Defining the functional relationships between proteins is critical for understanding virtually all aspects of cell biology. Large-scale identification of protein complexes has provided one important step towards this goal; however, even knowledge of the stoichiometry, affinity and lifetime of every protein-protein interaction would not reveal the functional relationships between and within such complexes. Genetic interactions can provide functional information that is largely invisible to protein-protein interaction data sets. Here we present an epistatic miniarray profile (E-MAP) consisting of quantitative pairwise measurements of the genetic interactions between 743 Saccharomyces cerevisiae genes involved in various aspects of chromosome biology (including DNA replication/repair, chromatid segregation and transcriptional regulation). This E-MAP reveals that physical interactions fall into two well-represented classes distinguished by whether or not the individual proteins act coherently to carry out a common function. Thus, genetic interaction data make it possible to dissect functionally multi-protein complexes, including Mediator, and to organize distinct protein complexes into pathways. In one pathway defined here, we show that Rtt109 is the founding member of a novel class of histone acetyltransferases responsible for Asf1-dependent acetylation of histone H3 on lysine 56. This modification, in turn, enables a ubiquitin ligase complex containing the cullin Rtt101 to ensure genomic integrity during DNA replication.

The SET domain proteins, SUV39 and G9a have recently been shown to be histone methyltransferases speci®c for lysines 9 and 27 (G9a only) of histone 3 (H3). The SET domains of the Saccharomyces cerevisiae Set1 and Drosophila trithorax proteins are closely related to each other but distinct from SUV39 and G9a. We characterized the complex associated with Set1 and Set1C and found that it is comprised of eight members, one of which, Bre2, is homologous to the trithorax-group (trxG) protein, Ash2. Set1C requires Set1 for complex integrity and mutation of Set1 and Set1C components shortens telomeres. One Set1C member, Swd2/Cpf10 is also present in cleavage polyadenylation factor (CPF). Set1C methylates lysine 4 of H3, thus adding a new speci®city and a new subclass of SET domain proteins known to methyltransferases. Since methylation of H3 lysine 4 is widespread in eukaryotes, we screened the databases and found other Set1 homologues. We propose that eukaryotic Set1Cs are H3 lysine 4 methyltransferases and are related to trxG action through association with Ash2 homologues. Keywords: chromatin/epigenetic/histone methyltransferase/telomere/trithorax-group IntroductionMembers of the trithorax group (trxG) have been identi®ed by genetic screens of Drosophila for mutations that suppress phenotypes caused by disregulation of polycomb-group (PcG) action or mimic loss-of-function homeotic mutant phenotypes. As expected from these complex genetic screens, the trxG appears to encompass several subclasses of gene regulatory factors (Kennison, 1995). One subclass involves chromatin remodelling activity. The realization that the trxG member Brahma (BRM) is a homologue of Saccharomyces cerevisiae Swi2/Snf2 (Peterson and Herskowitz, 1992;Carlson and Laurent, 1994;Elfring et al., 1994) led to the de®nition of the SWI/SNF complex as a chromatin remodelling machine (Cote et al., 1994;Logie and Peterson, 1997) and the identi®cation of another trxG member, moira, as a further component of the Drosophila SWI/SNF complex (Papoulas et al., 1998). Another trxG subclass encompasses the DNA binding proteins, zeste and GAGA factor. Although these proteins act independently, both appear to play similar roles in the stabilization of higher-order chromatin looping (Chen and Pirrotta, 1993;Katsani et al., 1999). A third subclass within the trxG (called here trxG3) that remains poorly understood includes trithorax itself, ash1 and ash2 (Shearn, 1989).Insight into the potential molecular actions of trxG3 members came from the identi®cation of several domains within their protein sequences (Mazo et al., 1990;Stassen et al., 1995;Adamson and Shearn, 1996;Tripoulas et al., 1996). Both trithorax (Trx) and Ash1 include a SET domain, which was identi®ed through its occurrence in the chromatin factors, Su(var)3-9, enhancer of zeste [E(Z)] and Trx (Jones and Gelbart, 1993;Tschiersch et al., 1994). All three trxG3 members also include one or more PHD ®ngers (Aasland et al., 1995) and Ash2 includes a SPRY domain (Ponting et al., 1997). Of these domains, the S...

