In human cancers, epigenetic silencing is a common mechanism by which tumor suppressor genes are inactivated, and is associated with aberrant methylation of promoter region CpG islands and local changes in chromatin structure. However, the mechanisms that drive this event are unclear. TMS1 (Target of Methylation-mediated Silencing) is an example of a gene that undergoes epigenetic silencing in a significant proportion of breast and other cancers. TMS1 encodes a bipartite protein that contains the pyrin (PYD) and caspase (CARD) recruitment domains and promotes cell apoptosis and inflammation. The role of TMS1 suggests that its repression allows abnormal cells to escape apoptosis and proliferate, thus leading to cancer development. To gain further insight into the mechanisms underlying aberrant DNA methylation and epigenetic silencing in cancer, we used TMS1 as a model gene to study the role of histone modifications in gene silencing. Chromatin immunoprecipitation was used to profile histone modifications across the TMS1 locus in normal diploid fibroblasts and cancer cell lines that were either umethylated and expressed TMS1 or methylated and silenced for TMS1. Histone variant H2A.Z exhibited a localization profile at TMS1 that was similar in cells methylated or unmethylated for TMS1 while histone H3 di- or tri-methylated at lysine 4 were found at higher levels across the TMS1 locus in unmethylated as compared to methylated cells. Histone H4 acetylated at lysine 16 (H4-K16Ac) was enriched at TMS1 in unmethylated compared to methylated cells. Interestingly, we observed two peaks of H4-K16Ac flanking the CpG island in unmethylated cells. In contrast, histone H4 trimethylated at lysine 20 (H4-K20me3) was absent across the TMS1 locus in unmethylated cells but showed localized enrichment near the transcription start site in methylated cells. To understand the contribution of H4-K16Ac to TMS1 regulation, we used RNAi to transiently silence hMOF, the HAT responsible for H4-K16 acetylation, in TMS1-positive MCF7 cells. hMOF down-regulation led to a decrease in H4-K16 Ac globally and specifically across the TMS1 locus. hMOF down-regulation also resulted in a decrease in both TMS1 mRNA and protein levels without affecting DNA methylation at the TMS1 CpG island. Silencing of TMS1 was transient in this setting and was fully reversed upon hMOF re-expression. These results indicate that H4-K16 Ac is necessary to maintain TMS1 gene activity and suggest a mechanism where stable epigenetic silencing marked by DNA methylation is preceded by an intermediate reversibly silent state characterized by hypoacetylated histones and an unmethylated CpG island. Considering that the global loss of H4 K16 Ac is a hallmark of cancer, identification of the subset of genes that are specifically affected by this histone mark will lead to novel insights into epigenetic silencing.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA