Figure 1. Reporter constructs and their activity in human and mouse myoblasts and myotubes.

(A) The distal end of chromosome 4 is shown above a schematic of an EcoRI fragment cloned from an FSHD-affected individual (λ42). λ42 contains two full D4Z4 units (green and pink rectangles), and two partial D4Z4 units on either end. The DUX4 open reading frame is shown as a yellow colored rectangle with homeoboxes shown as black boxes within each D4Z4 repeat. The approximate location of the TATA box (TACAA) and the transcription start site (bent arrow) are indicated. The restriction enzymes SfoI and ApoI were used to clone the non-coding region. The location of previously identified miRNA fragments from D4Z4 are shown as blue lines [3] (B) The non-coding region of D4Z4 was isolated from the second repeat of λ42 and placed upstream or downstream of the indicated reporter genes (D4Z4→eGFP, sense promoter driving eGFP; eGFP←D4Z4, antisense promoter driving eGFP; eGFP←D4Z4→dsRED, DUX4 promoter driving dsRED in the sense direction and eGFP in the antisense direction. (C) Control and FSHD Human myoblasts were differentiated into myotubes and assayed for DUX4 expression by RT-PCR. Locations of primers 1 and 2 to detect DUX4 transcripts are shown along with common splice sites within the DUX4 transcripts. (D) Mouse and Human myoblasts transduced with a lentivirus vector encoding D4Z4→eGFP were sorted by flow cytometry, and expanded in culture. The cells were seeded at equal densities, switched to either myoblast proliferation medium or myotube differentiation medium for 72 hours, and imaged by fluorescence microscopy. Scale bars = 50 µm. Images were taken at the same time with the same exposure settings.