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. CUT&RUN was performed following the published protocol by Skene et al (2018). On day 21 of neuronal differentiation, WT or FXS patient-derived induced neurons were plated at 150K/cm2. Neurons were treated for 7 days with 5-aza-dC, if indicated, and 14 days with DMSO or 500 nM 2HE-5NMe, if indicated. 300,000 FXS neurons were harvested, washed and bound to beads. Neurons-beads were divided into three aliquots for determining binding locations of IgG control, H3K4me3, and H3K27me3. Neurons were permeabilized and the appropriate primary antibodies were utilized on each aliquot separately. Protein A-MNase fusion protein (gifted by the Henikoff lab) was then added to bind to primary antibody locations. Targeted digestion was activated with addition of calcium and occurred for 5 minutes. Cut fragments were then released from insoluble nuclear chromatin and extracted using NucleoSpin columns (Takara) following manufacturer’s instructions. Size selection was performed using AMPure XP beads (Agencourt) before library preparation with the Kapa Hyper Prep Kit (Roche) following manufacturer’s instructions. End repair and A-tailing were performed before adapter ligation with TruSeq (Illumina) indices. The libraries were amplified and then quality control was performed to ensure that primer dimers were negligible. 150 base pair-end sequencing was performed with HiSeq by the Genomics Core Facility of the WCM Core Laboratories Center. We used an alignment strategy that eliminates reads that align to any mouse transcripts, since some mouse feeder cells may have remained in the sample used for CUT&RUN. Paired-end reads were aligned to human genome version GRCh38.p13 using Bowtie2. Reads were normalized for sizes of libraries. Reads were then mapped onto a list of all protein-coding gene promoters (-1000 to +1000 of MANE Select transcription start sites from GRCh38.p13).