Charles Boone

The Genetic Landscape of a Cell

Making Connections Genetic interaction profiles highlight cross-connections between bioprocesses, providing a global view of cellular pleiotropy, and enable the prediction of genetic network hubs. Costanzo et al. (p. 425) performed a pairwise fitness screen covering approximately one-third of all potential genetic interactions in yeast, examining 5.4 million gene-gene pairs and generating quantitative profiles for ∼75% of the genome. Of the pairwise interactions tested, about 3% of the genes investigated interact under the conditions tested. On the basis of these data, a reference map for the yeast genetic network was created. A genome-wide interaction map of yeast identifies genetic interactions, networks, and function. A genome-scale genetic interaction map was constructed by examining 5.4 million gene-gene pairs for synthetic genetic interactions, generating quantitative genetic interaction profiles for ~75% of all genes in the budding yeast, Saccharomyces cerevisiae. A network based on genetic interaction profiles reveals a functional map of the cell in which genes of similar biological processes cluster together in coherent subsets, and highly correlated profiles delineate specific pathways to define gene function. The global network identifies functional cross-connections between all bioprocesses, mapping a cellular wiring diagram of pleiotropy. Genetic interaction degree correlated with a number of different gene attributes, which may be informative about genetic network hubs in other organisms. We also demonstrate that extensive and unbiased mapping of the genetic landscape provides a key for interpretation of chemical-genetic interactions and drug target identification.

Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map

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.

Exploration of the Function and Organization of the Yeast Early Secretory Pathway through an Epistatic Miniarray Profile

We present a strategy for generating and analyzing comprehensive genetic-interaction maps, termed E-MAPs (epistatic miniarray profiles), comprising quantitative measures of aggravating or alleviating interactions between gene pairs. Crucial to the interpretation of E-MAPs is their high-density nature made possible by focusing on logically connected gene subsets and including essential genes. Described here is the analysis of an E-MAP of genes acting in the yeast early secretory pathway. Hierarchical clustering, together with novel analytical strategies and experimental verification, revealed or clarified the role of many proteins involved in extensively studied processes such as sphingolipid metabolism and retention of HDEL proteins. At a broader level, analysis of the E-MAP delineated pathway organization and components of physical complexes and illustrated the interconnection between the various secretory processes. Extension of this strategy to other logically connected gene subsets in yeast and higher eukaryotes should provide critical insights into the functional/organizational principles of biological systems.

Cotranscriptional Set2 Methylation of Histone H3 Lysine 36 Recruits a Repressive Rpd3 Complex

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.

Navigating the Chaperone Network: An Integrative Map of Physical and Genetic Interactions Mediated by the Hsp90 Chaperone

Physical, genetic, and chemical-genetic interactions centered on the conserved chaperone Hsp90 were mapped at high resolution in yeast using systematic proteomic and genomic methods. Physical interactions were identified using genome-wide two hybrid screens combined with large-scale affinity purification of Hsp90-containing protein complexes. Genetic interactions were uncovered using synthetic genetic array technology and by a microarray-based chemical-genetic screen of a set of about 4700 viable yeast gene deletion mutants for hypersensitivity to the Hsp90 inhibitor geldanamycin. An extended network, consisting of 198 putative physical interactions and 451 putative genetic and chemical-genetic interactions, was found to connect Hsp90 to cofactors and substrates involved in a wide range of cellular functions. Two novel Hsp90 cofactors, Tah1 (YCR060W) and Pih1 (YHR034C), were also identified. These cofactors interact physically and functionally with the conserved AAA(+)-type DNA helicases Rvb1/Rvb2, which are key components of several chromatin remodeling factors, thereby linking Hsp90 to epigenetic gene regulation.

