KMT2C/KMT2D-dependent H3K4me1 mediates DNA replication timing and origin firing
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ABSTRACT:
PROVIDER: PRJNA1164200 | ENA |
REPOSITORIES: ENA
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Project description:Mammalian genomes are replicated in a precise, cell-type-specific order during the S phase, a process that correlates with local transcriptional activity, chromatin modifications, and chromatin architecture. However, the causal relationships between these features and DNA replication timing (RT), particularly during cell fate transitions, remain largely unknown. Here, we employed machine learning to quantify chromatin features — including epigenetic marks, histone variants, and chromatin architectural factors — that best predict local RT under steady-state conditions and RT changes during early embryonic stem (ES) cell differentiation. We found that approximately one-third of the genome exhibits RT changes during differentiation. Chromatin features collectively predicted steady-state RT and RT changes with high accuracy. Notably, histone H3 lysine 4 monomethylation (H3K4me1), catalyzed by KMT2C/D, emerged as a top predictor. Genetic deletion of Kmt2c/d (but not Kmt2c alone) or their enzymatic activities erased genome-wide RT dynamics during cell differentiation. Sites that typically gain H3K4me1 in a KMT2C/D-dependent manner during differentiation failed to transition towards earlier RT, often without affecting transcriptional activation. Further analysis at KMT2C/D binding sites revealed a local requirement for KMT2C/D in promoting DNA replication origin firing. Our findings identify KMT2C/D-dependent H3K4me1 as a functional regulator of RT and origin firing, highlighting a causal relationship between the epigenome and DNA replication that is largely independent of transcription. These insights should be relevant to various diseases associated with KMT2C/D mutations. Mammalian genomes are replicated in a precise, cell-type-specific order during the S phase, a process that correlates with local transcriptional activity, chromatin modifications, and chromatin architecture. However, the causal relationships between these features and DNA replication timing (RT), particularly during cell fate transitions, remain largely unknown. Here, we employed machine learning to quantify chromatin features — including epigenetic marks, histone variants, and chromatin architectural factors — that best predict local RT under steady-state conditions and RT changes during early embryonic stem (ES) cell differentiation. We found that approximately one-third of the genome exhibits RT changes during differentiation. Chromatin features collectively predicted steady-state RT and RT changes with high accuracy. Notably, histone H3 lysine 4 monomethylation (H3K4me1), catalyzed by KMT2C/D, emerged as a top predictor. Genetic deletion of Kmt2c/d (but not Kmt2c alone) or their enzymatic activities erased genome-wide RT dynamics during cell differentiation. Sites that typically gain H3K4me1 in a KMT2C/D-dependent manner during differentiation failed to transition towards earlier RT, often without affecting transcriptional activation. Further analysis at KMT2C/D binding sites revealed a local requirement for KMT2C/D in promoting DNA replication origin firing. Our findings identify KMT2C/D-dependent H3K4me1 as a functional regulator of RT and origin firing, highlighting a causal relationship between the epigenome and DNA replication that is largely independent of transcription. These insights should be relevant to various diseases associated with KMT2C/D mutations.
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Project description:KMT2C and KMT2D are two of the most frequently mutated genes in bladder cancer and in histologically normal urothelium. In this study, we developed mouse models to investigate the molecular mechanism of Kmt2c/d loss in urothelial tumorigenesis.
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Project description:Genes encoding the histone H3 lysine 4 (H3K4) methyltransferases KMT2C and KMT2D are subject to deletion and mutation in pancreatic ductal adenocarcinomas (PDAC). We examined the functional and transcriptional consequences of loss of these methyltransferases in patients with PDAC. Patients with low KMT2C and KMT2D expression demonstrated a much-improved outcome compared with those expressing high levels. RNA-seq analysis of KMT2C or KMT2D loss in three cell lines (PANC1, SUIT2 and COLO357) identified 31 and 124 differentially expressed genes respectively, with 19 genes common to both methlytransferases. Gene set enrichment analysis, and correlation with publicly available patient gene expression (GEP) datasets, highlighted significant reductions in pathways relating to cell-cycle and cell growth, where gene expression correlated with KMT2C/D depletion. Furthermore, loss of Kmt2d in three murine cell lines increased sensitivity to the nucleoside analogue 5-fluorouracil (5FU). These experiments support critical, non-redundant, roles for KMT2D and KMT2C in PDAC, where depletion impacts cell-cycle to reduce cell proliferation, enhance response to specific chemotherapy, which may improve patient survival.
