We then used motif load to address whether TFBSs proximal to transcription start sites (TSSs) are more constrained compared to more distant regulatory regions. We found this to be the case in human, but not Drosophila (Figure 3d; see Discussion). CTCF binding sites in both species were a notable exception, tolerating the lowest mutational load at locations 500 bp to 1 kb from TSSs, but not closer to the TSS (Figure 3d, bottom panel), suggesting that the putative role of CTCF in establishing chromatin domains [42] is particularly important in proximity of gene promoters.
