(A) UCSC genome browser images illustrating normalized tag counts for Klf5 (post-differentiation day 0, 2, 5), MyoD (myotube, differentiated for 2 days) and Mef2D (myotube, differentiated for 5 days) at Myog loci in differentiating C2C12 myotubes. (B) Heatmap for binding of Klf5 (post-differentiation day 0, 2, and 5), MyoD (myotubes, differentiated for 2 days) and Mef2D (myotubes, differentiated for 5 days) within 2 kb around the center of the top 1000 Klf5-binding sites on day 5. The Klf5 binding sites were subdivided into 2 groups based on the presence of significant MyoD binding peaks in myotubes within 500 bp of the Klf5 binding regions. The distribution of Klf5 tag densities in each subgroup were shown as histogram. (C) Distribution of Klf5 tag densities in the vicinity of MyoD-bound loci in myoblasts differentiated for 0, 2 and 5 days. (D) Distribution of Klf5 tag densities in the vicinity of Mef2D-bound loci in myoblasts differentiated for 0, 2 and 5 days. (E) Venn diagram showing the overlap between the 12,769 MyoD peaks, 11,193 Mef2D peaks and 4382 Klf5 peaks identified by ChIP-seq. Note over half the Klf5 peaks (2224 out of 4382 peaks) are also bound by MyoD and/or Mef2. (F) Direct interaction of Klf5 and MyoD or Mef2 shown by co-immunoprecipitation. Whole cell extract from C2C12 cells differentiated for 5 days were immunoprecipitated using anti-MyoD or anti-Mef2 antibody. The interaction between Klf5 and MyoD or Mef2 is shown compared to the 1% Input or control samples without primary antibodies. (G) de novo motifs identified in regions bound by Klf5 in myotubes after differentiation for 5 days. (H). HEK-293T cells were co-transfected with Myog-luciferase; Klf5, MyoD, and Mef2D expression plasmids, as indicated; and a control CMV-renilla luciferase plasmid. Luciferase activities relative to the basal level of Myog-luciferase cotransfected with empty expression vectors are shown. Data represent means ± SEM. (**p<0.01, ***p<0.001, ****p<0.0001). Representative data from three individual experiments are shown.