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Project description:Histone lysine methyltransferases KMT2C and KMT2D are among the most commonly mutated genes in the highly metastatic TNBC subtype of breast cancer. However, it is not known if mutations of either of these genes similarly effect epigenomic and transcriptomic landscape or if a specific downstream target might influence metastases. Here, we generated heterogenous Kmt2c or Kmt2d KO murine TNBC cell lines side-by-side and performed in vivo metastases assay in syngeneic immunocompetent mice. Deficiency for either Kmt2c or Kmt2d, both, induced brain metastases from formerly non-metastatic cells. scRNAseq showed activation of pro-inflammatory pathways but conversely also increase of immune checkpoint blocking genes. Interestingly, histone mass spectrometry revealed changes of H3K27 but not the main substrate H3K4. However, ChIPseq for both, H3K4 and H3K27 modifications showed significant changes compared to wildtype cells. Strikingly, genome occupancy of H3K27me3 was reduced while H3K27 demethylase KDM6A was enriched on genomes of KO cells. Integration with gene expression data revealed significant correlations with histone and KDM6A ChIPseq, identifying them as a main driver of Kmt2c or Kmt2d KO-specific gene regulation. Although our datasets revealed more unique than shared signatures, we found Mmp3 being a common target upon Kmt2c or Kmt2d KO. Indeed, downregulation of Mmp3 reversed induction of Kmt2c and Kmt2d KO-dependent brain metastases. Finally, we found that Kdm6a knockdown reduces Mmp3 levels, again, leading to reduction of brain metastases of Kmt2c or Kmt2d KO cells.
2024-05-07 | MSV000094701 | MassIVE
Project description:Histone lysine methyltransferases KMT2C and KMT2D are among the most commonly mutated genes in the highly metastatic TNBC subtype of breast cancer. However, it is not known if mutations of either of these genes similarly effect epigenomic and transcriptomic landscape or if a specific downstream target might influence metastases. Here, we generated heterogenous Kmt2c or Kmt2d KO murine TNBC cell lines side-by-side and performed in vivo metastases assay in syngeneic immunocompetent mice. Deficiency for either Kmt2c or Kmt2d, both, induced brain metastases from formerly non-metastatic cells. scRNAseq showed activation of pro-inflammatory pathways but conversely also increase of immune checkpoint blocking genes. Interestingly, histone mass spectrometry revealed changes of H3K27 but not the main substrate H3K4. However, ChIPseq for both, H3K4 and H3K27 modifications showed significant changes compared to wildtype cells. Strikingly, genome occupancy of H3K27me3 was reduced while H3K27 demethylase KDM6A was enriched on genomes of KO cells. Integration with gene expression data revealed significant correlations with histone and KDM6A ChIPseq, identifying them as a main driver of Kmt2c or Kmt2d KO-specific gene regulation. Although our datasets revealed more unique than shared signatures, we found Mmp3 being a common target upon Kmt2c or Kmt2d KO. Indeed, downregulation of Mmp3 reversed induction of Kmt2c and Kmt2d KO-dependent brain metastases. Finally, we found that Kdm6a knockdown reduces Mmp3 levels, again, leading to reduction of brain metastases of Kmt2c or Kmt2d KO cells.
