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 â??rewritingâ?? 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 methylation changes induced by MBD2 knock-down we profiled three stably transfected scrambled control clones and three MBD2 knockdown clones on Illumina HumanMethylation450K arrays. Differential methylation analysis was carried out to identified CpG sites hypo-/hyper-methylated as a result of MBD2 knockdown.

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.

Project description:DNA methylation is critical for development and is strongly associated with gene regulation. Variation in the DNA methylome between closely related species may reveal unique functional adaptation. We have implemented a novel inter-primate DNA methylation genome-wide analysis between human, chimpanzee and rhesus macaque to identify human species-specific Differentially Methylated Regions (human s-DMRs) in orthologous loci. We analysed the peripheral blood cell DNA methylomes of these primates and identified 22,758 hypomethylated and 15,858 hypermethylated human s-DMRs. These s-DMRs are globally enriched within weak promoter, enhancer and transcribed regions via comparison with ChromHMM segmentation. Human s-DMRs, (both hypo- and hypermethylated) are found to be more prevalent in CpG Island shores than within the islands themselves (?2 P = 1.80 x 10-32). Examining human-specific Transcription Factor Binding Site motif change within CpG islands, we show gain and loss, in hypomethylated and hypermethylated s-DMRs, respectively, of CTCF motifs. Epigenetically the most divergent human-specific locus was the immunological Leukotriene B4 receptor (LTB4R, aka BLT1 receptor), due to collocating hypomethylated s-DMRs within the promoter CpG island and shore, as well as inverse increased gene body methylation. This gene is vital in host immune responses and associated with the pathogenesis of a wide range of human inflammatory diseases. This finding was supported by additional neutrophil-only DNA methylome and lymphoblastoid H3K4me3 chromatin comparative data. Functional investigation of the consequences of these epigenetic differences identified this receptor to have increased expression, and have a higher response to the LTB4 ligand in human versus rhesus macaque peripheral blood mononuclear cells. This result further emphasises the exclusive nature of the human immunological system, its divergent adaptation even from closely related primates, and the power of comparative epigenomics to identify and understand human uniqueness. DNA methylome analysis of pooled Human, Chimpanzee and Macaque

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Homo sapiens

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