Transcriptional Profiling of Saccharomyces cerevisiae Reveals the Impact of Variation of a Single Transcription Factor on Differential Gene Expression in 4NQO, Fermentable, and Nonfermentable Carbon Sources

Cellular metabolism can change the potency of a chemical’s tumorigenicity. 4-nitroquinoline-1-oxide (4NQO) is a tumorigenic drug widely used on animal models for cancer research. Polymorphisms of the transcription factor Yrr1 confer different levels of resistance to 4NQO in Saccharomyces cerevisiae. To study how different Yrr1 alleles regulate gene expression leading to resistance, transcriptomes of three isogenic S. cerevisiae strains carrying different Yrr1 alleles were profiled via RNA sequencing (RNA-Seq) and chromatin immunoprecipitation coupled with sequencing (ChIP-Seq) in the presence and absence of 4NQO. In response to 4NQO, all alleles of Yrr1 drove the expression of SNQ2 (a multidrug transporter), which was highest in the presence of 4NQO resistance-conferring alleles, and overexpression of SNQ2 alone was sufficient to over- come 4NQO-sensitive growth. Using shape metrics to refine the ChIP-Seq peaks, Yrr1 strongly associated with three loci including SNQ2. In addition to a known Yrr1 target SNG1, Yrr1 also bound upstream of RPL35B; however, overexpression of these genes did not confer 4NQO resistance. RNA-Seq data also implicated nucleotide synthesis pathways including the de novo purine pathway, and the ribonuclease reductase path- ways were downregulated in response to 4NQO. Conversion of a 4NQO-sensitive allele to a 4NQO-resistant allele by a single point mutation mimicked the 4NQO-resistant allele in phenotype, and while the 4NQO resistant allele increased the expression of the ADE genes in the de novo purine biosynthetic pathway, the mutant Yrr1 increased expression of ADE genes even in the absence of 4NQO. These same ADE genes were only increased in the wild-type alleles in the presence of 4NQO, indicating that the point mutation activated Yrr1 to upregulate a pathway normally only activated in response to stress. The various Yrr1 alleles also influenced growth on different carbon sources by altering the function of the mitochondria. Hence, the complement to 4NQO resistance was poor growth on nonfermentable carbon sources, which in turn varied depending on the allele of Yrr1 expressed in the isogenic yeast. The oxidation state of the yeast affected the 4NQO toxicity by altering the reactive oxygen species (ROS) generated by cellular metabolism. The integra- tion of RNA-Seq and ChIP-Seq elucidated how Yrr1 regulates global gene transcription in response to 4NQO and how various Yrr1 alleles confer differential resistance to 4NQO. This study provides guidance for further investigation into how Yrr1 regulates cellular responses to 4NQO, as well as transcriptomic resources for further analysis of transcription factor variation on carbon source utilization

Publisher Correction: The long non-coding RNA HOXB-AS3 regulates ribosomal RNA transcription in NPM1-mutated acute myeloid leukemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Author Correction: The long non-coding RNA HOXB-AS3 regulates ribosomal RNA transcription in NPM1-mutated acute myeloid leukemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Transcriptional Profiling of Saccharomyces cerevisiae Reveals the Impact of Variation of a Single Transcription Factor on Differential Gene Expression in 4NQO, Fermentable, and Nonfermentable Carbon Sources

