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.

Acetylation of Saccharomyces cerevisiae histone H3 on K56 by the histone acetyltransferase (HAT) Rtt109 is important for repairing replication-associated lesions. Rtt109 purifies from yeast in complex with the histone chaperone Vps75, which stabilizes the HAT in vivo. A whole-genome screen to identify genes whose deletions have synthetic genetic interactions with rtt109⌬ suggests Rtt109 has functions in addition to DNA repair. We show that in addition to its known H3-K56 acetylation activity, Rtt109 is also an H3-K9 HAT, and we show that Rtt109 and Gcn5 are the only H3-K9 HATs in vivo. Rtt109's H3-K9 acetylation activity in vitro is enhanced strongly by Vps75. Another histone chaperone, Asf1, and Vps75 are both required for acetylation of lysine 9 on H3 (H3-K9ac) in vivo by Rtt109, whereas H3-K56ac in vivo requires only Asf1. Asf1 also physically interacts with the nuclear Hat1/Hat2/Hif1 complex that acetylates H4-K5 and H4-K12. We suggest Asf1 is capable of assembling into chromatin H3-H4 dimers diacetylated on both H4-K5/12 and H3-K9/56.

Background: Transcription is disruptive to chromatin structure and can expose cryptic promoters. Results: We identify those factors that might regulate cryptic transcription from within inactive and transcribed locations. Conclusion: Nucleosome shielding prevents cryptic transcription, and replication-independent histone replacement is co-operatively mediated by three H3-H4 chaperones. Significance: Understanding how cryptic transcription is regulated and lost histones replaced is of fundamental importance.

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SUMMARY Condensin is a central regulator of mitotic genome structure, with mutants showing poorly condensed chromosomes and profound segregation defects. Here we identify NCT complex, comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02), Casein Kinase II (CKII) and several TAFs, as a regulator of condensin function. We show that NCT and condensin bind similar genomic regions, but only briefly co-localize during the periods of chromosome condensation and decondensation. This pattern of NCT binding at the core centromere, the region of maximal condensin enrichment, tracks the abundance of acetylated histone H4, as regulated by the Hat1-Mis16 acetyltransferase complex and recognized by the first Nrc1 bromodomain. Strikingly, mutants in NCT or Hat1-Mis16 restore the formation of segregation-competent chromosomes in cells containing defective condensin. These results are consistent with a model where NCT targets CKII to chromatin in a cell cycle-directed manner to modulate the activity of condensin during chromosome condensation and decondensation.