Additional file 7: Figure S5. of BET bromodomain inhibitors and agonists of the beta-2 adrenergic receptor identified in screens for compounds that inhibit DUX4 expression in FSHD muscle cells

FSHD myoblasts grown continuously in low dose BETi maintain suppressed DUX4 target gene expression but do not re-establish D4Z4 repeat DNA methylation. (A) DUX4 target gene expression in MB200 FSHD2 myoblasts grown for three weeks in media containing DMSO or 100 nM (+)-JQ1 compared to gene expression in 54-6 control (non-FSHD) myoblasts. (B) Structure of the D4Z4 repeat unit showing regions analyzed by bisulfate sequencing in this study (ADS3747 and ADS1454) relative to the DUX4 open reading frame (ORF). ADS3747 spans positions 665-708 and ADS1454 spans positions 2230-2361 with respect to the start of the KpnI site. The locations of previously published methylation-sensitive restriction sites and regions analyzed by bisulfite sequencing (DR1, DR2 and DR3) are indicated. (C-D) Average percent methylation across 9 CpG sites within ADS3747 (C) or 10 CpG sites within ADS1454 (D) in 54-6 control (non-FSHD, Normal), 54-2 FSHD1, and MB200 FSHD2 myoblasts, as well as MB200 FSHD2 myoblasts grown for three weeks in media containing DMSO or 100 nM (+)-JQ1. (E) Slow recovery of DUX4 target gene expression after BETi withdrawal. MB200 FSHD2 myoblasts grown for 3 weeks in 100 nM (+)-JQ1 were split and seeded onto culture plates in the absence of drug at ~10% confluence to allow for continued growth. ZSCAN4 mRNA levels in untreated MB200 FSHD2 myoblasts (Control), MB200 FSHD2 myoblasts maintained continuously in drug (JQ1) and MB200 FSHD2 myoblasts grown for the indicated times after compound withdrawal are shown. Error bars in (A) and (E) indicate the standard deviation from the mean of three biological replicates. (PDF 122 kb)