Insights into TOR function and rapamycin response: chemical genomic profiling by using a high-density cell array method

Abstract

With the advent of complete genome sequences, large-scale functional analyses are generating new excitement in biology and medicine. To facilitate genomewide functional analyses, we developed a high-density cell array with quantitative and automated readout of cell fitness. Able to print at > x 10 higher density on a standard microtiter plate area than currently possible, our cell array allows single-plate screening of the complete set of Saccharomyces cerevisiae gene-deletion library and significantly reduces the amount of small molecules and other materials needed for the study. We used this method to map the relation between genes and cell fitness in response to rapamycin, a medically important natural product that targets the eukaryotic kinase Tor. We discuss the implications for pharmacogenomics and the uncharted complexity in genotype-dependent drug response in molecularly targeted therapies. Our analysis leads to several basic findings, including a class of gene deletions that confer better fitness in the presence of rapamycin. This result provides insights into possible therapeutic uses of rapamycin/CCI-779 in the treatment of neurodegenerative diseases (including Alzheimer's, Parkinson's, and Huntington's diseases), and cautions the possible existence of similar rapamycin-enhanceable mutations in cancer. It is well established in yeast that although TOR2 has a unique rapamycin-insensitive function, TOR1 and TOR2 are interchangeable in the rapamycin-sensitive functions. We show that even the rapamycin-sensitive functions are distinct between TOR1 and TOR2 and map the functional difference to a approximately 120-aa region at the N termini of the proteins. Finally, we discuss using cell-based genomic pattern recognition in designing electronic or optical biosensors.

Fig. 1.

Chemical genomic screening by using a high-density cell array. (a) A microarraying robot was used to print the full S. cerevisiae single-gene deletion library on yeast extract/peptone/dextrose agar containing ≈0.5 μg of rapamycin (final concentration = 100 nM). Nine thousand six hundred strains can be arrayed on a standard one-well plate area. The yellow arrow points to the rapamycin-resistant fpr1Δ, which is missing the rapamycin receptor FKBP12. (b) The known TOR signaling components (adapted from ref. 20) were colored according to deletion strain phenotypes identified from our screen. Red, rapamycin-resistant when deleted; green, rapamycin-hypersensitive when deleted.

Fig. 2.

Gene Ontology (go) mapping of 396 genes that demonstrated altered sensitivity to rapamycin. RsWr, rapamycin-hypersensitive and wortmannin-resistant (when deleted); RrWs, rapamycin-resistant and wortmannin-hypersensitive; R, rapamycin-hypersensitive or rapamycin-resistant.

Fig. 3.

Circular clustergram of the 35 genes that individually contributed to rapamycin sensitivity as well as exhibited significant (>3-fold) changes in gene expression upon rapamycin treatment. The inner circle represents transcript profiling data, decrease is in green, increase in red; the outer circle represents functional profiling data, rapamycin-hypersensitive deletion is in green, rapamycin-resistant deletion in red.

Fig. 4.

TOR1 and TOR2 are different in their rapamycin-sensitive function. (a) Schematic representation of plasmids and cell dilutions used for experiments in b and e. (b) Tor1SR is ≈1,000-fold more active than Tor2SR in vps16Δ cells although they are equally active in most (including wild-type) cells. (c) Chromosomal duplication of TOR genes in yeast. (d) Sequence alignment of Tor1 and Tor2 revealed the nonhomologous domain of ≈100 amino acids at the N termini. (e) Sequence junction and activity of the chimeric Tor2-Tor1SR protein, and although it carries only 131 amino acids of Tor2, behaves like Tor2SR and is unable to rescue rapamycin inhibition of either endogenous Tor protein in vps16Δ mutant.