H3K9 methylation-independent activity for HPL-2/HP1 in heterochromatin foci, gene repression, and organogenesis
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PROVIDER: PRJNA1167308 | ENA |
REPOSITORIES: ENA
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Project description:Differentiated cells rely on segregation of the genome into heterochromatin and euchromatin, but the mechanisms that establish these domains in vivo are incompletely understood. The current models suggest that heterochromatin is marked by histone H3 lysine 9 methylation (H3K9me), which recruits heterochromatin protein 1 (HP1), to compact chromatin and repress transcription. In C. elegans, the SETDB1 homolog MET-2 is essential for H3K9me-mediated gene silencing; however, MET-2 also localizes to heterochromatic foci independently of H3K9me and exhibits non-catalytic functions that support both germline and somatic development. Here we extend these findings by examining the classical H3K9me reader HP1. We show that HPL-2/HP1 deficient in methyl-lysine binding or in the absence of H3K9me can still repress transcription, promote organogenesis and localize to heterochromatin foci. Whereas activity remains in the absence of the HPL-2:H3K9me interaction, complete loss of met-2 and hpl-2 shows synergistic phenotypes for organogenesis and gene de-repression, underscoring the existence of additional, H3K9me-independent functions. HPL-2 and MET-2 require distinct binding partners for their localization and activity: MET-2 associates with the disordered protein LIN-65/ATF7IP, while HPL-2 depends on the multi-zinc finger protein LIN-13, which interacts with the HPL-2 chromoshadow domain. Our findings suggest that HPL-2 and MET-2 operate in parallel pathways, localizing to heterochromatic foci and promoting gene silencing and organogenesis, with H3K9me acting as a reinforcing, but non-essential, element in these processes.
2025-07-01 | GSE278459 | GEO
Project description:Histone post-translational modifications (PTMs) segregate genomes into heterochromatin and euchromatin by recruiting readers, including HP1 proteins that bind H3 lysine 9 methylation (H3K9me). Here, we report H3K9me-independent functions for the HP1 orthologue HPL-2 and the H3K9 methyltransferase MET-2/SETDB1 in C. elegans. Both proteins co-localize in H3K9me-containing sub-nuclear foci independently. Their combined loss additively disrupts gene repression and organogenesis. HPL-2 remains functional in the absence of H3K9me: HPL-2 deficient for H3K9me-binding still localizes to heterochromatin foci, restricts cell fate decisions and represses developmental genes. MET-2, catalytically active or not, requires the disordered protein LIN-65 to form foci and silence genes in vivo, while HPL-2 requires a multi-zinc finger ligand, LIN-13. Molecular dynamics simulations and in vitro assays suggest that these cofactor-driven condensate assemblies underlie the H3K9me-independent formation of heterochromatic foci in vivo. We define parallel, H3K9me-independent pathways through which MET-2 and HPL-2 can repress genes essential for organogenesis, reinforcing H3K9me-dependent mechanisms.
2026-08-31 | GSE345142 | GEO
Project description:Heterochromatin protein 1 (HP1) is a conserved chromatin-associated factor implicated in the establishment and maintenance of H3K9me-marked heterochromatin, potentially through phase separation–mediated condensation. Whether HP1 promotes heterochromatin condensation primarily through its dimerization or liquid–liquid phase separation (LLPS) remains unresolved. Using the C. elegans HP1 orthologue HPL-2 and a combined in vitro–in vivo approach, we systematically dissected the molecular determinants of HPL-2 function in heterochromatin condensation. Through specific mutants, we demonstrate that HPL-2 dimerization is essential for condensing H3K9me chromatin arrays in vitro and for maintaining H3K9me heterochromatin foci in C. elegans embryos. We further show that HPL-2 dimerization is sufficient to mediate segregation of H3K9me from unmodified chromatin arrays in vitro, forming spatially distinct H3K9me-enriched condensates. Surprisingly, HPL-2 mutants defective in heterochromatin foci formation cause only minor transcriptional changes among genes associated with H3K9me-marked heterochromatin, implying that HP1-dependent heterochromatin foci and gene silencing are not tightly coupled in vivo. Nonetheless, these mutant C. elegans exhibit profound physiological and developmental defects. Our findings establish dimerization as the principal molecular mechanism of HP1-driven H3K9me-chromatin condensation, elucidate an auxiliary role of LLPS, and reveal the uncoupling between HP1-dependent heterochromatin and transcriptional regulation.
