S.cerevisiae INO80 is required for promoter proximal nucleosome remodeling.
Ontology highlight
ABSTRACT: During transcription, nucleosomes are evicted from regulatory and coding regions yet chromatin structure is stable. Restoration of chromatin structure involves concerted action of chromatin modifying activities. Our analysis demonstrates a genome wide function of the INO80 remodeling complex for stable repositioning of the nucleosome immediately proximal to the transcription initiation site. INO80 dependent remodeling of the promoter proximal nucleosomes has a global repressive role. Recruitment of INO80 to proximal nucleosomes overlaps with the elongating Polymerase II complex assembly. The amount of associated Polymerase II at start sites correlates with INO80 recruitment for inducible and constantly transcribed genes. Furthermore, at highly inducible promoters INO80 is required for repression of bidirectional transcription. Therefore, we suggest a function for INO80 after transcription initiation to achieve Polymerase II dependent reassembly of promoter proximal nucleosomes.
Project description:The INO80 complex is a chromatin remodeler that regulates DNA replication, repair, and transcription. Although the INO80 complex plays a crucial role in various chromatin-associated processes, the mechanism of its recruitment to specific genomic loci is not well understood. Here we used a native ChIP-MS approach to quantitatively profile modifications present on nucleosomes co-purified with INO80 from MNAse-digested HeLa chromatin.
Project description:The INO80 complex is a chromatin remodeler that regulates DNA replication, repair, and transcription. Although the INO80 complex plays a crucial role in various chromatin-associated processes, the mechanism of its recruitment to specific genomic loci is not well understood. Here we used a native ChIP-MS approach to quantitatively profile modifications present on nucleosomes co-purified with INO80 from MNAse-digested HeLa chromatin.
Project description:The INO80 protein is the main catalytic subunit of the INO80-chromatin remodeling complex, which is critical for DNA repair and transcription regulation in murine spermatocytes. In this study, we explored the role of INO80 in silencing genes on meiotic sex chromosomes in male mice. INO80 immunolocalization at the XY body in pachytene spermatocytes suggested a role for INO80 in the meiotic sex body. Subsequent deletion of Ino80 resulted in high expression of sex-linked genes. Furthermore, the active form of RNA polymerase II at the sex body of Ino80-null pachytene spermatocytes indicates incomplete inactivation of sex-linked genes. A reduction in the recruitment of initiators of meiotic sex chromosome inhibition (MSCI) argues for INO80-facilitated recruitment of DNA repair factors required for silencing sex-linked genes. This role of INO80 is independent of a common INO80 target H2A.Z. Instead, in the absence of INO80, a reduction in chromatin accessibility at DNA repair sites occurs on the sex chromosomes. These data suggest a role for INO80 in DNA repair factor localization, thereby facilitating the silencing of sex-linked genes during the onset of pachynema.
Project description:The INO80 protein is the main catalytic subunit of the INO80-chromatin remodeling complex, which is critical for DNA repair and transcription regulation in murine spermatocytes. In this study, we explored the role of INO80 in silencing genes on meiotic sex chromosomes in male mice. INO80 immunolocalization at the XY body in pachytene spermatocytes suggested a role for INO80 in the meiotic sex body. Subsequent deletion of Ino80 resulted in high expression of sex-linked genes. Furthermore, the active form of RNA polymerase II at the sex chromosomes of Ino80-null pachytene spermatocytes indicates incomplete inactivation of sex-linked genes. A reduction in the recruitment of initiators of meiotic sex chromosome inhibition (MSCI) argues for INO80-facilitated recruitment of DNA repair factors required for silencing sex-linked genes. This role of INO80 is independent of a common INO80 target, H2A.Z. Instead, in the absence of INO80, a reduction in chromatin accessibility at DNA repair sites occurs on the sex chromosomes. These data suggest a role for INO80 in DNA repair factor localization, thereby facilitating the silencing of sex-linked genes during the onset of pachynema.
Project description:Our understanding of transcription by RNA polymerase II (Pol II) is limited by our knowledge of the factors that mediate this critically important process. Here we describe the identification of NDF, a nucleosome destabilizing factor that facilitates Pol II transcription in chromatin. NDF has a PWWP motif, interacts with nucleosomes near the dyad, partially disassembles nucleosomes in an ATP-independent manner, and enhances transcription by Pol II through nucleosomes in vitro and in cell nuclei. NDF affects gene expression and co-localizes with H3K36me3 over transcribed regions of a subset of active genes with a preference for longer genes relative to shorter genes. Moreover, induction of transcription leads to the recruitment of NDF over gene bodies. In humans, NDF is present in all tested tissue types, is essential in stem cells, and is frequently overexpressed in breast cancer. Thus, NDF is a factor of primary importance for Pol II transcription in chromatin.
