H3K9 promotes under-replication of pericentromeric heterochromatin in Drosophila salivary gland polytene chromosomes
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ABSTRACT:
PROVIDER: PRJNA516646 | ENA |
REPOSITORIES: ENA
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Project description:We replaced the endogenous histones of Drosophila melanogaster with either histones containing an H3K9R mutation or histones containing an H4K16R mutation to interrogate direct functions for histone residues in salivary gland endoreplication. We performed genomic DNA sequencing in HWT (Histone Wild Type) control, H3K9R, and H4K16R females. We found that H3K9 promotes under-replication of pericentromeric heterochromatin but not along chromosome arms and H4K16 is dispensable for under-replication.
2019-02-21 | GSE125505 | GEO
Project description:Chromatin structure and its organization contributes to the proper regulation and timing of DNA replication. Yet, the precise mechanism by which chromatin contributes to DNA replication remains incompletely understood. This is particularly true for cell types that rely on polyploidization as a developmental strategy for growth and high biosynthetic capacity. During Drosophila larval development, cells of the salivary gland undergo endoreplication, repetitive rounds of DNA synthesis without intervening cell division, resulting in ploidy values of ~1350C. S phase of these endocycles displays a reproducible pattern of early and late replicating regions of the genome resulting from the activity of the same replication initiation factors that are used in diploid cells. However, unlike diploid cells, the latest replicating regions of polyploid salivary gland genomes, composed primarily of pericentric heterochromatic enriched in H3K9 methylation, are not replicated each endocycle, resulting in under-replicated domains with reduced ploidy. Here, we employ a histone gene replacement strategy in Drosophila to demonstrate that mutation of a histone residue important for heterochromatin organization and function (H3K9) but not mutation of a histone residue important for euchromatin function (H4K16), disrupts proper endoreplication in Drosophila salivary gland polyploid genomes thereby leading to DNA copy gain in pericentric heterochromatin. These findings reveal that H3K9 is necessary for normal levels of under-replication of pericentric heterochromatin and suggest that under-replication at pericentric heterochromatin is mediated through H3K9 methylation.
| S-EPMC6409945 | biostudies-literature
Project description:Many repetitive DNA elements are packaged in heterochromatin, but depend on occasional transcription to maintain long-term silencing. The factors that promote transcription of repeat elements in heterochromatin are largely unknown. Here, we show that DOT1L, a histone methyltransferase that modifies lysine 79 of histone H3 (H3K79), is required for transcription of major satellite repeats to maintain pericentromeric heterochromatin (PCH), and that this function is essential for preimplantation development. DOT1L is a transcriptional activator at single-copy genes but does not have a known role in repeat element transcription. We show that H3K79me3 is specifically enriched at repetitive elements, that loss of DOT1L compromises pericentromeric major satellite transcription, and that this function depends on interaction between DOT1L and the chromatin remodeler SMARCA5. DOT1L inhibition causes chromosome breaks and cell cycle defects, and leads to embryonic lethality. Together, our findings uncover a vital new role for DOT1L in transcriptional activation of heterochromatic repeats.
2023-05-26 | PXD028824 | Pride
Project description:Histone H3 lysine 9 (H3K9) methylation is a central epigenetic modification that defines heterochromatin from unicellular to multicellular organisms. In mammalian cells, H3K9 methylation can be catalyzed by at least six distinct SET domain enzymes: Suv39h1/Suv39h2, Eset1/Eset2 and G9a/Glp. We used mouse embryonic fibroblasts (MEFs) with a conditional mutation for Eset1 and introduced progressive deletions for the other SET domain genes by CRISPR/Cas9 technology. Compound mutant MEFs for all 6 SET domain methyltransferase (KMT) genes lack all H3K9 methylation states, derepress nearly all families of repeat elements and display genomic instabilities. Strikingly, the 6KO H3K9 KMT MEFs no longer maintain heterochromatin organization and have lost electron-dense heterochromatin. This is the first analysis of H3K9 methylation deficient mammalian chromatin and reveals a crucial function for H3K9 methylation in protecting heterochromatin organization and genome integrity.
2021-05-27 | PXD018175 | Pride
Project description:We replaced the endogenous histones of Drosophila melanogaster with histones containing an H3K9R mutation to interrogate the role of H3K9 in heterochromatin formation and function. We queried heterochromatin formation through Formaldhyde Assisted Isolation of Regulatory Elements coupled with sequencing to examine nucleosome occupancy and Heterochromatin Protein 1a ChIP sequencing to determine the localization of the major reader of H3K9me. We found that regions of pericentromeric heterochromatin exhibit decreased HP1a and nucleosome occupancy in H3K9R mutants. To examine potential consequences of these changes of chromatin architecture, we performed total RNA-seq. In H3K9R mutants we observed increased levels of transposon and pIRNA cluster transcripts; however, the protein-coding transcriptome was similar to controls.
