The combined manipulation of histone acetylation and cytosine methylation in chromatin presents another strategy for gene-targeted therapy through epigenetic modification. These two epigenetic processes are linked as was shown by Nan et al.57 and Jones et al.58 (see p. 155) who showed that the repressive chromatin structure associated with dense methylation was also associated with histone deacetylation. Methylated DNA binds the transcriptional repressor MeCP2 at the MBD, which recruits the Sin 3A/histone deacetylase complex to form tran-scriptionally repressive chromatin. This process was reversed by trichostatin A, a specific inhibitor of histone deacetylase.
Since little was known about the importance of methylation relative to histone deacetylation in the inhibition of gene transcription, Cameron et al. examined this question.137 They found that trichostatin alone did not reactivate several hy-permethylated genes [MLH1, TIMP3, CDKN2B (INK4B, p15), and CDKN2A (INK4, p16)] under conditions that allowed reactivation of nonmethylated genes. These findings suggested that dense CpG island methylation in gene promoter regions was dominant over histone deacetylation in maintaining gene repression. They then induced partial CpG island demethylation by treatment with the demethylating agent 5-aza-20-deoxycytidine in the presence or absence of his-tone deacetylase inhibition. They observed robust expression (4-fold increase) of the genes tested by combined drug treatment (trichostatin plus 5-aza-20-deoxycytidine) in an experiment in which low-level reactivation was seen with 5-aza-20-deoxycytidine treatment alone. These results indicated that histone de-acetylation may not be needed to maintain a silenced transcriptional state, but histone deacetylase has a role in silencing when levels of DNA methylation are reduced. Bisulfite sequencing showed that the increase in gene expression brought about by the combination of the two drugs occurred with retention of extensive methylation in the genes tested. They also found that inhibition of deacetylase activity can induce gene expression without a large-scale change from repressive to accessible chromatin in agreement with the work of others. Taken together the data suggested that decreased methylation is a prerequisite for transcription following histone deacetylase inhibition.
In experiments similar to those of Cameron et al., Chiurazzi and colleagues examined the relative roles of methylation and histone deacetylation in silencing
the FMR1 gene in fragile X syndrome. , 9 Hypermethylation of CGG repeats in this disorder silences the FMR1 gene to cause the absence of the FMR1 protein that subsequently leads to mental retardation (see p. 154). In their first paper, Chiurazzi et al. found that the demethylating agent 5-aza-2'-deoxycytidine partially restored FMR1 protein expression in B-lymphoblastoid cell lines obtained from fragile X patients confirming the role of FMR1 promoter hypermethylation in the pathogenesis of fragile X syndrome.138 In their second paper, they found that combining 5-aza-20-deoxycytidine with histone deacetylase inhibitors such as 4-phenylbutyrate, sodium butyrate, or trichostatin resulted in a 2- to 5-fold increase in FMR1 mRNA levels over that obtained with 5-aza-2'-deoxycytidine alone. The marked synergistic effect observed revealed that both histone hy-peracetylation and DNA demethylation participate in regulating FMR1 activity. These results may help pave the way for future attempts at pharmacologically restoring mutant FMR1 activity in vivo.139
Methylation and histone deacetylation thus appear to act as layers for epige-netic silencing. Cameron et al. believe that one function of DNA methylation may be to firmly ''lock'' genes into a silenced chromatin state.137 They suggested that this effect may be involved in transcriptional repression of methylated inactive X chromosomal genes and imprinted alleles. They proposed that to achieve maximal gene reactivation, it might be necessary to block simultaneously both DNA methylation and histone deacetylation, both of which are essential to the formation and maintenance of repressive chromatin.
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