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 site-specific chromatin incorporation of eukaryotic histone variant H2A.Z is driven by the multi-component chromatin remodeling complex SWR1/SRCAP/ p400. The budding yeast SWR1 complex replaces the H2A-H2B dimer in the canonical nucleosome with the H2A.Z-H2B dimer, but the mechanism governing the directionality of H2A-to-H2A.Z exchange remains elusive. Here, we use single-molecule force spectroscopy to dissect the disassembly/ reassembly of H2A-nucleosome and H2A.Z-nucleosome. We find that the N-terminal 1-135 residues of yeast SWR1-complex-protein-2 (previously termed Swc2-Z) facilitate the disassembly of nucleosomes containing H2A but not H2A.Z. The Swc2-mediated nucleosome disassembly/reassembly requires the inherently unstable H2A-nucleosome, whose instability is conferred by three H2A α2-helix residues Gly47, Pro49 and Ile63 as they selectively weaken the structural rigidity of H2A-H2B dimer. It also requires Swc2-ZN (residues 1-37) that directly anchors to H2A-nucleosome and functions in the SWR1-catalyzed H2A.Z replacement in vitro and yeast H2A.Z deposition in vivo. Our findings providecrucial insights into how SWR1 complex discriminates between the H2A-nucleosome and H2A.Z-nucleosome, establishing a simple paradigm for the governace of unidirectional H2A.Z exchange.
Project description:The Set2-Rpd3S pathway is important for the control of transcription memory. Mutation of components of this pathway results in cryptic transcription initiation within the coding region of approximately 30% of yeast genes. Specifically, deletion of the Set2 histone methyltransferase or Rco1, a component of the Rpd3S histone deacetylase complex leads to hyperacetylation of certain open reading frames (ORFs). We used this mutant as a system to study the role of histone modifications and co-activator recruitment in preinitiation complex (PIC) formation. Specifically, we looked at the dependence of promoters on the bromodomain-containing RSC complex and the Bdf1 protein. We found that the dependence of cryptic promoters for these proteins varied. Overall, our data indicates that cryptic promoters are independently regulated, and their activation is dependent on factors that govern gene activation at canonical promoters. Two-sample experiment: chromatin immunoprecipitation (ChIP) sample vs. input. Biological replicates: 3 independent ChIP samples for each yeast strain.
Project description:Chromatin plays roles in processes governed by different time scales. To assay the dynamic behaviour of chromatin in living cells, we used genomic tiling arrays to measure histone H3 turnover in G1-arrested S. cerevisiae at single-nucleosome resolution over 4% of the genome, and over the entire genome at lower (~265 bp) resolution. We find that nucleosomes at promoters are replaced more rapidly than at coding regions, and that replacement rates over coding regions correlate with polymerase density. In addition, rapid histone turnover is found at known chromatin boundary elements. These results suggest that rapid histone turnover serves to functionally separate chromatin domains and prevent spread of histone states. Keywords: Chip-chip, time course, histone turnover Ratios between Gal-induced H3-Flag and constitutive H3-Myc at 8 time points for unsynchronized yeast. Hybridization to high-resolution printed arrays of ~4% of the yeast genome.
Project description:Chromatin plays roles in processes governed by different time scales. To assay the dynamic behaviour of chromatin in living cells, we used genomic tiling arrays to measure histone H3 turnover in G1-arrested S. cerevisiae at single-nucleosome resolution over 4% of the genome, and over the entire genome at lower (~265 bp) resolution. We find that nucleosomes at promoters are replaced more rapidly than at coding regions, and that replacement rates over coding regions correlate with polymerase density. In addition, rapid histone turnover is found at known chromatin boundary elements. These results suggest that rapid histone turnover serves to functionally separate chromatin domains and prevent spread of histone states. Keywords: Chip-chip, time course, histone turnover Ratios between Gal-induced H3-Flag and constitutive H3-Myc at 5 time points for G1-arrested yeast. Hybridization to high-resolution printed arrays of ~4% of the yeast genome.
