A Light-Inducible Strain for Genome-Wide Histone Turnover Profiling in Neurospora crassa
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ABSTRACT:
ORGANISM(S): Neurospora crassa
PROVIDER: GSE143603 | GEO | 2021/01/01
REPOSITORIES: GEO
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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:Polycomb group (PcG) proteins assemble into chromatin modifying complexes that regulate stem cell identity and multicellular development. The trimethylation of lysine 27 on histone H3 (H3K27me3) is catalyzed by the conserved Polycomb Repressive Complex 2 (PRC2). In Neurospora crassa, the typically-subtelomeric pattern of H3K27me3 depends on other chromatin regulators including the H3K36 methyltransferase ASH1, the chromatin remodeling enzyme IMITATION SWITCH, and components of constitutive heterochromatin including the H3K9 methyltransferase DEFECTIVE IN METHYLATION-5 (DIM-5) and the H3K9me3-binding protein HETEROCHROMATIN PROTEIN 1 (HP1). How constitutive heterochromatin impacts PRC2 activity is unclear. We performed a genetic screen to identify histone deacetylase genes required to repress PRC2-methylated genes in N. crassa. We found that HISTONE DEACETYLASE-1 (HDA-1) is required for transcriptional repression in PcG-repressed domains and for normal patterns of H3K27me3. In the absence of HDA-1, H3K27me3 is lost from typical PRC2-methylated domains and instead is enriched at a subset of constitutive heterochromatin domains normally marked for H3K9me3. In addition to altered H3K27me3 patterns, HDA-1-deficient mutants displayed aberrant patterns of H3K9me3, H3K36me3, and lysine acetylation (Kac). Mutants lacking the HP1/HDA-1-interacting protein CHROMO DOMAIN PROTEIN-2 (CDP-2) displayed a similar but non-identical phenotype, and we observed variable patterns of aberrant H3K7me3 enrichment in genetically identical ∆hda-1 strains, raising the possibility that loss of HDA-1 activity leads to progressive changes in H3K27me3 enrichment. Indeed, a newly constructed ∆hda-1 deletion strain displayed a near-wild type H3K27me3 chromatin modification profile that decayed over multiple strain passages corresponding to hundreds of nuclear divisions. Together, our data indicate that HDA-1 is a critical regulator of epigenome stability in N. crassa.
2025-01-16 | GSE286916 | GEO
Project description:Polycomb group (PcG) proteins assemble into chromatin modifying complexes that regulate stem cell identity and multicellular development. The trimethylation of lysine 27 on histone H3 (H3K27me3) is catalyzed by the conserved Polycomb Repressive Complex 2 (PRC2). In Neurospora crassa, the typically-subtelomeric pattern of H3K27me3 depends on other chromatin regulators including the H3K36 methyltransferase ASH1, the chromatin remodeling enzyme IMITATION SWITCH, and components of constitutive heterochromatin including the H3K9 methyltransferase DEFECTIVE IN METHYLATION-5 (DIM-5) and the H3K9me3-binding protein HETEROCHROMATIN PROTEIN 1 (HP1). How constitutive heterochromatin impacts PRC2 activity is unclear. We performed a genetic screen to identify histone deacetylase genes required to repress PRC2-methylated genes in N. crassa. We found that HISTONE DEACETYLASE-1 (HDA-1) is required for transcriptional repression in PcG-repressed domains and for normal patterns of H3K27me3. In the absence of HDA-1, H3K27me3 is lost from typical PRC2-methylated domains and instead is enriched at a subset of constitutive heterochromatin domains normally marked for H3K9me3. In addition to altered H3K27me3 patterns, HDA-1-deficient mutants displayed aberrant patterns of H3K9me3, H3K36me3, and lysine acetylation (Kac). Mutants lacking the HP1/HDA-1-interacting protein CHROMO DOMAIN PROTEIN-2 (CDP-2) displayed a similar but non-identical phenotype, and we observed variable patterns of aberrant H3K7me3 enrichment in genetically identical ∆hda-1 strains, raising the possibility that loss of HDA-1 activity leads to progressive changes in H3K27me3 enrichment. Indeed, a newly constructed ∆hda-1 deletion strain displayed a near-wild type H3K27me3 chromatin modification profile that decayed over multiple strain passages corresponding to hundreds of nuclear divisions. Together, our data indicate that HDA-1 is a critical regulator of epigenome stability in N. crassa.
2025-01-16 | GSE286914 | 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:Gene inactivation via heterochromatization is important for genome integrity and prevention of unwanted transcripts with dysregulation in cancer. Repeat sequences are subject to heterochromatin inactivation and, in Neurospora, Repeat-Induced-Point mutation (RIP). The initiating factors behind both are poorly understood. We resolve the paradoxical observation that newly introduced Repeat-Linker-Repeat (RLR) constructs require RID alone for RIP, while genomic repeats are RIPed in the absence of RID, showing that eu- and hetero- chromatic repeats are handled differently, the latter requiring DIM-2 as well. The differences between mechanisms associated with older and newer duplicates caution against extrapolation from mechanisms inferred from model experimental systems to all genomically relevant processes. Additionally, while chromatin status affects RIP, RID we also show via lexA tethering is the nucleation centre for a transition from eu- to hetero- chromatin, in an HDA-1 dependent fashion. Constitutive heterochromatin by contrast is HDA1 independent and depends on HDA-1 paralogs. RID is thus a dual function initiator of both RIP and the transition to heterochromatin.
