide. Using this resource we found surprising differences compared to the nucleosome organization in the distantly related yeast Saccharomyces cerevisiae [the cerevisiae data has been published by others (PMID: 17873876) and the raw data is deposited at ArrayExpress(E-MEXP-1172)]. DNA sequence guides nucleosome positioning differently, e.g., poly(dA:dT) elements are not enriched in S. pombe nucleosome-depleted regions (NDRs). Regular nucleosomal arrays emanate more asymmetrically, i.e., mainly co-directionally with transcription, from promoter NDRs, but promoters harbouring the histone variant H2A.Z show regular arrays also upstream. Regular nucleosome phasing in S. pombe has a very short repeat length of 154 base pairs, and requires a remodeler, Mit1, conserved in humans but not found in S. cerevisiae. Nucleosome positioning mechanisms are evidently not universal but evolutionarily plastic.
2010-02-01 | GSE16040 | GEO
Project description:Impact of Nucleosomal DNA Sequences on SWR Remodeler Activity and Histone H2A.Z Targeting
Project description:Here we investigated the stability of nucleosomal modifications during pull-down affinity purification with HeLa nuclear extracts. Unmodified di-nucleosomes and di-nucleosomes decorated with H3K4me3K9acK14acK18acK23acK27ac, H4K5acK8acK12acK16acK20me2 and incorporating histone variant H2A.Z were incubated with HeLa nuclear extract or buffer alone for 4 hours at 4 degrees Celsius. The relative abundances of nucleosomal modifications were quantified using LC-MS.
Project description:The chromatin remodelers (CRs) SWI/SNF and RSC function in evicting promoter nucleosomes at highly expressed yeast genes, particularly those activated by transcription factor Gcn4. Ino80 remodeling complex (Ino80C) can establish nucleosome-depleted regions (NDRs) in reconstituted chromatin, and was implicated in removing histone variant H2A.Z from the -1 and +1 nucleosomes flanking NDRs; however, Ino80C’s function in transcriptional activation in vivo is not well understood. Analyzing the cohort of Gcn4-induced genes in ino80Δ mutants has uncovered a role for Ino80C on par with SWI/SNF in evicting promoter nucleosomes and transcriptional activation. Compared to SWI/SNF, Ino80C generally functions over a wider region, spanning the -1 and +1 nucleosomes, NDR, and proximal genic nucleosomes, at genes highly dependent on its function. Defects in nucleosome eviction in ino80Δ cells are frequently accompanied by reduced promoter occupancies of TBP, and diminished transcription; and Ino80 is enriched at genes requiring its remodeler activity. Importantly, nuclear depletion of Ino80 impairs promoter nucleosome eviction even in a mutant lacking H2A.Z. Thus, Ino80C acts widely in the yeast genome together with RSC and SWI/SNF in evicting promoter nucleosomes and enhancing transcription, all in a manner at least partly independent of H2A.Z editing.
Project description:The classic view of nucleosome organization at active promoters is that two well-positioned nucleosomes flank a nucleosome-depleted region (NDR). However, this view has been recently challenged by contradictory reports as to whether a distinct set of wider (≳150 bp) NDRs instead contain unusually unstable Micrococcal Nuclease-sensitive “fragile” particles, thought to be nucleosomal because of their size. To determine the composition of fragile particles we introduce CUT&RUN.ChIP, in which targeted nuclease cleavage and release is followed by chromatin immunoprecipitation. We find that fragile particles represent the occupancy and action of the RSC nucleosome remodeler acting on dynamically unwrapped nucleosomal intermediates. We also find that general regulatory factors (GRFs) bind to partially unwrapped nucleosomal intermediates at NDRs. We propose that RSC-engagement and its action cause nucleosomes to unravel, and subsequent binding of GRFs constitute a dynamic cycle of nucleosome deposition and clearance at the subset of wide Pol II promoter NDRs.
Project description:Histone variant H2A.Z-containing nucleosomes are incorporated at most eukaryotic promoters. This incorporation is mediated by the conserved SWR1 complex, which replaces histone H2A in canonical nucleosomes with H2A.Z in an ATP-dependent manner. Here, we show that promoter-proximal nucleosomes are highly heterogeneous for H2A.Z in Saccharomyces cerevisiae, with substantial representation of nucleosomes containing one, two, or no H2A.Z molecules. SWR1-catalyzed H2A.Z replacement in vitro occurs in a stepwise and unidirectional fashion, one H2A.Z-H2B dimer at a time, producing heterotypic nucleosomes as intermediates and homotypic H2A.Z nucleosomes as end products. The ATPase activity of SWR1 is specifically stimulated by H2A-containing nucleosomes without ensuing histone H2A eviction. Remarkably, further addition of free H2A.Z-H2B dimer leads to hyperstimulation of ATPase activity, eviction of nucleosomal H2A-H2B and deposition of H2A.Z-H2B. These results suggest that the combination of H2A-containing nucleosome and free H2A.Z-H2B dimer acting as both effector and substrate for SWR1 governs the specificity and outcome of the replacement reaction. Total nucleosomes from MNase-treated nuclear extracts were fractionated by sequential immunoprecipitation into homotypic H2A/H2A (AA), heterotypic H2A/H2A.Z (AZ), and homotypic H2A.Z/H2A.Z (ZZ) nucleosomes.
Project description:Goal of this project was the identification of chromatin interacting proteins whose binding is differentially regulated by various combinatorial chromatin modifications found in different chromatin states such as promoters, enhancers and heterochromatin. To achieve this, recombinant modified nucleosomes representing different chromatin states were assembled from canonical histones, H2A.Z and modified ligated H3/H4 histone proteins and biotinylated nucleosomal DNAs. Assembled nucleosomes were immobilized on streptavidin-coated beads via biotinylated di-nucleosomal 601 DNA and used for nucleosome affinity purifications to identify proteins regulated by chromatin modifications from SILAC-labelled HeLaS3 nuclear extracts (Arg10 and Lys8). SILAC affinity purifications were carried out in "forward" (heavy extract on modified nucleosome and light extract on unmodified nucleosome) and "reverse" (light extract on modified nucleosome and heavy extract on unmodified nucleosome) label-swap experiments and protein abundances were quantified by MaxQuant. Initial trial experiments with biotinylated mono-, di- and tetra- 601 nucleosomes are also deposited along with the data for the di-nucleosome experiments. See also: Bartke et al., 2010. Nucleosome-interacting proteins regulated by DNA and histone methylation. Cell 143, 470–484; doi:10.1016/j.cell.2010.10.012. The H4K20me2 samples from this experiment were previously deposited with identifier PXD009281. These were published in: Nakamura et al., 2019. H4K20me0 recognition by BRCA1-BARD1 directs homologous recombination to sister chromatids. Nature Cell Biology 21, 311–318; doi: 10.1038/s41556-019-0282-9.