Figure 5.

Recruitment of dCas9-KRAB to the DUX4 promoter or exon 1 represses the D4Z4 locus in facioscapulohumeral muscular dystrophy (FSHD) myocytes. Chromatin immunoprecipitation (ChIP) assays were performed using FSHD myogenic cultures infected with combinations of lentiviral supernatants expressing either dCas9-KRAB or individual sgRNAs targeting the DUX4 promoter (#6–8) or exon 1 (#3–5). Following infection, cells were induced to differentiate for ~40 hours, as in Figures 2 and 3. Chromatin was immunoprecipitated using antibodies specific for (a) KAP1, (b) HP1α, (c) HP1β, (d) H3K27ac, or (e) the elongating form of RNA Pol II (Pol II-PS2), and analyzed by qPCR using primers to the (f) p13-E11 region of 4q35 or exon 1, intron 1, or exon 3 of DUX4. Location of primers is shown in Figure 1a. In cases where enrichment of the specific factor was observed across the DUX4 locus, an off-target region was also assessed. Data are presented as fold enrichment of the target region by each specific antibody normalized to α-histone H3, with enrichment for the mock-infected cells set to 1. For all panels, each bar represents the average of at least three independent ChIP experiments.