Mus musculus
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA288306 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR2079680_1.fastq.gz | Fastqsanger.gz | |||
| SRR2079680_2.fastq.gz | Fastqsanger.gz | |||
| SRR2079690_1.fastq.gz | Fastqsanger.gz | |||
| SRR2079690_2.fastq.gz | Fastqsanger.gz | |||
| SRR2079700_1.fastq.gz | Fastqsanger.gz |
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Project description:We report the generation and analysis of high-throughput DNA methylation profiles at nucleotide resolution in a subset of targeted gene trap mouse mutants. Using high-throughput sequencing of bisulfite treated DNA, we generated DNA methylation percentage for CpG islands, and LacZ (reporter) gene in mice with the apparent silencing of the targeted gene promoter reflected by reduced reporter mRNA level. These results were contrasted with findings for a set of mutants with no silencing or CpG methylation following targeted mutagenesis using the same gene trap vector. Our findings supports the hypothesis that presence of the exogenous DNA in the targeting vector may influence the expression of genes in close proximity or may lead to promoter silencing of the target where the promoter is marked by CpG methylation. Examination of CpG methylation profiles in Knock-out and wild type mice We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. CpG Islands (samples labeled as CpG) and trans gene (samples labeled as LacZ) were amplified after Bisulfite treatment. Please note that the same gDNA was used to amplify CpG Island (Gene_CpG_KO ) and LacZ (Gene_LacZ_KO) reporter for the same gene. PCR product of amplification was gel separated, isolated and pooled. After libraries were prepared and sequenced, the alignment was performed. CpG island and LacZ alignments were done separately resulting in three different Processed Data files per gene investigated: Gene_CpG_KO, Gene_LacZ_KO and Gene_CpG_WT. LacZ reference is included in the submission, but is also available for download from KOMP Phenotype website (www.kompphenotype.org). Also please note that the libraries were prepared using Illumina TruSeq RNA Sample Prep Kit starting from adapter binding step as samples were double stranded Bisulfite treated DNA amplicons. So the library preparation was done as with RNASeq, but samples investigated were bisulfite treated.
2015-07-28 | E-GEOD-70317 | biostudies-arrayexpress
Project description:We report the generation and analysis of high-throughput DNA methylation profiles at nucleotide resolution in a subset of targeted gene trap mouse mutants. Using high-throughput sequencing of bisulfite treated DNA, we generated DNA methylation percentage for CpG islands, and LacZ (reporter) gene in mice with the apparent silencing of the targeted gene promoter reflected by reduced reporter mRNA level. These results were contrasted with findings for a set of mutants with no silencing or CpG methylation following targeted mutagenesis using the same gene trap vector. Our findings supports the hypothesis that presence of the exogenous DNA in the targeting vector may influence the expression of genes in close proximity or may lead to promoter silencing of the target where the promoter is marked by CpG methylation.
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Project description:In mouse development, long-term silencing by CpG island DNA methylation is specifically targeted to germline genes, however the molecular mechanisms of this specificity remain unclear. Here we demonstrate that the transcription factor E2F6, a member of the polycomb repressive complex 1.6 (PRC1.6), is critical to target and initiate epigenetic silencing at germline genes in early embryogenesis. Genome-wide, E2F6 binds preferentially to CpG islands in embryonic cells. E2F6 cooperates with MGA to silence a subgroup of germline genes in mouse embryonic stem cells and in vivo, a function that critically depends on the E2F6 marked box domain. Inactivation of E2f6 leads to a failure to deposit CpG island DNA methylation at these genes during implantation. Furthermore, E2F6 is required to initiate epigenetic silencing in early embryonic cells but becomes dispensable for the maintenance in differentiated cells. Our findings elucidate the mechanisms of epigenetic targeting of germline genes and provide a paradigm for how transient repression signals by DNA-binding factors in early embryonic cells are translated into long term epigenetic silencing during mammalian development.
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Project description:Cancer is characterised by DNA hypermethylation and gene silencing of CpG island-associated promoters, including tumour suppressor genes The methyl-CpG-binding domain (MBD) family of proteins bind to methylated DNA and can aid in the meditation of gene silencing by interaction with histone deacetylases and histone methyltransferases. However the mechanisms responsible for eliciting CpG island hypermethylation in cancer, and the potential role that MBD may proteins play in modulation of the methylome remain unclear. Our previous work demonstrated that MBD2 preferentially binds to the hypermethylated GSTP1 promoter CpG island in prostate cancer cells. Here, we use functional genetic approaches to investigate if MBD2 plays an active role in promoting DNA methylation. First, we show that loss of MBD2 results in inhibition of both maintenance and spread of de novo methylation of a transfected construct containing the GSTP1 promoter CpG island in prostate cancer cells and Mbd2-/- mouse fibroblasts. De novo methylation was rescued by transient expression of Mbd2 in Mbd2-/- cells. Second, we show that MBD2 depletion triggers significant hypomethylation genome-wide in prostate cancer cells with concomitant loss of MBD2 binding at promoter and enhancer regulatory regions. Finally, CpG islands and shores that become hypomethylated after MBD2 depletion in LNCaP cancer cells show significant hypermethylation in clinical prostate cancer, highlighting a potential active role of MBD2 in promoting cancer specific hypermethylation. Importantly, co-immunoprecipiation of MBD2 reveals that MBD2 associates with DNA methyltransferase (DNMT) enzymes 1 and 3A. Together our results demonstrate that MBD2 plays a critical role in “rewriting” the cancer methylome at specific regulatory regions.
