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Functional analysis with a barcoder yeast gene overexpression system.

ABSTRACT: Systematic analysis of gene overexpression phenotypes provides an insight into gene function, enzyme targets, and biological pathways. Here, we describe a novel functional genomics platform that enables a highly parallel and systematic assessment of overexpression phenotypes in pooled cultures. First, we constructed a genome-level collection of ~5100 yeast barcoder strains, each of which carries a unique barcode, enabling pooled fitness assays with a barcode microarray or sequencing readout. Second, we constructed a yeast open reading frame (ORF) galactose-induced overexpression array by generating a genome-wide set of yeast transformants, each of which carries an individual plasmid-born and sequence-verified ORF derived from the Saccharomyces cerevisiae full-length EXpression-ready (FLEX) collection. We combined these collections genetically using synthetic genetic array methodology, generating ~5100 strains, each of which is barcoded and overexpresses a specific ORF, a set we termed "barFLEX." Additional synthetic genetic array allows the barFLEX collection to be moved into different genetic backgrounds. As a proof-of-principle, we describe the properties of the barFLEX overexpression collection and its application in synthetic dosage lethality studies under different environmental conditions.

SUBMITTER: Douglas AC

PROVIDER: S-EPMC3464120 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Functional analysis with a barcoder yeast gene overexpression system.

Douglas Alison C AC Smith Andrew M AM Sharifpoor Sara S Yan Zhun Z Durbic Tanja T Heisler Lawrence E LE Lee Anna Y AY Ryan Owen O Göttert Hendrikje H Surendra Anu A van Dyk Dewald D Giaever Guri G Boone Charles C Nislow Corey C Andrews Brenda J BJ

G3 (Bethesda, Md.) 20121001 10

Systematic analysis of gene overexpression phenotypes provides an insight into gene function, enzyme targets, and biological pathways. Here, we describe a novel functional genomics platform that enables a highly parallel and systematic assessment of overexpression phenotypes in pooled cultures. First, we constructed a genome-level collection of ~5100 yeast barcoder strains, each of which carries a unique barcode, enabling pooled fitness assays with a barcode microarray or sequencing readout. Sec ...[more]

PMID: 23050238

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Project description:In Schizosaccharomyces pombe, over 90% of transcription factor genes are nonessential. Moreover, the majority do not exhibit significant growth defects under optimal conditions when deleted, complicating their functional characterization and target gene identification. Here, we systematically overexpressed 99 transcription factor genes with the nmt1 promoter. Screening the overexpression array revealed that 64 transcription factor genes exhibited reduced fitness when ectopically expressed. Cell cycle defects were also often observed. We further investigated three uncharacterized transcription factor genes (toe1+-toe3+) which displayed cell elongation when overexpressed. Ectopic expression of toe1+ resulted in a G1 delay while toe2+ and toe3+ overexpression produced an accumulation of septated cells with abnormalities in septum formation and nuclear segregation, respectively. Transcriptome profiling and ChIP-chip analysis of the transcription factor overexpression strains indicated that Toe1 activates target genes of the pyrimidine-salvage pathway, while Toe3 regulates target genes involved in polyamine synthesis. We also found that ectopic expression of the putative target genes SPBC3H7.05c, and dad5+ and SPAC11D3.06 could recapitulate the cell cycle phenotypes of toe2+ and toe3+ overexpression, respectively. Furthermore, the phenotypes of toe1+and toe2+ overexpression could be suppressed by deletion of the putative target genes urg2+ and SPAC1399.04c, and SPBC3H7.05c, SPACUNK4.15 and rds1+, respectively. This study implicates new transcription factors and metabolism genes in cell cycle regulation and demonstrates the potential of systematic overexpression analysis to elucidate the function and target genes of transcription factors in S. pombe. We generated 3 overexpression microarrays and 2 deletion microarray with dye swaps, 3 deletion microarrays with drug treatments with dye swaps, 1 deletion single replicate microarray experiment, and 3 single replicate chIP-chip experiments. The effect of the mutant strains were all compared to wild type or empyty vector strains except for two experiments one that looked at the toe1 mutant with and without chlorpromazine and another that looked at wild type with and without chlorpromazine.

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Functional analysis with a barcoder yeast gene overexpression system.

Publications

Functional analysis with a barcoder yeast gene overexpression system.

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