  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 ~47% of domains shared
between D. melanogaster and the other fly species. These diverging
DNA-contacting residues, found in ~66% of the D. melanogaster C2H2-ZF genes in
our analysis and corresponding to ~24% 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.
