There is an enrichment of these putative regulatory regions close to gene TSS (within 1 kb) as compared with the locations of all possible 1-kb regions on our custom ChIP-chip array (p < 1 × 10− 14, Hypergeometric test; Fig. 2). Despite this skew toward proximal promoter regions, about 80% of regions bound by MyoG at 48 h with H3K4me1 or H3K27Ac modifications fall outside of traditional promoters (1 kb from TSS) and, strikingly, 25% of such putative CRMs are more than 20 kb away from any TSS. Interestingly, genomic regions occupied by different TFs and with different binding dynamics during differentiation differ in their distances from gene TSS. Genomic regions that either lose or gain MyoG binding from 0 h to 48 h tend to be located further from TSSs than those that have constant MyoG binding at 0 and 48 h (p = 1.39 × 10− 4 for constant binding versus gain of binding at click here 48 h; Kolmogorov–Smirnov (KS) test; Fig. 2A). Increased distance from gene promoters is also observed for regions that change in both MyoG and SRF binding between 0 and 48 h of differentiation as compared with those that are bound by both MyoG and SRF at both timepoints. In addition, regions bound by all 3 of these TFs (MyoG, MyoD, SRF) tend to lie closer to genes than regions with only one or two TFs bound (p < 2.2 × 10− 16 for MyoG at 48 h vs. MyoG, SRF, and MyoD at 48 h; KS test; Fig. 2B). For 69% of the regions with enriched MyoG binding at 48 h and H3K4me1 or H3K27Ac marks in myoblasts, the closest gene is not known to be involved in myogenic differentiation and is not differentially expressed during this process. In fact, 90% of such putative CRMs are located outside of 1 kb muscle gene promoter regions and 56% are more than 20 kb away from a muscle gene TSS (Fig. 2C). Furthermore, for 30% of these MyoG-bound, H3K4me1 or H3K27Ac modified regions, associating with the closest muscle gene along the linear chromosome requires skipping over intervening non-muscle genes. We specifically included 100 genomic regions on our custom ChIP-chip array design that contained 287 high-scoring PhylCRM predictions of myogenic CRMs with clusters of evolutionarily conserved myogenic TF binding site matches, but were located far (> 50 kb) from any annotated muscle gene TSS. These distant predicted CRMs exhibited MyoG binding and enhancer-associated histone marks at a somewhat lower frequency (26%) than PhylCRM-predicted CRMs < 50 kb away from muscle gene TSS (35%). However, permutation testing showed that this level of overlap was greater than expected (p = 0.02) for randomly selected genomic regions represented on the array and much greater than expected (p < 1 × 10− 4) for genomic regions with the same median distance to the nearest TSS of any protein-coding gene (21.8 kb).