In cancer, the DNA methylation patterns of the genome are altered with frequent hypermethylation and gene silencing of CpG island associated promoters, including tumour suppressor genes, in parallel with hypomethylation of repeat DNA sequences and gene activation. Critical, yet unanswered questions in cancer biology remain regarding the balance of hyper- and hypo-methylation in normal and cancer cells and the potential role that CpG binding proteins play in controlling the DNA methylation landscape. DNA methylation-induced suppression occurs through the action of methylated DNA binding domain (MBD) proteins. Notably, methylated-DNA binding (MBD) proteins are proposed to mediate silencing of gene expression by interacting with histone deacetylases (HDACs) and histone methyltransferases (HMTases) to induce repressive chromatin modifications. Furthermore, we previously have also shown that the methyl-binding domain protein MBD2 plays a critical role in aberrant de novo DNA methylation in the prostate cancer cell line LNCaP.
Here, to further investigate the role of MBD2 in promoting DNA hypermethylation and transcriptional repression in cancer, we performed stable shRNA knockdown of MBD2 in LNCaP cells. Upon MBD2 knockdown, we detected an increase in expression of typically hypermethylated genes (eg NPR3 and EN1). Using MBD2 chromatin immunoprecipitation (ChIP) analysis, we show loss of MBD2 occupancy at these CpG island promoters. Furthermore, we performed Illumina 450k arrays and clonal bisulphite sequencing and found that these CpG islands show a loss of methylation in MBD2 KD LNCaP cells. To further investigate the molecular mechanisms, we performed MBD2 co-immunoprecipitation on LNCaP cells and show that MBD2 is associated with canonical protein partners, HDAC1 as well as a novel binding partner DNMT1. Importantly, we demonstrate that MBD2 plays a critical role in the spread of DNA hypermethylation in cancer, through its recruitment of DNA methyltransferase enzymes DNMT1, and conversely, reduction of MBD2 results in hypomethylation of target sites suggesting that MBD2 may be a potential therapeutic target in cancer treatment.