Intercalation of a new tier of transcription regulation into an ancient circuit

Abstract

Changes in gene regulatory networks are a major source of evolutionary novelty. Here we describe a specific type of network rewiring event, one that intercalates a new level of transcriptional control into an ancient circuit. We deduce that, over evolutionary time, the direct ancestral connections between a regulator and its target genes were broken and replaced by indirect connections, preserving the overall logic of the ancestral circuit but producing a new behaviour. The example was uncovered through a series of experiments in three ascomycete yeasts: the bakers' yeast Saccharomyces cerevisiae, the dairy yeast Kluyveromyces lactis and the human pathogen Candida albicans. All three species have three cell types: two mating-competent cell forms (a and α) and the product of their mating (a/α), which is mating-incompetent. In the ancestral mating circuit, two homeodomain proteins, Mata1 and Matα2, form a heterodimer that directly represses four genes that are expressed only in a and α cells and are required for mating. In a relatively recent ancestor of K. lactis, a reorganization occurred. The Mata1-Matα2 heterodimer represses the same four genes (known as the core haploid-specific genes) but now does so indirectly through an intermediate regulatory protein, Rme1. The overall logic of the ancestral circuit is preserved (haploid-specific genes ON in a and α cells and OFF in a/α cells), but a new phenotype was produced by the rewiring: unlike S. cerevisiae and C. albicans, K. lactis integrates nutritional signals, by means of Rme1, into the decision of whether or not to mate.

Figure 1. The core hsgs are not directly regulated by a1–α2 in K. lactis

a, The expression profiles of the set of 12 hsgs identified in K. lactis. Note that phosphate starvation induces expression of the hsgs and is required to identify these genes. For example, when starved for phosphate, the heterotrimeric G protein genes are expressed in a and α cells at levels about fivefold higher than in a/α cells. b, A comparison of hsgs defined by transcriptional profiling in S. cerevisiae (Sc), K. lactis (Kl) (panel a) and C. albicans (Ca) (ref. , and B.B.T., Q. M. Mitrovich, F. M. De La Vega, C. K. Monighetti and A.D.J., unpublished observations) shows a conserved subset of hsgs (GPA1, STE4, STE18, FAR1), which we refer to as the core hsgs (bold in a and b). c, ChIP enrichment profiles from experiments using haemagglutinin (HA)-tagged MATa1 a/α cells (magenta), HA-tagged MATα2 a/α cells (blue) and, as a control, untagged a/α cells (green). The ChIP enrichment was determined by hybridization to a tiling microarray. The location of the a1–α2 motif in the RME1 promoter is indicated by the orange star. The genes (tan boxes) are all transcribed in the reverse direction. Data were visualized with MochiView. d, The K. lactis a1–α2 motif determined from the ChIP-chip data. For comparison, the S. cerevisiae and C. albicans motifs (derived from published ChIP data4,6) are also shown.

Figure 2. RME1 is a direct activator of hsg expression and is required for K. lactis mating

a, The K. lactis Rme1 motif found by a de novo search of the 12 Kl hsgs and the S. cerevisiae motif derived from two experimentally characterized binding sites,. b, The set of 19 genes repressed twofold or greater relative to wild type when RME1 is absent and the cells are phosphate-starved. In bold are the core hsgs. c, Rme1 is a direct regulator of the core hsgs. ChIP of Rme1 was performed in K. lactis c-Myc-tagged RME1 a cells (blue and green lines, two biological replicates) and untagged, control a cells (orange line). The immunoprecipitated DNA was hybridized to a tiling microarray. The genes (tan boxes) above the line are transcribed in the forward direction and those below are transcribed in the reverse direction. The location of the Kl Rme1 motif is indicated by a purple star. d, Rme1 is required only in K. lactis to respond to mating pheromone. Wild-type or RME1 knockout a cells were exposed to either α mating pheromone or a mock treatment of dimethylsulphoxide (DMSO). Mating projections form readily when both wild-type and Δrme1 S. cerevisiae and C. albicans cells are exposed to mating pheromone. Only the K. lactis Δrme1 a cells were unable to respond to the presence of α mating pheromone. e, Rme1 is required for mating in K. lactis but not in S. cerevisiae nor C. albicans. Quantitative mating assays were performed by mating wild-type or Δrme1 a cells to wild-type α cells. In S. cerevisiae and C. albicans the percentage of a cells that was able to mate is similar for wild-type and Δrme1. K. lactis Δrme1 a cells are mating incompetent; no mating products were isolated from the Δrme1 a × wild-type α mating.

Figure 3. Overexpression of RME1 is sufficient for hsg expression in the absence of nutrient starvation

a, In the overexpression strain (pLAC4-RME1), RME1 transcription is induced by galactose-containing medium, a condition that does not cause expression of the heterotrimeric G proteins or pheromone response in wild-type (WT) cells. A strain using the empty pLAC4 vector was used as a control. The transcripts were measured relative to ACT1 transcript levels by RT–quantitative PCR (means and s.d., n = 3). In the absence of a starvation signal the hsgs, but not CKB1 (a non-hsg control), are upregulated when RME1 is overexpressed. b, RME1 overexpression allows cells to respond to mating pheromone in the absence of a starvation signal. K. lactis a cells that contained only the endogenous RME1 copy and an empty pLAC4 vector (WT), the endogenous copy of RME1 and RME1 driven by the pLAC4 promoter (WT + pLAC4-RME1) or only RME1 driven by the pLAC4 promoter (Δrme1 1pLAC4-RME1) were grown in YEP-galactose and exposed to α mating pheromone. Wild-type cells were unable to form mating projections in the absence of a starvation signal, but both strains overexpressing RME1 (pLAC4-RME1) formed mating projections in the absence of a starvation signal.

Figure 4. A simplified model for the evolution of regulation of core hsgs in three yeasts

In all three species the core hsgs are repressed by a1–α2; thus, they are ON in a and α cells and OFF in a/α cells. In S. cerevisiae and C. albicans the repression is direct (a1–α2 binds to the promoters of these genes), but in K. lactis it is indirect, through Rme1. The circuit rewiring in the K. lactis lineage has resulted in a new mating behaviour; this species is able to mate only when starved. We show that this behaviour is due to the intercalation of Rme1, which is upregulated by starvation in K. lactis.