Differences in transcriptional regulatory networks underlie much of the phenotypic variation observed across organisms. Changes to cis-regulatory elements are widely believed to be the predominant means by which regulatory networks evolve, yet examples of regulatory network divergence due to transcription factor (TF) variation have also been observed. To systematically ascertain the extent to which TFs contribute to regulatory divergence, we analyzed the evolution of the largest class of metazoan TFs, Cys2-His2 zinc finger (C2H2-ZF) TFs, across 12 Drosophila species spanning ~45 million years of evolution. Remarkably, we uncovered that a significant fraction of all C2H2-ZF 1-to-1 orthologs in flies exhibit variations that can affect their DNA-binding specificities. In addition to loss and recruitment of C2H2-ZF domains, we found diverging DNA-contacting residues in ~44% of domains shared between D. melanogaster and the other fly species. These diverging DNA-contacting residues, found in ~70% of the D. melanogaster C2H2-ZF genes in our analysis and corresponding to ~26% of all annotated D. melanogaster TFs, show evidence of functional constraint: they tend to be conserved across phylogenetic clades and evolve slower than other diverging residues. These same variations were rarely found as polymorphisms within a population of D. melanogaster flies, indicating their rapid fixation. The predicted specificities of these dynamic domains gradually change across phylogenetic distances, suggesting stepwise evolutionary trajectories for TF divergence. Further, whereas proteins with conserved C2H2-ZF domains are enriched in developmental functions, those with varying domains exhibit no functional enrichments. Our work suggests that a subset of highly dynamic and largely unstudied TFs are a likely source of regulatory variation in Drosophila and other metazoans. Author Summary The phenotypic differences observed between closely related organisms are thought to be due largely to changes in regulatory networks. Changes in transcriptional networks can occur via mutations in cis binding sites, for which there are numerous known examples, as well as via binding specificity variation in transcription factors (TFs), a less studied phenomenon that has been observed primarily in multi-gene families. Though large-scale experimental studies ascertaining the extent to which TFs contribute to regulatory network variation across organisms are lacking and would be time-consuming, computational methods can begin to address this challenge. Here, we present a systematic, large-scale analysis of DNA-binding specificity evolution in TF orthologs by computationally leveraging specific features of Cys_(2)-His_(2)zinc finger proteins, the largest class of TFs in animals and major components of their regulatory programs. We find not only that divergence of DNA-binding residues in 1-to-1 orthologous C2H2-ZFs is pervasive but also that these changes show evidence of functional constraint and occur in a gradual, evolutionarily viable manner. We conclude that the diversification of orthologous TFs has most likely played a major and largely unstudied role in gene regulatory network evolution in metazoans.
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