Methyl-CpG-Binding Protein MBD2 plays a critical role in maintenance and spread of DNA methylation of CpG islands and shores in cancer (methylation)
ABSTRACT: 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. 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 “rewriting” 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:To investigate the reciprocal modulation between microRNA (miRNA) and DNA methylation via exploring the correlation between miR-373 and methyl-CpG-binding domain protein (MBD)2.MiR-373 expression was examined using the TaqMan miRNA assay. Methylation of miR-373 was investigated using methylation-specific polymerase chain reaction, and recruitment of methyl binding proteins was studied using the chromatin immunoprecipitation assay. Mutation analysis was conducted using the QuikChange™ Site-Directed Mutagenesis kit. The activity of miR-373 gene promoter constructs and targeting at MBD2-three prime untranslated region (3'UTR) by miR-373 were evaluated by a dual-luciferase reporter gene assay.In hilar cholangiocarcinoma, miR-373 decreased and was closely associated with poor cell differentiation, advanced clinical stage, and shorter survival. The promoter-associated CpG island of miR-373 gene was hypermethylated and inhibited expression of miR-373. MBD2 was up-regulated and enriched at the promoter-associated CpG island of miR-373. Methylation-mediated suppression of miR-373 required MBD2 enrichment at the promoter-associated CpG island, and miR-373 negatively regulated MBD2 expression through targeting the 3'UTR.MiR-373 behaves as a direct transcriptional target and negative regulator of MBD2 activity through a feedback loop of CpG island methylation.
Project description:The methyl-CpG binding domain (MBD) proteins are key molecules in the interpretation of DNA methylation signals leading to gene silencing. We investigated their binding specificity at the constitutively methylated region of a CpG island containing the bidirectional promoter of the Breast cancer predisposition gene 1, BRCA1, and the Near BRCA1 2 (NBR2) gene. In HeLa cells, quantitative chromatin immunoprecipitation assays indicated that MBD2 is associated with the methylated region, while MeCP2 and MBD1 were not detected at this locus. MBD2 depletion (approximately 90%), mediated by a transgene expressing a small interfering RNA (siRNA), did not induce MeCP2 or MBD1 binding at the methylated area. Furthermore, the lack of MBD2 at the BRCA1-NBR2 CpG island is associated with an elevated level of NBR2 transcripts and with a significant reduction of induced-DNA-hypomethylation response. In MBD2 knockdown cells, transient expression of a Mbd2 cDNA, refractory to siRNA-mediated decay, shifted down the NBR2 mRNA level to that observed in unmodified HeLa cells. Variations in MBD2 levels did not affect BRCA1 expression despite its stimulation by DNA hypomethylation. Collectively, our data indicate that MBD2 has specific targets and its presence at these targets is indispensable for gene repression.
Project description:DNA hypermethylation is a common epigenetic abnormality in cancer and may serve as a useful marker to clone cancer-related genes as well as a marker of clinical disease activity. To identify CpG islands methylated in prostate cancer, we used methylated CpG island amplification (MCA) coupled with representational difference analysis (RDA) on prostate cancer cell lines. We isolated 34 clones that corresponded to promoter CpG islands, including 5 reported targets of hypermethylation in cancer. We confirmed the data for 17 CpG islands by COBRA and/or pyrosequencing. All 17 genes were methylated in at least 2 cell lines of a 21-cancer cell line panel containing prostate cancer, colon cancer, leukemia, and breast cancer. Based on methylation in primary tumors compared to normal adjacent tissues, NKX2-5, CLSTN1, SPOCK2, SLC16A12, DPYS and NSE1 are candidate biomarkers for prostate cancer (methylation range 50%-85%). The combination of NSE1 or SPOCK2 hypermethylation showed a sensitivity of 80% and specificity of 95% in differentiating cancer from normal. Similarly NKX2-5, SPOCK2, SLC16A12, DPYS and GALR2 are candidate biomarkers for colon cancer (methylation range 60%-95%) and GALR2 hypermethylation showed a sensitivity of 85% and specificity of 95%. Finally, SLC16A12, GALR2, TOX, SPOCK2, EGFR5 and DPYS are candidate biomarkers for breast cancer (methylation range 33%-79%) with the combination of EGFR5 or TOX hypermethylation showing a sensitivity of 92% and specificity of 92%. Expression analysis for eight genes that had the most hypermethylation confirmed the methylation associated silencing and reactivation with 5-aza-2'-deoxycytidine treatment. Our data identify new targets of transcriptional silencing in cancer, and provide new biomarkers that could be useful in screening for prostate cancer and other cancers.