Cellular metabolism can change the potency of a chemical’s tumorigenicity. 4-nitroquinoline-1-oxide (4NQO) is a tumorigenic drug widely used on animal models for cancer research. Polymorphisms of the transcription factor Yrr1 confer different levels of resistance to 4NQO in Saccharomyces cerevisiae. To study how different Yrr1 alleles regulate gene expression leading to resistance, transcriptomes of three isogenic S. cerevisiae strains carrying different Yrr1 alleles were profiled via RNA sequencing (RNA-Seq) and chromatin immunoprecipitation coupled with sequencing (ChIP-Seq) in the presence and absence of 4NQO. In response to 4NQO, all alleles of Yrr1 drove the expression of SNQ2 (a multidrug transporter), which was highest in the presence of 4NQO resistance-conferring alleles, and overexpression of SNQ2 alone was sufficient to overcome 4NQO-sensitive growth. Using shape metrics to refine the ChIP-Seq peaks, Yrr1 strongly associated with three loci including SNQ2. In addition to a known Yrr1 target SNG1, Yrr1 also bound upstream of RPL35B; however, overexpression of these genes did not confer 4NQO resistance. RNA-Seq data also implicated nucleotide synthesis pathways including the de novo purine pathway, and the ribonuclease reductase pathways were downregulated in response to 4NQO. Conversion of a 4NQO-sensitive allele to a 4NQO-resistant allele by a single point mutation mimicked the 4NQO-resistant allele in phenotype, and while the 4NQO resistant allele increased the expression of the ADE genes in the de novo purine biosynthetic pathway, the mutant Yrr1 increased expression of ADE genes even in the absence of 4NQO. These same ADE genes were only increased in the wild-type alleles in the presence of 4NQO, indicating that the point mutation activated Yrr1 to upregulate a pathway normally only activated in response to stress. The various Yrr1 alleles also influenced growth on different carbon sources by altering the function of the mitochondria. Hence, the complement to 4NQO resistance was poor growth on nonfermentable carbon sources, which in turn varied depending on the allele of Yrr1 expressed in the isogenic yeast. The oxidation state of the yeast affected the 4NQO toxicity by altering the reactive oxygen species (ROS) generated by cellular metabolism. The integration of RNA-Seq and ChIP-Seq elucidated how Yrr1 regulates global gene transcription in response to 4NQO and how various Yrr1 alleles confer differential resistance to 4NQO. This study provides guidance for further investigation into how Yrr1 regulates cellular responses to 4NQO, as well as transcriptomic resources for further analysis of transcription factor variation on carbon source utilization

Genetic variation in Dip5, an amino acid permease, and Pdr5, a multiple drug transporter, regulates glyphosate resistance in S. cerevisiae

S. cerevisiae from different environments are subject to a wide range of selective pressures, whether intentional or by happenstance. Chemicals classified by their application, such as herbicides, fungicides and antibiotics, can affect non-target organisms. First marketed as RoundUpTM, glyphosate is the most widely used herbicide. In plants, glyphosate inhibits EPSPS, of the shikimate pathway, which is present in many organisms but lacking in mam- mals. The shikimate pathway produces chorismate which is the precursor to all the aromatic amino acids, para-aminobenzoic acid, and Coenzyme Q10. Crops engineered to be resis- tant to glyphosate contain a homolog of EPSPS that is not bound by glyphosate. Here, we show that S. cerevisiae has a wide-range of glyphosate resistance. Sequence comparison between the target proteins, i.e., the plant EPSPS and the yeast orthologous protein Aro1, predicted that yeast would be resistant to glyphosate. However, the growth variation seen in the subset of yeast tested was not due to polymorphisms within Aro1, instead, it was caused by genetic variation in an ABC multiple drug transporter, Pdr5, and an amino acid permease, Dip5. Using genetic variation as a probe into glyphosate response, we uncovered mecha- nisms that contribute to the transportation of glyphosate in and out of the cell. Taking advan- tage of the natural genetic variation within yeast and measuring growth under different conditions that would change the use of the shikimate pathway, we uncovered a general transport mechanism of glyphosate into eukaryotic cells

Genetic variation in Dip5, an amino acid permease, and Pdr5, a multiple drug transporter, regulates glyphosate resistance in S. cerevisiae

