Extensive DNA-binding specificity divergence of a conserved transcription regulator

Abstract

The DNA sequence recognized by a transcription regulator can be conserved across large evolutionary distances. For example, it is known that many homologous regulators in yeasts and mammals can recognize the same (or closely related) DNA sequences. In contrast to this paradigm, we describe a case in which the DNA-binding specificity of a transcription regulator has changed so extensively (and over a much smaller evolutionary distance) that its cis-regulatory sequence appears unrelated in different species. Bioinformatic, genetic, and biochemical approaches were used to document and analyze a major change in the DNA-binding specificity of Matα1, a regulator of cell-type specification in ascomycete fungi. Despite this change, Matα1 controls the same core set of genes in the hemiascomycetes because its DNA recognition site has evolved with it, preserving the protein-DNA interaction but significantly changing its molecular details. Matα1 and its recognition sequence diverged most dramatically in the common ancestor of the CTG-clade (Candida albicans, Candida lusitaniae, and related species), apparently without the aid of a gene duplication event. Our findings suggest that DNA-binding specificity divergence between orthologous transcription regulators may be more prevalent than previously thought and that seemingly unrelated cis-regulatory sequences can nonetheless be homologous. These findings have important implications for understanding transcriptional network evolution and for the bioinformatic analysis of regulatory circuits.

Fig. 1.

Significant divergence of the αsg cis-regulatory sequence between C. albicans and S. cerevisiae. (A) PSSM for the S. cerevisiae clade αsg cis-regulatory sequence (Sc) was derived using MEME from 27 sequences identified in either the promoters of known S. cerevisiae αsgs (42) or the promoters of the orthologous genes in S. mikatae, S. paradoxus, and S. bayanus. The PSSM for the C. albicans clade αsg cis-regulatory sequence (Ca) was derived using MEME from 12 sequences that originated from either C. albicans αsg promoter sequences (10) or promoters of the orthologous genes in C. tropicalis and C. dubliniensis. (B) Alignments of the S. cerevisiae Matα1 motif to the unknown motif within the C. albicans αsg cis-regulatory sequence (Left) and the αsg Mcm1 motif from S. cerevisiae and C. albicans (Right). Motif alignments and E values were calculated using MochiView (30), which quantifies similarities between motifs by using an algorithm derived from Gupta et al. (32).

Fig. 2.

C. albicans (Ca) Matα1 activates transcription by binding to the C. albicans αsg cis-regulatory sequences. (A) A C. albicans αsg cis-regulatory sequence taken from the α-mating pheromone gene was inserted into a basal promoter construct upstream of a β-gal reporter (pLG669Z). The same C. albicans αsg cis-regulatory sequence was also mutated to alter the residues at the position where Matα1 binds to the S. cerevisiae cis-regulatory sequence (Ca-Δ). These constructs were introduced into S. cerevisiae MATa cells (MATa cells lack S. cerevisiae MATα1). In the two right lanes, strains also contain a 415-translation elongation factor promoter (TEF) plasmid modified to express a codon-changed C. albicans Matα1 (the codon changes were necessary because C. albicans decodes the CUG codon as serine and most other species, including S. cerevisiae, decode it as a leucine). Reporter activity was monitored using β-galactosidase assays. For each sample, n = 5 and error bars represent SE. (B) Electrophoretic mobility gel shift assays were performed using S. cerevisiae cell extracts. The labeled oligonucleotide used in this experiment was the C. albicans αsg cis-regulatory sequence described in A. Extracts were prepared from an S. cerevisiae MATa strain containing a galactose-inducible copy of the codon-changed C. albicans Matα1. Each lane contains 5 mg of protein from cell extracts. Galactose induction was performed overnight on samples in lanes 2 and 4 (lanes 1 and 3 are grown in glucose, turning off C. albicans Matα1 expression). In lanes 3 and 4, an N-terminal peptide antibody against C. albicans Matα1 (Bethyl Laboratories) was used to confirm that DNA-binding activity was attributable to the C. albicans Matα1 protein.

Fig. 3.

