Research Article: Conservation and Evolution of Cis-Regulatory Systems in Ascomycete Fungi

Date Published: December 9, 2004

Publisher: Public Library of Science

Author(s): Audrey P Gasch, Alan M Moses, Derek Y Chiang, Hunter B Fraser, Mark Berardini, Michael B Eisen

Abstract: Relatively little is known about the mechanisms through which gene expression regulation evolves. To investigate this, we systematically explored the conservation of regulatory networks in fungi by examining the cis-regulatory elements that govern the expression of coregulated genes. We first identified groups of coregulated Saccharomyces cerevisiae genes enriched for genes with known upstream or downstream cis-regulatory sequences. Reasoning that many of these gene groups are coregulated in related species as well, we performed similar analyses on orthologs of coregulated S. cerevisiae genes in 13 other ascomycete species. We find that many species-specific gene groups are enriched for the same flanking regulatory sequences as those found in the orthologous gene groups from S. cerevisiae, indicating that those regulatory systems have been conserved in multiple ascomycete species. In addition to these clear cases of regulatory conservation, we find examples of cis-element evolution that suggest multiple modes of regulatory diversification, including alterations in transcription factor-binding specificity, incorporation of new gene targets into an existing regulatory system, and cooption of regulatory systems to control a different set of genes. We investigated one example in greater detail by measuring the in vitro activity of the S. cerevisiae transcription factor Rpn4p and its orthologs from Candida albicans and Neurospora crassa. Our results suggest that the DNA binding specificity of these proteins has coevolved with the sequences found upstream of the Rpn4p target genes and suggest that Rpn4p has a different function in N. crassa.

Partial Text: The diversity of modern organisms reflects and arises from an underlying molecular diversity that is only beginning to be understood. In recent years, much focus has been given to the evolution of protein coding regions, under the assumption that diversification of protein function has driven the evolution of organismal form and function. Nevertheless, the relative dearth of species-specific genes, and the seeming abundance of functionally homologous proteins in many different genomes, suggest additional mechanisms of diversification. One mechanism likely to play a significant role is variation in gene expression (Monod and Jacob 1961; Wilson et al. 1974). Subtle alterations in the timing, location, and levels of protein synthesis can have considerable consequences at both the molecular and organismal level (Averof and Patel 1997; Gompel and Carroll 2003; Lee et al. 2003). Despite the likely importance of variation in gene expression, relatively little is known about the evolution of gene-expression regulation or how this evolution contributes to organismal diversification.

We began by systematically characterizing known cis-regulatory elements and their gene targets in the well-studied yeast S. cerevisiae. We compiled a catalog of known and predicted S. cerevisiaecis-regulatory elements (Dataset S1) in two ways. First, we retrieved 80 known consensus transcription factor-binding sites from the literature, based in part on information summarized on the Yeast Proteasome Database (Costanzo et al. 2001) and the Saccharomyces Genome Database (Weng et al. 2003). The majority of these sequences have been experimentally defined. Six others were identified by virtue of their conservation in the 3′ untranslated regions of closely related Saccharomyces species (Kellis et al. 2003), and five downstream elements were computationally predicted from mRNA immunoprecipitation experiments (Gerber et al. 2004). In addition to these known consensus sequences, we used the program MEME (Bailey and Elkan 1994) to identify 597 upstream sequence motifs common to groups of predicted coregulated genes (see below). Genes that contained one or more instance of each of these sequences in the 1,000-bp upstream or 500-bp downstream regions were identified as described in Materials and Methods.

The ascomycete fungi represent nearly 75% of all fungal species, and their diversity is evident by their unique morphologies, life styles, environmental interactions, and niches (Ainsworth et al. 2001). This diversity has been shaped by over a billion years of evolution (Berbee and Taylor 1993; Heckman et al. 2001) and has almost certainly been affected by variation in gene expression. To explore the evolution of gene-expression regulation in these fungi, we have examined the cis-regulatory networks of 14 ascomycete species whose genomes have been sequenced, using a framework that is not dependent on multiple alignments of orthologous regulatory regions. We have identified probable cis-acting sequences in each of these species by applying motif search and discovery methods to the flanking regions of orthologs of coregulated S. cerevisiae genes. Our ability to identify such sequences in the same gene groups from multiple species strongly suggests that the coregulation of those genes has been conserved. Examples from our analysis indicate that in many cases the genes’ coregulation is governed by a conserved regulatory system, while other examples suggest that some regulatory networks have evolved. These examples provide insights into the functional constraints that underlie the evolution of gene-expression regulation, as summarized below.