No abstract

Epigenetic inactivation of the RASSF1A tumor suppressor by CpG island methylation was frequently detected in cancer. However, the mechanisms of this aberrant DNA methylation are unknown. In the RASSF1A promoter, we characterized four Sp1 sites, which are frequently methylated in cancer. We examined the functional relationship between DNA methylation, histone modification, Sp1 binding, and RASSF1A expression in proliferating human mammary epithelial cells. With increasing passages, the transcription of RASSF1A was dramatically silenced. This inactivation was associated with deacetylation and lysine 9 trimethylation of histone H3 and an impaired binding of Sp1 at the RASSF1A promoter. In mammary epithelial cells that had overcome a stress-associated senescence barrier, a spreading of DNA methylation in the CpG island promoter was observed. When the RASSF1A-silenced cells were treated with inhibitors of DNA methyltransferase and histone deacetylase, binding of Sp1 and expression of RASSF1A reoccurred. In summary, we observed that histone H3 deacetylation and H3 lysine 9 trimethylation occur in the same time window as gene inactivation and precede DNA methylation. Our data suggest that in epithelial cells, histone inactivation may trigger de novo DNA methylation of the RASSF1A promoter and this system may serve as a model for CpG island inactivation of tumor suppressor genes.Epigenetic modifications are hallmarks for changes in expression pattern. Abnormal DNA methylation of promoter is a main mechanism for the inactivation of tumor suppressor genes and therefore of fundamental importance for the understanding of the etiology of cancer (24). Gene promoters are often located in CpG-rich DNA regions, so-called CpG islands (3). Active promoters are associated with unmethylated CpG islands and open chromatin structure for transcription regulators, whereas inactive promoters are characterized by a repressed chromatin structure and hypermethylated CpGs. Acetylation of lysines at histone H3 is associated with active chromatin, and deacetylation results in a repressed chromatin structure (23). Methylation of histone lysine 9 residue of histone H3 is observed at promoters of inactive genes. MethylCpG binding proteins provide a link between methylated DNA and hypoacetylated histones by recruiting histone deacetylases (25, 32). DNA methyltransferases, which are responsible for the maintenance of methylated DNA and necessary for the establishment of newly methylated promoters, were shown to be associated with proteins including retinoblastoma protein (Rb), E2F1, histone deacetylases, histone methyltransferase, and a transcriptional repressor (13,14,35,37). It has been proposed that the DNA methylation may be directed by alterations in the chromatin structure (2). Several studies indicate an altered DNA methylation pattern when components of the chromatin remodeling system, such as SNF2-like factors, were mutated (11,16,22). In Neurospora crassa and in Arabidopsis thaliana, it has been shown that a repressive chromatin modificat...