Molecular analysis of FMR1 reactivation in fragile-X induced pluripotent stem cells and their neuronal derivatives.

Dear Editor, Patient-specific induced pluripotent stem (iPS) cells, generated from somatic cells of disease-affected individuals, hold a tremendous potential for studying disease mechanisms and for drug screening approaches using cell types previously not available (Yamanaka, 2008). Fragile-X (FX) syndrome belongs to the autism spectrum disorders, and is the most common cause of inherited mental retardation with the prevalence of 1/3600 (Crawford et al., 2001). It is nearly always caused by silencing of the FMR1 gene due to abnormal CGG repeat expansions in the 5′-UTR of the gene (Verkerk et al., 1991). Abnormal CGG repeat expansion of over 200 repeats leads to transcriptional silencing and CpG methylation of the gene 5′-UTR and the gene promoter (Sutcliffe et al., 1992). The silencing of the FMR1 gene and the heterochromatinization of the gene promoter were shown to be developmentally dependent (Eiges et al., 2007). In undifferentiated human FX embryonic stem cells (FX-ES cells) derived from affected blastocyst-stage embryos, FMR1 is expressed and gene silencing occurs only upon differentiation (Eiges et al., 2007). Recently, we have generated 11 FX-iPS cell lines from three different FX patients (Urbach et al., 2010). In FX-iPS cells, and contrary to FX-ES cells, the gene is transcriptionally silent both in the pluripotent and differentiated states (Urbach et al., 2010). The absence of FMR1 expression in FX-iPS cells is accompanied by DNA methylation and histone modifications indicative of heterochromatin in the gene promoter (Urbach et al., 2010). Although FX-iPS cells cannot recapitulate the developmentally dependent silencing of FMR1, they represent a unique genetic model system that harbors a completely silent FMR1 locus to study disease phenotypes, and for potential drug screening using the most suitable cells in the context of the disease and the affected individual. Here, we wished to evaluate the reactivation of FMR1 in FX-iPS cells and their differentiated neuronal derivatives by epigenetic modulating drugs. We set out to investigate the ability of several chromatin remodeling drugs to reactivate FMR1 gene expression. Histone deacetylase inhibitor trichostatin-A (TSA) had no significant effect on FMR1 reactivation in FX-iPS cells (Supplementary Figure S1). Next, we wished to evaluate the effect of the demethylating agent 5azacytidine (5-azaC) on FX-iPS cells. Interestingly, 5-azaC was able to robustly reactivate FMR1 gene expression in FX-iPS cells (Figure 1A and Supplementary Figures S1 and S2). Multiple experiments performed on three FX-iPS cell clones at high concentrations have consistently shown that following 5-azaC administration, FMR1 gene expression is restored, with percent levels ranging between 15% and 45% of the WT levels (Figure 1A). A 10-mM 5-azaC treatment resulted in the highest expression levels of FMR1 (Figure 1A and Supplementary Figure S1). A combination of 5-azaC and TSA treatment culminated in a slight increase in FMR1 expression compared with 5-azaC treatment alone (Supplementary Figure S1). When wild type (WT)-ES cells were treated with 1-mM 5-azaC, FMR1 gene expression did not change (Supplementary Figure S3). The 5-azaC concentrations we used are in accordance with physiological levels found in plasma of patients suffering from solid tumors and hematologic malignancies that were treated with 5-azaC (Rudek et al., 2005). Since neurons are the cell type most afflicted in FX syndrome, we further examined the potential of 5-azaC to restore FMR1 expression in FX-neurons. To generate neurons from FX-iPS cells, we used an established protocol (Schuldiner et al., 2001) for neuronal induction from pluripotent cells. We thoroughly characterized the FX-neurons by various means (Supplementary Figures S4–S6). Following 5-azaC treatment, an increase in FMR1 transcript mRNA level could be detected in FX-neurons, albeit with less efficiency than in FX-iPS cells (Figure 1A). Of note, following 5-azaC treatment, some of the undifferentiated FX-iPS cells underwent cell death or differentiation, as observed by the presence of floating cells, or a change in the morphology of some of the undifferentiated colonies. However, many pluripotent colonies were still present after 7 days of treatment. This intriguing phenomenon was not observed with neuronal cells, suggesting a unique effect of 5-azaC on pluripotent stem cells, possibly by inhibiting DNMT3a and DNMT3b which are predominantly expressed in pluripotent stem cells and not in somatic cells. We next assessed whether FMR1 expression persists after withdrawal of 5-azaC treatment. FX-iPS and FX-neuronal cells were treated for 7 days with 5-azaC, and kept in culture for another 7 days without 5-azaC. As expected, the treatment resulted in reactivation of FMR1 expression, which persists at similar levels even after drug withdrawal, as judged by real-time PCR analysis (Figure 1B). Immunostaining for FMR1 protein (FMRP) showed that 5-azaC treated FX-iPS cultures express detectable levels of FMRP, indicating reactivation in the translational level as well (Figure 1C). In order to determine the 180 | Journal of Molecular Cell Biology (2012), 4, 180–183 doi:10.1093/jmcb/mjs007 Published online March 19, 2012