Extensive DNA-binding specificity divergence of the Matα1 protein. (A) αsg cis-regulatory sequence of the promoter for the α-mating pheromone from C. albicans (Ca) or from S. cerevisiae (Sc) was inserted into a basal promoter construct (pLG669z). These constructs were introduced into S. cerevisiae MATα Δmatα1 cells along with a 415-TEF plasmid modified to express S. cerevisiae MATα1 (columns 2 and 5) or a 415-TEF plasmid modified to express the codon-changed C. albicans MATα1 (columns 3 and 6). Reporter activity was monitored using β-galactosidase assays. For each sample, n = 5 and error bars represent SE. (B) Electrophoretic mobility gel shift assays were performed using S. cerevisiae cell extracts. The labeled oligonucleotide used in this experiment was either the C. albicans αsg cis-regulatory sequence (lanes 4–6) or S. cerevisiae αsg cis-regulatory sequence (lanes 1–3), both of which are described in A. Extracts were prepared from either S. cerevisiae MATa cells containing a galactose-inducible copy of C. albicans MATα1 or S. cerevisiae MATα cells containing a galactose-inducible copy of the S. cerevisiae MATα1 (p415GAL). Galactose induction was performed overnight on samples in lanes 2, 3, 5, and 6 (lanes 1 and 4 are grown in glucose). Each lane contains 5 mg of protein from cell extracts. (C) To create the Ca/Sc hybrid construct, the Matα1-binding site from the Ca reporter construct was used to replace the Matα1-binding site in the Sc reporter construct. To create the Sc/Ca hybrid construct, the Matα1-binding Saccharomyces site from the Sc reporter construct was used to replace the Matα1-binding site in the Ca reporter construct. Reporter activity was monitored using β-galactosidase assays.

Fig. 4.

DNA-binding specificity of the C. albicans Matα1 protein evolved after the divergence of S. cerevisiae and C. albicans. (A) Orthologs of the S. cerevisiae and C. albicans αsgs were mapped across 38 genome-sequenced yeasts (10, 11, 13, 28, 46, 48). Where a clear ortholog could be detected, the promoters of these orthologs were scanned with either the S. cerevisiae or C. albicans clade αsg cis-regulatory sequence PSSM (created as described in Fig. 1A). Maximum log₁₀ odds scores are shown. Darker shades of orange indicate a stronger match to the PSSM. One-to-one orthologs become more difficult to detect with greater evolution distance, hence, the small number of orthologs identified in the filamentous fungi (e.g., S. sclerotiorum, A. terreus). (B) PSSM for the filamentous fungi αsg cis-regulatory sequence was derived using MEME from nine sequences identified in the promoters of αsg orthologs in the filamentous fungi species U. reesii, C. immitis, F. graminea, A. terreus, A. nidulans, and S. sclerotiorum. (C) Putative αsg cis-regulatory sequence from the promoter of the STE3 ortholog in the filamentous fungi species U. reesii (FF) was placed into the basal promoter construct (pLG669z). The same construct was mutated at the position of the putative Matα1 motif (FFΔ). S. cerevisiae Matα1 was supplied by the endogenous copy within a MATα strain (columns 1 and 2), and C. albicans Matα1 was supplied from expression of p415TEF within an S. cerevisiae MATa strain (columns 3 and 4). Reporter activity was monitored using β-galactosidase assays. For each sample, n = 5 and error bars represent SE.

Fig. 5.

Matα1 DNA-binding specificity has continued to diverge within the CTG-clade. Three putative αsg cis-regulatory sequences were identified by MEME in the promoters of C. lusitaniae αsg orthologs. The αsg cis-regulatory sequence of the promoter for the α-mating pheromone (MFα1) from C. lusitaniae (Cl) was inserted into a basal promoter construct (pLG669z), and the C. lusitaniae Matα1 was expressed from a 415-TEF plasmid. Plasmids were transformed into an S. cerevisiae MATα Δmatα1 strain. Reporter activity was monitored using β-galactosidase assays. For each sample, n = 5 and error bars represent SE.