Summary The sequence specificity of DNA-binding proteins is the primary mechanism by which the cell recognizes genomic features. Here, we describe systematic determination of yeast transcription factor DNA-binding specificities. We obtained binding specificities for 112 DNA-binding proteins representing 19 distinct structural classes, one-third of which have not been previously reported. Several newly discovered binding sequences have striking genomic distributions relative to transcription start sites, supporting their biological relevance and suggesting a role in promoter architecture. Among these are Rsc3 binding sequences, containing the core CGCG, which are found preferentially ~100 bp upstream of transcription start sites. Mutation of RSC3 results in a dramatic increase in nucleosome occupancy in hundreds of proximal promoters containing a Rsc3 binding element, but has little impact on promoters lacking Rsc3 binding sequences, indicating that Rsc3 plays a broad role in targeting nucleosome exclusion at yeast promoters.

The dimorphic switch from a single-cell budding yeast to a filamentous form enables Saccharomyces cerevisiae to forage for nutrients and the opportunistic pathogen Candida albicans to invade human tissues and evade the immune system. We constructed a genome-wide set of targeted deletion alleles and introduced them into a filamentous S. cerevisiae strain, Σ1278b. We identified genes involved in morphologically distinct forms of filamentation: haploid invasive growth, biofilm formation, and diploid pseudohyphal growth. Unique genes appear to underlie each program, but we also found core genes with general roles in filamentous growth, including MFG1 (YDL233w), whose product binds two morphogenetic transcription factors, Flo8 and Mss11, and functions as a critical transcriptional regulator of filamentous growth in both S. cerevisiae and C. albicans.

We describe a fluorescent reporter system that exploits the functional genomic tools available in budding yeast to systematically assess consequences of genetic perturbations on gene expression. We used our Reporter-Synthetic Genetic Array (R-SGA) method to screen for regulators of core histone gene expression. We discovered that the histone chaperone Rtt106 functions in a pathway with two other chaperones, Asf1 and the HIR complex, to create a repressive chromatin structure at core histone promoters. We found that activation of histone (HTA1) gene expression involves both relief of Rtt106-mediated repression by the activity of the histone acetyltransferase Rtt109 and restriction of Rtt106 to the promoter region by the bromodomain-containing protein Yta7. We propose that the maintenance of Asf1/HIR/Rtt106-mediated repressive chromatin domains is the primary mechanism of cell-cycle regulation of histone promoters. Our data suggest that this pathway may represent a chromatin regulatory mechanism that is broadly used across the genome.

Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology, and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors, including the CAF-1 complex, Spt10, and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning, and these data provide a valuable resource for future studies.