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Epigenetically Silenced Genes Found Using Methylated DNA Immunoprecipitation in Colon Cancer Cells Deficient in DNMTs

ABSTRACT: CpG island promoter hypermethylation of tumor suppressor genes is a common hallmark of human cancer, and new large-scale epigenomic technologies might be useful in our attempts to define the complete DNA hypermethylome of tumor cells. Here we report a functional search for hypermethylated CpG islands using the colorectal cancer cell line HCT-116, in which two major DNA methyltransferases, DNMT1 and DNMT3b, have been genetically disrupted (DKOcells). Using methylated DNA immunoprecipitation (MeDIP) methodology in conjunction with promoter microarray analyses we found that DKO cells experience a significant loss of hypermethylated CpG islands. Further characterization of these candidate sequences demonstrates CpG island promoter hypermethylation and silencing of genes with potentially important roles in tumorigenesis, such as the Ras guanine-nucleotide release factor RASGRF2, the apoptosis-associated basic helixloop transcription factor BHLHB9, and the homeobox gene HOXD1. Hypermethylation of these genes occurs already in premalignant lesions and accumulates during tumorigenesis. Thus, our results demonstrate the usefulness of DNMT genetic disruption strategies combined with MeDIP in searching for unknown hypermethylated candidate genes in human cancer that might aid our understanding of the biology of the disease and be of potential translational use. Keywords: Human Proximal Promoter Array; DNA Methylation

ORGANISM(S): Homo sapiens

PROVIDER: GSE9267 | GEO | 2008/01/01

SECONDARY ACCESSION(S): PRJNA102899

REPOSITORIES: GEO

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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 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.

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Epigenetically Silenced Genes Found Using Methylated DNA Immunoprecipitation in Colon Cancer Cells Deficient in DNMTs

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