Barcode sequencing screen identifies SUB1 as a regulator of yeast pheromone inducible genes

Anna Sliva, Zheng Kuang, Pamela B. Meluh, Jef D. Boeke

Research output: Contribution to journalArticle

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 identifiedmutants 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.

Original languageEnglish (US)
Pages (from-to)881-892
Number of pages12
JournalG3: Genes, Genomes, Genetics
Volume6
Issue number4
DOIs
StatePublished - 2016
Externally publishedYes

Fingerprint

Pheromones
Yeasts
Genes
RNA
Mitogen-Activated Protein Kinases
Flow Cytometry
Single-Cell Analysis
Membrane Proteins
Genome
Technology
Population

Keywords

  • Mating
  • SUB1
  • Transcription

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology
  • Genetics(clinical)

Cite this

Barcode sequencing screen identifies SUB1 as a regulator of yeast pheromone inducible genes. / Sliva, Anna; Kuang, Zheng; Meluh, Pamela B.; Boeke, Jef D.

In: G3: Genes, Genomes, Genetics, Vol. 6, No. 4, 2016, p. 881-892.

Research output: Contribution to journalArticle

Sliva, Anna ; Kuang, Zheng ; Meluh, Pamela B. ; Boeke, Jef D. / Barcode sequencing screen identifies SUB1 as a regulator of yeast pheromone inducible genes. In: G3: Genes, Genomes, Genetics. 2016 ; Vol. 6, No. 4. pp. 881-892.
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