2024-04-24 | GSE237390 | GEO
Project description:Histone lysine methyltransferases KMT2C and KMT2D are among the most commonly mutated genes in the highly metastatic TNBC subtype of breast cancer. However, it is not known if mutations of either of these genes similarly effect epigenomic and transcriptomic landscape or if a specific downstream target might influence metastases. Here, we generated heterogenous Kmt2c or Kmt2d KO murine TNBC cell lines side-by-side and performed in vivo metastases assay in syngeneic immunocompetent mice. Deficiency for either Kmt2c or Kmt2d, both, induced brain metastases from formerly non-metastatic cells. scRNAseq showed activation of pro-inflammatory pathways but conversely also increase of immune checkpoint blocking genes. Interestingly, histone mass spectrometry revealed changes of H3K27 but not the main substrate H3K4. However, ChIPseq for both, H3K4 and H3K27 modifications showed significant changes compared to wildtype cells. Strikingly, genome occupancy of H3K27me3 was reduced while H3K27 demethylase KDM6A was enriched on genomes of KO cells. Integration with gene expression data revealed significant correlations with histone and KDM6A ChIPseq, identifying them as a main driver of Kmt2c or Kmt2d KO-specific gene regulation. Although our datasets revealed more unique than shared signatures, we found Mmp3 being a common target upon Kmt2c or Kmt2d KO. Indeed, downregulation of Mmp3 reversed induction of Kmt2c and Kmt2d KO-dependent brain metastases. Finally, we found that Kdm6a knockdown reduces Mmp3 levels, again, leading to reduction of brain metastases of Kmt2c or Kmt2d KO cells.
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Project description:Histone lysine methyltransferases KMT2C and KMT2D are among the most commonly mutated genes in the highly metastatic TNBC subtype of breast cancer. However, it is not known if mutations of either of these genes similarly effect epigenomic and transcriptomic landscape or if a specific downstream target might influence metastases. Here, we generated heterogenous Kmt2c or Kmt2d KO murine TNBC cell lines side-by-side and performed in vivo metastases assay in syngeneic immunocompetent mice. Deficiency for either Kmt2c or Kmt2d, both, induced brain metastases from formerly non-metastatic cells. scRNAseq showed activation of pro-inflammatory pathways but conversely also increase of immune checkpoint blocking genes. Interestingly, histone mass spectrometry revealed changes of H3K27 but not the main substrate H3K4. However, ChIPseq for both, H3K4 and H3K27 modifications showed significant changes compared to wildtype cells. Strikingly, genome occupancy of H3K27me3 was reduced while H3K27 demethylase KDM6A was enriched on genomes of KO cells. Integration with gene expression data revealed significant correlations with histone and KDM6A ChIPseq, identifying them as a main driver of Kmt2c or Kmt2d KO-specific gene regulation. Although our datasets revealed more unique than shared signatures, we found Mmp3 being a common target upon Kmt2c or Kmt2d KO. Indeed, downregulation of Mmp3 reversed induction of Kmt2c and Kmt2d KO-dependent brain metastases. Finally, we found that Kdm6a knockdown reduces Mmp3 levels, again, leading to reduction of brain metastases of Kmt2c or Kmt2d KO cells.
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Project description:KMT2C and KMT2D are two of the frequently mutated epigenetic modifiers in urothelial cancer. To compare the histone post-translational modification with Kmt2c/d loss, we performed mass spectrometry analysis of histones from Kmt2c/d WT and dKO urothelial cells.
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Project description:The chromatin-based rules governing the selection and activation of replication origins remain to be elucidated. It is believed that DNA replication initiates from open chromatin domains, thus replication origins residing in regulatory elements that are located at open and active chromatin. However, we report here that lysine specific demethylase 1 (LSD1), which biochemically catalyzes H3K4me1/2 demethylation to favor chromatin condensation, interacts with the DNA replication machinery. We found that LSD1 level peaks in early S phase. We demonstrated that LSD1 promotes DNA replication by facilitating origin firing in euchromatic regions and through regulating replication timing. Indeed, euchromatic zones enriched in H3K4me2 are the preferred sites for pre-RC binding in early replication. Remarkably, LSD1 deficiency leads to a genome-wide switch from early to late in replication timing. We showed that LSD1-promoted DNA replication is mechanistically linked to the loading of TICRR (TopBP1-Interacting Checkpoint and Replication Regulator) onto the pre-RC and subsequent recruitment of the initiator Cdc45 during origin firing. Together, these results reveal an unexpected role for LSD1 in euchromatic origin firing and replication timing.
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