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. 2019 Feb 26;17(2):e3000153. doi: 10.1371/journal.pbio.3000153

Fig 6. Pax3 cooperates with Six4 and Tead2 to activate the myogenic program.

Fig 6

(A) Number of ChIP-seq peaks identified upon PAX3 immunoprecipitation in primary myoblasts, 1-day, and 6-day iPax3-induced cells. Venn diagram indicates the overlap between these 3 datasets. (B) Selected transcription factor motifs enriched at Pax3-bound loci from 1-day, 6-day, and primary myoblasts. Distribution of the motifs across 500 bp from the peak center is reported below. (C) Six4 and Runx1 bind at a subset of 6-day Pax3 loci. Fewer loci were also bound by Jun, Tead1-4, and Usf1 proteins. k-means clustering was generated using published ChIP-seq data (from myogenic cell line C2C12). (D) Distribution of ChIP-seq reads for Six4, Jun, Tead1/4, Runx1, and Usf1 1-day and 6-day Pax3 genomic binding data in cluster 1 (from panel C) generated using k-means clustering. Curves show overlapping of Six4 and Runx1 binding at Pax3 peaks (±3 kb from peak center). (E) IGV track displaying genomic occupancy for Pax3, Six4, Runx1, Jun, Tead1/4, and Usf1 at the Cdon locus. Dashed red square indicates Pax3-bound site characterized by Six4, Runx1, Jun, and Tead1/4 occupancy. (F-G) Knockdown of Six4 or Tead2 in iPax3 PDGFRα+FLK1− cells impairs Pax3 transcriptional activity and ultimately myogenic differentiation. Upon Pax3 induction, day 5 EB cells were analyzed by qRT-PCR with the indicated probes (F) or assayed for terminal differentiation (G). Graph represents mean + SD from at least 3 independent experiments. MYHC (red); nuclei (blue). Bar: 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001. Numerical values are available in S1 Data. AP1, activator protein 1; Cdon, cell adhesion molecule–related/down-regulated by oncogenes; ChIP-seq, chromatin immunoprecipitation sequencing; Ctrl, control; EB, embryoid body; Gapdh, glyceraldehyde 3-phosophate dehydrogenase; IgG, immunoglobulin G; IGV, Integrative Genomics Viewer; iPax3, inducible-Pax3; Lfng, lunatic fringe; Megf10, multiple epidermal growth factor–like domains protein 10; Myf5, myogenic factor 5; MYHC, myosin heavy chain; Pax3, paired box 3; qRT-PCR, quantitative reverse transcription PCR; RFX, regulatory factor X; Runx1, Runt-related transcription factor 1; shSCR, scramble control; shSix4, Six4 knockdown; shTead2, Tead2 knockdown; Six4, sine oculis-related homeobox 4; Tead1/2/4, TEA domain family member 1/2/4; Tot, total; Usf1, upstream stimulatory factor 1.