Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer

Densely methylated DNA associates with transcriptionally repressive chromat in characterized by the presence of underacetylated histones(1,2). Recently , these two epigenetic processes have been dynamically linked. The methyl-C pG-binding protein MeCP2 appears to reside in a complex with histone deacet ylase activity(3,4). MeCP2 can mediate formation of transcriptionally repre ssive chromatin on methylated promoter templates in vitro, and this process can be reversed by trichostatin A (TSA), a specific inhibitor of histone d eacetylase(3-5). Little is known, however, about the relative roles of meth ylation and histone deacetylase activity in the stable inhibition of transc ription on densely methylated endogenous promoters, such as those for silen ced alleles of imprinted genes(6), genes on the female inactive X chromosom e(7) and tumour-suppressor genes inactivated in cancer cells(8,9). We show here that the hypermethylated genes MLH1, TIMP3 (TIMP3), CDKN2B (INK4B, p15 ) and CDKN2A (INK4, p16) cannot be transcriptionally reactivated with TSA a lone in tumour cells in which we have shown that TSA alone can upregulate t he expression of non-methylated genes. Following minimal demethylation and slight gene reactivation in the presence of low dose 5-aza-2'deoxycytidine (5Aza-dC), however, TSA treatment results in robust re-expression of each g ene. TSA does not contribute to demethylation of the genes, and none of the treatments alter the chromatin structure associated with the hypermethylat ed promoters. Thus, although DNA methylation and histone deacetylation appe ar to act as synergistic layers for the silencing of genes in cancer, dense CpG island methylation is dominant for the stable maintenance of a silent state at these loci.