Abstract
In fragile X syndrome (FXS), expansion of a CGG repeat tract in the 5′-untranslated region of the FMR1 gene to >200 repeats causes transcriptional silencing by inducing heterochromatin formation. Understanding the mechanism of FMR1 silencing is important as gene reactivation is a potential treatment approach for FXS. To date, only the DNA demethylating drug 5-azadeoxycytidine (AZA) has proved effective at gene reactivation; however, this drug is toxic. The repressive H3K9 methylation mark is enriched on the FMR1 gene in FXS patient cells and is thus a potential druggable target. However, its contribution to the silencing process is unclear. Here, we studied the effect of small molecule inhibitors of H3K9 methylation on FMR1 expression in FXS patient cells. Chaetocin showed a small effect on FMR1 gene reactivation and a synergistic effect on FMR1 mRNA levels when used in combination with AZA. Additionally, chaetocin, BIX01294 and 3-Deazaneplanocin A (DZNep) were able to significantly delay the re-silencing of AZA-reactivated FMR1 alleles. These data are consistent with the idea that H3K9 methylation precedes DNA methylation and that removal of DNA methylation is necessary to see the optimal effect of histone methyl-transferase (HMT) inhibitors on FMR1 gene expression. Nonetheless, our data also show that drugs targeting repressive H3K9 methylation marks are able to produce sustained reactivation of the FMR1 gene after a single dose of AZA.
Highlights
Fragile X syndrome (FXS) is the leading cause of inherited intellectual disability, which affects approximately 1 in 5000 males and 1 in 4000 to 8000 females [1]
In addition to DNA methylation and hypoacetylated histones, the silenced fragile X mental retardation 1 (FMR1) gene in fragile X syndrome (FXS) patient cells is associated with the marks of facultative heterochromatin, histone H3 di-methylated at lysine 9 (H3K9me2) and histone H3 tri-methylated at lysine 27 (H3K27me3), as well as the marks of constitutive
Previous studies have shown that treatment with the DNA methylation inhibitor AZA can partially reactivate the FMR1 gene in FXS-patient-derived lymphoblastoid and fibroblast cell lines [7,10,32,33]
Summary
Fragile X syndrome (FXS) is the leading cause of inherited intellectual disability, which affects approximately 1 in 5000 males and 1 in 4000 to 8000 females [1]. In addition to DNA methylation and hypoacetylated histones, the silenced FMR1 gene in FXS patient cells is associated with the marks of facultative heterochromatin, histone H3 di-methylated at lysine 9. In lymphoblastoid cell lines derived from individuals carrying a transcriptionally active FM allele, H3K9me was present without DNA methylation [14,15] This suggests that this mark might be essential, it is not sufficient for FMR1 gene silencing. Treatment with inhibitors of H3K9 methylation without first removing DNA methylation was not very effective, with only chaetocin showing some effect on FMR1 reactivation in FXS patient cells. BIX01294, chaetocin and DZNep delayed re-silencing of reactivated alleles in FXS lymphoblastoid cells These results are consistent with previous studies that suggested that H3K9 methylation occurs prior to DNA methylation [13,14]. Our data provide further support that targeting both DNA methylation and histone methylation may be required for efficient reactivation of the FMR1 gene
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