With the increasing knowledge accumulating on the many functions of the FMRP protein, especially at the synaptic level, numerous pharmacological trials have been performed to try and compensate for the altered function of specific neuronal receptors [reviewed in Bagni and Oostra, 2013]. One of the most advanced clinical trials used a metabotropic glutamate receptor antagonist with promising results [Jacquemont et al., 2011]. However, considering the large number of mRNAs targeted by FMRP and the various known dysregulated pathways, including the GABAergic pathway [reviewed in Bagni and Oostra, 2013], it would be better if one could turn on again the FMR1 gene silenced by the full mutation. In fact, the existence of rare UFM individuals, known since the report of Smeets et al. , suggested us the possibility of pharmacologically restoring FMR1 transcription. DNA demethylation can be obtained with 5′-azaC or, more efficiently, with 5′-aza-2′-deoxycytidine (5-azadC) that is incorporated as analog of deoxycitidine during cell replication and irreversibly blocks DNA methyltransferases [Jackson-Grusby et al., 1997]. In 1998 we first achieved in vitro reactivation of the FMR1 full mutation by treating fragile X lymphoblastoid cells with 5-azadC [Chiurazzi et al., 1998], detecting mRNA by RT-PCR and FMRP protein by immunocytochemistry in a fraction of treated cells (5–10%). The lower efficiency of mRNA translation, due to the CGG expansion [Feng et al., 1995], probably accounts for the apparent discrepancy between mRNA and protein levels. One year later [Chiurazzi et al., 1999] we combined 5-azadC treatment with various histone deacetylase (HDAC) inhibitors (butyrate, phenylbutyrate and trichostatin A) and observed a synergistic effect on FMR1 reactivation by semiquantitative RT-PCR. However, HDAC inhibitors alone were unable to induce any reactivation at all [Chiurazzi et al., 1999], suggesting that DNA methylation is dominant over histone hypoacetylation at the FMR1 locus, as also reported for other heavily methylated genes [Cameron et al., 1999]. These initial observations were confirmed by measuring FMR1 transcript with real-time RT-PCR and confirming DNA demethylation at the FMR1 promoter after 5-azadC treatment [Pietrobono et al., 2002]. We then investigated histone modifications in the promoter, exon 1 and exon 16 of FMR1 by chromatin immunoprecipitation (ChIP) before and after pharmacological treatment of FXS lymphoblasts with 5-azadC and acetyl-L-carnitine [Tabolacci et al., 2005]. This latter compound can efficiently increase histone acetylation, but is not sufficient for FMR1 reactivation when used alone [Tabolacci et al., 2005]. It is worth noting that, although acetylcarnitine does not reactivate FMR1 full mutations, it does have a significant clinical effect on fragile X patients and improves their adaptive and social behavior [Torrioli et al., 2008]. We also tried valproic acid (VPA), since it had been reported to increase histone acetylation and to demethylate DNA, but again only modest reactivation was obtained with apparently no DNA demethylation [Tabolacci et al., 2008b]. On the contrary, 5-azadC induced both histone acetylation and increased methylation of lysine 4 of histone H3 (H3-K4), while partly reducing methylation of lysine 9 of histone H3 (H3-K9) [Tabolacci et al., 2005]. These epigenetic changes, induced by 5-azadC, appeared to restore a euchromatic configuration of the FMR1 promoter in treated FXS cells (see Fig. 2), effectively transforming a methylated full mutation into an unmethylated full mutation (UFM) [Pietrobono et al., 2005; Tabolacci et al., 2008a]. 5-azadC effects increased with dose and treatment time [Chiurazzi et al., 1999; Pietrobono et al., 2002], but 4 weeks after treatment was discontinued we observed regain of DNA methylation in the FMR1 promoter and loss of transcription [Pietrobono et al., 2002]. Further follow-up experiments are warranted in order to understand if some clones actually remained demethylated or if all cells reverted to the heterochromatic status.
Ten years ago we first discussed the potential of pharmacological intervention in order to reactivate silenced genes or to increase the expression of specific genes in order to treat epigenetic disorders [Chiurazzi and Neri, 2003]. Some genes may simply require histone hyperacetylation to be turned on or to upregulate their transcriptional output. This the case of the ALDPL1 [Kemp et al., 1998] and SMN2 genes [Chang et al., 2001; Andreassi et al., 2004], involved in adrenoleukodystrophy and spinal muscular atrophy, respectively. Transcriptional activation could partially compensate for disease causing mutations in these two genes. Other genes that, like the fully mutated FMR1, have a methylated CpG island, require also DNA demethylation in order to resume transcription. We have actually shown that 5-azadC treatment is also effective in turning on the FAM11A gene associated with the Xq28 folate-sensitive fragile site, that is transcriptionally silent in FRAXF full mutations [Shaw et al., 2002]. The same effect might be expected for the other fragile sites due to a CGG repeat expansion followed by DNA methylation (e.g., FRAXE, FRA16A, etc.) mentioned earlier.
An obvious concern that arises when considering the clinical use of 5-azadC is its toxicity. In fact, while 5-azaC and 5-azadC are generally well tolerated in hematological malignancies [Gnyszka et al., 2013], the effects of a long-term treatment are unknown. A second obstacle is the apparent requirement for cell division in order to be effective; interestingly, at least two reports suggest that 5-azadC may require minimal or no incorporation in DNA to effectively reduce levels of the maintenance DNA methyltransferase DNMT1 [Ghoshal et al., 2005; Patel et al., 2010]. But the major objection to using drugs like 5-azadC or HDAC inhibitors is that their action is likely to be unspecific and genome-wide: however, a microarray screening of 10,814 genes by Suzuki et al.  showed that a very limited set of genes are actually transcriptionally upregulated by treatment with 5-azadC (51 genes) and/or trichostatin A (23 genes). Furthermore, our unpublished observations suggest that 5-azadC induces DNA demethylation only in limited regions and typically “respects,” for example differentially methylated regions of imprinted genes, as if certain regions were more susceptible to reactivation. However, in certain cases a genome-wide effect would be actually desirable as in cancer treatments [Gnyszka et al., 2013] or in treatment of polyglutamine expansion disorders, where global histone hypoacetylation seems to play a prominent role [Steffan et al., 2001; Bodai et al., 2012; Pirooznia and Elefant, 2013; Valor et al., 2013].
However, the ultimate therapy for “local” epigenetic disorders such as FXS or imprinting disorders may not rely on small molecules such as DNMT or HDAC inhibitors but on small RNAs designed for a targeted modulation of gene silencing or reactivation [Ackley et al., 2013]. In fact, if sufficient knowledge on the specific epimutation, efficient siRNAs and delivery vectors were available, siRNA-based therapeutics may eventually become a reality [Angart et al., 2013].