Gene duplication and the evolution of ribosomal protein gene regulation in yeast

Abstract

Coexpression of genes within a functional module can be conserved at great evolutionary distances, whereas the associated regulatory mechanisms can substantially diverge. For example, ribosomal protein (RP) genes are tightly coexpressed in Saccharomyces cerevisiae, but the cis and trans factors associated with them are surprisingly diverged across Ascomycota fungi. Little is known, however, about the functional impact of such changes on actual expression levels or about the selective pressures that affect them. Here, we address this question in the context of the evolution of the regulation of RP gene expression by using a comparative genomics approach together with cross-species functional assays. We show that an activator (Ifh1) and a repressor (Crf1) that control RP gene regulation in normal and stress conditions in S. cerevisiae are derived from the duplication and subsequent specialization of a single ancestral protein. We provide evidence that this regulatory innovation coincides with the duplication of RP genes in a whole-genome duplication (WGD) event and may have been important for tighter control of higher levels of RP transcripts. We find that subsequent loss of the derived repressor led to the loss of a stress-dependent repression of RPs in the fungal pathogen Candida glabrata. Our comparative computational and experimental approach shows how gene duplication can constrain and drive regulatory evolution and provides a general strategy for reconstructing the evolutionary trajectory of gene regulation across species.

Fig. 1.

The evolutionary history of RP genes and their regulators IFH1 and CRF1. (A) Shown are the key regulators previously associated with RP gene promoters in S. cerevisiae. Regulators are shown with no specific orientation along the promoter. Rap1 directly binds to RP promoters (4) whereas Ifh1 and Crf1 interact with RP promoters via Fhl1 (–15). It is still unclear whether Sfp1 and Hmo1 affect RP gene expression through direct binding (18, 19) or indirectly (6, 33). (B) IFH1 and CRF1 are paralogs originating from an ancestral WGD (20, 21, 23). (Left) A phylogenetic tree of Hemiascomycota fungi that diverged before and after a WGD event (red star), the number of RP genes found in each genome (in parentheses), and the presence (check) or absence (X mark) of an IFH-like or CRF-like gene in these species (Right). Although IFH1 was retained in all lineages after the WGD (red star), CRF1 was lost in C. glabrata, consistent with the pattern of paralogous RP retention. IFH1 retains sequence features similar to those of its non post-WGD orthologs, and CRF1 has lost an acidic N terminus region (white box) but has retained a conserved FHB domain (13) (gray box). (C) A Venn diagram of the RP genes retained in duplicate in each of the three post-WGD species. Nearly all paralogous RP copies were lost in C. glabrata, whereas S. cerevisiae and S. castellii have retained a significant portion of them. (D) (Left) In S. cerevisiae, IFH1 (blue) induces RP gene expression whereas CRF1 (red) is a stress-responsive repressor (13), and both interact with FHL1 (gray). (Right) Hypothetical roles of the IFH1/CRF1 ancestor as solely an activator (blue, suggesting neofunctionalization of CRF1) or as both an activator and a repressor (blue/red, consistent with subfunctionalization).

Fig. 2.

RP and IFH1/CRF1 expression in all species. Shown are the log₂ fold-changes in expression levels of ribosomal protein (RP, Center) and ribosome biogenesis (RiBi, Left) genes in each of the species at each time point, relative to time point 0. Gray, individual genes; black, mean and standard deviation of the whole set. (Right) The change in expression levels in each species’ copy of IFH1 (blue) and CRF1 (red) or their preduplication orthologs (gray) during the peak of each species’ response to stress (peak times for species are from top to bottom: 30, 15, 30, 45, 30, and 30 min).

Fig. 3.

cis- and trans-regulatory evolution of RP genes across pre- and post-WGD species. (A) Representative cis-regulatory motifs (columns) found to be enriched in RP promoters in at least one species (rows) and the enrichments of RP promoters associated with the motif in each species (dark purple, P < 10⁻¹⁰; black, P = 1). The total number of annotated RPs in each species is denoted next to the species name. (B) A model of an evolutionary trajectory of IFH1/CRF1 functional roles. The pre-WGD ortholog of IFH1/CRF1 performed dual roles, both inducing and repressing RP expression levels in a condition-dependent manner. After duplication, each paralog specialized to act as a separate activator (IFH1) and repressor (CRF1). The repressor was subsequently lost in the lineage leading to C. glabrata.