The gag-pol protein of Rous sarcoma virus (RSV), the precursor to the enzymes responsible for reverse transcription and integration, is expressed from two genes that lie in different translational reading frames by ribosomal frameshifting. Here, we localize the site of frameshifting and show that the frameshifting reaction is mediated by slippage of two adjacent tRNAs by a single nucleotide in the 5' direction. The gag terminator, which immediately follows the frameshift site, is not required for frameshifting. Other suspected retroviral frameshift sites mediate frameshifting when placed at the end of RSV gag. Mutations in RSV pol also affect synthesis of the gag-pol protein in vitro. The effects of these mutations best correlate with the potential to form an RNA stem-loop structure adjacent to the frameshift site. A short sequence of RSV RNA, 147 nucleotides in length, containing the frameshift site and stem-loop structure, is sufficient to direct frameshifting in a novel genetic context.

Boundary elements hinder the spread of heterochromatin, yet these sites do not fully account for the preservation of adjacent euchromatin. Histone variant H2A.Z (Htz1 in yeast) replaces conventional H2A in many nucleosomes. Microarray analysis revealed that HTZ1-activated genes cluster near telomeres. The reduced expression of most of these genes in htz1Delta cells was reversed by the deletion of SIR2 (sir2Delta) suggesting that H2A.Z antagonizes telomeric silencing. Other Htz1-activated genes flank the silent HMR mating-type locus. Their requirement for Htz1 can be bypassed by sir2Delta or by a deletion encompassing the silencing nucleation sites in HMR. In htz1Delta cells, Sir2 and Sir3 spread into flanking euchromatic regions, producing changes in histone H4 acetylation and H3 4-methylation indicative of ectopic heterochromatin formation. Htz1 is enriched in these euchromatic regions and acts synergistically with a boundary element to prevent the spread of heterochromatin. Thus, euchromatin and heterochromatin each contains components that antagonize switching to the opposite chromatin state.

The conserved histone variant H2A.Z functions in euchromatin to antagonize the spread of heterochromatin. The mechanism by which histone H2A is replaced by H2A.Z in the nucleosome is unknown. We identified a complex containing 13 different polypeptides associated with a soluble pool of H2A.Z in Saccharomyces cerevisiae. This complex was designated SWR1-Com in reference to the Swr1p subunit, a Swi2/Snf2-paralog. Swr1p and six other subunits were found only in SWR1-Com, whereas six other subunits were also found in the NuA4 histone acetyltransferase and/or the Ino80 chromatin remodeling complex. H2A.Z and SWR1 were essential for viability of cells lacking the EAF1 component of NuA4, pointing to a close functional connection between these two complexes. Strikingly, chromatin immunoprecipitation analysis of cells lacking Swr1p, the presumed ATPase of the complex, revealed a profound defect in the deposition of H2A.Z at euchromatic regions that flank the silent mating type cassette HMR and at 12 other chromosomal sites tested. Consistent with a specialized role for Swr1p in H2A.Z deposition, the majority of the genome-wide transcriptional defects seen in swr1Δ cells were also found in htz1Δ cells. These studies revealed a novel role for a member of the ATP-dependent chromatin remodeling enzyme family in determining the region-specific histone subunit composition of chromatin in vivo and controlling the epigenetic state of chromatin. Metazoan orthologs of Swr1p (Drosophila Domino; human SRCAP and p400) may have analogous functions.

In S. cerevisiae, histone variant H2A.Z is deposited in euchromatin at the flanks of silent heterochromatin to prevent its ectopic spread. We show that H2A.Z nucleosomes are found at promoter regions of nearly all genes in euchromatin. They generally occur as two positioned nucleosomes that flank a nucleosome-free region (NFR) that contains the transcription start site. Astonishingly, enrichment at 5' ends is observed not only at actively transcribed genes but also at inactive loci. Mutagenesis of a typical promoter revealed a 22 bp segment of DNA sufficient to program formation of a NFR flanked by two H2A.Z nucleosomes. This segment contains a binding site of the Myb-related protein Reb1 and an adjacent dT:dA tract. Efficient deposition of H2A.Z is further promoted by a specific pattern of histone H3 and H4 tail acetylation and the bromodomain protein Bdf1, a component of the Swr1 remodeling complex that deposits H2A.Z.