Project description:Centromeres are maintained epigenetically by the presence of CENP-A, an evolutionarily-conserved histone H3 variant, which directs kinetochore assembly and hence, centromere function. To identify factors that promote assembly of CENP-A chromatin, we affinity selected solubilised fission yeast CENP-ACnp1 chromatin. All subunits of the Ino80 complex were enriched, including the auxiliary subunit Hap2. In addition to a role in maintenance of CENP-ACnp1 chromatin integrity at endogenous centromeres, Hap2 is required for de novo assembly of CENP-ACnp1 chromatin on naïve centromere DNA and promotes H3 turnover on centromere regions and other loci prone to CENP-ACnp1 deposition. Prior to CENP-ACnp1 chromatin assembly, Hap2 is required for transcription from centromere DNA. These analyses suggest that Hap2-Ino80 destabilises H3 nucleosomes on centromere DNA through transcription-mediated histone H3 turnover, driving the replacement of resident H3 nucleosomes with CENP-ACnp1 nucleosomes. These inherent properties define centromere DNA by directing a program that mediates CENP-ACnp1 assembly on appropriate sequences.
Project description:Metazoan transcription is controlled through either coordinated recruitment of transcription machinery to the gene promoter, or subsequently, through regulated pausing of RNA polymerase II (Pol II) in early elongation. We report that a key difference between genes that use these distinct regulatory strategies lies in the chromatin architecture specified by their DNA sequences. Pol II pausing is prominent at highly-regulated genes whose sequences inherently disfavor nucleosome formation within the gene, but favor nucleosomal occlusion of the promoter. Pausing of polymerase maintains these genes in an active state by inhibiting the formation of repressive promoter chromatin. In contrast, promoters of housekeeping genes that lack paused Pol II are deprived of nucleosomes regardless of polymerase binding, but show higher nucleosome occupancy downstream. Our results suggest that the âdefaultâ chromatin state of a gene instructs its regulation, and that highly-regulated promoters have evolved to encourage competition between nucleosomes and paused Pol II for promoter occupancy. All experiments were done using two channels per chip, comparing DNA immunoprecipitated by the indicated antibody to matching input chromatin used for affinity purification. Where appropriate, replicate data sets were averaged.
Project description:Global warming imposes a major threat to plant growth and crop production. In some plants including Arabidopsis thaliana, elevated temperatures induce a series of morphological and developmental adjustments, termed thermomorphogenesis to facilitate plant cooling under high-temperature conditions. Plant thermal response is suppressed by histone variant H2A.Z. At warm temperatures, H2A.Z is evicted from nucleosomes at thermo-responsive genes, resulting in their activation. However, the mechanisms that regulate H2A.Z eviction and subsequent transcription activation are largely unknown. Here, we show that the ino80 chromatin-remodeling complex (ino80-C) promotes thermomorphogenesis and activates the expression of thermo-responsive and auxin-related genes. ino80-C associates with PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), a potent regulator in thermomorphogenesis, and mediates temperature-induced H2A.Z eviction at PIF4 targets. Moreover, ino80-C directly interacts with COMPASS-like and transcription elongation factors to promote active histone modification Histone H3 lysine 4 trimethylation (H3K4me3) and RNA Polymerase II (RNA Pol II) elongation, leading to the thermal induction of transcription. Notably, transcription elongation factors are required for the eviction of H2A.Z at PIF4 targets, suggesting the cooperation of ino80-C and transcription elongation in H2A.Z removal. Our results demonstrate that the (PIF4)-(ino80-C)-(COMPASS-like)-(transcription elongator) module controls plant thermal response, and establish a link between H2A.Z eviction and active transcription.
Project description:Global warming imposes a major threat to plant growth and crop production. In some plants including Arabidopsis thaliana, elevated temperatures induce a series of morphological and developmental adjustments, termed thermomorphogenesis to facilitate plant cooling under high-temperature conditions. Plant thermal response is suppressed by histone variant H2A.Z. At warm temperatures, H2A.Z is evicted from nucleosomes at thermo-responsive genes, resulting in their activation. However, the mechanisms that regulate H2A.Z eviction and subsequent transcription activation are largely unknown. Here, we show that the ino80 chromatin-remodeling complex (ino80-C) promotes thermomorphogenesis and activates the expression of thermo-responsive and auxin-related genes. ino80-C associates with PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), a potent regulator in thermomorphogenesis, and mediates temperature-induced H2A.Z eviction at PIF4 targets. Moreover, ino80-C directly interacts with COMPASS-like and transcription elongation factors to promote active histone modification Histone H3 lysine 4 trimethylation (H3K4me3) and RNA Polymerase II (RNA Pol II) elongation, leading to the thermal induction of transcription. Notably, transcription elongation factors are required for the eviction of H2A.Z at PIF4 targets, suggesting the cooperation of ino80-C and transcription elongation in H2A.Z removal. Our results demonstrate that the (PIF4)-(ino80-C)-(COMPASS-like)-(transcription elongator) module controls plant thermal response, and establish a link between H2A.Z eviction and active transcription.
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.