Transcriptional Reactivation of the FMR1 Gene. A Possible Approach to the Treatment of the Fragile X Syndrome

Abstract

Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability, caused by CGG expansion over 200 repeats (full mutation, FM) at the 5' untranslated region (UTR) of the fragile X mental retardation 1 (FMR1) gene and subsequent DNA methylation of the promoter region, accompanied by additional epigenetic histone modifications that result in a block of transcription and absence of the fragile X mental retardation protein (FMRP). The lack of FMRP, involved in multiple aspects of mRNA metabolism in the brain, is thought to be the direct cause of the FXS phenotype. Restoration of FMR1 transcription and FMRP production can be obtained in vitro by treating FXS lymphoblastoid cell lines with the demethylating agent 5-azadeoxycytidine, demonstrating that DNA methylation is key to FMR1 inactivation. This concept is strengthened by the existence of rare male carriers of a FM, who are unable to methylate the FMR1 promoter. These individuals produce limited amounts of FMRP and are of normal intelligence. Their inability to methylate the FMR1 promoter, whose cause is not yet fully elucidated, rescues them from manifesting the FXS. These observations demonstrate that a therapeutic approach to FXS based on the pharmacological reactivation of the FMR1 gene is conceptually tenable and worthy of being further pursued.

Keywords: DNA methylation; FMR1 gene; Fragile X syndrome; drug treatments; epigenetic therapy; histone modifications.

Figure 1

Structure of the fragile X mental retardation 1 (FMR1) gene (top) and its various allelic forms. The polymorphic CGG sequence is placed upstream of exon 1 in the 5′ untranslated region (UTR). Based on the CGG expansion four different classes of alleles are shown, with their transcriptional activity indicated by the arrow: normal (WT); premutated (PM) with a higher transcription (bold arrow) and slight decrease of translation associated to the fragile X-associated tremor/ataxia syndrome (FXTAS) end fragile X-associated primary ovarian insufficiency (FXPOI) phenotypes; unmethylated full mutation (UFM), similar to PM for transcription and translation, without an apparent phenotype; methylated full mutation (MFM) leading to absence of transcript and fragile X mental retardation protein (FMRP) and consequently to fragile X syndrome (FXS).

Figure 2

Schematic representation of the metabotropic glutamate receptor (mGluR) theory. In normal dendrites FMRP inhibits translation of pre-existing mRNAs after mGluR stimulation, with normal ionotropic glutamatergic receptor (AMPAR) endocytosis. FXS dendrite appears thinner compared to normal, because mGluR stimulation by glutamate causes an excess of local protein synthesis with exaggerated long term depression (LTD) due to a higher AMPAR endocytosis activity. The use of a mGluR inhibitor leads to a normalization of LTD in absence of FMRP.

Figure 3

Epigenetic changes induced by 5-azadeoxycytidine (5-azadC) treatment. The use of 5-azadC on different FXS lymphoblasts produce FMR1 transcript reactivation as shown by RT-PCR, and a partial rescue of translation demonstrated by immunocytochemistry (Panel A). Reactivation was quantified by real time PCR and the percentage of demethylated clones was evaluated by bisulphite sequencing of the CpG island of the FMR1 promoter region (Panel B). The effects of 5-azadC on histone marks (acetylation of H3 and H4, increased methylation of H3K4) are reported in panel C. Data derived from Refs. [42,65,66].

Figure 4

Long-lasting effect of 5-azadC and methylome analysis after treatment. In A are reported the transcription and translation results of a recent study [66]. The upper panel reports the scheme of 5-azadC treatment with the relative time points. Relative quantification of FMR1-mRNA by RT-PCR after treatments with 5-azadC of 2 different FXS lymphoblastoid cell lines showed increased FMR1-mRNA expression at T3 (8 days after last drug administration), decreasing at T6 (18 days after last drug administration) and T7 (22 days after last drug administration). Western blot with antibody against FMRP and GAPDH on FXS cell extracts demonstrated that after treatment the expression of FMRP was restored and disappeared after 22 days (T7) from the end of the treatment (Panel A). Panel B. Bisulphite sequencing of the methylation boundary including the CpG island of the FMR1 promoter region before (top-left) and after (bottom-left) treatment with 5-azadC of WT cells showed no substantial modification of the methylation profile, while an almost complete demethylation of the promoter region, not affecting the methylation boundary was observed in FXS cells (top right). The methylation boundary is indicated by the arrow. Dendrogram of the methylation profile of the analyzed samples demonstrated a clustering of untreated samples compare to treated ones. The heat map shows some changes in the methylation profile after treatment, that however do not reach statistical significance (p > 0.05). Data derived from ref. [67].

Figure 5

Major epigenetic modifications at the FMR1 locus after histone acetylating treatments. In anormally active WT allele a permissive euchromatic configuration is present (bottom), while in methylated full mutation (FXS) the heterochromatic configuration does not allow transcription (top). The use of L-acetylcarnitine (LAC) on FXS induces an increase of H3 and H4 acetylation without DNA demethylation and transcriptional reactivation (middle left). Valproic acid (VPA) treatment shows a slight transcriptional activity with hyperacetylation of H3 and H4 and methylation of H3K4, while H3K9 methylation remains unmodified. MBD, methyl-binding domain protein; HD, histone deacethylases; Ac, histone acetyl groups.