H3K9me3 ChIP-seq from Neurospora crassa wild type (WT) and dim-3 (severely reduced H3K9me3 levels) strains
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ABSTRACT:
ORGANISM(S): Neurospora crassa
PROVIDER: GSE61173 | GEO | 2015/03/22
SECONDARY ACCESSION(S): PRJNA260401
REPOSITORIES: GEO
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Project description:We report the placement of cytosine methylation from the filamentous fungus Neurospora crassa in wild type and dim-3 strains by bisulfite-sequencing. Compared to a wild type strain, the dim-3 strain has a global reduction in cytosine methylation, and this reduction in cytosine methylation is exacerbated by the supplementation of histidine to the growth medium. This global reduction in cytosine methylation results from a causative mutation in importin alpha (NUP-6), a component of the nuclear transport machinery, which severely reduces the level of the heterochromatic mark H3K9me3. NUP-6(dim-3) compromises the heterochromatic localization of several components of the DCDC, a histone H3K9 methyltransferase complex, which in turn reduces the level of Heterochromatin Protein-1 (HP1) found at heterochromatin and compromises the direct interaction between HP1 and the DNA methyltransferase dim-2. To determine whether the reduced level of cytosine methylation, as assayed by Southern blotting heterochromatic regions following digestion of genomic DNA with methylation-sensitive restriction endonucleases, is globally reduced at the A:T rich, repetitive DNA comprising heterochromatin, we performed Bisulfite-seq on a dim-3 strain (grown either in minimal medium or medium supplemented with histidine) and compared that to a wild type strain (grown in minimum medium; histidine does not reduce the levels of cytosine methylation in a wild type strain, as assayed by Southern blotting).
2015-03-22 | GSE61174 | GEO
Project description:Eukaryotic genomes are organized into chromatin domains with distinct three-dimensional arrangements resulting from nucleic acid and protein factor interactions within the physical constraints of the nucleus. It is of obvious interest to determine interactions between various chromosomal regions defined by these nuclear constraints, and to identify important factors that limit the interactions. We used chromosome conformation capture (3C) followed by high-throughput sequencing (HiC) to improve our understanding of Neurospora crassa genome organization and to examine if known components of heterochromatin machinery influence nuclear organization. In heterochromatin establishment, DIM-5 tri-methylates histone H3 on lysine 9 (H3K9me3), and this mark is subsequently bound by Heterochromatin Protein-1 (HP1). NUP-6 is required to target DIM-5 to incipient A:T rich DNA and the dim-3 mutant of NUP-6 is deficient in DIM-5 localization. We performed HiC on chromatin from wildtype, Δdim-5, Δhpo, and dim-3 strains. The genome configuration of wild type nuclei revealed strong intra- and inter-chromosomal associations between both constitutive and facultative heterochromatic domains, with the strongest interactions among the centromeres, telomeres and interspersed heterochromatin. We found that loss of the H3K9me3 mark, as well as loss of HP1, minimally altered the chromatin organization found at these strongly interacting heterochromatic regions. Surprisingly, dim-3 chromatin was highly disorganized suggesting NUP-6 plays key role(s) in genome structure. Thus, our datasets suggest that while heterochromatic regions are critical to defining the genome conformation in Neurospora, non-canonical protein factors may play a key role in maintaining this organization.
