Unmasking of epigenetically silenced candidate tumor suppressor genes by removal of methyl-CpG-binding domain proteins.

Methyl-cytosine-phosphate-guanine (CpG)-binding domain (MBD) proteins are bound to hypermethylated promoter CpG islands of tumor suppressor genes in human cancer cells, although a direct causal relationship at the genome-wide level between MBD presence and gene silencing remains to be demonstrated. To this end, we have inhibited the expression of MBD proteins in HeLa cells by short hairpin RNAs; and studied the functional consequences of MBD depletion using microarray-based expression analysis in conjunction with extensive bisulfite genomic sequencing and chromatin immunoprecipitation. The removal of MBDs results in a release of gene silencing associated with a loss of MBD occupancy in 5'-CpG islands without any change in the DNA methylation pattern. Our results unveil new targets for epigenetic inactivation mediated by MBDs in transformed cells, such as the cell adhesion protein gamma-parvin and the fibroblast growth factor 19, where we also demonstrate their bona fide tumor suppressor features. Our data support a fundamental role for MBD proteins in the direct maintenance of transcriptional repression of tumor suppressors and identify new candidate genes for epigenetic disruption in cancer cells.

Regional methylation of the 5' end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells.

In mammalians, demethylation of specific promoter regions often correlates with gene activation; inversely, dense methylation of CpG islands leads to gene silencing, probably mediated by methyl-CpG binding proteins. In cell lines and cancers, inhibition of tissue-specific genes and tumor suppressor genes expression seems to be related to such hypermethylation. The 5' end of the breast cancer predisposition gene BRCA1 is embedded in a large CpG island of approximately 2.7 kb in length. In human sporadic breast cancers, the down-regulation of BRCA1 does not seem to be related to BRCA1 gene alterations. Southern blot analysis and the bisulfite sequencing method indicate that the BRCA1 CpG island is regionally methylated in all human tissues analyzed and unmethylated in the gametes, suggesting a role for DNA methylation in the control of gene expression. We have therefore investigated the potential role of methyl-CpG binding proteins in the regulation of BRCA1 gene expression. In vitro, partial methylation of constructs containing this region strongly inhibits gene expression in the presence of MeCP2 protein. Moreover, in the five human cell lines analyzed, chemically induced hypomethylation is associated with BRCA1 gene activation. These data suggest that methyl-CpG binding proteins might be associated with the control of BRCA1 gene expression and that methyl-DNA binding proteins may participate in the regulation of gene expression in mammalian cells.