Polyphosphate loss promotes SNF/SWI- and Gcn5-dependent mitotic induction of PHO5 - PubMed (original) (raw)

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

Daniel W Neef et al. Mol Cell Biol. 2003 Jun.

Abstract

Approximately 800 transcripts in Saccharomyces cerevisiae are cell cycle regulated. The oscillation of approximately 40% 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 phm3delta 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.

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Figures

FIG. 1.

FIG. 1.

Mitotic induction of PHO5 requires PHO2, PHO4, and PHO81. (A) Northern analysis of wild-type (WT), _pho81_Δ, _pho2_Δ, and _pho4_Δ cultures at various times following release from synchronization with α-factor. The wild-type strain was analyzed in parallel with each null strain as a positive control. (B) Transcript levels of PHO5 (normalized to TCM1) from panel A. (C) Flow cytometric analysis of Sytox Green-stained cells. The x and y axes indicate the fluorescence intensity and numbers of cells analyzed (which was identical in all panels), respectively. 1N and 2N refer to haploid and diploid DNA contents, respectively. (D) Total rAPase activities of asynchronous YPD cultures. The means ± 1 standard deviation from three independent experiments are shown.

FIG. 2.

FIG. 2.

PHO5 activation is SNF/SWI and Gcn5 dependent. (A) Northern analysis of synchronous cultures of wild-type (WT), snf2/_swi2_Δ, and _gcn5_Δ strains released from nocodazole arrest in YPD medium. (B) Normalized PHO5 transcript levels from panel A. (C) Flow cytometric analysis of wild-type cells in panel A. (D) Total rAPase activities of asynchronous YPD cultures (n = 3; mean ± 1 standard deviation). (E) Time course of PHO5 activation. Asynchronous cultures grown on defined Pi-free medium with 13.4 mM Pi added back were starved for Pi and assayed for total rAPase activity at the indicated times.

FIG. 3.

FIG. 3.

PHO5 mitotic activation is repressed by addition of orthophosphate (Pi). (A) Northern analysis of α-factor-synchronized cultures. Single colonies from a YPD plate were inoculated into YPD overnight cultures and then diluted in YPD or YPD + Pi (13.4 mM) for 12 or 55 h (as indicated on the left) prior to α-factor arrest. Cells grown for 6 h in defined Pi-free medium with (+) or without (−) Pi were included as a positive control in the Northern analysis of the samples grown for 55 h in YPD + Pi. (B) Normalized PHO5 transcript levels from panel A.

FIG. 4.

FIG. 4.

Repression of PHO5 mitotic expression by added Pi is time dependent. (A) Total rAPase activities of asynchronous cultures. Cultures were grown overnight in either YPD (bars 1 and 2) or YPD + Pi (bars 3 and 4) and then washed and resuspended in YPD (bars 1 and 4) or YPD + Pi (bars 2 and 3) for an additional 6 h, as indicated (n = 3; mean ± 1 standard deviation). (B) Time course of decrease in total rAPase activity following addition of Pi. Cells were plated and pregrown on YPD and transferred to YPD (control) or YPD + Pi. The percentage of activity of the control (growth in parallel in YPD) is plotted as a function of time.

FIG. 5.

FIG. 5.

Loss of polyP leads to derepression of PHO5. (A) Total rAPase activities of wild-type (WT), _phm1_Δ, _phm2_Δ, _phm3_Δ, and _phm4_Δ strains. Cells were grown in YPD and diluted in YPD or YPD + Pi medium for 6 h prior to assay of rAPase activity (n = 3; mean ± 1 standard deviation). The severity of loss of polyP accumulation increases from left to right and parallels the extent of disruption of the Phm/Vtc complex, with _phm3_Δ and _phm4_Δ strains having no detectable polyP or Phm/Vtc complex (31, 33). (B) Analysis of polyP levels. Wild-type (PHM3) or _phm3_Δ strains were grown for 2 days on plates and then for 24 h in liquid YPD-low Pi, YPD, or YPD + Pi medium containing a low (L; lane 6), moderate (M; lanes 1, 3, and 5), or high (H; lanes 2 and 4) concentration of Pi, respectively. For lanes 1 to 4, after the 24-h incubation period, internal aliquots of each culture were assayed for rAPase activity and polyP levels by the metachromatic absorbance shift method (as indicated at the bottom; ND, nondetectable). The overplus samples (lanes 5 to 6) received additional treatments of Pi starvation followed by Pi addition (see Materials and Methods) before rAPase and polyP levels were determined. PolyP was also visualized in the gel after electrophoresis of equal amounts (10 μg) of total RNA and staining with toluidine blue O dye, a basic dye that binds polyanions. RNA species at the top of the gel are indicated on the right.

FIG. 6.

FIG. 6.

SNF/SWI and Gcn5 are required for polyP accumulation. Steady-state levels of polyP from wild-type (WT), _gcn5_Δ, and snf2/_swi2_Δ strains were measured (n = 3; mean ± 1 standard deviation) by the metachromatic shift method after asynchronous growth in YPD (the same conditions used for rAPase activity measurements in Fig. 2D).

FIG. 7.

FIG. 7.

PolyP levels fluctuate during the cell cycle. (A) Flow cytometric analysis of α-factor-synchronized cultures. (B) Analysis of polyP and mRNA levels. Total RNA and polyP were isolated at 15-min intervals following release from α-factor arrest. PolyP was analyzed as for Fig. 5B, except that the samples were treated with RNase A prior to electrophoresis (polyP gel) and metachromatic shift quantification of polyP levels of an internal fraction (graph at bottom). Northern analysis of PHO5 and TCM1 mRNA levels of internal aliquots of RNA-polyP not treated with RNase A (middle) are plotted versus time and compared to polyP levels at the bottom.

FIG. 8.

FIG. 8.

Mitotic activation of PHO5 is increased in _phm3_Δ strains. (A) Northern analysis of α-factor-synchronized cultures of wild-type (WT) and _phm3_Δ cells. (B) Normalized PHO5 transcript levels from panel A.

FIG. 9.

FIG. 9.

Deletion of PHM3 increases the rate of PHO5 activation. (A) Northern analysis of RNA isolated during a time course of PHO5 activation. Wild-type (WT) and _phm3_Δ strains grown on defined Pi-free medium with 13.4 mM Pi added back (to build up stores of polyP in the wild-type strain) were then starved for Pi by being washed and resuspended in Pi-free medium, and RNA was isolated at the indicated times. The total rAPase activities obtained from an internal aliquot of each culture are indicated below each lane. (B) Absolute PHO5 transcript levels from panel A. The thick trend line indicates the initial, linear period of transcript accumulation.

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