Engineered SMCHD1 and D4Z4 mutations reveal roles of D4Z4 heterochromatin disruption and feedforward DUX4 network activation in FSHD

Abstract

Facioscapulohumeral dystrophy (FSHD) is commonly associated with contraction of D4Z4 repeats on chromosome 4q (FSHD1). Mutations in the SMCHD1 gene are linked to both minor cases with no prominent repeat loss (FSHD2) and severe cases of FSHD1. Abnormal upregulation of the transcription factor DUX4, encoded in the D4Z4 repeat, is believed to play a central role in FSHD. However, defining the disease mechanism has been hampered by the heterogeneity of patient-derived cells, difficulty to detect DUX4 in patient myocytes, and limited animal models because D4Z4 repeats are primate-specific. To overcome these limitations, we engineered isogenic human skeletal myoblast lines with D4Z4 and/or SMCHD1 mutations. We found a highly synergistic effect of double mutations on triggering two key disease processes, D4Z4 heterochromatin disruption and cross-stimulation of DUX4 targets, such as histone H3.X/Y and LEUTX transcription factor. Thus, engineered human myocyte models provide unique insights into the molecular mechanisms underpinning FSHD.