Epigenetic crosstalk between DNA demethylation and histone acetylation

Abnormal methylation patterns such as regional hypermethylation and genomic hypomethylation often result in transcriptional changes of critical genes that are central to the progression of human cancers. It is therefore important to identify the mechanisms that are responsible for the alterations in order to identify proper pharmacological targets. This thesis examines whether specific cellular factors are involved in establishing the state of DNA hypomethylation in cancer cells and whether changes in chromatin structure could affect DNA methylation. MBD2 is a protein that has been previously characterized to possess distinct transcription activities; it can either function as a methylation-dependent transcription repressor, a methylation-independent transcription activator, as well as an inducer of DNA demethylation. Chapters 3 and 4 demonstrate that MBD2 induces gene-specific DNA demethylation in pancreatic and bladder cancer cells by recruiting transcriptional activator AP-2, Sp1 and the histone acetyltransferase CBP to the associated promoters. These results substantiate the idea that demethylation induced by MBD2 might facilitate the recruitment of transcription factors to the gene to activate its expression. Histone deacetylase (HDAC) inhibitors are drugs designed to target chromatin modification. In chapter 5, we showed that increasing histone acetylation by HDAC inhibitor TSA was associated with a significant decrease in global methylation. TSA also induces histone acetylation, DNA demethylation and expression of specific methylated tumor suppressor genes, such as E-CADHERIN and RARbeta2 in different human cancer cell lines. Our findings provide evidence for a reversible crosstalk between histone acetylation and DNA demethylation, which has significant implications on the use of HDAC inhibitors in clinic and on our understanding of the processes leading to deregulated genomic methylation patterns in cancer. Targeting promoter hypermethylation of tumor suppressor genes is identified as critical event for altering states of methylation in cancer cells. However, it is unclear how are these methylation patterns stably preserved to maintain transcriptional silencing of the targeted genes. In chapter 6, we found that MeCP2, a methylated DNA binding protein, has the ability to protect its target sequence against pharmacologically-induced DNA demethylation. We used deletion analysis to delineate the functional domains of MeCP2 required to suppress demethylation. Our results indicate that both the methylated DNA binding domain (MBD) and C-terminal domains of MeCP2 are required for the protein to protect methylation of the associated transcript. Interestingly, deletion analysis separates the effects of MeCP2 on gene expression, histone acetylation and DNA demethylation, suggesting this protein can modulate chromatin modifications and DNA methylation through distinct functional units.