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Chromatin Assembly Factor 1 is Required for Normal Gene Repression and Facultative Heterochromatin Formation in Neurospora crassa [RNA-seq]

ABSTRACT: Formation of facultative heterochromatin by Polycomb Repressive Complex 2 (PRC2) is a well conserved epigenetic mechanism that functions to control transcriptional dynamics across eukaryotes. PRC2 catalyzes histone H3 lysine 27 tri-methylation (H3K27me3), a histone post-translational modification that helps maintain stable gene repression. Gene repression by PRC2 is important for organismal development and defects in PRC2 function are associated with human ailments, such as Weaver syndrome and various cancers. In this study, we reveal the replication-dependent histone chaperone, Chromatin Assembly Factor 1 (CAF-1), is required for normal facultative heterochromatin formation in model fungus N. crassa. CAF-1 deficiency results in widespread misregulation of gene expression, particularly within facultative heterochromatin domains, which is accompanied by a rearrangement of H3K27me3 patterns. Regional losses of H3K27me3 are associated with reduced levels of ASH-1 catalyzed H3K36 methylation, and gains of histone modifications associated with active transcription. Furthermore, analysis of a double mutant deficient for both CAF-1 and PRC2 activity revealed that these complexes play distinct roles in maintaining gene repression and facultative heterochromatin. Collectively, these results shed light on CAF-1's role in maintaining a repressive chromatin environment.

ORGANISM(S): Neurospora crassa

PROVIDER: GSE314682 | GEO | 2025/12/26

REPOSITORIES: GEO

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Chromatin Assembly Factor 1 is Required for Normal Gene Repression and Facultative Heterochromatin Formation in Neurospora crassa [ChIP-seq]

Project description:Formation of facultative heterochromatin by Polycomb Repressive Complex 2 (PRC2) is a well conserved epigenetic mechanism that functions to control transcriptional dynamics across eukaryotes. PRC2 catalyzes histone H3 lysine 27 tri-methylation (H3K27me3), a histone post-translational modification that helps maintain stable gene repression. Gene repression by PRC2 is important for organismal development and defects in PRC2 function are associated with human ailments, such as Weaver syndrome and various cancers. In this study, we reveal the replication-dependent histone chaperone, Chromatin Assembly Factor 1 (CAF-1), is required for normal facultative heterochromatin formation in model fungus N. crassa. CAF-1 deficiency results in widespread misregulation of gene expression, particularly within facultative heterochromatin domains, which is accompanied by a rearrangement of H3K27me3 patterns. Regional losses of H3K27me3 are associated with reduced levels of ASH-1 catalyzed H3K36 methylation, and gains of histone modifications associated with active transcription. Furthermore, analysis of a double mutant deficient for both CAF-1 and PRC2 activity revealed that these complexes play distinct roles in maintaining gene repression and facultative heterochromatin. Collectively, these results shed light on CAF-1's role in maintaining a repressive chromatin environment.

2025-12-26 | GSE314683 | GEO

Neurospora crassa genome organization requires subtelomeric facultative heterochromatin

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.

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IMITATION SWITCH is required for normal chromatin structure and gene repression in PRC2 target domains

Project description:Polycomb Group (PcG) proteins are part of an epigenetic cell memory system that plays essential roles in multicellular development, stem cell biology, X-chromosome inactivation, and cancer. In animals, plants, and many fungi, Polycomb Repressive Complex 2 (PRC2) catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) to assemble transcriptionally repressed facultative heterochromatin. PRC2 is structurally and functionally conserved in the model fungus Neurospora crassa, and recent work in this organism has generated insights into PRC2 control and function. To identify new components of the facultative heterochromatin pathway, we performed a targeted screen of Neurospora deletion strains lacking individual ATP-dependent chromatin remodeling enzymes. We found the Neurospora homolog of IMITATION SWITCH (ISW) is critical for normal transcriptional repression, nucleosome organization, and establishment of typical histone methylation patterns in facultative heterochromatin domains. We also found that stable interaction between PRC2 and chromatin depends on ISW. A functional ISW ATPase domain is required for gene repression and H3K27 methylation. ISW homologs interact with accessory proteins to form multiple complexes with distinct functions. Using proteomics and molecular approaches, we identified three distinct Neurospora ISW-containing complexes. A triple mutant lacking three ISW-accessory factors and disrupting multiple ISW complexes led to widespread upregulation of PRC2 target genes and altered H3K27 methylation patterns, similar to an ISW-deficient strain. Taken together, our data show that ISW is a key component of the facultative heterochromatin pathway in Neurospora and that distinct ISW complexes perform an apparently overlapping role to regulate chromatin structure and gene repression at PRC2 target domains.