2016-05-19 | GSE71024 | GEO
Project description:Cytosine methylation, a fundamental form of epigenetic regulation, is found in many eukaryotes and plays a significant role in cancer and other diseases. Using the genetically tractable model organism Neurospora crassa, we have identified genes that when mutated, cause the strains to be defective in methylation (dim). The process of DNA methylation in Neurospora has been shown to be dependent on DCDC, a five-member complex that directs the histone methyltransferase DIM-5 to tri-methylate Lysine 9 on histone H3 (H3K9me3). This mark is recognized by HP1, which directs DIM-2 to methylate DNA. In contrast, the HCHC complex employs HDA-1, CDP-2, HP1, and CHAP to deacetylate that same residue on H3. While we know a good deal about DNA methylation, it is still unclear whether we have identified all the genes involved in the process. Thus, we continued our search for dim mutants, using a selection for reactivation of silenced drug resistance genes. Interestingly, we predominantly identified known dim genes, including dim-5, dim-7, dim-8, dim-9, hpo, chap, cdp-2, and hda-1. Using a Sanger sequencing-based approach, we identified mutations in these known dim genes, presumably responsible for the Dim- phenotype; many mutations were unique point mutants that could compromise protein activity or structure, or impact protein-protein interactions. For mutants in which the gene was not placed into a complementation group, we employed a bulk segregant analysis and whole genome sequencing approach to identify additional mutations in known DNA methylation genes, including hH3 and dim-1, as well as a novel dim mutant: dim-10, a fungal-specific protein that may work with DCDC for H3K9me3 catalysis. Not only will this dim mutant collection be a useful resource to investigate the roles of these dim genes and their protein products in DNA methylation, but the isolation of a novel dim gene by a forward genetics approach provides an exciting avenue of research into how incipient heterochromatin formation is achieved.
2020-09-21 | GSE100770 | GEO
Project description:Methylated lysine 27 on histone H3 (H3K27me) marks repressed "facultative heterochromatin", including developmentally regulated genes in plants and animals. The mechanisms responsible for localization of H3K27me are largely unknown, perhaps in part because of the complexity of epigenetic regulatory networks. We used a relatively simple model organism bearing both facultative and constitutive heterochromatin, Neurospora crassa, to explore possible interactions between elements of heterochromatin. In higher eukaryotes, reductions of H3K9me3 and DNA methylation in constitutive heterochromatin have been variously reported to cause redistribution of H3K27me3. In Neurospora, we found that elimination of any member of the DCDC H3K9 methylation complex (DIM-5, DIM-7, CUL4, DDB1/DIM-8 or DIM-9) caused massive changes in the distribution of H3K27me; regions of facultative heterochromatin lost H3K27me3 while regions that are normally marked by H3K9me3 became methylated at H3K27. Elimination of DNA methylation by deletion of the DNA methyltransferase gene, dim-2, had no obvious effect on the distribution of H3K27me. Elimination of HP1, which "reads" H3K9me3, also caused major changes in the distribution of H3K27me, indicating that HP1 is important for normal localization of facultative heterochromatin. Because loss of HP1 caused redistribution of H3K27me2/3 but not H3K9me3, these normally non-overlapping marks became superimposed. Indeed, mass spectrometry revealed substantial cohabitation of H3K9me3 and H3K27me2 on H3 molecules from an hpo strain. Loss of H3K27me machinery (e.g. the methyltransferase SET-7) did not impact constitutive heterochromatin, but partially rescued the slow growth of the DCDC mutants, suggesting that the poor growth of these mutants is partly attributable to ectopic H3K27me. Altogether, our findings with Neurospora clarify interactions of facultative and constitutive heterochromatin in eukaryotes.
2015-11-03 | GSE68897 | GEO
Project description:Facultative heterochromatin in the filamentous fungus Neurospora crassa is identified by the repressive histone mark H3K27me3 and is primarily subtelomeric, while constitutive heterochromatin, marked by the DIM-5-catalzyed H3K9me3, is found at centromeres, telomeres, and smaller dispersed regions. In strains lacking constitutive heterochromatin (e.g., Δdim-5), H3K27me2/3 relocalizes to the regions formerly marked by H3K9me3. H3K27me3 is catalyzed by the SET-7 histone methyltransferase subunit of the Polycomb Repressive Complex 2 (PRC2); another PRC2 member, Neurospora p55 (NPF) regulates subtelomeric H3K27me2/3. Despite the de-repression of >70 genes, a Δset-7 strain has no distinguishable phenotype. To investigate the facultative heterochromatin contribution to genome organization, we performed high-throughput “chromosome conformation capture” (Hi-C) on mutants with impacted H3K27me2/3 deposition. A Δset-7 strain has decreased inter-/intra-subtelomeric contacts among others; this pattern is mirrored in a Δnpf strain, which lacks subtelomeric H3K27me3. In a Δset-7 strain, telomere bundles were often uncoupled from the nuclear membrane and de-repressed genes were subtelomeric. The chromosome conformation of a Δset-7;Δdim-5 double mutant was similar to Δset-7, suggesting that facultative heterochromatin relocalization does not compensate for H3K9me3 loss and rescue the Neurospora genome organization in strains with defective constitutive heterochromatin.