Mammalian Dock180 and ELMO proteins, and their homologues in Caenorhabditis elegans and Drosophila melanogaster, function as critical upstream regulators of Rac during development and cell migration. The mechanism by which Dock180 or ELMO mediates Rac activation is not understood. Here, we identify a domain within Dock180 (denoted Docker) that specifically recognizes nucleotide-free Rac and can mediate GTP loading of Rac in vitro. The Docker domain is conserved among known Dock180 family members in metazoans and in a yeast protein. In cells, binding of Dock180 to Rac alone is insufficient for GTP loading, and a Dock180 ELMO1 interaction is required. We can also detect a trimeric ELMO1 Dock180 Rac1 complex and ELMO augments the interaction between Dock180 and Rac. We propose that the Dock180 ELMO complex functions as an unconventional two-part exchange factor for Rac.

SUMMARY The fungus Cryptococcus neoformans is a leading cause of mortality and morbidity among HIV-infected individuals. We utilized the completed genome sequence and optimized methods for homologous DNA replacement using high-velocity particle bombardment to engineer 1,201 gene knockout mutants. We screened this resource in vivo for proliferation in murine lung tissue and in vitro for three well-recognized virulence attributes — polysaccharide capsule formation, melanization, and growth at body temperature. We identified dozens of previously uncharacterized genes that affect these known attributes as well as 40 infectivity mutants without obvious defects in these traits. The latter mutants affect predicted regulatory factors, secreted proteins, and immune-related factors, and represent powerful tools for elucidating novel virulence mechanisms. In particular, we describe a GATA family transcription factor that inhibits phagocytosis by murine macrophages independently of the capsule, indicating a previously unknown mechanism of innate immune modulation.

Functional genomic studies in Saccharomyces cerevisiae have contributed enormously to our understanding of cellular processes. Their full potential, however, has been hampered by the limited availability of reagents to systematically study essential genes and the inability to quantify the small effects of most gene deletions on growth. Here we describe the construction of a library of hypomorphic alleles of essential genes and a high-throughput growth competition assay to measure fitness with unprecedented sensitivity. These tools dramatically increase the breadth and precision with which quantitative genetic analysis can be performed in yeast. We illustrate the value of these approaches by using genetic interactions to reveal new relationships between chromatin-modifying factors and to create a functional map of the proteasome. Finally, by measuring the fitness of strains in the yeast deletion library, we addressed an enigma regarding the apparent prevalence of gene dispensability and found that most genes do contribute to growth.A fundamental challenge of the post-genomic era is to assign functions to genes and to understand how gene networks are organized to execute diverse cellular processes. The budding yeast S. cerevisiae has served as a premier eukaryotic model system for this task [1][2][3][4][5] , and a library of strains in which each nonessential yeast gene is deleted has been an important tool for these efforts 1 . This resource has made it possible to assess the contribution of each nonessential gene to any measurable phenotype.These genomic approaches can be extended by systematically measuring genetic interactions 5 . Genetic interactions describe how the phenotype associated with compromising one gene is modulated by perturbing a second gene. Such effects can be defined quantitatively Correspondence should be addressed to J.S.W. (weissman@cmp.ucsf.edu). 6 These authors contributed equally to this work.Note: Supplementary information is available on the Nature Methods website. HHMI Author Manuscript HHMI Author Manuscript HHMI Author Manuscriptas the difference between the observed magnitude of a phenotype for a double mutant and that expected if the genes do not interact, as given by ε= W AB − W A × W B , where ε is the interaction score and W X refers to the phenotype seen in genetic background X (refs. 6,7 ). Theoretical considerations and empirical experience have established the utility of genetic interactions in defining functional relationships between genes 2,3,6,8 . Early studies focused on 'synthetic sick or lethal' or 'negative' interactions 3 , in which the phenotype associated with perturbing two genes simultaneously is more severe than expected given the phenotypes of the individual gene perturbations (ε < 0). More recently, interactions in which the phenotype of a double mutant is less severe than expected (ε > 0, 'alleviating' or 'positive' interactions) have proven highly valuable as they often occur between genes that have closely related functions-for example, between ...

SUMMARY The chromatin architecture of eukaryotic gene promoters is generally characterized by a nucleosome-free region (NFR) flanked by at least one H2A.Z variant nucleosome. Computational predictions of nucleosome positions based on thermodynamic properties of DNA-histone interactions have met with limited success. Here we show that the action of the essential RSC remodeling complex in S. cerevisiae helps explain the discrepancy between theory and experiment. In RSC-depleted cells, NFRs shrink such that the average positions of flanking nucleosomes move toward predicted sites. Nucleosome positioning at distinct subsets of promoters additionally requires the essential Myb family proteins Abf1 and Reb1, whose binding sites are enriched in NFRs. In contrast, H2A.Z deposition is dispensable for nucleosome positioning. By regulating H2A.Z deposition using a steroid-inducible protein splicing strategy, we show that NFR establishment is necessary for H2A.Z deposition. These studies suggest an ordered pathway for the assembly of promoter chromatin architecture.