Research Article: The Evolution of Combinatorial Gene Regulation in Fungi

Date Published: February 26, 2008

Publisher: Public Library of Science

Author(s): Brian B Tuch, David J Galgoczy, Aaron D Hernday, Hao Li, Alexander D Johnson, Laurence D Hurst

Abstract: It is widely suspected that gene regulatory networks are highly plastic. The rapid turnover of transcription factor binding sites has been predicted on theoretical grounds and has been experimentally demonstrated in closely related species. We combined experimental approaches with comparative genomics to focus on the role of combinatorial control in the evolution of a large transcriptional circuit in the fungal lineage. Our study centers on Mcm1, a transcriptional regulator that, in combination with five cofactors, binds roughly 4% of the genes in Saccharomyces cerevisiae and regulates processes ranging from the cell-cycle to mating. In Kluyveromyces lactis and Candida albicans, two other hemiascomycetes, we find that the Mcm1 combinatorial circuits are substantially different. This massive rewiring of the Mcm1 circuitry has involved both substantial gain and loss of targets in ancient combinatorial circuits as well as the formation of new combinatorial interactions. We have dissected the gains and losses on the global level into subsets of functionally and temporally related changes. One particularly dramatic change is the acquisition of Mcm1 binding sites in close proximity to Rap1 binding sites at 70 ribosomal protein genes in the K. lactis lineage. Another intriguing and very recent gain occurs in the C. albicans lineage, where Mcm1 is found to bind in combination with the regulator Wor1 at many genes that function in processes associated with adaptation to the human host, including the white-opaque epigenetic switch. The large turnover of Mcm1 binding sites and the evolution of new Mcm1–cofactor interactions illuminate in sharp detail the rapid evolution of combinatorial transcription networks.

Partial Text: The recent genome sequencing and annotation of the major model organisms established that organismal complexity does not scale in a simple way with gene count. This discordance is consistent with earlier proposals that “tinkering” with gene regulation may be a particularly powerful mode of evolution [1–3]. In principle, changes in when and where, and thereby in what combinations, genes are expressed can help to explain changes in organismal complexity over longer time scales. Over shorter time scales, the contributions of changes in gene regulation to phenotypic variation have been clearly demonstrated [4,5]. For example, small changes in gene regulation underlie the gain and loss of wing spots in Drosophila species [6] and armor in stickleback fish [7].

In this work, we have tracked the evolution of combinatorial gene regulation by the highly conserved transcriptional regulator Mcm1 and each of its known cofactors across the ascomycete fungal lineage. Our analysis shows that the genes regulated by Mcm1 have changed considerably over the evolutionary time scales represented by this lineage; our results reveal many more differences than similarities in the Mcm1 circuitry. Regulation by Mcm1 is more pervasive in K. lactis and C. albicans, where 12% of all genes are bound, than in S. cerevisiae, where 4% of genes are bound. The fraction of genes shared as targets between all three species is very low (13%–18%), and we have demonstrated that this is due to both substantial gain and loss of Mcm1 binding sites along each branch of this phylogeny (Figure 2B). The extensive amount of gain and loss observed is consistent with recent studies in mammals [16] and closely related yeasts [17] and suggests the following three possibilities: (1) there is a richness of selective advantages offered in the dynamic rewiring of gene regulatory networks, (2) there are a large number of neutral alternatives to gene regulation by Mcm1, or (3) selection on gene expression is weak. The latter possibility seems at odds with other observations such as the large fraction of genes devoted to transcriptional regulation in S. cerevisiae (∼3%), the greater-than-expected number of transcriptional regulators retained after the whole genome duplication (∼6% versus ∼3%), and the considerable conservation found in many S. cerevisiae promoters [54,55]. Additionally, the fact that many of the Mcm1 sites are enriched at functionally related genes and often found in tandem with cofactor motifs argues strongly against the hypothesis that a large number of these sites are fortuitous and nonfunctional. Gauging the relative contributions of selection versus neutral drift on the gene regulatory networks will be an exciting challenge for future research [56].

Detailed methods can be found in Text S1, Figure S1–S10, and Table S1–S3. The information can also be found in one complete file, Protocol S1.