Functional Discovery via a Compendium of Expression Profiles

1. Functional Discovery via a Compendium of Expression Profiles Hughes et al.

2. Questions • Can groups of coregulated genes be identified? • Do mutations that affect similar processes have the same profiles? • Can cellular functions of uncharacterized genes be predicted?

3. Approach • All experiments were carried out under the same growth condition – SC media 2% glucose 30°C, mid-log phase • Did a parallel growth assay for the 198 bar-coded mutants (used custom-made arrays) • Identified biological noise using 63 control experiments • 276 deletion mutants (151 in duplicate) • 11 tetracycline-regulatable alleles of essential genes • 13 well-characterized compounds

4. Distribution of mutants used

5. Parallel growth assay • 198 mutants were pooled and grown. • Seven time points taken over 20 doublings. • Relative abundance of each strain was determined using a custom made microarray.

6. Results

7. Control experiments

8. 300 experiment compendium data set • Major clusters include • Mating • Ergosterol biosynthesis • Mitochondrial respiration • PKC/calcineurin activated genes • Ribosome/Translation • MAPK signaling • Cell wall maintenance In general different mutants that affect the same cellular process display related transcript profiles

9. Can cellular functions of uncharacterized ORF’s be predicted? • Compare profiles of knows with unknowns • Ergosterol biosynthesis • Cell wall maintenance • Mitochondrial respiration • Protein synthesis

10. YER044c/ERG28 • ERG28 clusters with other ERG genes • ERG28 not essential exhibits slow growth • Sterols accumulate in the mutant • Mutant has reduced ergosterol • Mutant growth phenotype suppressed by ERG28 and hERG28 (novel gene)

11. Dicyclonine, topical anesthetic • Profile mapped with sterol profiles • GC analysis indicated buildup of fecosterol, indicating inhibition of Erg2p • ERG2/erg2∆ mutant is hypersensitive • Overexpression of ERG2 increases resistance • Maybe inhibits phosphate transport in humans

12. YER083c/Cell wall function • No transcripts could be identified that were induced only by mutants involved in cell wall function • However, when they look at known cell wall genes they found YER083c • YER083c exhibits characteristics of cell wall mutants • Slow growth • Hypersensitive to calcofluor white • Increased lysis rate

13. Mitochondrial-mutant profiles • Two types • Compromise mitochondrial function • Involved in iron regulation • Three unclassified genes fell in these clusters • Co-regulated with mitochondrial ribosomal genes

14. Mitochondrial-mutant profiles

15. Protein synthesis • Can low magnitude changes be biologically significant? • Found 3 uncharacterized ORFs that cluster with ribosome subuints • Mutants exhibit slow growth and reduced protein synthesis

16. Conclusions • In general different mutants that affect the same cellular process have similar transcript profiles, whether it is a direct or indirect interaction. • 4 of the 15 clusters were driven by transcriptional changes in the same genes that drove the clustering in the control experiments. • Low magnitude changes do contribute biologically significant results

17. In the future • Because you are most likely to see differences if growth is affected under the conditions tested. A panel of conditions needs to be developed. • Pooled, bar-coded mutants • Phenotypic macroarrays