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2025-06-24 | PXD058532 | Pride

Specific cPRC1 complexes are co-opted to mediate oncogenic gene repression in diffuse midline glioma

Project description:Summary Diffuse midline glioma (DMG) is a fatal childhood brain tumour characterised primarily by mutant histone H3 (H3K27M). H3K27M causes a global reduction in Polycomb Repressive Complex 2 (PRC2)-mediated H3K27me3 by inhibiting PRC2 enzymatic activity. Paradoxically, PRC2 is essential in DMG tumour cells where residual complex activity is required for oncogenic gene repression, although the molecular mechanisms acting downstream of PRC2 in this context are poorly understood. Here, we’ve discovered this oncogenic gene repression is mediated by specific canonical PRC1 (cPRC1) formations. By combining CRISPR screening, biochemical and chromatin mapping approaches with functional perturbations we show that cPRC1 complexes containing CBX4 and PCGF4 drive oncogenic gene repression downstream of H3K27me3 in DMG cells. Remarkably, the altered H3K27me3 modification landscape characteristic of these tumours rewires the distribution of cPRC1 complexes on chromatin. CBX4 and PCGF4 containing cPRC1 accumulate at sites of H3K27me3 while other cPRC1 formations are displaced. Despite accounting for <5% of cPRC1 complexes in DMG, CBX4/PCGF4-containing complexes predominate as gene repressors. Our findings link the altered distribution of H3K27me3 with imbalanced cPRC1 function, promoting oncogenic gene repression in DMG cells, revealing new disease mechanisms and highlighting potential therapeutic opportunities in this incurable childhood brain tumour.

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2025-06-16 | GSE271752 | GEO

Macromolecular interactions dictate Polycomb-mediated epigenetic repression [RNA-seq]

2025-06-16 | GSE271648 | GEO

Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

Project description:Polycomb Repressive Complex 2 (PRC2) plays an essential role in development by catalysing trimethylation of histone H3 lysine 27 (H3K27me3), resulting in gene repression. PRC2 consists of two sub-complexes, PRC2.1 and PRC2.2, in which the PRC2 core associates with distinct ancillary subunits such as MTF2 and JARID2, respectively. Both MTF2, present in PRC2.1, and JARID2, present in PRC2.2, play a role in core PRC2 recruitment to target genes in mouse embryonic stem cells (mESCs). However, it remains unclear how these distinct sub-complexes cooperate to establish H3K27me3 domains. Here, we combine a range of Polycomb mutant mESCs with chemical inhibition of PRC2 catalytic activity, to systematically dissect their relative contributions to PRC2 binding to target loci. We find that PRC2.1 and PRC2.2 mediate two distinct paths for recruitment, with mutually reinforced binding. Part of the cross-talk between PRC2.1 and PRC2.2 occurs via their catalytic product H3K27me3, which is bound by the PRC2 core-subunit EED, thereby mediating a positive feedback. Strikingly, removal of either JARID2 or H3K27me3 only has a minor effect on PRC2 recruitment, whereas their combined ablation largely attenuates PRC2 recruitment. This strongly suggests an unexpected redundancy between JARID2 and EED-H3K27me3-mediated recruitment of PRC2. Furthermore, we demonstrate that all core PRC2 recruitment occurs through the combined action of MTF2-mediated recruitment of PRC2.1 to DNA and PRC1-mediated recruitment of JARID2-containing PRC2.2. Both axes of binding are supported by EED-H3K27me3 positive feedback, but to a different degree. Finally, we provide evidence that PRC1 and PRC2 mutually reinforce reciprocal binding. Together, these data disentangle the interdependent and cooperative interactions between Polycomb complexes that are important to establish Polycomb repression at target sites.

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