TFforge utilizes large-scale binding site divergence to identify transcriptional regulators involved in phenotypic differences.

Changes in gene regulation are important for phenotypic and in particular morphological evolution. However, it remains challenging to identify the transcription factors (TFs) that contribute to differences in gene regulation and thus to phenotypic differences between species. Here, we present TFforge (Transcription Factor forward genomics), a computational method to identify TFs that are involved in the loss of phenotypic traits. TFforge screens an input set of regulatory genomic regions to detect TFs that exhibit a significant binding site divergence signature in species that lost a particular phenotypic trait. Using simulated data of modular and pleiotropic regulatory elements, we show that TFforge can identify the correct TFs for many different evolutionary scenarios. We applied TFforge to available eye regulatory elements to screen for TFs that exhibit a significant binding site decay signature in subterranean mammals. This screen identified interacting and co-binding eye-related TFs, and thus provides new insights into which TFs likely contribute to eye degeneration in these species. TFforge has broad applicability to identify the TFs that contribute to phenotypic changes between species, and thus can help to unravel the gene-regulatory differences that underlie phenotypic evolution.