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Multiple signaling pathways control the cellular response to O2 levels
Stephen D. Willis2 and Mark J. Hickman1,2
Departments of 1Biological Sciences and 2Chemistry & BiochemistryRowan University
Four potential O2-responsive signaling pathways
Future Directions
Genes that participate in the response
The gene expression response to O2 withdrawal
• Cells respond to environmental O2 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 O2 levels because yeast shares many signaling pathways with humans
• The response is complex, as at least 19 genes make up four O2-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 O2 levels
• Global gene expression after O2 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 O2 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 NatR
NatMXPCR 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 dsc1D haploids.
Dissection of a dsc6Δ/DSC6 diploid shows dsc6D haploids are not viable. Only DSC6 haploids grew.
mga2Δ spt23Δ
Wildtype
upc2Δ
ecm22Δ
upc2Δ ecm22Δ
mga2Δ
spt23Δ
hap1Δ
dsc1Δ
dsc2Δ
dsc3Δ
dsc5Δ
- O2 +O2
sterols/heme/membrane
fluidity?
heme unsaturated fatty acids?
Upc2
Hap1
Mga2
DAN1 OLE1HEM13
heme/carbon source?
Hap2/3/4/5complex
COX6
O2-dependentmolecule/condition
Transcription factor(s)
Example of regulated gene
Spt23Ecm22Rox1 Mot3
Hog1Signalingprotein
CYC1
??
Deleting genes
Chromosome
• The dsc genes were first identified to be part of an O2-dependent signaling complex in S. pombe (Stewart, 2011)
• To test growth of dsc mutants, a dscD / 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 O2 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# cells
RNA-Seq analysis of gene expression
• The global gene expression response to O2 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 N2 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 O2 levels
TetradsTetrads
Spores Spores
100-fold increased
100-fold decreased
no changeHickman lab website.