SIRT1 Inhibition Alleviates Gene Silencing in Fragile X Mental Retardation Syndrome

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Abstract
Expansion of the CGG•CCG-repeat tract in the 5′ UTR of the FMR1 gene to >200 repeats leads to heterochromatinization of the promoter and gene silencing. This results in Fragile X syndrome (FXS), the most common heritable form of mental retardation. The mechanism of gene silencing is unknown. We report here that a Class III histone deacetylase, SIRT1, plays an important role in this silencing process and show that the inhibition of this enzyme produces significant gene reactivation. This contrasts with the much smaller effect of inhibitors like trichostatin A (TSA) that inhibit Class I, II and IV histone deacetylases. Reactivation of silenced FMR1 alleles was accompanied by an increase in histone H3 lysine 9 acetylation as well as an increase in the amount of histone H4 that is acetylated at lysine 16 (H4K16) by the histone acetyltransferase, hMOF. DNA methylation, on the other hand, is unaffected. We also demonstrate that deacetylation of H4K16 is a key downstream consequence of DNA methylation. However, since DNA methylation inhibitors require DNA replication in order to be effective, SIRT1 inhibitors may be more useful for FMR1 gene reactivation in post-mitotic cells like neurons where the effect of the gene silencing is most obvious. Fragile X syndrome is the leading cause of heritable intellectual disability. The affected gene, FMR1, encodes FMRP, a protein that regulates the synthesis of a number of important neuronal proteins. The causative mutation is an increase in the number of CGG•CCG-repeats found at the beginning of the FMR1 gene. Alleles with >200 repeats are silenced. The silencing process involves DNA methylation as well as modifications to the histone proteins around which the DNA is wrapped in vivo. Treatment with 5-azadeoxycytidine, a DNA methyltransferase inhibitor, reactivates the gene. However, this reagent is toxic and since no DNA demethylase has been found in humans, methylation inhibitors are not useful in cells like neurons that no longer divide. We show here that splitomicin is also able to reactivate the Fragile X allele. It does so by inhibiting a protein deacetylase, SIRT1, thus favoring the action of another enzyme, hMOF that reverses the SIRT1 modification. We also found that 5-azadeoxycytidine acts, at least in part, by reversing the effect of SIRT1. However, since splitomicin reactivation occurred without DNA demethylation, DNA replication is not necessary for its efficacy. Thus, unlike DNA methylation inhibitors, SIRT1 inhibitors may be able to reactivate Fragile X alleles in neurons.