S. cerevisiae from different environments are subject to a wide range of selective pressures, whether intentional or by happenstance. Chemicals classified by their application, such as herbicides, fungicides and antibiotics, can affect non-target organisms. First marketed as RoundUpTM, glyphosate is the most widely used herbicide. In plants, glyphosate inhibits EPSPS, of the shikimate pathway, which is present in many organisms but lacking in mam- mals. The shikimate pathway produces chorismate which is the precursor to all the aromatic amino acids, para-aminobenzoic acid, and Coenzyme Q10. Crops engineered to be resis- tant to glyphosate contain a homolog of EPSPS that is not bound by glyphosate. Here, we show that S. cerevisiae has a wide-range of glyphosate resistance. Sequence comparison between the target proteins, i.e., the plant EPSPS and the yeast orthologous protein Aro1, predicted that yeast would be resistant to glyphosate. However, the growth variation seen in the subset of yeast tested was not due to polymorphisms within Aro1, instead, it was caused by genetic variation in an ABC multiple drug transporter, Pdr5, and an amino acid permease, Dip5. Using genetic variation as a probe into glyphosate response, we uncovered mecha- nisms that contribute to the transportation of glyphosate in and out of the cell. Taking advan- tage of the natural genetic variation within yeast and measuring growth under different conditions that would change the use of the shikimate pathway, we uncovered a general transport mechanism of glyphosate into eukaryotic cells

Genetic variation effects growth inhibition by glyphosate.

<p><b>A</b>. Shikimate pathway produces the precursor for phenylalanine, tyrosine, tryptophan, <i>para</i>-Aminobenzoic acid (PABA), folic acid and Coenzyme Q10. The canonical target of glyphosate is EPSPS in plants and Aro1 is the yeast homolog of EPSPS. PABA and 4-hydroxylbenzoate can be converted to Coenzyme Q10. <b>B</b>. Serial dilution of genetically diverse yeast on rich media (YPD) with dilutions of glyphosate as indicated (1% vol/ vol is equivalent to 78 mM). <b>C</b>. Serial dilution of genetically diverse yeast on minimal media with glyphosate. Aromatic amino acids, tryptophan (W), tyrosine (Y) and phenylalanine (F) were added to YM plates to make WYF.</p

Genetic Linkage analysis of glyphosate sensitivity in glyphosate on minimal media with and without aromatic amino acids.

<p><b>A</b>. Genetic linkage of sensitivity of YJM789 to 0.15% glyphosate in yeast minimal media supplemented with aromatic amino acids (WYF). LOD score (y-axis) was mapped across the yeast genome (x-axis). The LOD significant levels (alpha = 0.05) was 3.65 and was marked by a red dashed line. <b>B</b>. Genetic linkage of sensitivity of YJM789 to 0.15% glyphosate in yeast minimal media (YM). LOD score (y-axis) was mapped across the yeast genome (x-axis). The LOD significant levels (alpha = 0.05) was 3.47 and was marked by a red dashed line. <b>C</b>. The genomic loci under the peak located on chromosome 16 contains 14 genes. Genes encoded on the top strand are in yellow and genes encoded on the bottom strand are blue. <b>D</b>. Serial dilutions of S288c (GSY147), YJM789 and BY4741 with <i>DIP5</i> knocked out grown on YM (HULM), WYF, aspartic acid (D) with glyphosate at the concentrations indicated. Histidine, uracil, leucine and methionine (HULM) were supplemented for growth of BY4741. <b>E</b>. Serial dilutions of S288c (GSY147) x YJM789 hybrids. The entire <i>DIP5</i> coding region in either parent was deleted (Δ), crossed, and the resulting hemizygotes were grown on solid media YM, WYF, aspartic acid (D) with glyphosate at the indicated concentrations and supplemented.</p

Genetic variation effects growth inhibition by glyphosate.

<p><b>A</b>. Shikimate pathway produces the precursor for phenylalanine, tyrosine, tryptophan, <i>para</i>-Aminobenzoic acid (PABA), folic acid and Coenzyme Q10. The canonical target of glyphosate is EPSPS in plants and Aro1 is the yeast homolog of EPSPS. PABA and 4-hydroxylbenzoate can be converted to Coenzyme Q10. <b>B</b>. Serial dilution of genetically diverse yeast on rich media (YPD) with dilutions of glyphosate as indicated (1% vol/ vol is equivalent to 78 mM). <b>C</b>. Serial dilution of genetically diverse yeast on minimal media with glyphosate. Aromatic amino acids, tryptophan (W), tyrosine (Y) and phenylalanine (F) were added to YM plates to make WYF.</p