2026-08-25 | GSE339336 | GEO
Project description:Heterochromatin protein 1 (HP1) is a conserved chromatin-associated factor implicated in the establishment and maintenance of H3K9me-marked heterochromatin, potentially through phase separation–mediated condensation. Whether HP1 promotes heterochromatin condensation primarily through its dimerization or liquid–liquid phase separation (LLPS) remains unresolved. Using the C. elegans HP1 orthologue HPL-2 and a combined in vitro–in vivo approach, we systematically dissected the molecular determinants of HPL-2 function in heterochromatin condensation. Through specific mutants, we demonstrate that HPL-2 dimerization is essential for condensing H3K9me chromatin arrays in vitro and for maintaining H3K9me heterochromatin foci in C. elegans embryos. We further show that HPL-2 dimerization is sufficient to mediate segregation of H3K9me from unmodified chromatin arrays in vitro, forming spatially distinct H3K9me-enriched condensates. Surprisingly, HPL-2 mutants defective in heterochromatin foci formation cause only minor transcriptional changes among genes associated with H3K9me-marked heterochromatin, implying that HP1-dependent heterochromatin foci and gene silencing are not tightly coupled in vivo. Nonetheless, these mutant C. elegans exhibit profound physiological and developmental defects. Our findings establish dimerization as the principal molecular mechanism of HP1-driven H3K9me-chromatin condensation, elucidate an auxiliary role of LLPS, and reveal the uncoupling between HP1-dependent heterochromatin and transcriptional regulation.
2026-08-25 | GSE342086 | GEO
Project description:HP1 can act independently of H3K9 methylation to promote heterochromatin focus formation, gene repression, and organogenesis [CUT&Tag]
| PRJNA1518890 | ENA
Project description:Histone H3 lysine 9 (H3K9) methylation and heterochromatin protein 1 (HP1) are well conserved epigenetic silencing mark and its reader molecule, and crucial for heterochromatin formation. However, the details of the importance of H3K9 methylation and HP1 in heterochromatin formation still remain unclear. One of the reason is the redundancy problem, as there are multiple reader molecules for H3K9 methylation, including HP1, and HP1 itself functions as a hub that recruits various effector molecules. To overcome the redundancy issue, we took synthetic biology approach and introduced H3K9 methylation and HP1 into budding yeast Saccharomyces cerevisiae, which does not have this system, and examined its impact on transcription and chromatin compaction. We observed that mammalian H3K9 methyltransferase can induce genome wide H3K9 di- and tri-methylation (H3K9me2,3) in budding yeast, and that HP1 accumulates over the H3K9 methylated regions. However, H3K9 methylation occurred mainly in the gene body region of the genes and excluded around TSS where H3K9ac pre-exists. Correspondingly, expression of H3K9 methyltransferase and HP1 did not affect transcription in budding yeast, including repression. ATAC-seq analysis also showed no impact on chromatin accessibility, and Hi-C-seq analysis of chromatin 3D structure revealed no significant differences. These results suggest that even though H3K9 methylation and recruitment of HP1 play essential roles in epigenetic regulation of heterochromatin, they are not sufficient to build up heterochromatin, at least at gene body regions, and further participation of effector molecules, including downstream factors of HP1, is required.
2024-02-03 | GSE224639 | GEO
Project description:Histone H3 lysine 9 (H3K9) methylation and heterochromatin protein 1 (HP1) are well conserved epigenetic silencing mark and its reader molecule, and crucial for heterochromatin formation. However, the details of the importance of H3K9 methylation and HP1 in heterochromatin formation still remain unclear. One of the reason is the redundancy problem, as there are multiple reader molecules for H3K9 methylation, including HP1, and HP1 itself functions as a hub that recruits various effector molecules. To overcome the redundancy issue, we took synthetic biology approach and introduced H3K9 methylation and HP1 into budding yeast Saccharomyces cerevisiae, which does not have this system, and examined its impact on transcription and chromatin compaction. We observed that mammalian H3K9 methyltransferase can induce genome wide H3K9 di- and tri-methylation (H3K9me2,3) in budding yeast, and that HP1 accumulates over the H3K9 methylated regions. However, H3K9 methylation occurred mainly in the gene body region of the genes and excluded around TSS where H3K9ac pre-exists. Correspondingly, expression of H3K9 methyltransferase and HP1 did not affect transcription in budding yeast, including repression. ATAC-seq analysis also showed no impact on chromatin accessibility, and Hi-C-seq analysis of chromatin 3D structure revealed no significant differences. These results suggest that even though H3K9 methylation and recruitment of HP1 play essential roles in epigenetic regulation of heterochromatin, they are not sufficient to build up heterochromatin, at least at gene body regions, and further participation of effector molecules, including downstream factors of HP1, is required.
