Barcode Sequencing Screen Identifies SUB1 as a Regulator of Yeast Pheromone Inducible Genes

Abstract

The yeast pheromone response pathway serves as a valuable model of eukaryotic mitogen-activated protein kinase (MAPK) pathways, and transcription of their downstream targets. Here, we describe application of a screening method combining two technologies: fluorescence-activated cell sorting (FACS), and barcode analysis by sequencing (Bar-Seq). Using this screening method, and pFUS1-GFP as a reporter for MAPK pathway activation, we readily identified mutants in known mating pathway components. In this study, we also include a comprehensive analysis of the FUS1 induction properties of known mating pathway mutants by flow cytometry, featuring single cell analysis of each mutant population. We also characterized a new source of false positives resulting from the design of this screen. Additionally, we identified a deletion mutant, sub1Δ, with increased basal expression of pFUS1-GFP. Here, in the first ChIP-Seq of Sub1, our data shows that Sub1 binds to the promoters of about half the genes in the genome (tripling the 991 loci previously reported), including the promoters of several pheromone-inducible genes, some of which show an increase upon pheromone induction. Here, we also present the first RNA-Seq of a sub1Δ mutant; the majority of genes have no change in RNA, but, of the small subset that do, most show decreased expression, consistent with biochemical studies implicating Sub1 as a positive transcriptional regulator. The RNA-Seq data also show that certain pheromone-inducible genes are induced less in the sub1Δ mutant relative to the wild type, supporting a role for Sub1 in regulation of mating pathway genes. The sub1Δ mutant has increased basal levels of a small subset of other genes besides FUS1, including IMD2 and FIG1, a gene encoding an integral membrane protein necessary for efficient mating.

Keywords: SUB1; mating; transcription.

Figure 1

Overview of expected pheromone response pathway mutants. Control strains (gray), mutant uninduced (blue), and mutant induced with α-factor (red). The three wild-type controls were run with every experiment, in gray: wild-type Gfp^–, Uninduced (“Un Gfp^basal”), and Induced (“In Gfp⁺”).

Figure 2

Overview of the screening strategy. (A) Reporter construct. (B) Overview of FACS sorting transformed YKO library. (C) FACS profile of YKO library overlay of no α-factor (blue), and the sorted mutant populations “Un Gfp^–” and “Un Gfp⁺”. (D) FACS profile of YKO library overlay of +α-factor (red), and the sorted populations “in Gfp^–”, “In Gfp^basal”, and “In Gfp⁺⁺” over wild-type controls (gray).

Figure 3

Known mutants in the mating pathway, and color-coded summary model. (A) FUS1-GFP of known mating pathway mutants. (B) Color-coded model of mutants in the pheromone response pathway based on mutant FUS1-GFP expression.

Figure 4

sub1Δ and hog1Δ mutants have higher pFUS1-GFP expression. sub1Δ and hog1Δ each have about a twofold higher basal FUS1 expression than wild type; sub1Δ hog1Δ has about threefold higher basal level (wild type, three replicates; sub1Δ, five replicates; hog1Δ, 4 replicates; sub1Δ hog1Δ, three replicates).

Figure 5

RNA-Seq of sub1Δ mutant. (A) Volcano plot: RNA-Seq in vegetative growth conditions of sub1Δ and wild type shows that most genes have unchanged expression; most genes with changed expression relative to wild type are left-shifted, consistent with an overall positive role of SUB1 on transcription. (B) FIG1 and IMD2 have significantly higher basal levels in sub1Δ, FUS1 has 1.2-fold higher basal levels in the sub1Δ mutant that, while not statistically significant, nevertheless agrees with the modest increase in GFP expression observed. (C) Concordance at the top (CAT) plot, showing that the RNA expression profile of a sub1Δ mutant in α-factor-induced conditions is indistinguishable from wild type, while the RNA expression profile of these two strains is significantly different in high sorbitol conditions (yellow area represents the 99.9% critical region for the null hypothesis of no correspondence). (D) Alpha-factor-inducible genes induce to a lower extent in a sub1Δ mutant compared to wild type, log2fold ≥ 2; padj < 0.01, about 90 genes.

Figure 6

ChIP-Seq of Sub1 in α-factor induced conditions. (A) Sub1 exhibits increased binding at the promoters of ∼76 pheromone induced genes (circled in green). This plot was generated by first selecting peaks that increased with p-value < 0.05. Then the genes near these peaks were identified. (B) Sub1 increases binding at the promoter of FUS1 upon α-factor induction. (C) Sub1 increases binding at the promoter of FIG1 upon α-factor induction.