The yeast histone deacetylase Rpd3 can be recruited to promoters to repress transcription initiation. Biochemical, genetic, and gene-expression analyses show that Rpd3 exists in two distinct complexes. The smaller complex, Rpd3C(S), shares Sin3 and Ume1 with Rpd3C(L) but contains the unique subunits Rco1 and Eaf3. Rpd3C(S) mutants exhibit phenotypes remarkably similar to those of Set2, a histone methyltransferase associated with elongating RNA polymerase II. Chromatin immunoprecipitation and biochemical experiments indicate that the chromodomain of Eaf3 recruits Rpd3C(S) to nucleosomes methylated by Set2 on histone H3 lysine 36, leading to deacetylation of transcribed regions. This pathway apparently acts to negatively regulate transcription because deleting the genes for Set2 or Rpd3C(S) bypasses the requirement for the positive elongation factor Bur1/Bur2.

One of the earliest marks of a double-strand break (DSB) in eukaryotes is serine phosphorylation of the histone variant H2AX at the carboxy-terminal SQE motif to create gammaH2AX-containing nucleosomes. Budding-yeast histone H2A is phosphorylated in a similar manner by the checkpoint kinases Tel1 and Mec1 (ref. 2; orthologous to mammalian ATM and ATR, respectively) over a 50-kilobase region surrounding the DSB. This modification is important for recruiting numerous DSB-recognition and repair factors to the break site, including DNA damage checkpoint proteins, chromatin remodellers and cohesins. Multiple mechanisms for eliminating gammaH2AX as DNA repair completes are possible, including removal by histone exchange followed potentially by degradation, or, alternatively, dephosphorylation. Here we describe a three-protein complex (HTP-C, for histone H2A phosphatase complex) containing the phosphatase Pph3 that regulates the phosphorylation status of gammaH2AX in vivo and efficiently dephosphorylates gammaH2AX in vitro. gammaH2AX is lost from chromatin surrounding a DSB independently of the HTP-C, indicating that the phosphatase targets gammaH2AX after its displacement from DNA. The dephosphorylation of gammaH2AX by the HTP-C is necessary for efficient recovery from the DNA damage checkpoint.

Although cellular behaviors are dynamic, the networks that govern these behaviors have been mapped primarily as static snapshots. Using an approach called differential epistasis mapping, we have discovered widespread changes in genetic interaction among yeast kinases, phosphatases, and transcription factors as the cell responds to DNA damage. Differential interactions uncover many gene functions that go undetected in static conditions. They are very effective at identifying DNA repair pathways, highlighting new damage-dependent roles for the Slt2 kinase, Pph3 phosphatase, and histone variant Htz1. The data also reveal that protein complexes are generally stable in response to perturbation, but the functional relations between these complexes are substantially reorganized. Differential networks chart a new type of genetic landscape that is invaluable for mapping cellular responses to stimuli.One of the most basic approaches to understanding gene function relies on the identification of genetic interactions, which occur when the phenotypic effects of one gene depend on the presence of a second. Recently, a number of technologies have been developed to systematically map genetic interaction networks over large sets of genes in budding yeast (1-3) and other model organisms (4,5 To gain insight into how genetic networks are altered by stress, we assembled a large genetic interactome with and without perturbation by the DNA-damaging agent methyl methane-sulfonate (MMS). Using the technique of epistatic miniarray profiles (E-MAP) (8), genetic interactions were interrogated among a set of 418 yeast genes selected to provide broad coverage of the cellular signaling and transcriptional machinery, including nearly all yeast kinases, phosphatases, and transcription factors, as well as known DNA repair factors ( fig. S1 and table S1). About 80,000 double-mutant strains were generated from all pairwise mutant combinations of the 418 genes, in which mutations were complete gene deletions (nonessential genes) or hypomorphic alleles (essential genes) as appropriate. Double-mutant combinations were grown with or without 0.02% MMS, and their colony sizes were analyzed statistically to compute a genetic interaction score (S score) in each condition (9), which indicates whether the strain was healthier or sicker than expected (positive or negative S, respectively) (10).From established score thresholds for positive and negative interactions (S ≥ +2.0, S ≤ −2.5) (9) we identified two genetic networks: a set of 1905 interactions for the untreated condition, and a set of 2297 interactions under MMS. Analysis of these "static" genetic maps showed strong associations with physical interaction networks of various kinds. For example, gene pairs with either positive or negative genetic interactions were highly enriched for proteins known to physically interact. In addition, both maps were enriched for known kinase-and phosphatase-substrate pairs, as well as transcription factor-target pairs ( fig. S2). The correspondence to physical...

Summary Reversible protein phosphorylation is a signaling mechanism involved in all cellular processes. To create a systems view of the signaling apparatus in budding yeast, we generated an E-MAP (epistatic miniarray profile) comprised of 100,000 pair-wise, quantitative genetic interactions, including virtually all protein kinases and phosphatases and key cellular regulators. Quantitative genetic interaction mapping reveals factors working in compensatory pathways (negative genetic interactions; e.g. synthetic lethality) or those operating in linear pathways (positive genetic interactions; e.g. suppression). Within kinases, phosphatases, and their substrates, we found an enrichment of positive genetic interactions. To develop a global view of the signaling apparatus, we isolated “triplet genetic motifs” and assembled these into a higher-order map. The resulting network view provides new insights into signaling pathway regulation, and revealed a link between the cell cycle kinase, Cak1, the Fus3 MAP kinase, and a pathway that regulates chromatin integrity during transcription by RNA polymerase II.

To date, cross-species comparisons of genetic interactomes have been restricted to small or functionally related gene sets, limiting our ability to infer evolutionary trends. To facilitate a more comprehensive analysis, we constructed a genome-scale epistasis map (E-MAP) for the fission yeast Schizosaccharomyces pombe, providing phenotypic signatures for ~60% of the non-essential genome. Using these signatures, we generated a catalogue of 297 functional modules, and assigned function to 144 previously uncharacterised genes, including mRNA splicing and DNA damage checkpoint factors. Comparison with an integrated genetic interactome from the budding yeast Saccharomyces cerevisiae revealed a hierarchical model for the evolution of genetic interactions, with conservation highest within protein complexes, lower within biological processes, and lowest between distinct biological processes. Despite the large evolutionary distance and extensive rewiring of individual interactions, both networks retain conserved features and display similar levels of functional cross-talk between biological processes, suggesting general design principles of genetic interactomes.