Over‐expressing DUX4 induces pathways regulating myogenesis and apoptosis

Facioscapulohumeral muscular dystrophy (FSHD) is linked to the deletion of the D4Z4 arrays at chromosome 4q35 subtelomeric region. Each D4Z4 array contains a double homeobox 4 (DUX4) open reading frame. We previously showed that the DUX4 functioned as a transcription factor and regulated expression of paired‐like homeodomain transcription factor 1 (PITX1) which was identified specifically upregulated in FSHD by expression profiling. In this study, we identified additional downstream targets and regulatory pathways of DUX4 by expression profiling C2C12 myoblasts transfected with expression vectors of DUX4. Expression vectors of DUX4 homologues, namely DUX4c and DUX1, and insertless vectors were used as controls. The expression profiling study was conducted using the Affymetrix 430_2 microarrays. The data was analyzed using Affymetrix MAS 5.0 followed by t test using Genespring GX11. We identified 948 genes to be differentially expressed in the cells expressing the DUX4 gene compared to the insertless control (p < 0.05), while 787 and 901 genes were differentially expressed in cells transfected with DUX4c and DUX1 expression vectors, respectively. The 5 top ranked canonical pathways affected by DUX4 identified using the Ingenuity Pathway Analysis were IGF‐1 signaling, ovarian cancer signaling, PTEN signaling, estrogen dependent breast cancer signaling and docosahexaenoic acid signaling. The data showed that DUX4 over‐expression lead to activation of pathways involved in cell cycle, proliferation, and apoptosis. The down‐regulation of genes involved in IGF1 pathway and the activation of the PTEN pathway suggested a negative regulatory effect of DUX4 on myofiber differentiation, which might contribute to the FSHD pathology.