DNA Methylation, Mechanisms of FMR1 Inactivation and Therapeutic Perspectives for Fragile X Syndrome

Abstract

Among the inherited causes of intellectual disability and autism, Fragile X syndrome (FXS) is the most frequent form, for which there is currently no cure. In most FXS patients, the FMR1 gene is epigenetically inactivated following the expansion over 200 triplets of a CGG repeat (FM: full mutation). FMR1 encodes the Fragile X Mental Retardation Protein (FMRP), which binds several mRNAs, mainly in the brain. When the FM becomes methylated at 10-12 weeks of gestation, the FMR1 gene is transcriptionally silent. The molecular mechanisms involved in the epigenetic silencing are not fully elucidated. Among FXS families, there is a rare occurrence of males carrying a FM, which remains active because it is not methylated, thus ensuring enough FMRPs to allow for an intellectual development within normal range. Which mechanisms are responsible for sparing these individuals from being affected by FXS? In order to answer this critical question, which may have possible implications for FXS therapy, several potential epigenetic mechanisms have been described. Here, we focus on current knowledge about the role of DNA methylation and other epigenetic modifications in FMR1 gene silencing.

Keywords: DNA methylation; FMR1 gene; epigenetic modifications; fragile X syndrome; gene expression signatures.

Figure 1

Representative model of DNA methyltransferase 1 (DNMT1) interaction with the FMR1 locus: In FMR1 transcriptionally inactive allele, as in Fragile X syndrome (FXS) individuals, DNMT1 (in pink) freely binds DNA and methylates it (violet dots), preventing transcription through RNA polymerase (in blue) (top panel). In FMR1 transcriptionally active alleles, namely CTRL, PM and UFM, RNA polymerase transcribes RNA (pink line) and DNMT1 binds to nascent RNA and R-loop structures, preventing DNA methylation (bottom panel). Created with BioRender.com.

Figure 2

Differences of FMR1 methylation during differentiation of human embryonic stem cells (hESCs) derived from normal and FXS individuals: Both normal and FXS hESC have an unmethylated FMR1 promoter and similar FMR1 expression levels. Upon differentiation, the FMR1 promoter and the CGG expansion become hypermethylated in FXS hESCs, with the subsequent decrease of the FMR1 expression level. Created with BioRender.com.

Figure 3

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) genome and epigenome editing strategies: (1) FXS iPSCs were edited with CRISPR/Cas9 to remove the CGG repeats in the FMR1 gene [84,85]. (2) Targeted demethylation of CGG repeats was achieved by Cas9-Tet1 [86]. (3) Transcriptional activator VP16 fused to Cas9 robustly enhanced FMR1 transcription [87]. All these CRISPR/Cas9 strategies resulted in FMR1 transcription and Fragile X Mental Retardation Protein (FMRP) production by edited FXS cells. Created with BioRender.com.