ChIP_Seq analysis in cultured mTSC cells with or without Dox
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE196222 | GEO | 2023/06/28
REPOSITORIES: GEO
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Project description:To detect the direct target genes of H4K20me1, H4K20me3, and H3K36me3 in hTSCs, hTSCs are collected and subjected to ChIP-Seq. After aligned to human hg38 by HISAT2, peaks are called by MACS2. Our results show that methylation of H4K20 mediated by PR-SET7 as the key regulator of hTSCs, indicating the essential role of PR-SET7. This ChIP-Seq data provides fundamental information for our further physiological study of PR-SET7.
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Project description:To detect the gene profiles in WT and Pr-set7 KO mTSCs, mTSCs are collected and subjected to RNA-Seq. After aligned to mouse mm10 by HISAT2, RPKM value was calculated by Edger. Our results show that Pr-set7 as the key regulator for mTSCs. There are also some genes differentially expressed after Pr-set7 KO, some of the DEGs were further confirmed by qPCR, the DEGs were associated with viral mimic inflammatory activities, antiviral innate immune response, genomic instability, and programmed cell death, indicating the essential role of Pr-set7. This RNA-Seq data provides fundamental information for our further physiological study of Pr-set7.
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Project description:The methylation state of lysine 20 on histone H4 (H4K20) has been linked to cell cycle progression, Origin Recognition Complex (ORC) binding, and replication origin regulation. Monomethylation of H4K20 (H4K20me1) is mediated by the cell cycle-regulated histone methyltransferase PR-Set7, which is essential for genome integrity and cell cycle progression. PR-Set7 depletion in mammalian cells results in defective S-phase progression and the accumulation of DNA damage, which could be partially attributed to a defect in pre-Replication Complex (pre-RC) formation and origin activity. However, these studies were limited to a handful of mammalian origins, and it remains unclear how PR-Set7 and H4K20 methylation impact the replication program on a genomic scale. Using Drosophila Kc167 cells, we employed genetic, cytological, and genomic approaches to better understand the role of PR-Set7 and H4K20 methylation in regulating DNA replication and governing genome stability. We find that depletion of Drosophila PR-Set7 and loss of H4K20me1 result in the accumulation of DNA damage and an ATR-dependent cell cycle arrest. The cell cycle arrest occurs during the second S-phase following loss of PR-Set7 activity, suggesting that accumulation of nascent H4K20 is recalcitrant to the DNA replication program. Deregulation of H4K20 methylation had no impact on origin activation throughout the genome; instead, we found that the DNA damage marker, phosphorylated H2A.v (γ-H2A.v), accumulated specifically in late replicating domains in the absence of PR-Set7. This suggests that the molecular basis for the cell cycle arrest and accumulation of DNA damage resulting from loss of PR-Set7 is stochastic fork collapse within late replicating domains.
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Project description:The ability of neural stem cells (NSCs) to switch between quiescence and proliferation is crucial for brain development and homeostasis. Increasing evidence suggest that variants of histone lysine methyltransferases including KMT5A are associated with neurodevelopmental disorders. However, the function of KMT5A/Pr-set7/SETD8 in the central nervous system is not well established. Here, we show that Drosophila Pr-Set7 is a novel regulator of NSC reactivation. Loss-of-function of pr-set7 causes a delay in NSC reactivation and loss of H4K20 monomethylation in the brain. Through NSC-specific in vivo profiling, we demonstrate that Pr-set7 binds to the promoter region of cyclin dependent kinase 1 (cdk1) and Wnt pathway transcriptional co-activator earthbound1/jerky (ebd1). Further validation indicates that Pr-set7 is required for the expression of cdk1 and ebd1 in the brain. Similar to Pr-set7, Cdk1 and Ebd1 promote NSC reactivation. Moreover, Cdk1 upregulates the Ebd1 levels in NSCs, while Ebd1 appears to downregulate Cdk1 expression, suggesting a negative feedback regulation. Finally, overexpression of Cdk1 and Ebd1 significantly suppressed NSC reactivation defects observed in pr-set7-depleted brains. Therefore, Pr-set7, the sole H4K20 methyltransferase, promotes NSC reactivation through regulating Wnt signaling and cell-cycle progression. Given the conservation of Pr-set7, our findings may contribute to the understanding of mammalian KMT5A/PR-SET7/SETD8 in NSC proliferation and associated neurodevelopmental disorders.
