Polyphosphate Loss Promotes SNF/SWI- and Gcn5-Dependent Mitotic Induction of PHO5

Abstract

Approximately 800 transcripts in Saccharomyces cerevisiae are cell cycle regulated. The oscillation of ϳ40% of these genes, including a prominent subclass involved in nutrient acquisition, is not understood. To address this problem, we focus on the mitosis-specific activation of the phosphate-responsive promoter, PHO5. We show that the unexpected mitotic induction of the PHO5 acid phosphatase in rich medium requires the transcriptional activators Pho4 and Pho2, the cyclin-dependent kinase inhibitor Pho81, and the chromatin-associated enzymes Gcn5 and Snf2/Swi2. PHO5 mitotic activation is repressed by addition of orthophosphate, which significantly increases cellular polyphosphate. Polyphosphate levels also fluctuate inversely with PHO5 mRNA during the cell cycle, further substantiating an antagonistic link between this phosphate polymer and PHO5 mitotic regulation. Moreover, deletion of PHM3, required for polyphosphate accumulation, leads to premature onset of PHO5 expression, as well as an increased rate, magnitude, and duration of PHO5 activation. Orthophosphate addition, however, represses mitotic PHO5 expression in a phm3⌬ strain. Thus, polyphosphate per se is not necessary to repress PHO transcription but, when present, replenishes cellular phosphate during nutrient depletion. These results demonstrate a dynamic mechanism of mitotic transcriptional regulation that operates mostly independently of factors that drive progression through the cell cycle.Coordination of cell growth and division is essential to all living organisms and is innately tied to the cell division cycle. Periodic increases in certain transcripts at distinct cell cycle phases can meet specific, one-time requirements. For instance, nucleotide biosynthetic and histone genes are activated prior to and during S phase, respectively, to ensure adequate substrate concentrations for chromosomal duplication (9, 16). In addition, the sequential activation and proteolytic destruction of cyclins that partner with cyclin-dependent kinase (CDK) activities drives progression through the cell cycle (29). In contrast to this posttranslational mode of cell cycle control, it is clear that much cell cycle regulation occurs at the level of initiation of transcription by RNA polymerase II. In Saccharomyces cerevisiae, three major classes of transcriptional activators, MBF and SBF, Swi5/Ace2, and Mcm1-associated factors, predominantly regulate various gene clusters at the G 1 /S transition; M and the M/G 1 boundary; and G 1 , M, or M/G 1 , respectively (42).Spellman et al. (42) identified ca. 800 genes exhibiting cell cycle oscillation. While the regulation of ϳ500 of these genes can be ascribed to the three known classes of cell cycle transactivators, the mode of regulation of the rest is not understood. Many of these ϳ300 genes participate in nutrient acquisition, and their transcripts show peak expression in M or M/G 1 . For instance, mitotic expression has been observed for genes involved in phosphate metabolism (28, 35) encoding low-affinity (PHO89) and hi...

Isolation of an activator-dependent, promoter-specific chromatin remodeling factor

“…Induction by phosphate starvation rather than PHO80 deletion in chd1Δ isw1Δ cells significantly delayed but did not completely abolish production of acid phosphatase, presumably because of the weak PHO5 mitotic induction pathway (Fig. S5) (30). We conclude that Chd1 directly participates in the activation of PHO5 and in the remolding of promoter nucleosomes in vivo.…”

“…SDS-PAGE and mass spectrometry of the fractions from the final gel filtration step revealed three coeluting sets of polypeptides ( Fig. 4A), corresponding to the chromatin remodeling complex RSC (bands due to Sth1 and Rsc2, fractions 23-28), DNA polymerase ϵ (bands due to Pol2 and Dpb2, fractions [30][31][32][33], and the chromatin remodeling ATPase Chd1 with Sub1 (fractions [32][33][34][35]. MNase analysis of PHO5 chromatin remodeling revealed a peak of Pho4-dependent, promoter-specific activity in fractions 32-35 ( Fig.…”

The Histone Chaperone Asf1 Increases the Rate of Histone Eviction at the Yeast PHO5 and PHO8 Promoters

“…We and others observed in the past that the absence of chromatinrelated factors like Gcn5 or Snf2, although not essential for chromatin opening at the PHO5 promoter, could lead to a delay in the induction process 3 (7,30,36,37). We also tested this possibility for the above histone chaperone mutant strains by monitoring induction kinetics.…”

“…However, the absence of Snf2 leads to a marked kinetic delay in PHO5 induction 3 (7,36,37), indicating a significant role of the SWI/SNF complex in chromatin remodeling at the PHO5 promoter. We wanted to test whether the SWI/SNF complex cooperates with Asf1 in this remodeling process and, if so, whether they function exclusively in the same pathway or whether Asf1 also has a role in the alternative pathway through which PHO5 promoter chromatin is remodeled in the absence of Snf2.…”

“…Dhasarathy and Kladde (36) showed recently that the cofactors Gcn5 and Snf2, which are not necessary for complete PHO5 induction and chromatin opening under fully inducing conditions 3 (7,30,36,37), become essential under submaximal inducing conditions. The submaximal induction conditions were generated by allowing various amounts of residual phosphate in the induction medium.…”