hMLH1 promoter methylation and silencing in primary endometrial cancers are associated with specific alterations in MBDs occupancy and histone modifications

Abstract

Objective-To investigate the relationship between hMLH1 promoter methylation and changes in chromatin composition. To study how the occupancy of methyl CpG binding domain proteins (MBDs) and histone acetylation/methylation in hMLH1 promoter may participate in hMLH1 silencing.Methods-64 endometrial cancer samples were screened for hMLH1 mRNA expression. hMLH1 promoter methylation status was confirmed by methylation-specific PCR in cancers with high and low levels of hMLH1 expression. Chromatin immunoprecipitation was performed to compare the MBD occupancy and histone modifications between the methylated/silenced and unmethylated/active hMLH1 genes in multiple primary endometrial cancers.Results-We demonstrated that MeCP2, MBD1 and MBD2, but not MBD3 and MBD4, specifically bind to methylated hMLH1 promoters. Hyperacetylated histones H3 and H4 were found to be associated with the unmethylated and transcriptionally active hMLH1 promoters. While H3 lysine-4 methylation was present in unmethylated hMLH1 promoters, H3 lysine-9 methylation was found exclusively in methylated promoters. Western blot analysis showed that similar global levels of MBDs and histones were present in the two cancer groups with high and low hMLH1 expression.Conclusions-A distinct combination of MBDs and histone modification is associated with the silencing of the hMLH1 gene. The changes in hMLH1 chromatin composition are closely related to methylation status of hMLH1 promoters. These changes are not accounted by the global expression levels of MBDs and histones in endometrial cancers.

Modulation of histone methylation and MLH1 gene silencing by hexavalent chromium

“…Here we analyzed the changes of H3K9me2 and H3K4me3 occupancy at the MLH1 promoter during chromate-induced silencing of the active MLH1 gene which differs from studies that re-activated silenced MLH1 gene in cancer cells. Our findings demonstrate that the enrichment of H3K9me2 at MLH1 promoter is highly correlated with MLH1 gene inactivation supporting the previous reports that H3K9me2 is important for both initiation and maintenance of gene silencing (McGarvey et al, 2006; Xiong et al, 2006). We do not know whether acute exposure of chromate is able to induce DNA hypermethylation in MLH1 promoter.…”

“…Recently, other factors including histone modification (McGarvey et al , 2006; Xiong et al , 2006; Meng et al , 2007) and nucleosome occupancy (Lin et al, 2007) at the promoter region have been implicated in the regulation of MLH1 gene expression. Enrichment of the repressive histone marks, H3K9me1, H3K9me2, H3K9me3, H3K27me2, and H3K27me3 were found at the hypermethylated MLH1 promoter in MLH1 silenced cells compared with the unmethylated active promoter in MLH1 expressing cells (McGarvey et al, 2006).…”

“…Enrichment of the repressive histone marks, H3K9me1, H3K9me2, H3K9me3, H3K27me2, and H3K27me3 were found at the hypermethylated MLH1 promoter in MLH1 silenced cells compared with the unmethylated active promoter in MLH1 expressing cells (McGarvey et al, 2006). Treatment of MLH1 silenced cells with 5-aza-2′-deoxycytidine (5-Aza-dC), a DNA methyltransferase inhibitor, not only reactivated MLH1 expression in those cells but also enriched the active histone marks H3K9Ac and H3K4me2 in its promoter (McGarvey et al, 2006; Xiong et al, 2006). Interestingly, the occupancy of H3K9me2 in the promoter was strikingly reduced after 5-Aza-dC treatment while the other repressive marks H3K9me3, H3K27me2 and H3K27me3 remained unchanged (McGarvey et al, 2006).…”

Hypoxia-Induced Epigenetic Regulation and Silencing of the BRCA1 Promoter

“…It has been reported that the methyl DNA binding proteins, MDB2 and MeCP2, bind not just to densely methylated re- on May 10, 2018 by guest http://mcb.asm.org/ gions of DNA but also to isolated methyl CpG dinucleotides (45,54). Hence, we also tested by ChIP for the presence of MDB2 and MeCP2 at the BRCA1 promoters in MCF-7 cells exposed to hypoxia.…”

Acquired vorinostat resistance shows partial cross-resistance to ‘second-generation’ HDAC inhibitors and correlates with loss of histone acetylation and apoptosis but not with altered HDAC and HAT activities

“…Recently, we reported the generation of two MDR-independent, vorinostat-resistant sublines [12]: these were the DNA mismatch repair (MMR)-proficient HCT116ch3 colorectal adenocarcinoma cell line (supplemented with chromosome 3 harboring the wildtype copy of the MLH1 gene to compensate for the MLH1 gene truncating mutation present in the parental MMRdeficient HCT116 cell line) and the MMR-deficient HCT116ch2 cell line (supplemented with the MLH1-irrelevant chromosome 2 for chromosome balance). Although a relationship between MLH1 expression and histone acetylation has been suggested [13,14], this vorinostat-induced resistance was independent of the presence or absence of MLH1 protein. Noteworthy, these vorinostat-resistant sublines were cross-resistant to the HDAC inhibitor trichostatin A but retained sensitivity to non-HDAC inhibitor-type anticancer agents.…”