Investigating mechanisms of pathogenesis in facioscapulohumeral muscular dystrophy
Facioscapulohumeral muscular dystrophy (FSHD) is a rare disease with characteristic weakness in facial and periscapular muscles which progresses to additional muscle groups. FSHD is caused by the misexpression of the embryonic transcription factor DUX4 in muscle cells. In 95% of FSHD patients, a series of macrorepeats preceding DUX4 is contracted and derepressed in part through loss of DNA methylation. In the remaining FSHD patients, the repeats are derepressed but not contracted, and 80% of these patients have a mutation in SMCHD1, which regulates DNA methylation in these repeats. DUX4 is involved in zygotic genome activation (ZGA) when it activates a number of transcription factors and chromatin remodelers, such as DUXA, LEUTX and ZSCAN4, as well as long terminal repeats (LTRs), such as ERVL-MaLRs, which are also activated in FSHD. DUX4 expression in patient muscle cells is sparse (0.5% of myotube nuclei), but its expression in only a few nuclei is sufficient to activate target gene expression in multiple nuclei within a multinucleated muscle cell, which is sustained when DUX4 is no longer present. My work has focused on understanding progression of FSHD at a molecular level both into different muscle groups and following DUX4 activation.I used single nucleus RNA sequencing to understand the contribution of individual nuclei to gene dysregulation following DUX4 expression. I identified nuclei with native expression of DUX4, as well as two populations of nuclei with high and low expression of DUX4-induced genes. The high group appears to perpetuate pathogenesis and has higher expression of genes related to the cell cycle despite the nuclei coming from cells in G0. I also found that DUX4 is coexpressed with only a subset of its target genes, while the DUX4 homolog DUXA is expressed with a wider set of targets. To understand why certain muscle groups are commonly or less affected in FSHD, I assayed DNA methylation and gene expression in different muscle groups. Genes induced during myogenesis in FSHD have higher expression in commonly affected muscle groups despite their promoters having high DNA methylation. Muscle groups differ in expression and DNA methylation of transcription factors key to developmental patterning and specification that may contribute to susceptibility to FSHD. Finally, I explored the role of DUXA as a potential regulator of DUX4 target genes following their initial activation. I found that DUXA depletion is sufficient to lower expression of DUX4 target genes including LTRs. I also identified a set of genes which are induced along with DUX4 during myogenesis in FSHD2 that are not induced following DUXA depletion. I have thus identified a candidate regulator of FSHD gene dysregulation and candidate contributors to differential muscle group susceptibility in FSHD.