1. Multiple signaling pathways control the cellular response to O 2 levels Stephen D. Willis 2 and Mark J. Hickman 1,2 Departments of 1 Biological Sciences and 2 Chemistry & Biochemistry Rowan University Four potential O 2 -responsive signaling pathways Future Directions Genes that participate in the response The gene expression response to O 2 withdrawal Cells respond to environmental O 2 levels by changing gene expression Disruption of this response contributes to human cancer and cardiovascular disease The yeast S. cerevisiae is useful for studying response to O 2 levels because yeast shares many signaling pathways with humans The response is complex, as at least 19 genes make up four O 2 -responsive signaling pathways Genetic makeup of these signaling pathways is not understood Here, the signaling genes were deleted to test their role in the response to O 2 levels Global gene expression after O 2 withdrawal was monitored using RNA-Seq Our work has revealed both unexpected growth phenotypes and signaling redundancy for the different deletion strains Summary In this heatmap, each row represents the expression of a specific gene over time and in different strains The color and intensity of each bar shows mRNA expression relative to expression at time 0 in WT (see color key below) One conclusion from observing the wild- type response is that genes have varying kinetics and magnitudes, though the genes can be grouped based on similar responses The UPC2 or MGA2 signaling genes were thought to play a significant role in the response to O 2 withdrawal, but deleting each gene had a minor effect on the response Perhaps, as seen above with growth, each signaling gene has to be deleted with its paralog to have a major impact on gene expression References Hickman, M.J., Spatt, D. and Winston, F. 2011. A hypoxic- induction pathway mediated by the Hog1 MAPK in Saccharomyces cerevisiae. Genetics: 188: 325. Stewart E.V., Christine C. Nwosu, Zongtian Tong, Assen Roguev, Timothy D. Cummins, Dong-Uk Kim, Jacqueline Hayles, Han-Oh Park, Kwang-Lae Hoe, David W. Powell, Nevan J. Krogan, and Peter J. Espenshade. 2011. Yeast SREBP Cleavage Activation Requires the Golgi Dsc E3 Ligase Complex. Molecular Cell 42: 160–171. Each gene to be deleted (geneX) was replaced by recombination (shown by dotted lines) with a selectable marker such as NatMX (encoding for resistance to Nourseothricin) The NatMX gene was amplified by PCR and introduced into cells by transformation Replacement was selected by Nat R NatMX PCR product geneX Qiagen RNeasy Kit Illumina TruSeq mRNA Sample Prep Kit Frozen cell pellet Total RNA cDNA library FASTQ file Illumina sequencing Aligned reads Tophat alignment Expression level of transcript Cufflinks transcript assembly Growth phenotypes of dsc mutants Dissection of a dsc1Δ/DSC1 diploid shows normal growth of dsc1  haploids. Dissection of a dsc6Δ/DSC6 diploid shows dsc6  haploids are not viable. Only DSC6 haploids grew. mga2Δ spt23Δ Wildtype upc2Δ ecm22Δ upc2Δ ecm22Δ mga2Δ spt23Δ hap1Δ dsc1Δ dsc2Δ dsc3Δ dsc5Δ - O 2 +O 2 sterols/heme/me mbrane fluidity? hemeunsaturated fatty acids? Upc2 Hap1 Mga2 DAN1OLE1HEM13 heme/carb on source? Hap2/3/4/5 complex COX6 O 2 -dependent molecule/condit ion Transcription factor(s) Example of regulated gene Spt23Ecm22 Rox 1 Mot3 Hog1 Signaling protein CYC1 ?? Deleting genes Chromosome The dsc genes were first identified to be part of an O 2 -dependent signaling complex in S. pombe (Stewart, 2011) To test growth of dsc mutants, a dsc  / DSC diploid was sporulated giving rise to four haploid spores. The four spores of each tetrad are arranged in a vertical column. Multiple tetrads are depicted. Each tetrad shows the expected 2:2 DSC:dscΔ ratio. Diverse growth phenotypes of signaling mutants Normal growth was observed in all mutants while O 2 is present. The simultaneous deletion of UPC2 and ECM22 prevented growth in hypoxia. Deleting each deletion alone had no growth effect. UPC2 and ECM22 are paralogs, so our results are consistent with these genes functioning redundantly. MGA2 is required for hypoxic growth. However, its paralog, SPT23, is not. The deletion of HAP1 or any of the DSC genes does not have an effect on hypoxic growth. # cells RNA-Seq analysis of gene expression The global gene expression response to O 2 levels was studied We hypothesized that deleting a signaling gene would disrupt a particular signaling pathway and thus the portion of the response controlled by that pathway Yeast cultures were grown hypoxically by continuously flushing with N 2 gas After 0, 5, 10, 30, 60, 120, 180, and 240 minutes of hypoxia, yeast were processed as depicted Delete all 19 signaling genes Create simultaneous deletions to test for genetic interactions and for pathway architecture Test mutants for growth phenotypes and for global gene expression Ultimately, characterize the many signaling pathways that mediate the global response to O 2 levels Tetrads Spores 100-fold increased 100-fold decreased no change Hickman lab website.