Sustained epigenetic reactivation in fragile X neurons with an RNA-binding small molecule [CUT&RUN]

Fragile X syndrome (FXS) is a disease of pathologic epigenetic silencing induced by RNA. In FXS, an expanded CGG-repeat tract in the FMR1 gene induces epigenetic silencing during embryogenesis. FMR1 silencing can be reversed with 5-aza-deoxyctidine (5-aza-dC), a nonspecific epigenetic reactivator; however, continuous administration of 5-aza-dC is problematic due to its toxicity. We describe an approach to restore FMR1 expression in FXS neurons by transient treatment with 5-aza-dC, followed by treatment with 2HE-5NMe, which binds the CGG-repeat expansion in the FMR1 mRNA and blocks the resilencing of the FMR1 gene after withdrawal of 5-aza-dC. Genome-wide profiling of histone marks shows that 2HE-5Nme maintains FMR1 in a reactivated state. FMR1 reactivation in neurons results in re-expression of FMRP and reversal of FXS-associated dendritic spine defects. These results demonstrate that an RNA-binding small molecule can achieve gene-specific epigenetic control, and provide an approach for restoration of FMRP in FXS neurons.