2025-03-28 | PXD060025 | Pride
Project description:We report the placement of the H3K9me3 heterochromatin mark from the filamentous fungus Neurospora crassa in wild type and dim-3 strains. The dim-3 strain has a global reduction in H3K9me3, which results from a causative mutation in importin alpha (NUP-6), a component of the nuclear transport machinery. NUP-6(dim-3) compromises the heterochromatic localization of several components of the DCDC, a histone H3K9 methyltransferase complex, from their sub-nuclear chromatin targets despite appropriate nuclear transport. To determine whether the reduced level of global H3K9me3 observed from isolated histones localizes to A:T rich DNA (genomic DNA destined to become heterochromatin), we performed H3K9me3 ChIP-seq on a dim-3 strain and compared that to a wild type strain.
2015-03-22 | GSE61173 | GEO
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:Polycomb group proteins assemble into chromatin modifying complexes that regulate stem cell identity and multicellular development. The trimethylation of lysine 27 on histone H3 (H3K27me3) is catalyzed by the conserved Polycomb Repressive Complex 2 (PRC2). In Neurospora crassa, the typically-subtelomeric pattern of H3K27me3 depends on other chromatin regulators including the H3K36 methyltransferase ASH1, the chromatin remodeling enzyme IMITATION SWITCH, and components of constitutive heterochromatin including the H3K9 methyltransferase DEFECTIVE IN METHYLATION-5 and the H3K9me3-binding protein HETEROCHROMATIN PROTEIN 1 (HP1). How constitutive heterochromatin impacts PRC2 activity is unclear. HP1 forms multiple protein complexes including the HCHC complex, which removes acetyl groups from histones in constitutive heterochromatin, and contains the proteins Histone Deacetylase 1(HDA-1) and Chromodomain Protein-2 (CDP-2). To identify genes required for Polycomb repression in N. crassa, we performed a genetic screen and identified HDA-1 as necessary for PRC2-targeted gene silencing. In the absence of HDA-1, H3K27me2/3 is lost from the subtelomeres and aberrantly accumulates at constitutive heterochromatin domains normally enriched for H3K9me3; CDP-2 is also important for wildtype H3K27me3 localization. The H3K27me2/3 redistribution in Δhda-1 or Δcdp-2 strains is variable and incomplete, and mutant progeny obtained following a sexual cross displayed wild-type H3K27me3 patterns, consistent with HCHC loss leading to progressive changes in H3K27me3 enrichment. Indeed, a newly constructed ∆hda-1 deletion strain displays a wild-type H3K27me3 pattern that relocalizes to constitutive heterochromatic regions after sequential passages. Thus, HCHC-specific deacetylation prevents aberrant recruitment of PRC2 to constitutive heterochromatin.
2025-01-21 | GSE286537 | GEO
Project description:Polycomb group proteins assemble into chromatin modifying complexes that regulate stem cell identity and multicellular development. The trimethylation of lysine 27 on histone H3 (H3K27me3) is catalyzed by the conserved Polycomb Repressive Complex 2 (PRC2). In Neurospora crassa, the typically-subtelomeric pattern of H3K27me3 depends on other chromatin regulators including the H3K36 methyltransferase ASH1, the chromatin remodeling enzyme IMITATION SWITCH, and components of constitutive heterochromatin including the H3K9 methyltransferase DEFECTIVE IN METHYLATION-5 and the H3K9me3-binding protein HETEROCHROMATIN PROTEIN 1 (HP1). How constitutive heterochromatin impacts PRC2 activity is unclear. HP1 forms multiple protein complexes including the HCHC complex, which removes acetyl groups from histones in constitutive heterochromatin, and contains the proteins Histone Deacetylase 1(HDA-1) and Chromodomain Protein-2 (CDP-2). To identify genes required for Polycomb repression in N. crassa, we performed a genetic screen and identified HDA-1 as necessary for PRC2-targeted gene silencing. In the absence of HDA-1, H3K27me2/3 is lost from the subtelomeres and aberrantly accumulates at constitutive heterochromatin domains normally enriched for H3K9me3; CDP-2 is also important for wildtype H3K27me3 localization. The H3K27me2/3 redistribution in Δhda-1 or Δcdp-2 strains is variable and incomplete, and mutant progeny obtained following a sexual cross displayed wild-type H3K27me3 patterns, consistent with HCHC loss leading to progressive changes in H3K27me3 enrichment. Indeed, a newly constructed ∆hda-1 deletion strain displays a wild-type H3K27me3 pattern that relocalizes to constitutive heterochromatic regions after sequential passages. Thus, HCHC-specific deacetylation prevents aberrant recruitment of PRC2 to constitutive heterochromatin.
2025-01-21 | GSE286535 | 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