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

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. Overall design: WCM37 or SI-214 cells were plated on 35 mm dishes and differentiated as described above. The differentiated neurons were next incubated in 5-aza-dC (1 µM) for 7 days. Media was then used to wash out 5-aza-dC and then the neurons were incubated in media with 2HE-5NMe or vehicle (DMSO) for 14 days. Media was removed from cells and then RNA was extracted from the cells using Trizol (Invitrogen, 15596026) following the manufacturer's instructions. The RNA was precipitated using isopropanol, washed with 70% ethanol and resuspended in water. The RNA (100 ng) was then converted into a cDNA library using the SMART-seq mRNA LP (with UMIs) kit (Takara, 634762) following the manufacturer's instructions. The quality of the library was assessed using a High Sensitivity D5000 ScreenTape assay (Agilent) and then the cDNAs were sequenced using a NovaSeq 6000 with S1 Flow Cells (paired end, 2x50 bp).