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. 2001 Sep 3;20(17):4944-51.
doi: 10.1093/emboj/20.17.4944.

Increasing the rate of chromatin remodeling and gene activation--a novel role for the histone acetyltransferase Gcn5

S Barbaric  1 , J Walker, A Schmid, J Q Svejstrup, W Hörz
Affiliations

Increasing the rate of chromatin remodeling and gene activation--a novel role for the histone acetyltransferase Gcn5

S Barbaric et al. EMBO J. .

Abstract

Histone acetyltransferases (HATs) such as Gcn5 play a role in transcriptional activation. However, the majority of constitutive genes show no requirement for GCN5, and even regulated genes, such as the yeast PHO5 gene, do not seem to be affected significantly by its absence under normal activation conditions. Here we show that even though the steady-state level of activated PHO5 transcription is not affected by deletion of GCN5, the rate of activation following phosphate starvation is significantly decreased. This delay in transcriptional activation is specifically due to slow chromatin remodeling of the PHO5 promoter, whereas the transmission of the phosphate starvation signal to the PHO5 promoter progresses at a normal rate. Chromatin remodeling is equally delayed in a galactose-inducible PHO5 promoter variant in which the Pho4 binding sites have been replaced by Gal4 binding sites. By contrast, activation of the GAL1 gene by galactose addition occurs with normal kinetics. Lack of the histone H4 N-termini leads to a similar delay in activation of the PHO5 promoter. These results indicate that one important contribution of HATs is to increase the rate of gene induction by accelerating chromatin remodeling, rather than to affect the final steady-state expression levels.

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Figures

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Fig. 1. The kinetics of PHO5 induction is strongly dependent on Gcn5. (A) The time course of PHO5 induction was followed by measuring acid phosphatase activity at the indicated time points after transferring cells from phosphate-containing media (+Pi) to phosphate-free media (–Pi) in a wt strain (circles) and a Δgcn5 strain (squares). The scale of the time axes is different for the first 5 h (solid lines) and the remaining 13 h (broken lines). (B) Identical measurements were carried out with the Gcn5 HAT domain mutant strains KQL (solid squares) and YIA (open squares) (Gregory et al., 1998; Wang et al., 1998). The solid circles denote the wt strain.
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Fig. 2. The Gcn5-dependent delay in PHO5 expression occurs at the level of transcription. Total RNA was extracted at the indicated time points after transferring cells from phosphate-containing media (+Pi) to phosphate-free media (–Pi) in a wt and a Δgcn5 strain, blotted and hybridized with a PHO5 and an ACT1 coding region specific probe (load control). Autoradiographs are shown. The amounts of PHO5 mRNA in the Δgcn5 relative to the wt cells at the different time points (normalized for ACT1 expression) were calculated and are shown at the bottom. Expression of the highly expressed, constitutive ACT1 gene is not affected by a GCN5 deletion (Lee et al., 2000).
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Fig. 3. The rate of chromatin remodeling at the PHO5 promoter is strongly decreased in the absence of Gcn5. Nuclei isolated from cells of a wt strain (YS18, lanes 1, 2, 5, 6, 9 and 10) and a Δgcn5 strain (YS5319, lanes 3, 4, 7, 8, 11 and 12) 3 h after transfer to phosphate-free medium were treated for 30 min at 37°C with 50 U (odd numbered lanes) or 200 U (even numbered lanes) of the restriction enzyme indicated. In order to monitor the extent of cleavage, DNA was isolated, cleaved with HaeIII, analyzed on a 1.5% agarose gel, blotted and hybridized with a probe (Almer et al., 1986). The positions of the restriction sites with respect to the nucleosomal organization of the repressed PHO5 promoter are shown schematically beneath. In all cases, the appearance of the lower band represents accessibility of the corresponding site. The nucleosomes that are perturbed during normal activation are shown as open circles, the Pho4 binding sites as squares and the TATA box as T.
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Fig. 4. The kinetics of Pho4 accumulation in the nucleus following phosphate starvation is not affected by Gcn5. A wt and a Δgcn5 strain expressing a Pho4–GFP fusion protein were grown in high phosphate medium (+Pi) and at time zero transferred to phosphate-free medium (–Pi). Pho4 localization was monitored at the times indicated by fluorescence microscopy (left panel). 4′,6-diamidine-2-phenylindole (DAPI)-stained cells are shown in the center panel, and cells visualized by phase contrast microscopy on the right.
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Fig. 5. The effect of Gcn5 on the kinetics of PHO5 induction is maintained in pho80 cells. Expression of PHO5 in a pho80, pho4 strain (YS33, circles) and a pho80, pho4, gcn5 strain (YS53389, squares) carrying a plasmid with a PHO4 gene controlled by the GAL10 promoter (pKV701-PHO4) (Jayaraman et al., 1994), was followed by measuring acid phosphatase activity upon galactose induction in phosphate-free (A) or phosphate-containing media (B). The scale of the time axes is different for the first 8 h (solid lines) and the remaining 12 h (broken lines).
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Fig. 6. Effect of Gcn5 on induction of galactose-inducible promoters. (A) Activation of pPHO5-lacZ variant 33 [both Pho4 sites in the PHO5 promoter replaced by Gal4 binding sites (Ertinger, 1998)] was followed in a wt (YS18, squares) and a gcn5 strain (YS5189, circles) by measuring β-galactosidase activity. At time zero, galactose was added to the medium. The scale of the time axis is different for the first 2.5 h (solid lines) and the remaining 6 h (broken lines). (B) Galactose-mediated activation of the p416-GAL1-lacZ plasmid containing the GAL1 promoter was measured in the same two strains.
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Fig. 7. Effect of the histone H4 N-termini on the kinetics of PHO5 induction. The time course of PHO5 induction in strains PKY899 (HHF2; squares) and PKY813 [hhf2(del 4–28); circles] was followed by measuring acid phosphatase activity at the indicated time points after transferring cells from phosphate-containing media to phosphate-free media.
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