Methylation of Histone H3 by Set2 in Saccharomyces cerevisiae Is Linked to Transcriptional Elongation by RNA Polymerase II

ABSTRACT Set2 methylates Lys36 of histone H3. We show here that yeast Set2 copurifies with RNA polymerase II (RNAPII). Chromatin immunoprecipitation analyses demonstrated that Set2 and histone H3 Lys36 methylation are associated with the coding regions of several genes that were tested and correlate with active transcription. Both depend, as well, on the Paf1 elongation factor complex. The C terminus of Set2, which contains a WW domain, is also required for effective Lys36 methylation. Deletion of CTK1, encoding an RNAPII CTD kinase, prevents Lys36 methylation and Set2 recruitment, suggesting that methylation may be triggered by contact of the WW domain or C terminus of Set2 with Ser2-phosphorylated CTD. A set2 deletion results in slight sensitivity to 6-azauracil and much less β-galactosidase produced by a reporter plasmid, resulting from a defect in transcription. In synthetic genetic array (SGA) analysis, synthetic growth defects were obtained when a set2 deletion was combined with deletions of all five components of the Paf1 complex, the chromodomain elongation factor Chd1, the putative elongation factor Soh1, the Bre1 or Lge1 components of the histone H2B ubiquitination complex, or the histone H2A variant Htz1. SET2 also interacts genetically with components of the Set1 and Set3 complexes, suggesting that Set1, Set2, and Set3 similarly affect transcription by RNAPII.

Integration of chemical-genetic and genetic interaction data links bioactive compounds to cellular target pathways

Bioactive compounds can be valuable research tools and drug leads, but it is often difficult to identify their mechanism of action or cellular target. Here we investigate the potential for integration of chemical-genetic and genetic interaction data to reveal information about the pathways and targets of inhibitory compounds. Taking advantage of the existing complete set of yeast haploid deletion mutants, we generated drug-hypersensitivity (chemical-genetic) profiles for 12 compounds. In addition to a set of compound-specific interactions, the chemical-genetic profiles identified a large group of genes required for multidrug resistance. In particular, yeast mutants lacking a functional vacuolar H+-ATPase show multidrug sensitivity, a phenomenon that may be conserved in mammalian cells. By filtering chemical-genetic profiles for the multidrug-resistant genes and then clustering the compound-specific profiles with a compendium of large-scale genetic interaction profiles, we were able to identify target pathways or proteins. This method thus provides a powerful means for inferring mechanism of action.

Exploration of Essential Gene Functions via Titratable Promoter Alleles

Nearly 20% of yeast genes are required for viability, hindering genetic analysis with knockouts. We created promoter-shutoff strains for over two-thirds of all essential yeast genes and subjected them to morphological analysis, size profiling, drug sensitivity screening, and microarray expression profiling. We then used this compendium of data to ask which phenotypic features characterized different functional classes and used these to infer potential functions for uncharacterized genes. We identified genes involved in ribosome biogenesis (HAS1, URB1, and URB2), protein secretion (SEC39), mitochondrial import (MIM1), and tRNA charging (GSN1). In addition, apparent negative feedback transcriptional regulation of both ribosome biogenesis and the proteasome was observed. We furthermore show that these strains are compatible with automated genetic analysis. This study underscores the importance of analyzing mutant phenotypes and provides a resource to complement the yeast knockout collection.

Exploring genetic interactions and networks with yeast

The development and application of genetic tools and resources has enabled a partial genetic-interaction network for the yeast Saccharomyces cerevisiae to be compiled. Analysis of the network, which is ongoing, has already provided a clear picture of the nature and scale of the genetic interactions that robustly sustain biological systems, and how cellular buffering is achieved at the molecular level. Recent studies in yeast have begun to define general principles of genetic networks, and also pave the way for similar studies in metazoan model systems. A comparative understanding of genetic-interaction networks promises insights into some long-standing genetic problems, such as the nature of quantitative traits and the basis of complex inherited disease.

Formins direct Arp2/3-independent actin filament assembly to polarize cell growth in yeast

Formins have been implicated in the regulation of cytoskeletal structure in animals and fungi. Here we show that the formins Bni1 and Bnr1 of budding yeast stimulate the assembly of actin filaments that function as precursors to tropomyosin-stabilized cables that direct polarized cell growth. With loss of formin function, cables disassemble, whereas increased formin activity causes the hyperaccumulation of cable-like filaments. Unlike the assembly of cortical actin patches, cable assembly requires profilin but not the Arp2/3 complex. Thus formins control a distinct pathway for assembling actin filaments that organize the overall polarity of the cell.