Identification of SMCHD1 as a Novel Regulator of Alternative Splicing in FSHD2

Structural Maintenance of Chromosomes Flexible Hinge Domain Containing 1 (SMCHD1) is a non-canonical member of the structural maintenance of chromosomes (SMC) protein family involved in the regulation of chromatin structure, epigenetic regulation and transcription.  Mutations in SMCHD1 cause facioscapulohumeral muscular dystrophy type 2 (FSHD2), a rare genetic disorder characterized by progressive muscle weakness and wasting, believed to be caused by aberrant expression of DUX4 in muscle cells. Here we suggest a new role for SMCHD1 as a regulator of alternative splicing, and demonstrate how splicing alteration caused by SMCHD1 mutations lead to DUX4 expression and FSHD pathogenesis. Analyzing RNA-seq data from muscle biopsies of FSHD2 patients and FSHD2 mouse model found that hundreds of genes were alternatively spliced upon SMCHD1 mutation. At least 20% of alternatively spliced genes were associated with abnormalities of the musculature. Moreover, we show that alternatively spliced exons tend to be bound by SMCHD1, and SMCHD1 bound exons demonstrate slower elongation rate, suggesting SMCHD1 binding promotes exon exclusion by slowing RNAPII. Specifically, we discovered that SMCHD1 mutations promotes the splicing of the DNMT3B1 isoform of DNMT3B, which leads to hypomethylation of the D4Z4 region and DUX4 expression. These results suggest that alternative splicing regulated by SMCHD1 may play a major role in FSHD2 pathogenesis by promoting alternative splicing of different targets including DNMT3B, and highlight the potential for targeting alternative splicing as a therapeutic strategy for this disorder. We extracted RNA from Smchd1 null NSCs and performed next-generation sequencing, using RNA ScreenTape kit (catalog #5067-5576; Agilent Technologies), D1000 ScreenTape kit (catalog #5067-5582; Agilent Technologies), Qubit RNA HS Assay kit (catalog # Q32852; Invitrogen), and Qubit DNA HS Assay kit (catalog #32854; Invitrogen) were used for each specific step for quality control and quantity determination of RNA and library preparation. For mRNA library preparation: TruSeq RNA Library Prep Kit v2 was used (Illumina). In brief, 1 µg was used for the library construction; library was eluted in 20 µL of elution buffer. Libraries were adjusted to 10 mM, and then 10 µL (50%) from each sample was collected and pooled in one tube. Multiplex sample pool (1.5 pM including PhiX 1.5%) was loaded in NextSeq 500/550 High Output v2 kit (75 cycles cartridge and 150 cycles cartridge; Illumina). Run conditions were in paired end (43 × 43 bp and 80 × 80 bp, respectively) and loaded on NextSeq 500 System machine (Illumina).