Promoter CpG content regulates DNMT-dependent silencing dynamics
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ABSTRACT:
ORGANISM(S): Escherichia coli Cricetulus griseus
PROVIDER: GSE224403 | GEO | 2023/03/01
REPOSITORIES: GEO
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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.
2015-07-28 | GSE70317 | GEO
Project description:Most mammalian RNA Polymerase II initiation events occur at CpG islands, which are rich in CpGs and devoid of DNA methylation. Despite their relevance for gene regulation, it is unknown to what extent the CpG dinucleotide itself actually contributes to promoter activity. To address this question, we determined the transcriptional activity of a large number of chromosomally integrated promoter constructs and monitored binding of transcription factors assumed to play a role in CpG island activity. This revealed that CpG density significantly improves motif-based prediction of transcription factor binding. Our experiments also show that high CpG density alone is insufficient for transcriptional activity, yet results in increased transcriptional output when combined with particular transcription factor motifs. Yet, this CpG contribution to promoter activity is independent of DNA methyltransferase activity. Together this refines our understanding of mammalian promoter regulation as it shows that high CpG density within CpG islands directly contributes to an environment permissive for full transcriptional activity.
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Project description:We assayed CpG methylation in cerebral cortex of neurologically and psychiatrically normal human postmortem specimens, as well as mouse forebrain specimens. Cross-species human-mouse DNA methylation conservation analysis shows that DNA methylation is not correlated with sequence conservation. Instead, greater DNA methylation conservation is correlated with increasing CpG density. We identified key genomic features that can be targeted for identification of epigenetic loci that may be developmentally and evolutionarily conserved and wherein aberrations in DNA methylation patterns can confer risk for disease.
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Project description:Epigenetic modifications and nucleosome positioning play an important role in modulating gene expression. However, how the patterns of epigenetic modifications and nucleosome positioning are established around promoters is not well understood. Here, we have addressed these questions in a series of genome-wide experiments coupled to a novel bioinformatic analysis approach. Our data reveal a clear correlation between CpG density, promoter activity and accumulation of active or repressive histone marks. CGI boundaries define the chromatin promoter regions that will be epigenetically modified. CpG-rich promoters are targeted by histone modifications and histone variants, while CpG-poor promoters are regulated by DNA methylation. CGIs boundaries, but not transcriptional activity, are essential determinants of H2A.Z positioning in vicinity of the promoters, suggesting that the presence of H2A.Z is not related to transcriptional control. Accordingly, H2A.Z depletion has no impact on gene expression of arrested mouse embryonic fibroblasts. Therefore, the underlying DNA sequence, the promoter CpG density and, to a lesser extent, transcriptional activity, are key factors implicated in promoter chromatin architecture.
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Project description:We assayed CpG methylation in cerebral cortex of neurologically and psychiatrically normal human postmortem specimens, as well as mouse forebrain specimens. Cross-species human-mouse DNA methylation conservation analysis shows that DNA methylation is not correlated with sequence conservation. Instead, greater DNA methylation conservation is correlated with increasing CpG density. We identified key genomic features that can be targeted for identification of epigenetic loci that may be developmentally and evolutionarily conserved and wherein aberrations in DNA methylation patterns can confer risk for disease. Characterization of evolutionary signatures of DNA methylation in the brain
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Project description:The lysine-specific demethylase 2A gene (KDM2A) is ubiquitously expressed and its transcripts consist of several alternatively spliced forms, including KDM2A and the shorter form N782 that lacks the 3’ end encoding F-box and LRR. KDM2A binds to numerous CpG-rich genomic loci and regulates various cellular activities; however, the mechanism of the pleiotropic function is unknown. Here, we identify the mechanism of KDM2A played by its CXXC-PHD domain. KDM2A is necessary for a rapid proliferation of post-natal keratinocytes while its 3’ end eclipses the stimulatory effect. EGFP-N782 binds to chromatin together with the XRCC5/6 complex, and the CXXC-PHD domain regulates the CpG-rich IGFBPL1 promoter. In vitro, CXXC-PHD enhances binding of nuclear extract ORC3 to the CpG-rich promoter, but not to the AT-rich DIP2B promoter to which ORC3 binds constitutively. Furthermore, CXXC-PHD recruits 94 nuclear factors involved in replication, ribosome synthesis, and mitosis, including POLR1A to the IGFBPL1 promoter. This recruitment is unprecedented; however, the result suggests that these nuclear factors bind to their cognate loci, as substantiated by the result that CXXC-PHD recruits POLR1A to the rDNA promoter. We propose that CXXC-PHD promotes permissiveness for nuclear factors to interact, but involvement of the XRCC5/6 complex in the recruitment is undetermined.
