DNA-binding specificity changes in the evolution of forkhead transcription factors

Abstract

The evolution of transcriptional regulatory networks entails the expansion and diversification of transcription factor (TF) families. The forkhead family of TFs, defined by a highly conserved winged helix DNA-binding domain (DBD), has diverged into dozens of subfamilies in animals, fungi, and related protists. We have used a combination of maximum-likelihood phylogenetic inference and independent, comprehensive functional assays of DNA-binding capacity to explore the evolution of DNA-binding specificity within the forkhead family. We present converging evidence that similar alternative sequence preferences have arisen repeatedly and independently in the course of forkhead evolution. The vast majority of DNA-binding specificity changes we observed are not explained by alterations in the known DNA-contacting amino acid residues conferring specificity for canonical forkhead binding sites. Intriguingly, we have found forkhead DBDs that retain the ability to bind very specifically to two completely distinct DNA sequence motifs. We propose an alternate specificity-determining mechanism whereby conformational rearrangements of the DBD broaden the spectrum of sequence motifs that a TF can recognize. DNA-binding bispecificity suggests a previously undescribed source of modularity and flexibility in gene regulation and may play an important role in the evolution of transcriptional regulatory networks.

Keywords: protein–DNA interactions; transcription factor binding site motif.

Fig. 1.

DNA binding-site motifs bound by forkhead domain proteins. A representative member of each class of binding site discussed in the text is shown. Bold symbols are used to represent binding specificities in subsequent figures.

Fig. 2.

ML phylogenetic tree of forkhead domains. This compact tree was constructed for presentation purposes from a representative subset of phylogenetically informative species: metazoans mouse, fly, and sponge; choanoflagellates Salpingoeca rosetta and Monosiga brevicollis; Capsaspora owczarzaki and Sphaeroforma arctica from Ichthyosporea; S cerevisiae from Dikarya; Allomyces macrogynus from Blastocladiomycota; S. punctatus from Chytridiomycota; Mortierella verticillata from Mortierellomycotina; F. alba from Nucleariida; and Acanthamoeba castellanii from Amoebozoa. Nodes supported with strong likelihood ratios are indicated with red circles (aLRT ≥ 99%) or blue circles (aLRT ≥ 95%); bootstrap support values are shown for nodes with ≥80% support. Clades containing alternate binding specificities are highlighted in color (see text). Importantly, the groupings of subfamilies in this tree and the complete tree with all Fox domains are almost identical to each other (Fig. S2).

Fig. 3.

Detailed analysis of Fox3 and FoxN subfamilies. ML phylogenetic trees for Fox domains from a broader range of species for fungal Fox3 (A) and holozoan FoxN/R (B) clades. Red and blue circles indicate node support as in Fig. 2. Bold symbols represent binding capacity for different motif classes as defined in Fig. 1.

Fig. 4.

Biclustering of Fox domain binding data reveals multiple functional classes. E-score binding profiles were clustered both by protein (rows) and by contiguous 8-mer (columns) for any 8-mer bound (E score ≥ 0.35) by at least one assayed Fox protein. Fox domains fall into functional classes (bold symbols represent binding capacity for different motifs as defined in Fig. 1) that do not uniformly correlate with phylogeny (protein names are colored by phylogenetic grouping as in Fig. 2). Cluster 1 (black bar) comprises proteins specific only for the FkhP,S motifs, cluster 2 proteins are specific only for FHL variants, and cluster 3 proteins have more complex specificity; see text for details. Sequence motifs shown were generated by alignment of the indicated clusters of 8-mers and are for visualization purposes only.

Fig. 5.

Canonical Fox base-contacting residues do not explain most alternate specificity. (A) A previous cocrystal structure of mouse FoxK1 bound to the canonical FkhP site GTAAACA [Protein Data Bank ID code 2C6Y (10)]. The recognition helix is highlighted; side chains are shown in blue and labeled for those amino acids that make base-specific contacts in at least two existing structures. (B) Protein sequence alignment of the recognition helix (red underscore) and adjacent positions for a sample of Fox domains representing various specificity classes (bold symbols represent binding capacity for different motif classes as defined in Fig. 1). Numbers above alignment represent positions within the Pfam Fork_head domain HMM.