2024-02-03 | GSE224638 | GEO
Project description:Histone H3 lysine 9 (H3K9) methylation and heterochromatin protein 1 (HP1) are well conserved epigenetic silencing mark and its reader molecule, and crucial for heterochromatin formation. However, the details of the importance of H3K9 methylation and HP1 in heterochromatin formation still remain unclear. One of the reason is the redundancy problem, as there are multiple reader molecules for H3K9 methylation, including HP1, and HP1 itself functions as a hub that recruits various effector molecules. To overcome the redundancy issue, we took synthetic biology approach and introduced H3K9 methylation and HP1 into budding yeast Saccharomyces cerevisiae, which does not have this system, and examined its impact on transcription and chromatin compaction. We observed that mammalian H3K9 methyltransferase can induce genome wide H3K9 di- and tri-methylation (H3K9me2,3) in budding yeast, and that HP1 accumulates over the H3K9 methylated regions. However, H3K9 methylation occurred mainly in the gene body region of the genes and excluded around TSS where H3K9ac pre-exists. Correspondingly, expression of H3K9 methyltransferase and HP1 did not affect transcription in budding yeast, including repression. ATAC-seq analysis also showed no impact on chromatin accessibility, and Hi-C-seq analysis of chromatin 3D structure revealed no significant differences. These results suggest that even though H3K9 methylation and recruitment of HP1 play essential roles in epigenetic regulation of heterochromatin, they are not sufficient to build up heterochromatin, at least at gene body regions, and further participation of effector molecules, including downstream factors of HP1, is required.
2024-02-03 | GSE224640 | GEO
Project description:Heterochromatin formation in Schizosaccharomyces pombe requires the spreading of histone 3 (H3) Lysine 9 (K9) methylation (me) from nucleation centers by the H3K9 methylase, Suv39/Clr4, and the reader protein, HP1/Swi6. To accomplish this, Suv39/Clr4 and HP1/Swi6 have to associate with nucleosomes both nonspecifically, binding DNA and octamer surfaces and specifically, via recognition of methylated H3K9 by their respective chromodomains. However, how both proteins avoid competition for the same nucleosomes in this process is unclear. Here, we show that phosphorylation tunes the nucleosome affinity of HP1/Swi6 such that it preferentially partitions onto Suv39/Clr4’s trimethyl product rather than its unmethylated substrates. Preferential partitioning enables efficient conversion from di-to trimethylation on nucleosomes in vitro and H3K9me3 spreading in vivo. Together, our data suggests that phosphorylation of HP1/Swi6 creates a regime that relieves competition with the “read-write” mechanism of Suv39/Clr4 for productive heterochromatin spreading.
2025-12-22 | PXD057316 | Pride
Project description:Histone H3 lysine 9 (H3K9) methylation and heterochromatin protein 1 (HP1) are well conserved epigenetic silencing mark and its reader molecule, and crucial for heterochromatin formation. However, the details of the importance of H3K9 methylation and HP1 in heterochromatin formation still remain unclear. One of the reason is the redundancy problem, as there are multiple reader molecules for H3K9 methylation, including HP1, and HP1 itself functions as a hub that recruits various effector molecules. To overcome the redundancy issue, we took synthetic biology approach and introduced H3K9 methylation and HP1 into budding yeast Saccharomyces cerevisiae, which does not have this system, and examined its impact on transcription and chromatin compaction. We observed that mammalian H3K9 methyltransferase can induce genome wide H3K9 di- and tri-methylation (H3K9me2,3) in budding yeast, and that HP1 accumulates over the H3K9 methylated regions. However, H3K9 methylation occurred mainly in the gene body region of the genes and excluded around TSS where H3K9ac pre-exists. Correspondingly, expression of H3K9 methyltransferase and HP1 did not affect transcription in budding yeast, including repression. ATAC-seq analysis also showed no impact on chromatin accessibility, and Hi-C-seq analysis of chromatin 3D structure revealed no significant differences. These results suggest that even though H3K9 methylation and recruitment of HP1 play essential roles in epigenetic regulation of heterochromatin, they are not sufficient to build up heterochromatin, at least at gene body regions, and further participation of effector molecules, including downstream factors of HP1, is required.
2024-02-03 | GSE224636 | GEO