2016-10-27 | GSE82222 | GEO
Project description:H3K9me3-heterochromatin, compacted by HP1 proteins, restricts transcription factor binding and functions as a barrier to cell fate changes in development and reprogramming1,2. To investigate principles underlying H3K9me3-heterochromatin maintenance in mammalian cells, we applied degron engineering3,4 on mouse ESCs to conditionally deplete all three H3K9me3-methyltransferases within one hour and found an unexpectedly dynamic nature of heterochromatin maintenance. Following H3K9me3 methyltransferases degradation, HP1β dissociates from heterochromatin within three hours, and transcription factors quickly access the de-compacted heterochromatin, activating transcription of their targets, which then promotes H3K9me3 decay. Both passive dilution and active removal contribute to the H3K9me3 decay. Mathematical modeling reveals distinct H3K9me3 heterochromatin states with diverse chromatin features which influence initial H3K9me3 levels and the stability of H3K9me3 domains, thereby expanding our understanding of distinct heterochromatin domains. Rampant transposable element de-repression interferes with transcription of poised early developmental and signaling genes outside H3K9me3-domains, highlighting the intricate roles of dynamic H3K9me3-heterochromatin maintenance in safeguarding developmental programs. Our findings deconvolve the hierarchical relationships between H3K9me3 marks, methyltransferases, HP1 and transcription factors to reveal principles of H3K9me3-heterochromatin maintenance and how its remodeling enables transcription and cell fate change.
2024-09-19 | GSE233040 | GEO
Project description:H3K9me3-heterochromatin, compacted by HP1 proteins, restricts transcription factor binding and functions as a barrier to cell fate changes in development and reprogramming1,2. To investigate principles underlying H3K9me3-heterochromatin maintenance in mammalian cells, we applied degron engineering3,4 on mouse ESCs to conditionally deplete all three H3K9me3-methyltransferases within one hour and found an unexpectedly dynamic nature of heterochromatin maintenance. Following H3K9me3 methyltransferases degradation, HP1β dissociates from heterochromatin within three hours, and transcription factors quickly access the de-compacted heterochromatin, activating transcription of their targets, which then promotes H3K9me3 decay. Both passive dilution and active removal contribute to the H3K9me3 decay. Mathematical modeling reveals distinct H3K9me3 heterochromatin states with diverse chromatin features which influence initial H3K9me3 levels and the stability of H3K9me3 domains, thereby expanding our understanding of distinct heterochromatin domains. Rampant transposable element de-repression interferes with transcription of poised early developmental and signaling genes outside H3K9me3-domains, highlighting the intricate roles of dynamic H3K9me3-heterochromatin maintenance in safeguarding developmental programs. Our findings deconvolve the hierarchical relationships between H3K9me3 marks, methyltransferases, HP1 and transcription factors to reveal principles of H3K9me3-heterochromatin maintenance and how its remodeling enables transcription and cell fate change.
