Genetic and epigenetic mechamisms of FSHD pathogenesis

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Facioscapulohumeral dystrophy (FSHD) is one of the most prevalent muscular dystrophies. The majority of cases are associated with shortening of the D4Z4 repeat sequences on chromosome 4q (FSHD1) while mutations in the SMCHD1 transcriptional repressor gene are linked to a minor subset of FSHD patients (FSHD2). Mutations in SMCHD1 also greatly exacerbate the phenotype of FSHD1, thus acting as a modifier of the disorder's severity in FSHD1. Abnormal expression of the DUX4 gene present in the D4Z4 repeats is linked to the development of both FSHD1 and FSHD2. However, only a small percentage of patient muscle cells express DUX4 protein, which can also occasionally be observed in muscle cells from unaffected individuals. This suggests that DUX4 expression alone may not be sufficient for FSHD development. Furthermore, exactly how the DUX4 gene is upregulated only in a small number of patient muscle cells and how it contributes to FSHD development and progression are unclear. SMCHD1 is part of the histone H3 lysine 9-trimethylated (H3K9me3) “heterochromatin” structure that normally represses DUX4 expression, which is compromised in both FSHD1 and FSHD2 patient cells. We obtained evidence that H3K9me3 is specifically reduced not only at D4Z4 but also at other parts of the genome in FSHD cells. Furthermore, SMCHD1 mutations may have DUX4-independent effects on FSHD pathogenesis. Since D4Z4 repeats are not present in the mouse genome, patient muscle cells are essential for assessing FSHD-specific cellular changes. However, high-quality patient myoblasts are limited, and variability among samples with only a small subset of cells expressing DUX4 may exacerbate the averaging artifact of population analysis. We plan to take two complementary strategies to circumvent these issues and test our hypothesis: (1) development of clonal FSHD-modeling human myoblast lines, and (2) single-nucleus profiling of primary control and FSHD muscle cells. We propose a hypothesis that FSHD is a heterochromatin abnormality disorder, in which genome-wide changes of H3K9me3 and SMCHD1 function predispose to or initiate FSHD, and a small number of (DUX4- expressing) disease-driving cells dictate the progression of the phenotype. The Specific Aims of this project are (1) to generate “FSHD-modeling” human myoblast lines to establish the FSHD disorder mechanism(s) and to study D4Z4 chromatin regulation, (2) to perform genome-wide epigenetic and expression analyses at the single-nucleus level to understand the consequences of DUX4 upregulation and possibly identify FSHD-driving cells, and (3) to take a proteomics approach to identify the components of D4Z4 heterochromatic structure to further delineate the mechanism and consequence of its dysregulation in FSHD, which will be integrated into the analyses in Aims 1 and 2. The successful outcome of this project may lead to further understanding of the mechanism(s) underlying FSHD pathogenesis and the identification of potential new therapeutic targets and approaches for treatment.