Methyl-binding domain protein 2-dependent proliferation and survival of breast cancer cells.

Methyl cytosine binding domain protein 2 (MBD2) has been shown to bind to and mediate repression of methylated tumor suppressor genes in cancer cells, where repatterning of CpG methylation and associated gene silencing is common. We have investigated the role of MBD2 in breast cancer cell growth and tumor suppressor gene expression. We show that stable short hairpin RNA (shRNA)-mediated knockdown of MBD2 leads to growth suppression of cultured human mammary epithelial cancer lines, SK-BR-3, MDA-MB-231, and MDA-MB-435. The peak antiproliferative occurs only after sustained, stable MBD2 knockdown. Once established, the growth inhibition persists over time and leads to a markedly decreased propensity for aggressive breast cancer cell lines to form in vivo xenograft tumors in Bagg Albino (BALB)/C nu/nu mice. The growth effects of MBD2 knockdown are accompanied by derepression of tumor suppressor genes, including DAPK1 and KLK10. Chromatin immunoprecipitation assays and bisulfite sequencing show MBD2 binding directly to the hyper methylated and CpG-rich promoters of both DAPK1 and KLK10. Remarkably, the promoter CpG island-associated methylation of these genes remained stable despite robust transcriptional activation in MBD2 knockdown cells. Expression of a shRNA-resistant MBD2 protein resulted in restoration of growth and resilencing of the MBD2-dependent tumor suppressor genes. Our data suggest that uncoupling CpG methylation from repressive chromatin remodeling and histone modifications by removing MBD2 is sufficient to initiate and maintain tumor suppressor gene transcription and suppress neoplastic cell growth. These results show a role for MBD2 in cancer progression and provide support for the prospect of targeting MBD2 therapeutically in aggressive breast cancers.

The role of the epigenetic signal, DNA methylation, in gene regulation during erythroid development.

The sequence complexity of the known vertebrate genomes alone is insufficient to account for the diversity between individuals of a species. Although our knowledge of vertebrate biology has evolved substantially with the growing compilation of sequenced genomes, understanding the temporal and spatial regulation of genes remains fundamental to fully exploiting this information. The importance of epigenetic factors in gene regulation was first hypothesized decades ago when biologists posited that methylation of DNA could heritably alter gene expression [Holliday and Pugh, 1975. Science 187(4173), 226-232; Riggs, 1975. Cytogenet. and Cell Genet.14(1), 9-25; Scarano et al., 1967. Proc. Natl. Acad. Sci. USA 57(5), 1394-1400)]. It was subsequently shown that vertebrate DNA methylation, almost exclusively at the 5' position of cytosine in the dinucleotide CpG, played a role in a number of processes including embryonic development, genetic imprinting, cell differentiation, and tumorigenesis. At the time of this writing, a large and growing list of genes is known to exhibit DNA methylation-dependent regulation, and we understand in some detail the mechanisms employed by cells in using methylation as a regulatory modality. In this context, we revisit one of the original systems in which the role of DNA methylation in vertebrate gene regulation during development was described and studied: erythroid cells. We briefly review the recent advances in our understanding of DNA methylation and, in particular, its regulatory role in red blood cells during differentiation and development. We also address DNA methylation as a component of erythroid chromatin architecture, and the interdependence of CpG methylation and histone modification.