An epistasis map (E-MAP) was constructed in the fission yeast, Schizosaccharomyces pombe, by systematically measuring the phenotypes associated with pairs of mutations. This high-density, quantitative genetic interaction map focused on various aspects of chromosome function, including transcription regulation and DNA repair/replication. The E-MAP uncovered a previously unidentified component of the RNA interference (RNAi) machinery (rsh1) and linked the RNAi pathway to several other biological processes. Comparison of the S. pombe E-MAP to an analogous genetic map from the budding yeast revealed that, whereas negative interactions were conserved between genes involved in similar biological processes, positive interactions and overall genetic profiles between pairs of genes coding for physically associated proteins were even more conserved. Hence, conservation occurs at the level of the functional module (protein complex), but the genetic cross talk between modules can differ substantially.Genetic interactions report on the extent to which the function of one gene depends on the presence of a second. This phenomenon, known as epistasis, can be used for defining functional relationships between genes and the pathways in which the corresponding proteins function. Two main categories of genetic interactions exist: (i) negative (e.g., synthetic sickness/ lethality) and (ii) positive (e.g., suppression). We have developed a quantitative approach, termed epistasis map (E-MAP), allowing us to measure the whole spectrum of genetic interactions, both positive and negative (1,2). In budding yeast, Saccharomyces cerevisiae, it has been demonstrated that positive genetic interactions can identify pairs of genes whose products are physically associated and/or function in the same pathway (1,2), whereas negative interactions exist between genes acting on parallel pathways (3,4).We developed the Pombe Epistasis Mapper (PEM) approach (5) that allows high-throughput generation of double mutants in the fission yeast, Schizosaccharomyces pombe. Fission yeast is more similar to metazoans than is S. cerevisiae, owing to its large complex centromere structure, the restriction of spindle construction to mitotic entry, gene regulation by histone methylation and chromodomain heterochromatin proteins, gene and transposon regulation by the RNA interference (RNAi) pathway, and the widespread presence of introns in genes. To further study these processes and to try to understand how genetic interaction networks have evolved (6), we generated an E-MAP in S. pombe that focuses on nuclear function, designed to be analogous to one we created in budding yeast (2). An E-MAP in S. pombeUsing our PEM system (5), we generated a quantitative genetic interaction map in S. pombe, comprising ~118,000 distinct double mutant combinations among 550 genes involved in various aspects of chromosome function ( Fig. 1A and tables S1 and S4) (7). The genes on the map were chosen on the basis of a previous budding yeast E-MAP (1,2) and also included factor...

Set3 is one of two proteins in the yeast Saccharomyces cerevisiae that, like Drosophila Trithorax, contains both SET and PHD domains. We found that Set3 forms a single complex, Set3C, with Snt1, YIL112w, Sif2, Cpr1, and two putative histone deacetylases, Hos2 and NAD-dependent Hst1. Set3C includes NAD-dependent and independent deacetylase activities when assayed in vitro. Homology searches suggest that Set3C is the yeast analog of the mammalian HDAC3/SMRT complex. Set3C represses genes in early/middle of the yeast sporulation program, including the key meiotic regulators ime2 and ndt80. Whereas Hos2 is only found in Set3C, Hst1 is also present in a complex with Sum1, supporting previous characterizations of Hst1 and Sum1 as repressors of middle sporulation genes during vegetative growth. However, Hst1 is not required for meiotic repression by Set3C, thus implying that Set3C (−Hst1) and not Hst1-Sum1, is the meiotic-specific repressor of early/middle sporulation genes.

We developed a systematic approach to map human genetic networks by combinatorial CRISPR-Cas9 perturbations coupled to robust analysis of growth kinetics. We targeted all pairs of 73 cancer genes with dual-guide RNAs in three cell lines, altogether comprising 141,912 tests of interaction. Numerous therapeutically relevant interactions were identified and these patterns replicated with combinatorial drugs at 75% precision. Based on these results we anticipate cellular context will be critical to synthetic-lethal therapies.

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.

Set1 is the catalytic subunit and the central component of the evolutionarily conserved Set1 complex (Set1C) that methylates histone 3 lysine 4 (H3K4). Here we have determined protein/protein interactions within the complex and related the substructure to function. The loss of individual Set1C subunits differentially affects Set1 stability, complex integrity, global H3K4 methylation, and distribution of H3K4 methylation along active genes. The complex requires Set1, Swd1, and Swd3 for integrity, and Set1 amount is greatly reduced in the absence of the Swd1-Swd3 heterodimer. Bre2 and Sdc1 also form a heteromeric subunit, which requires the SET domain for interaction with the complex, and Sdc1 strongly interacts with itself. Inactivation of either Bre2 or Sdc1 has very similar effects. Neither is required for complex integrity, and their removal results in an increase of H3K4 mono-and dimethylation and a severe decrease of trimethylation at the 5 end of active coding regions but a decrease of H3K4 dimethylation at the 3 end of coding regions. Cells lacking Spp1 have a reduced amount of Set1 and retain a fraction of trimethylated H3K4, whereas cells lacking Shg1 show slightly elevated levels of both di-and trimethylation. Set1C associates with both serine 5-and serine 2-phosphorylated forms of polymerase II, indicating that the association persists to the 3 end of transcribed genes. Taken together, our results suggest that Set1C subunits stimulate Set1 catalytic activity all along active genes.

Analysis of the phosphoproteomes and the gene interaction networks of divergent yeast species defines the relative contribution of changes in protein phosphorylation pathways to the generation of phenotypic diversity.