Transcription factors (TFs) are crucial regulators of eukaryotic gene expression that bind to short stretches of DNA commonly known as TF-binding sites (TFBSs) and modify the expression of genes adjacent to these regions. TFs bind to TFBSs with differing affinities, resulting in longer binding times in some locations than others. Low-affinity TFBSs are predicted to be common, outnumbering high-affinity TFBSs and contributing greatly to overall expression. However, evaluating the importance of low-affinity TFBSs remains challenging because their individual contributions to gene regulation are relatively small and several low-affinity sites for different TFs can overlap (which can confound the interpretations of mutational effects). Furthermore, high-affinity sites are often prioritized over low-affinity sites as they are easier to identify in the DNA sequence, easier to validate experimentally, and more likely to be evolutionarily conserved and yield a phenotype when mutated.

A recent paper in Nature by Lim et al. demonstrated that affinity-increasing mutations in low-affinity TFBSs can affect organismal phenotypes, such as polydactyly in vertebrates. More specifically, within the ZRS enhancer the authors identified polymorphisms that regulate expression of Shh and cause polydactyly. They found that these mutations alter a low-affinity TFBS for ETS-1 and slightly increase affinity for ETS-1, which results in ectopic expression of Shh and polydactyly in mouse models.