Polyphosphate loss promotes SNF/SWI- and Gcn5-dependent mitotic induction of PHO5.

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 G1/S transition; M and the M/G1 boundary; and G1, M, or M/G1, respectively (42). Spellman et al. (42) identified ca. 800 genes exhibiting cell cycle oscillation. While the regulation of ∼500 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 ∼300 genes participate in nutrient acquisition, and their transcripts show peak expression in M or M/G1. For instance, mitotic expression has been observed for genes involved in phosphate metabolism (28, 35) encoding low-affinity (PHO89) and high-affinity (PHO84) transporters of inorganic orthophosphate (Pi), as well as repressible acid phosphatases (rAPases; PHO5, PHO11, and PHO12) and constitutive APase (PHO3). Based on the timing of their expression, the mitotically induced rAPase genes are grouped with 34 other genes comprising the MCM cluster (42). However, there is no evidence for a direct or indirect role of Mcm1, or another known DNA binding cell cycle regulator, in their transcriptional activation. Moreover, as all the experiments of Spellman et al. (42) were conducted in rich medium that contained Pi, it is unclear why these PHO genes, which ordinarily are induced upon Pi deprivation, are activated during specific cell cycle phases. Thus, PHO5, PHO11, and PHO12, as well as other cell cycle-regulated PHO genes, fall into a large group of genes involved in maintaining nutritional homeostasis for which it is not understood how cell cycle periodicity is accomplished or why it is needed. To date, studies of the mechanisms of yeast PHO gene activation have focused on regulatory events that occur in asynchronous cultures as a function of limiting Pi. By an as-yet-unknown mechanism, Pi limitation initiates a signal transduction cascade that activates Pho81, a CDK inhibitor. Pho81 inhibits the phosphorylation activity of the cyclin-CDK Pho80-Pho85 (39), leading to nuclear retention of the otherwise cytoplasmic helix-loop-helix factor Pho4, the primary PHO transactivator (22, 34). In the nucleus, Pho4, either by itself or as a heterodimer with Pho2, a nuclear homeodomain protein, activates >20 different genes in the PHO system (19, 33). These include several phosphate metabolism (PHM) genes, PHM1 (VTC2), PHM2 (VTC3), PHM3 (VTC4), PHM4 (VTC1), and PHM5 (PPN1). Phm1 to -4 form a biochemical complex in vivo, the integrity of which is disrupted by phm3 or phm4 null mutations (6, 31). As polyphosphate (polyP), a linear polymer of up to hundreds of Pi residues linked by high-energy phosphoanhydride bonds, is not detectable in such strains, the Phm/Vtc complex is proposed to be a polyP synthetase (33). However, this complex is also needed for the proper morphology of vacuoles (31), where 99% of the total cellular polyP is stored (44). PolyP is present in all organisms examined to date and, in S. cerevisiae, can reach concentrations as high as 120 mM in the vacuole, as much as 60% of the total phosphate content (23). It has been suggested that polyP functions as a phosphate reservoir, since its levels drop when cells need Pi, e.g., during log-phase growth or in Pi-limiting growth media (11, 41). It has also been suggested that polyP serves a cellular protective function in that it chelates cations, such as Ca2+, in the vacuole (23). However, yeast strains deficient in polyP accumulation are not sensitive to high concentrations of calcium (33). Finally, polyP levels increase as yeast cultures approach stationary phase for reasons and by mechanisms that are not clear (40, 47). Thus, while conditions and enzymes involved in polyP synthesis and hydrolysis in eukaryotes have been identified, its metabolic, regulatory, and physiological function(s) remain obscure. Relative to our understanding of the connection between nutrient sensing and the decision to execute Start (36), little is known about how nutrient levels influence regulation in other stages of the cell cycle. To gain insight into this problem, we have focused on elucidating both the molecular mechanisms and the physiological basis for M phase activation of PHO5. We report that the unexpected mitotic induction of PHO5 in rich medium is under the control of Pho4, Pho2, and Pho81. Increasing the metabolic pools of polyP represses mitotic expression of PHO5. Conversely, elimination of polyP by deletion of PHM3 leads to PHO5 activation. Moreover, we demonstrate that polyP levels are influenced by progression of the cell cycle, declining prior to and being replenished after M phase. Our results suggest that polyP is a dynamic phosphate reserve, and they define a regulatory influence of polyP on PHO mitotic gene expression. Further, our studies demonstrate that Pho4/Pho2 mediates a novel, cell cycle stage-specific mode of regulation that is fine tuned in response to nutrient availability and operates mostly independently of activities that drive cell cycle progression. More generally, our findings suggest that nutrient deficiencies may underlie the transcriptional periodicity of a large class of nutrient transporters that are cell cycle regulated.