Histone modifications and silencing prior to DNA methylation of a tumor suppressor gene

Abstract

We attempted to answer two central questions about epigenetic silencing of the tumor suppressor gene p16INK4a in this study: (1) whether the maintenance of associated histone modifications is dependent on DNA methylation and (2) whether such histone modifications can occur prior to DNA methylation. By coupling chromatin immunoprecipitation with gene targeting and the analysis of specific alleles, we found that elimination of DNA methylation from a p16INK4a allele resulted in profound changes in surrounding histones. After continued passage of such cells, methylation of histone H3 lysine-9 occurred in conjunction with re-silencing in the absence of DNA methylation. These results have important implications for understanding the biochemical events underlying the silencing of tumor suppressor genes and the resultant growth suppression. Significance The mechanisms underlying epigenetic silencing have been extensively investigated in various non-mammalian systems, but very little is known about this process in tumor suppressor genes in cancer cells. Our data show that the maintenance of epigenetic silencing and of associated histone modifications is dependent on DNA methylation. Because we used a genetic rather than pharmacologic approach to remove DNA methyl groups, the data leading to this conclusion were unambiguous. Moreover, we found that histone modifications associated with silencing of a tumor suppressor gene can occur independently of DNA methylation. These studies demonstrate similarities between the processes of epigenetic silencing of tumor suppressor genes and X inactivation and suggest that DNA methylation serves to “lock in” rather than initiate silencing. Introduction A variety of tumor suppressor genes have been shown to be silenced in human cancers (Jones and Baylin, 2002). In virtually all cases, the silencing is accompanied by DNA hypermethylation of the promoters of these genes, and DNA methylation has often been interpreted to be causally involved in the silencing. DNA methylation is also associated with silencing of genes on the inactive X chromosome. However, studies have shown that DNA methylation occurs only after X chromosome inactivation, excluding DNA methylation as the proximate cause (Heard et al., 1997; Migeon, 1994). Analogous results have not been described for tumor suppressor genes on the autosomes. A recently developed experimental system has given us the opportunity to evaluate the biochemical events underlying silencing in a temporal fashion. When both the DNA Methyltransferase 1 (DNMT1) and DNA Methyltransferase 3B (DNMT3B) genes are disrupted through gene targeting, virtually all DNA methyltransferase activity and DNA methylation are eliminated (Rhee et al., 2002). This situation is akin to that occurring in developing germ cells, wherein DNA methylation is erased, permitting parental imprinting (Razin et al., 1984; Reik et al., 2001). This system has given us a unique opportunity to address particular questions regarding the relationship between tumor suppressor gene silencing, histone modifications, and DNA methylation.