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Project description:Cancer is characterised by DNA hypermethylation and gene silencing of CpG island-associated promoters, including tumour suppressor genes The methyl-CpG-binding domain (MBD) family of proteins bind to methylated DNA and can aid in the meditation of gene silencing by interaction with histone deacetylases and histone methyltransferases. However the mechanisms responsible for eliciting CpG island hypermethylation in cancer, and the potential role that MBD may proteins play in modulation of the methylome remain unclear. Our previous work demonstrated that MBD2 preferentially binds to the hypermethylated GSTP1 promoter CpG island in prostate cancer cells. Here, we use functional genetic approaches to investigate if MBD2 plays an active role in promoting DNA methylation. First, we show that loss of MBD2 results in inhibition of both maintenance and spread of de novo methylation of a transfected construct containing the GSTP1 promoter CpG island in prostate cancer cells and Mbd2-/- mouse fibroblasts. De novo methylation was rescued by transient expression of Mbd2 in Mbd2-/- cells. Second, we show that MBD2 depletion triggers significant hypomethylation genome-wide in prostate cancer cells with concomitant loss of MBD2 binding at promoter and enhancer regulatory regions. Finally, CpG islands and shores that become hypomethylated after MBD2 depletion in LNCaP cancer cells show significant hypermethylation in clinical prostate cancer, highlighting a potential active role of MBD2 in promoting cancer specific hypermethylation. Importantly, co-immunoprecipiation of MBD2 reveals that MBD2 associates with DNA methyltransferase (DNMT) enzymes 1 and 3A. Together our results demonstrate that MBD2 plays a critical role in “rewriting” the cancer methylome at specific regulatory regions.
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Project description:Cancer is characterised by DNA hypermethylation and gene silencing of CpG island-associated promoters, including tumour suppressor genes The methyl-CpG-binding domain (MBD) family of proteins bind to methylated DNA and can aid in the meditation of gene silencing by interaction with histone deacetylases and histone methyltransferases. However the mechanisms responsible for eliciting CpG island hypermethylation in cancer, and the potential role that MBD may proteins play in modulation of the methylome remain unclear. Our previous work demonstrated that MBD2 preferentially binds to the hypermethylated GSTP1 promoter CpG island in prostate cancer cells. Here, we use functional genetic approaches to investigate if MBD2 plays an active role in promoting DNA methylation. First, we show that loss of MBD2 results in inhibition of both maintenance and spread of de novo methylation of a transfected construct containing the GSTP1 promoter CpG island in prostate cancer cells and Mbd2-/- mouse fibroblasts. De novo methylation was rescued by transient expression of Mbd2 in Mbd2-/- cells. Second, we show that MBD2 depletion triggers significant hypomethylation genome-wide in prostate cancer cells with concomitant loss of MBD2 binding at promoter and enhancer regulatory regions. Finally, CpG islands and shores that become hypomethylated after MBD2 depletion in LNCaP cancer cells show significant hypermethylation in clinical prostate cancer, highlighting a potential active role of MBD2 in promoting cancer specific hypermethylation. Importantly, co-immunoprecipiation of MBD2 reveals that MBD2 associates with DNA methyltransferase (DNMT) enzymes 1 and 3A. Together our results demonstrate that MBD2 plays a critical role in ârewritingâ the cancer methylome at specific regulatory regions. LNCaP prostate cancer cell line clones with reduced MBD2 expression were establised by using shRNA to MBD2 and scrambled control clones were established with scrambled control shRNA. To interrogate expression changes induced by MBD2 knock-down we profiled three stably transfected scrambled control clones and three MBD2 knockdown clones on Affymetrix HuGene 1.0ST expression arrays. Differential expression analysis was carried out to identified genes up-/down-regulated by MBD2 knockdown.
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Project description:RNA modifications are important regulators of transcript activity and an increasingly emerging body of data suggest that the epitranscriptome and its associated enzymes are altered in human tumors. In this study, the initial screening for genetic and epigenetic defects of 5-methylcytosine RNA methyltransferases in tranformed cell lines, identified the NOL1/NOP2/Sun domain family member 7 (NSUN7) as undergoing promoter CpG island hypermethylation-associated transcriptional silencing in a cancer-specific manner. NSUN7 epigenetic inactivation was common in liver malignant cells and we coupled bisulfite conversion of cellular RNA with next-generation sequencing (bsRNA-seq) to found the RNA targets of this poorly characterized putative methyltransferase. Using knock-out and restoration-of-function models, we observed that the mRNA of the coiled-coil domain containing 9B (CCDC9B) gene required NSUN7-mediated methylation for transcript instability. Most important, proteomics analyses determined that CCDC9B loss impaired protein levels of its partner, the MYC-regulator Influenza Virus NS1A Binding Protein (IVNS1ABP), creating sensitivity to bromodomain inhibitors in those liver cancer cells exhibiting NSUN7 epigenetic silencing. The DNA methylation-associated loss of NSUN7 was also observed in primary liver tumors where it was associated with poor overall survival and an immune-like signature, providing a potential attractive therapeutic niche for the current use of immune checkpoint inhibitors in hepatocellular carcinoma.
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