Project description:Secreted frizzled-related protein 1 (SFRP1), which is an extracellular inhibitor involved in Wnt signalling, is downregulated by promoter hypermethylation in the early stages of colorectal tumorigenesis. Polycomb (PCG) and methyl-CpG-binding domain (MBD) proteins that serve a role in epigenetic gene regulation. The aim of the present study was to determine the role of PCG and MBD proteins in the regulation of SFRP1 gene expression in colorectal cancer (CRC), specifically in CRC cell lines and the human embryo intestinal mucosa cell line CCC-HIE-2. The methylation status of the SFRP1 gene promoter were analysed using methylation-specific PCR (MSP), whereas SFRP1 mRNA expression was analysed using reverse transcription-quantitative PCR. The association between PCG and MBD proteins and the SFRP1 gene was assessed, where associated proteins were screened by chromatin immunoprecipitation and their expression were subsequently knocked down using RNA interference to determine their role in the regulation of SFRP1 gene expression. The SFRP1 promoter was demonstrated to be hypermethylated in CRC cell lines and partially methylated in the non-cancerous cell line CCC-HIE-2. SFRP1 mRNA expression was significantly lower in CRC cell lines compared with that of CCC-HIE-2 cells. The expression of PCGs enhancer of zeste homolog 2 (EZH2) and BMI1, along with MBD2, was indicated to be upregulated with SFRP1 methylation in HCT116 and SW480 cells. The SFRP1 promoter region was enriched with EZH2 in CCC-HIE-2 cells and enriched with EZH2 and MBD2 in SW480 cells, whereas none of the proteins examined were indicated on the SFRP1 promoter in HCT116 cells. The expression of SFRP1 was reactivated by MBD2 small interfering (si)RNA but not by EZH2 siRNA in SW480 cells, but combined MBD2 and EZH2 knockdown effectively restored SFRP1 gene expression without affecting the methylation status of the SFRP1 promoter. In conclusion, data from the present study revealed that MBD2 and EZH2 regulated SFRP1 expression without affecting the hypermethylation of SFRP1 in CRC cell lines. Instead, the regulation of SFRP1 expression may be through a distinct mechanism, which warrants further investigation.
Project description:14-3-3sigma proteins regulate numerous cellular processes that are important to cancer development. One of its biological roles involves G2 cell-cycle arrest following DNA damage. It has also been reported that the loss of 14-3-3sigma expression via CpG methylation may contribute to malignant transformation by impairing the G2 cell-cycle checkpoint function, thereby allowing an accumulation of genetic defects. However, how the CpG methylation-dependent silencing mechanism works in relation to promoter methylation associated with methyl-CpG-binding proteins (MeCPs) is still unclear. To better understand the mechanism, we first examined the methylation status of the 14-3-3sigma promoter-associated CpG islands and 14-3-3sigma gene expression in a subset of prostate cancer cell lines using methylation-specific PCR (MSP), an HhaI-based DNA methylation assay, and reverse transcription-PCR (RT-PCR). We found that the 14-3-3sigma expression is lost in LNCaP and Tramp-C1 prostate cancer cell lines and that this expression is restored after treatment with epigenetic silencing modifiers 5-aza-2'-deoxycytidine (5-aza) and trichostatin A (TSA). These results imply transcriptional silencing via promoter-associated CpG methylation. Chromatin immunoprecipitation analysis revealed that methyl-CpG-binding protein 2 (MBD2) is associated preferentially to the methylated CpG island in the 14-3-3sigma promoter in LNCaP and Tramp-C1 cells but not in 14-3-3sigma-expressing PC3 and DU145 cells, which contain an unmethylated CpG island in the 14-3-3sigma promoter region. The 14-3-3sigma gene silencing because of CpG methylation correlates with binding of MBD2. In addition, the activation of 14-3-3sigma gene expression by a combination of 5-aza and TSA also involves the release of the MBD2 from the 14-3-3sigma promoter-methylated CpG island in LNCaP and Tramp-C1 cells. Furthermore, MBD2 knockdown by siRNA stimulated 14-3-3sigma expression in LNCaP cells. We also investigated whether the loss of 14-3-3sigma expression in LNCaP and Tramp-C1 cells affects cell proliferation by MTT assays. Interestingly, we observed that 14-3-3sigma-inactivated LNCaP and Tramp-C1 cells had markedly decreased cell proliferation and protein expression of proliferation cell nuclear antigen (PCNA) after restoration of 14-3-3sigma expression with 5-aza and TSA treatment. On the other hand, the same treatment did not significantly affect 14-3-3sigma-active PC3 and DU145 cells, which normally express 14-3-3sigma. Finally, 14-3-3sigma knockdown by siRNA resulted in increased proliferation in PC3 and DU145 cells. These findings suggest that the transcriptional silencing of the 14-3-3sigma gene is caused by promoter CpG island methylation associated with MBD2, and that this may play an important role in prostate cancer progression during the invasive and metastatic stages of the disease.