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Project description:H4K20 monomethylation maintains genome integrity by regulating proper mitotic condensation, DNA damage response and replication licensing. Here we show that in non-dividing hepatic cells H4K20Me1 is specifically enriched in the gene bodies of active genes and dynamically regulated by the antagonistic action of PR-Set7 methylase and Phf8 demethylase. In liver-specific PR-Set7-deficient mice, reduced H4K20Me1 levels primarily affected RNAPII release from promoter-proximal regions. Glucose and fatty acid metabolic genes were highly sensitive to impaired RNAPII release. Downregulation of glycolytic genes resulted in an energy starvation condition partially compensated by AMPK activation and increased mitochondrial activity. This metabolic reprogramming generated a normoglycemic, but highly sensitized state, which upon different metabolic stress conditions, quickly aggravated into a senescent phenotype initiated by ROS overproduction and oxidative DNA damage. The results illustrate how defects in the general process of RNAPII transition into elongation phase can trigger specific metabolic changes, which lead to genome instability.
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Project description:To detect the gene profiles in si-NC and si-PR-SET7 KD hTSCs, hTSCs are collected and subjected to RNA-Seq. After aligned to mouse hg38 by HISAT2, RPKM value was calculated by Edger. Our results show that PR-SET7 as the key regulator for hTSCs. There are also some genes differentially expressed after PR-SET7 KD, some of the DEGs were further confirmed by qPCR, the DEGs were associated with viral mimic inflammatory activities, antiviral innate immune response, genomic instability, and programmed cell death, indicating the essential role of PR-SET7. This RNA-Seq data provides fundamental information for our further physiological study of PR-SET7.
2023-06-28 | GSE196223 | GEO
Project description:The role of histone modifications in DNA replication remains poorly understood. Here, we show that histone H4K20 monomethylation (me1) favors the assembly of the pre-replication complex on chromatin, an association that is increased when H4K20me1 is converted to trimethylation (H4K20me3). Remarkably, these histone methylations enhance origin activity, indicating a positive role in both origin licensing and activation. Genome-wide approaches revealed that H4K20me1 and H4K20me3 are enriched in distinct GC-rich regions, where their presence correlates with early-replicating and late-firing origins respectively. Accordingly, depletion of H4K20me1 at early origins delays the replication of active chromatin. Conversely, loss of H4K20me2/3 causes a drop in the licensing and activity of late-firing origins, slowing down the replication of heterochromatin. Altogether, these results suggest that the concerted activity of mammalian H4K20 methyltransferases and the resulting distribution of H4K20me states are essential to ensure the order and replication timing of different chromatin states.
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Project description:Histone methyl groups can be removed by histone demethylases. Although LSD1 and JmjC domain-containing proteins have been identified to be histone demethylases, enzymes responsible for many histone methylation states or sites are not identified. Here, we performed a high-content cell-based screening of a cDNA library containing 2,500 nuclear proteins and identified KDM10A as a histone demethylase specific for histone H4 lysine 20 (H4K20). Ectopic expression of KDM10A reduced the global levels of H4K20me1/2/3 in 293T cells. In vitro, KDM10A specifically demethylated H4K20me1/2/3 and generated formaldehyde. The enzymatic activity required Fe(II) and α-ketoglutarate as cofactors. Domain mapping indicated that the demethylase activity required its UBA domain. We also demonstrated that KDM10B, a protein homologous to KDM10A, had H4K20 demethylase activity in vitro and in vivo. ChIP-seq reveals that KDM10A/B demethylated H4K20 methylation at specific genomic loci in vivo. We further demonstrated that KDM10A/B activated the transcription of coding genes by demethylating H4K20me1 in the gene body, and the transcription of repetitive elements by demethylating H4K20me3 in intergenic regions. NMR analyses demonstrated that an HxxxE motif in the UBA domain is crucial for iron binding, mutation of these two residues abolished iron binding and the demethylase activity. Thus, we identified a family of UBA domain-containing proteins as histone demethylases.
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Project description:Histone methyl groups can be removed by histone demethylases. Although LSD1 and JmjC domain-containing proteins have been identified to be histone demethylases, enzymes responsible for many histone methylation states or sites are not identified. Here, we performed a high-content cell-based screening of a cDNA library containing 2,500 nuclear proteins and identified KDM10A as a histone demethylase specific for histone H4 lysine 20 (H4K20). Ectopic expression of KDM10A reduced the global levels of H4K20me1/2/3 in 293T cells. In vitro, KDM10A specifically demethylated H4K20me1/2/3 and generated formaldehyde. The enzymatic activity required Fe(II) and α-ketoglutarate as cofactors. Domain mapping indicated that the demethylase activity required its UBA domain. We also demonstrated that KDM10B, a protein homologous to KDM10A, had H4K20 demethylase activity in vitro and in vivo. ChIP-seq reveals that KDM10A/B demethylated H4K20 methylation at specific genomic loci in vivo. We further demonstrated that KDM10A/B activated the transcription of coding genes by demethylating H4K20me1 in the gene body, and the transcription of repetitive elements by demethylating H4K20me3 in intergenic regions. NMR analyses demonstrated that an HxxxE motif in the UBA domain is crucial for iron binding, mutation of these two residues abolished iron binding and the demethylase activity. Thus, we identified a family of UBA domain-containing proteins as histone demethylases.
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