Project description:We developed a system to study the DNA replication-independent turnover nucleosomes containing the histone variant H3.3 in mammalian cells. By measuring the genome-wide incorporation of H3.3 at different time points following epitope-tagged H3.3 expression, we find three categories of H3.3-nucleosome turnover in vivo: rapid turnover, intermediate turnover and, specifically at telomeres, slow turnover. Our data indicate that H3.3-containing nucleosomes at enhancers and promoters undergo a rapid turnover that is associated with active histone modification marks including H3K4me1, H3K4me3, H3K9ac, H3K27ac and the histone variant H2A.Z. The rate of turnover is negatively correlated with H3K27me3 at regulatory regions and with H3K36me3 at gene bodies. Examination of incorporation dynamics of histone variant H3.3
Project description:Transcription in eukaryotes correlates with major chromatin changes, including the replacement of old nucleosomal histones by new histones at the promoters of genes. The role of these histone exchange events in transcription remains unclear. In particular, the causal relationship between histone exchange and activator binding, preinitiation complex (PIC) assembly, and/or subsequent transcription remains unclear. Here, we provide evidence that histone exchange at gene promoters is not simply a consequence of PIC assembly or transcription but instead is mediated by activators. We further show that not all activators up-regulate gene expression by inducing histone turnover. Thus, histone exchange does not simply correlate with transcriptional activity, but instead reflects the mode of action of the activator. Last, we show that histone turnover is not only associated with activator function but also plays a role in transcriptional repression at the histone loci.
Project description:The histone variant H2A.Z is a hallmark of nucleosomes flanking the promoters of protein coding genes, and is often found in nucleosomes that also carry lysine 56- acetylated histone H3 (H3-K56Ac), a mark which promotes rapid replication- independent turnover of nucleosomes. Although H2A.Z and H3-K56Ac have been generally implicated in transcriptional activation, their exact contributions have remained elusive. Here we find that H3-K56Ac promotes RNA polymerase II occupancy at a large number of protein coding and noncoding loci, yet neither H3- K56Ac nor H2A.Z has a significant impact on steady state mRNA levels in yeast. Instead, broad effects of H3-K56Ac or H2A.Z on levels of both coding and noncoding RNAs (ncRNAs) are only revealed in the absence of the nuclear RNA exosome. H2A.Z is also necessary for expression of divergent, promoter-proximal ncRNAs in mouse embryonic stem cells, suggesting a conserved role for H2A.Z across eukaryotes. Finally, we show that H2A.Z functions with H3-K56Ac in chromosome folding, facilitating formation of chromosome interaction domains (CIDs). Our study suggests that H2A.Z and H3-K56Ac work in concert with the RNA exosome to control mRNA and ncRNA expression, perhaps in part by regulating higher order chromatin structures. 2 replicates of WT (CY1089), rtt109â (CY2210), rrp6â (CY2071) and one replicate of the W303 input (Sample 7). TableS5.xlsx contains the processed IP/input values for each ORF transcript.
Project description:The Set2-Rpd3S pathway is important for the control of transcription memory. Mutation of components of this pathway results in cryptic transcription initiation within the coding region of approximately 30% of yeast genes. Specifically, deletion of the Set2 histone methyltransferase or Rco1, a component of the Rpd3S histone deacetylase complex leads to hyperacetylation of certain open reading frames (ORFs). We used this mutant as a system to study the role of histone modifications and co-activator recruitment in preinitiation complex (PIC) formation. Specifically, we looked at the dependence of promoters on the bromodomain-containing RSC complex and the Bdf1 protein. We found that the dependence of cryptic promoters for these proteins varied. Overall, our data indicates that cryptic promoters are independently regulated, and their activation is dependent on factors that govern gene activation at canonical promoters.
Project description:The histone variant H2A.Z, which has been reported to have both activating and repressive effects on gene expression, is known to occupy nucleosomes at the 5’ ends of protein-coding genes. We now find that H2A.Z is also significantly enriched in gene coding regions and at the 3’ ends of genes in budding yeast, where it co-localises with histone marks associated with active promoters. By comparing H2A.Z binding to global gene expression in budding yeast strains engineered so that normally unstable transcripts are abundant, we show that H2A.Z is required for normal levels of antisense transcripts as well as sense ones. High levels of H2A.Z at antisense promoters are associated with decreased antisense transcript levels when H2A.Z is deleted, indicating that H2A.Z has an activating effect on antisense transcripts. Decreases in antisense transcripts affected by H2A.Z are accompanied by increased levels of paired sense transcripts. Therefore, the effect of H2A.Z on protein coding gene expression is a reflection of its importance for normal levels of both sense and antisense transcripts. Htz1 ChIP-seq in wild-type (WT) and rrp6Δ yeast, along with negative control ChIP-seq in htz1Δ and input control. Strand-specific transcriptomic profiles of WT, htz1Δ, rrp6Δ and htz1Δrrp6Δ. Replicates are present for all samples except the negative and input control ChIP samples.