2016-08-30 | GSE85374 | GEO
Project description:Histone post-translational modifications and the proteins that bind them are proposed to be drivers of 3D genome organization, but whether and how they do so remain unanswered. Here, we evaluate the contribution of H3K9-methylated constitutive heterochromatin to 3D genome organization in Drosophila tissues. We find that the predominant organizational feature of wildtype tissues is segregation of euchromatic chromosome arms from heterochromatic pericentromeres. Reciprocal perturbation of HP1a•H3K9 binding, using a point mutation in the HP1a chromodomain or replacement of the replication-dependent H3 with H3K9R mutant histones, revealed that HP1a binding to methylated H3K9 in constitutive heterochromatin is required to restrict long-range interactions between pericentromeres and chromosome arms. Surprisingly, self-association of pericentromeric heterochromatin is largely preserved upon disruption of HP1a•H3K9 binding despite loss of pericentromeric H3K9 methylation and HP1a occupancy. Thus, the HP1a•H3K9 interaction contributes to, but does not solely drive, segregation of euchromatin and heterochromatin inside the nucleus.
2024-05-24 | GSE236965 | GEO
Project description:Histone post-translational modifications and the proteins that bind them are proposed to be drivers of 3D genome organization, but whether and how they do so remain unanswered. Here, we evaluate the contribution of H3K9-methylated constitutive heterochromatin to 3D genome organization in Drosophila tissues. We find that the predominant organizational feature of wildtype tissues is segregation of euchromatic chromosome arms from heterochromatic pericentromeres. Reciprocal perturbation of HP1a•H3K9 binding, using a point mutation in the HP1a chromodomain or replacement of the replication-dependent H3 with H3K9R mutant histones, revealed that HP1a binding to methylated H3K9 in constitutive heterochromatin is required to restrict long-range interactions between pericentromeres and chromosome arms. Surprisingly, self-association of pericentromeric heterochromatin is largely preserved upon disruption of HP1a•H3K9 binding despite loss of pericentromeric H3K9 methylation and HP1a occupancy. Thus, the HP1a•H3K9 interaction contributes to, but does not solely drive, segregation of euchromatin and heterochromatin inside the nucleus.
2024-05-24 | GSE234276 | GEO
Project description:Histone post-translational modifications and the proteins that bind them are proposed to be drivers of 3D genome organization, but whether and how they do so remain unanswered. Here, we evaluate the contribution of H3K9-methylated constitutive heterochromatin to 3D genome organization in Drosophila tissues. We find that the predominant organizational feature of wildtype tissues is segregation of euchromatic chromosome arms from heterochromatic pericentromeres. Reciprocal perturbation of HP1a•H3K9 binding, using a point mutation in the HP1a chromodomain or replacement of the replication-dependent H3 with H3K9R mutant histones, revealed that HP1a binding to methylated H3K9 in constitutive heterochromatin is required to restrict long-range interactions between pericentromeres and chromosome arms. Surprisingly, self-association of pericentromeric heterochromatin is largely preserved upon disruption of HP1a•H3K9 binding despite loss of pericentromeric H3K9 methylation and HP1a occupancy. Thus, the HP1a•H3K9 interaction contributes to, but does not solely drive, segregation of euchromatin and heterochromatin inside the nucleus.
2024-05-24 | GSE233384 | GEO
Project description:Regional centromeres in Candida lusitaniae lack pericentromeric heterochromatin
| PRJNA291764 | ENA
Project description:In Drosophila salivary gland polytene chromosomes, a substantial portion of heterochromatin is underreplicated. The combination of mutations SuURES and Su(var)3-906 results in the polytenization of a substantial fraction of unique and moderately repeated sequences but has almost no effect on satellite DNA replication. The Rap1 interacting factor 1 (Rif) protein is a conserved regulator of replication timing, and in Drosophila, it affects underreplication in polytene chromosomes. We compared the morphology of pericentromeric regions and labeling patterns of in situ hybridization of heterochromatin-specific DNA probes between wild-type salivary gland polytene chromosomes and the chromosomes of Rif1 mutants and SuUR Su(var)3-906 double mutants. We show that, despite general similarities, heterochromatin zones exist that are polytenized only in the Rif1 mutants, and that there are zones that are under specific control of Su(var)3-9. In the Rif1 mutants, we found additional polytenization of the largest blocks of satellite DNA (in particular, satellite 1.688 of chromosome X and simple satellites in chromosomes X and 4) as well as partial polytenization of chromosome Y. Data on pulsed incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into polytene chromosomes indicated that in the Rif1 mutants, just as in the wild type, most of the heterochromatin becomes replicated during the late S phase. Nevertheless, a significantly increased number of heterochromatin replicons was noted. These results suggest that Rif1 regulates the activation probability of heterochromatic origins in the satellite DNA region.
| S-EPMC7349278 | biostudies-literature