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.