Histone Modifications Depict an Aberrantly Heterochromatinized FMR1 Gene in Fragile X Syndrome

Abstract

Fragile X syndrome is caused by an expansion of a polymorphic CGG triplet repeat that results in silencing of FMR1 expression. This expansion triggers methylation of FMR1's CpG island, hypoacetylation of associated histones, and chromatin condensation, all characteristics of a transcriptionally inactive gene. Here, we show that there is a graded spectrum of histone H4 acetylation that is proportional to CGG repeat length and that correlates with responsiveness of the gene to DNA demethylation but not with chromatin condensation. We also identify alterations in patient cells of two recently identified histone H3 modifications: methylation of histone H3 at lysine 4 and methylation of histone H3 at lysine 9, which are marks for euchromatin and heterochromatin, respectively. In fragile X cells, there is a decrease in methylation of histone H3 at lysine 4 with a large increase in methylation at lysine 9, a change that is consistent with the model of FMR1's switch from euchromatin to heterochromatin in the disease state. The high level of histone H3 methylation at lysine 9 may account for the failure of H3 to be acetylated after treatment of fragile X cells with inhibitors of histone deacetylases, a treatment that fully restores acetylation to histone H4. Using 5-aza-2'-deoxycytidine, we show that DNA methylation is tightly coupled to the histone modifications associated with euchromatin but not to the heterochromatic mark of methylation of histone H3 at lysine 9, consistent with recent findings that this histone modification may direct DNA methylation. Despite the drug-induced accumulation of mRNA in patient cells to 35% of the wild-type level, FMR1 protein remained undetectable. The identification of intermediates in the heterochromatinization of FMR1 has enabled us to begin to dissect the epigenetics of silencing of a disease-related gene in its natural chromosomal context.

The active FMR1 promoter is associated with a large domain of altered chromatin conformation with embedded local histone modifications

“…The promoter was also depleted for H3K9Me2, a modification that is correlated with inactive chromatin and that occurred at low levels throughout the locus. In fragile X cells, H3Ac, H4Ac, and H3K4Me2 at the FMR1 promoter were all reduced, whereas H3K9Me2 was increased at the transcription start site of FMR1, consistent with previous results (15).…”

“…Previous studies have analyzed histone modifications in only a small (Ϸ2 kb) region around the FMR1 promoter (14)(15)(16). In light of the findings presented above, we were interested in determining the relationship between histone modifications and the region of altered chromatin conformation detected by 3C.…”

“…The active promoter displays high levels of histone acetylation and H3K4 methylation, whereas the silenced promoter is enriched in H3K9 methylation (14)(15)(16). Further, expression of FMR1 was correlated with formation of DNaseI hypersensitive sites at the promoter, pointing to an open chromatin conformation at this site (18).…”

“…Silencing of FMR1 is caused by expansion of a CGG trinucleotide repeat located in the first exon of the gene (13). This genetic alteration triggers DNA methylation of the repeat plus local changes in histone modifications (14)(15)(16). When combined, these epigenetic alterations induce the silencing of FMR1, leading to the clinical features of fragile X syndrome, the most common form of inherited mental retardation (17).…”

Epigenetics, fragile X syndrome and transcriptional therapy

“…Kumari and Usdin [2010] also reported increased methylation of lysine 20 on histone 4 (H4K20) near the CGG expansion. All these epigenetic changes result in heterochromatinization of the FMR1 locus [Coffee et al, 2002;Tabolacci et al, 2005Tabolacci et al, , 2008a, preventing transcription and resulting in absence of the FMRP protein. An exhaustive overview of FMRP functions is provided by Bagni and Oostra [2013] in this same issue.…”