DNA methylation and histone modifications are two epigenetic mechanisms which influence gene transcription. In cancers, these epigenetic machineries are dysregulated leading to tumor suppressor gene (TSG) silencing. Our interests are to understand the players involved in maintaining the DNA methylation patterns that persist through cell division in cancer cells.

We and others have demonstrated that DNA hypermethylated TSGs contain specific histone modifications. We sought to determine whether altering histone modifications would affect DNA methylation patterns. Studies have shown a link between the polycomb group protein, Ezh2, and its associated histone mark, H3-K27me3, and DNA methylation. We therefore hypothesized that Ezh2 was important in maintaining DNA methylation at silenced TSGs. Upon depletion of H3-K27me3 by siRNA knockdown of Ezh2, we observed that TSG expression and DNA methylation remained unchanged. However, unmethylated low expressing genes increased in expression after Ezh2 knockdown. Therefore, this suggested that Ezh2 modulates expression of genes which are basally expressed, but is not necessary for maintaining TSG DNA methylation and silencing.

To further understand the players involved in maintaining TSG silencing we sought to better understand the function of the maintenance methyltransferase, Dnmt1. While initially identified for its methyltransferase activity, previous work has demonstrated that Dnmt1 lacking the catalytic domain also represses transcription of a reporter gene partially through interactions with histone deacetylases. Recently, other histone modifying enzymes have been shown to associate with Dnmt1. We hypothesized that Dnmt1, in addition to methylating DNA, acts as a scaffolding protein able to modulate expressions of genes partially through its interactions with other protein partners. Wildtype or catalytically inactive mutant DNMT1 was introduced into DNMT1-/- cell line and expression, DNA methylation, and chromatin were analyzed at specific loci. We found that a catalytically inactive Dnmt1 represses expression of the Dnmt1 target genes and that DNA methylation was not required for gene repression. Additionally, active histone modifications were lost upon recruitment of Dnmt1 to the promoters of the target genes. This illustrated that Dnmt1 represses gene expression without its catalytic activity and that the chromatin is affected upon recruitment of Dnmt1 protein to gene promoters.