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Project description:Understanding cell-type-specific epigenetic codes on a global level is a major challenge after the sequencing of the human genome has been completed. Here we applied methyl-CpG immunoprecipitation (MCIp) to obtain comparative methylation profiles of coding and noncoding genes in three human tissues, testis, brain, and monocytes. Forty-four mainly testis-specific promoters were independently validated using bisulfite sequencing or single-gene MCIp, confirming the results obtained by the MCIp microarray approach. We demonstrate the previously unknown somatic hypermethylation at many CpG-rich, testis-specific gene promoters, in particular in ampliconic areas of the Y chromosome. We also identify a number of miRNA genes showing tissue-specific methylation patterns. The comparison of the obtained tissue methylation profiles with corresponding gene expression data indicates a significant association between tissue-specific promoter methylation and gene expression, not only in CpG-rich promoters. In summary, our study highlights the exceptional epigenetic status of germ-line cells in testis and provides a global insight into tissue-specific DNA methylation patterns. Keywords: MCIp-on-Chip The promoter hypomethylation profiles of the two somatic tissues (monocytes and brain) were compared to human testis (reference). The set includes two hybridisations with independent testis samples for each comparison. Each comparison uses a set of two microarrays.
2007-08-07 | E-GEOD-5548 | biostudies-arrayexpress
Project description:DNA methylation changes in neuroblastoma, a clinically-heterogeneous pediatric tumor, have been described essentially in promoter regions. We analyzed the DNA methylome of neuroblastoma using high-density microarrays and observed differential methylation not only in promoters but also in intragenic and intergenic regions at both CpG and non-CpG sites. These epigenetic changes showed a non-random distribution relative functional chromatin domains, and targeted development and cancer-related genes, relevant for neuroblastoma pathogenesis. CCND1, a gene overexpressed in neuroblastoma, showed hypomethylation of gene-body and upstream regulatory regions. Furthermore, tumors with diverse clinical-risk showed clear differences affecting CpG and, remarkably, non-CpG sites. Non-CpG methylation was present in clinically-favorable tumors and affected genes such as ALK, where non-CpG methylation correlated with low gene expression. Finally, we identified CpG and non-CpG methylation signatures which correlated with patient’s age at time-points relevant for neuroblastoma clinical behavior, and targeted genes related to neural development and neural crest regulatory network We report on the first DNA methylomes of neuroblastoma tumors using high-density microarrays. DNA methylation changes in this pediatric tumor affected both CpG and non-CpG sites associated with developmental and cancer-related genes such as CCND1 and ALK. Our study also provides new insights into the molecular basis of the heterogeneous clinical behavior of neuroblastoma.
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Project description:DNA methylation changes in neuroblastoma, a clinically-heterogeneous pediatric tumor, have been described essentially in promoter regions. We analyzed the DNA methylome of neuroblastoma using high-density microarrays and observed differential methylation not only in promoters but also in intragenic and intergenic regions at both CpG and non-CpG sites. These epigenetic changes showed a non-random distribution relative functional chromatin domains, and targeted development and cancer-related genes, relevant for neuroblastoma pathogenesis. CCND1, a gene overexpressed in neuroblastoma, showed hypomethylation of gene-body and upstream regulatory regions. Furthermore, tumors with diverse clinical-risk showed clear differences affecting CpG and, remarkably, non-CpG sites. Non-CpG methylation was present in clinically-favorable tumors and affected genes such as ALK, where non-CpG methylation correlated with low gene expression. Finally, we identified CpG and non-CpG methylation signatures which correlated with patient’s age at time-points relevant for neuroblastoma clinical behavior, and targeted genes related to neural development and neural crest regulatory network We report on the first DNA methylomes of neuroblastoma tumors using high-density microarrays. DNA methylation changes in this pediatric tumor affected both CpG and non-CpG sites associated with developmental and cancer-related genes such as CCND1 and ALK. Our study also provides new insights into the molecular basis of the heterogeneous clinical behavior of neuroblastoma.
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