Project description:DNA methylation is one of the most important epigenetic mechanisms in regulating gene expression. Genome hypermethylation has been proposed as a critical mechanism in human malignancies. However, whole-genome quantification of DNA methylation of human malignancies has rarely been investigated, and the significance of the genome distribution of CpG methylation is unclear. We performed whole-genome methylation sequencing to investigate the methylation profiles of 13 prostate samples: 5 prostate cancers, 4 matched benign prostate tissues adjacent to tumor, and 4 age-matched organ-donor prostate tissues. Alterations of methylation patterns occurred in prostate cancer and in benign prostate tissues adjacent to tumor, in comparison with age-matched organ-donor prostates. More than 95% alterations of genome methylation occurred in sequences outside CpG islands. Only a small fraction of the methylated CpG islands had any effect on RNA expression. Both intragene and promoter CpG island methylations negatively affected gene expression. However, suppressions of RNA expression did not correlate with levels of CpG island methylation, suggesting that CpG island methylation alone might not be sufficient to shut down gene expression. Motif analysis revealed a consensus sequence containing Sp1 binding motif significantly enriched in the effective CpG islands.
Project description:Aberrant DNA methylation plays a significant role in nearly all human cancers and may contribute to disease progression to advanced phenotypes. Study of advanced prostate cancer phenotypes in the human disease is hampered by limited availability of tissues. We therefore took advantage of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model to study whether three different phenotypes of TRAMP tumors (PRIM, late-stage primary tumors; AIP, androgen-independent primary tumors; and MET, metastases) displayed specific patterns of CpG island hypermethylation using Restriction Landmark Genomic Scanning. Each tumor phenotype displayed numerous hypermethylation events, with the most homogeneous methylation pattern in AIP and the most heterogeneous pattern in MET. Several loci displayed a phenotype-specific methylation pattern; the most striking pattern being loci methylated at high frequency in PRIM and AIP but rarely in MET. Examination of the mRNA expression of three genes, BC058385, Goosecoid, and Neurexin 2, which exhibited nonpromoter methylation, revealed increased expression associated with downstream methylation. Only methylated samples showed mRNA expression, in which tumor phenotype was a key factor determining the level of expression. The CpG island in the human orthologue of BC058385 was methylated in human AIP but not in primary androgen-stimulated prostate cancer or benign prostate. The clinical data show a proof-of-principle that the TRAMP model can be used to identify targets of aberrant CpG island methylation relevant to human disease. In conclusion, phenotype-specific hypermethylation events were associated with the overexpression of different genes and may provide new markers of prostate tumorigenesis.
Project description:Cytosine methylation is an epigenetic and regulatory mark that functions in part through recruitment of chromatin remodeling complexes containing methyl-CpG binding domain (MBD) proteins. Two MBD proteins, Mbd2 and Mbd3, were previously shown to bind methylated or hydroxymethylated DNA, respectively; however, both of these findings have been disputed. Here, we investigated this controversy using experimental approaches and re-analysis of published data and find no evidence for methylation-independent functions of Mbd2 or Mbd3. We show that chromatin localization of Mbd2 and Mbd3 is highly overlapping and, unexpectedly, we find Mbd2 and Mbd3 are interdependent for chromatin association. Further investigation reveals that both proteins are required for normal levels of cytosine methylation and hydroxymethylation in murine embryonic stem cells. Furthermore, Mbd2 and Mbd3 regulate overlapping sets of genes that are also regulated by DNA methylation/hydroxymethylation factors. These findings reveal an interdependent regulatory mechanism mediated by the DNA methylation machinery and its readers.
Project description:CpG island arrays represent a high-throughput epigenomic discovery platform to identify global disease-specific promoter hypermethylation candidates along bladder cancer progression. DNA obtained from 10 pairs of invasive bladder tumours were profiled vs their respective normal urothelium using differential methylation hybridisation on custom-made CpG arrays (n=12 288 clones). Promoter hypermethylation of 84 clones was simultaneously shown in at least 70% of the tumours. SOX9 was selected for further validation by bisulphite genomic sequencing and methylation-specific polymerase chain reaction in bladder cancer cells (n=11) and primary bladder tumours (n=101). Hypermethylation was observed in bladder cancer cells and associated with lack of gene expression, being restored in vitro by a demethylating agent. In primary bladder tumours, SOX9 hypermethylation was present in 56.4% of the cases. Moreover, SOX9 hypermethylation was significantly associated with tumour grade and overall survival. Thus, this high-throughput epigenomic strategy has served to identify novel hypermethylated candidates in bladder cancer. In vitro analyses supported the role of methylation in silencing SOX9 gene. The association of SOX9 hypermethylation with tumour progression and clinical outcome suggests its relevant clinical implications at stratifying patients affected with bladder cancer.