Due to evolutionary conservation of biology, experimental knowledge captured from genetic studies in eukaryotic model organisms provides insight into human cellular pathways and ultimately physiology. Yeast chemogenomic profiling is a powerful approach for annotating cellular responses to small molecules. Using an optimized platform, we provide the relative sensitivities of the heterozygous and homozygous deletion collections for nearly 1800 biologically active compounds. The data quality enables unique insights into pathways that are sensitive and resistant to a given perturbation, as demonstrated with both known and novel compounds. We present examples of novel compounds that inhibit the therapeutically relevant fatty acid synthase and desaturase (Fas1p and Ole1p), and demonstrate how the individual profiles facilitate hypothesis-driven experiments to delineate compound mechanism of action. Importantly, the scale and diversity of tested compounds yields a dataset where the number of modulated pathways approaches saturation. This resource can be used to map novel biological connections, and also identify functions for unannotated genes. We validated hypotheses generated by global two-way hierarchical clustering of profiles for (i) novel compounds with a similar mechanism of action acting upon microtubules or vacuolar ATPases, and (ii) an un-annotated ORF, YIL060w, that plays a role in respiration in the mitochondria. Finally, we identify and characterize background mutations in the widely used yeast deletion collection which should improve the interpretation of past and future screens throughout the community. This comprehensive resource of cellular responses enables the expansion of our understanding of eukaryotic pathway biology.

The crystal structure of the ligand binding domain (LBD) of the estrogen-related receptor ␣ (ERR␣, NR3B1) complexed with a coactivator peptide from peroxisome proliferator-activated receptor coactivator-1␣ (PGC-1␣) reveals a transcriptionally active conformation in the absence of a ligand. This is the first x-ray structure of ERR␣ LBD, solved to a resolution of 2.5 Å, and the first structure of a PGC-1␣ complex. The putative ligand binding pocket (LBP) of ERR␣ is almost completely occupied by side chains, in particular with the bulky side chain of Phe 328 (corresponding to Ala 272 in ERR␥ and Ala 350 in estrogen receptor ␣). Therefore, a ligand of a size equivalent to more than ϳ4 carbon atoms could only bind in the LBP, if ERR␣ would undergo a major conformational change (in particular the ligand would displace H12 from its agonist position). The x-ray structure thus provides strong evidence for ligand-independent transcriptional activation by ERR␣. The interactions of PGC-1␣ with ERR␣ also reveal for the first time the atomic details of how a coactivator peptide containing an inverted LXXLL motif (namely a LLXYL motif) binds to a LBD. In addition, we show that a PGC-1␣ peptide containing this nuclear box motif from the L3 site binds ERR␣ LBD with a higher affinity than a peptide containing a steroid receptor coactivator-1 motif and that the affinity is further enhanced when all three leucine-rich regions of PGC-1␣ are present.Nuclear hormone receptors (NRs) 1 are transcription factors that control essential developmental and physiological pathways (1). Although the transcriptional activity of NRs is often regulated by specific ligands, several members of the superfamily have no known natural ligands and are therefore referred to as orphan NRs (2). Estrogen-related receptor ␣ (ERR␣; NR3B1) was the first orphan NR to be identified on the basis of its similarity with estrogen receptor ␣ (ER␣; NR3A1) (3). ERR␣ and its relatives ERR␤ (NR3B2) and ERR␥ (NR3B3) form a small family of orphan NRs that are evolutionarily related to the estrogen receptors ER␣ and ER␤. ERRs preferentially bind to DNA sites composed of a single half-site preceded by three nucleotides with the consensus sequence TNAAGGTCA, referred to as an ERR response element. It has been shown that ERR␣ also efficiently binds to estrogen response elements and that these receptors share common target genes (4). This observation was further supported by studies demonstrating cross-talk between the ER and ERR pathways (reviewed in Ref. 5). The most striking feature observed in the phenotype of mice lacking ERR␣ is their resistance to high fat diet-induced obesity and the impaired activity of enzymes implicated in lipid metabolism. This finding led to the hypothesis that ERR␣ could be implicated in obesity or metabolic diseases (6). A function of ERR␣ on bone metabolism has also been suggested (7,8). Finally, recent publications show that ERR␣ and ERR␥ are associated with biomarkers of breast cancer and further emphasize the importance of ER-ERR cross-talk (9...

Background: TORC2/Ypk1 regulates actin polarization and endocytosis via unknown effectors. Results: Pharmacological inhibition of TORC2 reveals that flippase kinases and biophysical properties of the plasma membrane are major effectors of TORC2. Conclusion: TORC2 regulates actin and endocytosis via multiple pathways, each with different signaling kinetics. Significance: Elucidation of TORC2 effector pathways in yeast will inform future studies in higher eukaryotes.

A chemicogenetic screen was performed in budding yeast mutants that have a weakened replication stress response. This identified an inhibitor of target of rapamycin (TOR) complexes 1 and 2 that selectively enhances the sensitivity of sgs1Δ cells to hydroxyurea and camptothecin. More importantly, the inhibitor has strong synthetic lethality in combination with either the break-inducing antibiotic Zeocin or ionizing radiation, independent of the strain background. Lethality correlates with a rapid fragmentation of chromosomes that occurs only when TORC2, but not TORC1, is repressed. Genetic inhibition of Tor2 kinase, or its downstream effector kinases Ypk1/Ypk2, conferred similar synergistic effects in the presence of Zeocin. Given that Ypk1/Ypk2 controls the actin cytoskeleton, we tested the effects of actin modulators latrunculin A and jasplakinolide. These phenocopy TORC2 inhibition on Zeocin, although modulation of calcineurin-sensitive transcription does not. These results implicate TORC2-mediated actin filament regulation in the survival of low levels of DNA damage.

No abstract