Abstract
BackgroundFragile X syndrome (FXS) is caused by CGG expansion over 200 repeats at the 5′ UTR of the FMR1 gene and subsequent DNA methylation of both the expanded sequence and the CpGs of the promoter region. This epigenetic change causes transcriptional silencing of the gene. We have previously demonstrated that 5-aza-2-deoxycytidine (5-azadC) treatment of FXS lymphoblastoid cell lines reactivates the FMR1 gene, concomitant with CpG sites demethylation, increased acetylation of histones H3 and H4 and methylation of lysine 4 on histone 3.ResultsIn order to check the specificity of the 5-azadC-induced DNA demethylation, now we performed bisulphite sequencing of the entire methylation boundary upstream the FMR1 promoter region, which is preserved in control wild-type cells. We did not observe any modification of the methylation boundary after treatment. Furthermore, methylation analysis by MS-MLPA of PWS/AS and BWS/SRS loci demonstrated that 5-azadC treatment has no demethylating effect on these regions. Genome-wide methylation analysis through Infinium 450K (Illumina) showed no significant enrichment of specific GO terms in differentially methylated regions after 5-azadC treatment. We also observed that reactivation of FMR1 transcription lasts up to a month after a 7-day treatment and that maximum levels of transcription are reached at 10–15 days after last administration of 5-azadC.ConclusionsTaken together, these data demonstrate that the demethylating effect of 5-azadC on genomic DNA is not random, but rather restricted to specific regions, if not exclusively to the FMR1 promoter. Moreover, we showed that 5-azadC has a long-lasting reactivating effect on the mutant FMR1 gene.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0060-x) contains supplementary material, which is available to authorized users.
Highlights
Fragile X syndrome (FXS) is caused by CGG expansion over 200 repeats at the 5′ UTR of the FMR1 gene and subsequent DNA methylation of both the expanded sequence and the CpGs of the promoter region
Urbach and colleagues showed that FMR1 locus in induced pluripotent stem cells derived from FXS individuals is hypermethylated, suggesting that its methylation, once established, is stable and not revertible through reprogramming techniques [13]
The methylation boundary is thought to have a role in chromatin remodelling of the FMR1 locus by recruiting a number of proteins [16], such as CTCF (CCCTC-binding factor), the first insulator protein found in mammals [19]
Summary
Fragile X syndrome (FXS) is caused by CGG expansion over 200 repeats at the 5′ UTR of the FMR1 gene and subsequent DNA methylation of both the expanded sequence and the CpGs of the promoter region. A similar epigenetic status can be induced by treatment of FXS cells with the DNA demethylating agent 5-aza-2-deoxycytidine (5-azadC), which causes histone changes (H3 and H4 hyperacetylation, H3K4 methylation) that precede DNA demethylation [9,10,11] In accordance with these results, silencing of FMR1 in human embryonic stem cells seems to begin from histone modifications prior to DNA methylation [12]. We showed that inhibitors of histone deacetylases potentiate the effect of 5-azadC, without allowing a substantial reactivation of the gene [22] These experiments suggest that DNA methylation is dominant over histone modifications in determining the transcriptional inactivation of the mutant FMR1 gene. This conclusion was confirmed by further experiments, studying the modest reactivating effect of valproic acid (VPA), which acts as inhibitor of histone deacetylases without DNA demethylation [23]
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