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: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: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: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:Chromosomes must correctly fold in eukaryotic nuclei for proper genome function. Eukaryotic organisms hierarchically organize their genomes: in the fungus Neurospora crassa, chromatin fiber loops compact into Topologically Associated Domain (TAD)-like structures that are anchored by heterochromatic region aggregates. However, insufficient information exists on how histone post-translational modifications, including acetylation, impact genome organization. In Neurospora, the HCHC (HDA-1, CDP-2, HP1, CHAP) complex deacetylates heterochromatic regions including centromeres: loss of individual HCHC members increases centromeric acetylation and cytosine methylation. Here, we evaluate the role of the HCHC complex on genome organization using chromosome conformation capture with high-throughput sequencing (Hi-C) in Δcdp-2 or Δchap deletion strains. CDP-2 loss increases interactions between intra- and inter-chromosomal heterochromatic regions, while removal of CHAP decreases heterochromatic region compaction. Individual HCHC mutants exhibit different histone PTM patterns genome-wide: in Dcdp-2, heterochromatic H4K16 acetylation is increased, yet some heterochromatic regions lose H3K9 trimethylation, which locally increases inter-heterochromatin contacts; CHAP loss produces minimal acetylation changes but increases H3K9me3 enrichment in heterochromatin. Furthermore, deletion of the DIM-2 DNA methyltransferase in a Δcdp-2 background causes extensive genome disorder, as heterochromatic-euchromatic contacts increase despite additional H3K9me3 enrichment. Our results highlight how enhanced cytosine methylation ensures heterochromatic compartmentalization when silenced regions are acetylated.
2023-07-03 | GSE232934 | GEO
Project description:Chromosomes must correctly fold in eukaryotic nuclei for proper genome function. Eukaryotic organisms hierarchically organize their genomes: in the fungus Neurospora crassa, chromatin fiber loops compact into Topologically Associated Domain (TAD)-like structures that are anchored by heterochromatic region aggregates. However, insufficient information exists on how histone post-translational modifications, including acetylation, impact genome organization. In Neurospora, the HCHC (HDA-1, CDP-2, HP1, CHAP) complex deacetylates heterochromatic regions including centromeres: loss of individual HCHC members increases centromeric acetylation and cytosine methylation. Here, we evaluate the role of the HCHC complex on genome organization using chromosome conformation capture with high-throughput sequencing (Hi-C) in Δcdp-2 or Δchap deletion strains. CDP-2 loss increases interactions between intra- and inter-chromosomal heterochromatic regions, while removal of CHAP decreases heterochromatic region compaction. Individual HCHC mutants exhibit different histone PTM patterns genome-wide: in Dcdp-2, heterochromatic H4K16 acetylation is increased, yet some heterochromatic regions lose H3K9 trimethylation, which locally increases inter-heterochromatin contacts; CHAP loss produces minimal acetylation changes but increases H3K9me3 enrichment in heterochromatin. Furthermore, deletion of the DIM-2 DNA methyltransferase in a Δcdp-2 background causes extensive genome disorder, as heterochromatic-euchromatic contacts increase despite additional H3K9me3 enrichment. Our results highlight how enhanced cytosine methylation ensures heterochromatic compartmentalization when silenced regions are acetylated.
2023-07-03 | GSE232933 | 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:Nucleosome turnover concomitant with incorporation of the replication-independent histone variant H3.3 is a hallmark of regulatory regions in the animal genome. In our current understanding, nucleosome turnover is universally linked to DNA accessibility and histone acetylation. In mouse embryonic stem cells, H3.3 is also highly enriched at interstitial heterochromatin, most prominently intracisternal-A particle endogenous retroviral elements. Interstitial heterochromatin is established over confined domains by the TRIM28/SETDB1 corepressor complex and has stereotypical features of repressive chromatin, such as H3K9me3 and recruitment of all HP1 isoforms. Here, we demonstrate that fast histone turnover and H3.3 incorporation is compatible with these hallmarks of heterochromatin. We identify Smarcad1 to be a chromatin remodeler that generates nucleosome-free regions which are subsequently filled with histone H3.3, generating a surprisingly dynamic heterochromatin state. Loss of histone H3.3 elicits a highly specific opening of interstitial heterochromatin demonstrating that in the wake of Smarcad1 remodeling, H3.3 is required to maintain minimal DNA accessibility by reassembling nucleosomes.
2020-08-28 | GSE149080 | GEO
Project description:One key aspect of epigenetic inheritance is that chromatin structure can be stably inherited through generations even after removal of the initial signal that establishes such structure. In fission yeast, the RNA interference (RNAi) machinery is critical for the targeting of histone methyltransferase Clr4 to repetitive DNA elements to establish a large domain of heterochromatin marked with histone H3 lysine 9 methylation (H3K9me). Subsequently during DNA replication, the deposition of parental histones containing H3K9me into daughter DNA strands is expected to recruit Clr4 to modify neighboring new histones, resulting in the inheritance of heterochromatin in both daughter cells. However, pericentric heterochromatin cannot be properly inherited in the absence of RNAi, suggesting the existence of mechanisms that counteract chromatin-based inheritance. Here we show that in the absence of certain components of the INO80 chromatin remodeling complex, pericentric heterochromatin can be properly inherited in RNAi mutants. The ability of INO80 to counter heterochromatin inheritance is attributed to one accessory subunit Iec5, which promotes histone turn over at heterochromatin regions, but has little effects on the ability of INO80 in regulating nucleosome positioning at heterochromatin, gene expression, or the DNA damage response. Slow histone turnover preserves parental histones at repeat regions to enhance epigenetic inheritance of heterochromatin not only in RNAi mutants but also in wild type cells. Our analyses identified a separation-of-function mutation of INO80 in regulating histone turnover at heterochromatin and demonstrate the important role of histone turnover in controlling heterochromatin inheritance and maintaining the proper heterochromatin landscape of the genome.
2020-11-18 | GSE150544 | GEO