How duplicated transcription regulators can diversify to govern the expression of nonoverlapping sets of genes

Abstract

The duplication of transcription regulators can elicit major regulatory network rearrangements over evolutionary timescales. However, few examples of duplications resulting in gene network expansions are understood in molecular detail. Here we show that four Candida albicans transcription regulators that arose by successive duplications have differentiated from one another by acquiring different intrinsic DNA-binding specificities, different preferences for half-site spacing, and different associations with cofactors. The combination of these three mechanisms resulted in each of the four regulators controlling a distinct set of target genes, which likely contributed to the adaption of this fungus to its human host. Our results illustrate how successive duplications and diversification of an ancestral transcription regulator can underlie major changes in an organism's regulatory circuitry.

Keywords: Candida albicans; gene duplication; gene regulation; molecular evolution; regulatory networks; transcription regulator.

Figure 1.

Recently duplicated C. albicans LYS transcription regulators bind to largely nonoverlapping sets of target genes. (A) Cladogram depicting the phylogenetic relationships among extant species of the Candida and Saccharomyces clades. The arrows to the right of the tree represent the homologs of S. cerevisiae LYS14 found in each species’ genome. Gene orthology assignments (represented by arrows of the same color) are based on synteny and the reconstructed phylogeny of the gene family (Supplemental Fig. 1). Red ovals in the branches of the cladogram represent the inferred single-gene duplication events that gave rise to the four C. albicans homologs. No “strict orthology” can be inferred between a particular Candida LYS gene and Saccharomyces LYS14 based on phylogenetic reconstructions (Supplemental Fig. 1) or synteny (therefore, the annotation of one of the Candida genes as LYS14 is misleading in this respect). The similarity in color between LYS144 and Saccharomyces LYS14 depicts simply the fact that the DNA sequences recognized by these homologous proteins most closely resemble one another. (B) Inferred relationships among the four LYS regulators (to the left) in C. albicans and the gene network (to the right) formed by the four regulators (purple, orange, blue, and green circles) and their target genes (small black circles) as mapped by ChIP. The distances separating the four Lys proteins are inversely proportional to the number of shared target genes (fewer shared targets, greater separation). Although some target genes are bound by more than one regulator, most of the targets are specific to only one of the four LYS regulators.

Figure 2.

DNA motifs preferred by C. albicans LYS transcription regulators. (A) DNA motifs derived from MITOMI and ChIP data sets. (B) Gel shift assays showing binding of the Lys proteins to their predicted binding sites. ³²P-labeled DNA fragments (∼0.4 nM) containing the predicted wild-type or mutant LYS-binding sites were incubated with increasing concentrations of purified Lys protein (0, 0.039, 0.156, 0.625, 2.5, 10, and 40 nM) for 30 min at room temperature in standard EMSA buffer and resolved in 6% polyacrylamide gels run with 0.5× TGE. Point mutations introduced in the binding sites are indicated in red.

Figure 3.

DNA-binding preferences among the four C. albicans Lys proteins. (A) Determination of the DNA-binding preferences of Lys14p through competition experiments. The purified C. albicans Lys14 protein was incubated with a radiolabeled DNA fragment containing its preferred binding site (LYS14a; i.e., the sequence consisting of repeats adjacent to each other but not overlapping). Increasing amounts of unlabeled competitor DNA fragments containing LYS14a-, LYS14b- (sequence with the repeats overlapping by 1 nt), LYS142-, LYS143-, or LYS144-binding sites were added to the reactions, and the mixtures were resolved by polyacrylamide gel electrophoresis. (B) Quantification of the assays shown in A and best-fit curves. (C) Summary of the binding preferences of the four proteins. (Top panel) The Lys14p-binding preferences are a summary of the data displayed in A and B. The bars show the inverse of the concentration of competitor DNA needed to achieve a 50% reduction in binding to the radiolabeled sequence, with the concentration of the cognate fragment set to a value of 1. Therefore, values <1 correspond to weaker binding. The middle and bottom panels summarize the corresponding experiments for Lys142p, Lys143p, and Lys144p; the images, quantification, and best-fit curves for these three proteins are shown in Supplemental Figures 5–7.

Figure 4.

Mcm1p promotes Lys144p binding to DNA. (A) Putative Mcm1p motif identified in the set of DNA sequences occupied in vivo by Lys144p. (B) Distribution of Lys144p and Mcm1p DNA-binding sites in a subset of the sequences occupied by Lys144p. Putative Lys144p sites are shown in purple (half-sites), whereas the predicted Mcm1p sites are underlined. Check marks to the right indicate whether Mcm1p has been found to bind in vivo to the respective target gene (Tuch et al. 2008). (C) Gel shift assays carried out with one of the sequences shown in B (ORF19.2077) and purified Lys144 and Mcm1 proteins. The open arrow corresponds to the DNA+Lys144p-bound complex, whereas the solid arrow indicates the location of the DNA+Lys144+Mcm1 tripartite complex.