| Title: | Barcode Sequencing Screen Identifies SUB1 as a Regulator of Yeast Pheromone Inducible Genes |
| Author(s): | Sliva A; Kuang Z; Meluh PB; Boeke JD; |
| Address: | "Institute for Systems Genetics, New York University Langone School of Medicine, New York 10016 Human Genetics Program, Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205. Institute for Systems Genetics, New York University Langone School of Medicine, New York 10016. Calico Life Sciences, Google Inc., San Francisco, California 94080. Institute for Systems Genetics, New York University Langone School of Medicine, New York 10016 Jef.Boeke@nyumc.org" |
| ISSN/ISBN: | 2160-1836 (Electronic) 2160-1836 (Linking) |
| 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, sub1Delta, 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 sub1Delta 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 sub1Delta mutant relative to the wild type, supporting a role for Sub1 in regulation of mating pathway genes. The sub1Delta 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: | "*DNA Barcoding, Taxonomic Gene Expression Gene Expression Regulation, Fungal/*drug effects Gene Knockout Techniques *Genes, Fungal Genes, Reporter Genetic Vectors/genetics Mating Factor/genetics/metabolism Models, Biological Mutation Pheromones/*pharmacol;" |
| Notes: | "MedlineSliva, Anna Kuang, Zheng Meluh, Pamela B Boeke, Jef D eng P30 CA016087/CA/NCI NIH HHS/ T32 GM007814/GM/NIGMS NIH HHS/ U54 GM103520/GM/NIGMS NIH HHS/ Research Support, N.I.H., Extramural England 2016/02/04 G3 (Bethesda). 2016 Apr 7; 6(4):881-92. doi: 10.1534/g3.115.026757" |