2024-09-19 | GSE233039 | GEO
Project description:H3K9me3-heterochromatin, compacted by HP1 proteins, restricts transcription factor binding and functions as a barrier to cell fate changes in development and reprogramming1,2. To investigate principles underlying H3K9me3-heterochromatin maintenance in mammalian cells, we applied degron engineering3,4 on mouse ESCs to conditionally deplete all three H3K9me3-methyltransferases within one hour and found an unexpectedly dynamic nature of heterochromatin maintenance. Following H3K9me3 methyltransferases degradation, HP1β dissociates from heterochromatin within three hours, and transcription factors quickly access the de-compacted heterochromatin, activating transcription of their targets, which then promotes H3K9me3 decay. Both passive dilution and active removal contribute to the H3K9me3 decay. Mathematical modeling reveals distinct H3K9me3 heterochromatin states with diverse chromatin features which influence initial H3K9me3 levels and the stability of H3K9me3 domains, thereby expanding our understanding of distinct heterochromatin domains. Rampant transposable element de-repression interferes with transcription of poised early developmental and signaling genes outside H3K9me3-domains, highlighting the intricate roles of dynamic H3K9me3-heterochromatin maintenance in safeguarding developmental programs. Our findings deconvolve the hierarchical relationships between H3K9me3 marks, methyltransferases, HP1 and transcription factors to reveal principles of H3K9me3-heterochromatin maintenance and how its remodeling enables transcription and cell fate change.
2024-09-19 | GSE231845 | GEO
Project description:H3K9me3-heterochromatin, compacted by HP1 proteins, restricts transcription factor binding and functions as a barrier to cell fate changes in development and reprogramming1,2. To investigate principles underlying H3K9me3-heterochromatin maintenance in mammalian cells, we applied degron engineering3,4 on mouse ESCs to conditionally deplete all three H3K9me3-methyltransferases within one hour and found an unexpectedly dynamic nature of heterochromatin maintenance. Following H3K9me3 methyltransferases degradation, HP1β dissociates from heterochromatin within three hours, and transcription factors quickly access the de-compacted heterochromatin, activating transcription of their targets, which then promotes H3K9me3 decay. Both passive dilution and active removal contribute to the H3K9me3 decay. Mathematical modeling reveals distinct H3K9me3 heterochromatin states with diverse chromatin features which influence initial H3K9me3 levels and the stability of H3K9me3 domains, thereby expanding our understanding of distinct heterochromatin domains. Rampant transposable element de-repression interferes with transcription of poised early developmental and signaling genes outside H3K9me3-domains, highlighting the intricate roles of dynamic H3K9me3-heterochromatin maintenance in safeguarding developmental programs. Our findings deconvolve the hierarchical relationships between H3K9me3 marks, methyltransferases, HP1 and transcription factors to reveal principles of H3K9me3-heterochromatin maintenance and how its remodeling enables transcription and cell fate change.
2024-09-19 | GSE270213 | GEO
Project description:Regulation of heterochromatin is critical for genome stability. Different states of methylated H3K9 have been discovered with distinct roles in heterochromatin formation and silencing. However, the control of the transition from H3K9me2 to H3K9me3 is still unclear. Here we investigate the role of the conserved bromodomain AAA-ATPase, Abo1, involved in maintaining the global nucleosome organization in fission yeast. We identified several key factors involved in heterochromatin silencing to interact genetically with Abo1: the histone deacetylase Clr3, the H3K9 methyltransferase Clr4, and the HP1 homologue Swi6. Cells lacking Abo1 display an imbalance of H3K9me2 and H3K9me3 in heterochromatin. In abo1∆ cells, the centromeric constitutive heterochromatin had increased H3K9me2 but decreased H3K9me3 levels compared to wild type. In contrast, facultative heterochromatin regions, show both reduced H3K9me2 and H3K9me3 levels in abo1∆. Genome-wide analysis showed that abo1∆ cells have silencing defects in both centromeres and subtelomeres, but not in a subset of heterochromatin islands. Our work uncovers a new role for Abo1 in supporting Clr4 activity and allowing for the transition of H3K9me2 to H3K9me3 in telomeric and centromeric heterochromatin. We used microarrays to detail the global gene expression in wilde typ (Hu2185) and abo1∆ (Hu2318) strain with three temperature induction: 25°C (cold stress), 30°C(standard cultivation) and 37°C heat stress. We found silencing defect under in heterochromatin in abo1∆ deletion in all three conditions.
2020-04-13 | GSE125910 | GEO