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. 2005 Sep;25(18):8179-90.
doi: 10.1128/MCB.25.18.8179-8190.2005.

Regulation of NuA4 histone acetyltransferase activity in transcription and DNA repair by phosphorylation of histone H4

Rhea T Utley  1 , Nicolas Lacoste, Olivier Jobin-Robitaille, Stéphane Allard, Jacques Côté
Affiliations

Regulation of NuA4 histone acetyltransferase activity in transcription and DNA repair by phosphorylation of histone H4

Rhea T Utley et al. Mol Cell Biol. 2005 Sep.

Abstract

The NuA4 complex is a histone H4/H2A acetyltransferase involved in transcription and DNA repair. While histone acetylation is important in many processes, it has become increasingly clear that additional histone modifications also play a crucial interrelated role. To understand how NuA4 action is regulated, we tested various H4 tail peptides harboring known modifications in HAT assays. While dimethylation at arginine 3 (R3M) had little effect on NuA4 activity, phosphorylation of serine 1 (S1P) strongly decreased the ability of the complex to acetylate H4 peptides. However, R3M in combination with S1P alleviates the repression of NuA4 activity. Chromatin from cells treated with DNA damage-inducing agents shows an increase in phosphorylation of serine 1 and a concomitant decrease in H4 acetylation. We found that casein kinase 2 phosphorylates histone H4 and associates with the Rpd3 deacetylase complex, demonstrating a physical connection between phosphorylation of serine 1 and unacetylated H4 tails. Chromatin immunoprecipitation experiments also link local phosphorylation of H4 with its deacetylation, during both transcription and DNA repair. Time course chromatin immunoprecipitation data support a model in which histone H4 phosphorylation occurs after NuA4 action during double-strand break repair at the step of chromatin restoration and deacetylation. These findings demonstrate that H4 phospho-serine 1 regulates chromatin acetylation by the NuA4 complex and that this process is important for normal gene expression and DNA repair.

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Figures

FIG. 1.
FIG. 1.
Phosphorylation of H4 serine 1 inhibits hyperacetylation of its tail by Esa1/NuA4. (A) Esa1 and NuA4 can independently acetylate each lysine residue of the histone H4 tail but prefer to do multiple acetylations. H4 tail peptides were synthesized with or without the acetyl moieties at the conserved lysines 5, 8, 12, and 16 as shown. All peptides are acetylated at the N terminus, as found in vivo. HAT assays were performed with 300 ng of the indicated peptide and either purified NuA4 complex or recombinant Esa1 (catalytic subunit of NuA4), incubated for 30 min at 30°C, spotted on Whatman filters, washed, and subjected to scintillation counting. Acetylation values were normalized to WT H4. The error bars indicate standard deviations. (B) Phosphorylation of serine 1 inhibits acetylation of the H4 N-terminal domain by Esa1 and NuA4. Peptides were synthesized with or without modifications, including phosphorylation of serine 1 (S1P), dimethylation of arginine 3 (R3M), and dimethylation of lysine 20 (K20M). HAT assays were carried out as for panel A.
FIG. 2.
FIG. 2.
NuA4 activity is recovered upon dephosphorylation of histone H4 at serine 1. (A) Lambda phosphatase assays were performed on S1P and control H4 peptides as outlined below the graph. When phosphatase (ppase) action occurred, NuA4 activity was restored to levels observed on the previously unphosphorylated peptide (H4). Controls were carried out to show that the inhibitor was effective and not affecting NuA4 under these conditions. NaV, sodium orthovanadate. The error bars indicate standard deviations. (B) A similar phosphatase treatment was performed on 0.5 μg of purified human core histones (CH) in the absence or presence of lambda phosphatase, and they were subjected to HAT assays with NuA4. While Western blotting showed the complete dephosphorylation of histone H4 serine 1, a reproducible 45% increase in NuA4 activity was observed.
FIG. 3.
FIG. 3.
Methylation of histone H4 arginine 3 alleviates the inhibitory effect of phospho-serine 1 on NuA4 activity. H4 tail peptides were synthesized with various combinations of modifications (S1P, R3M, and K5Ac) as shown. HAT assays were carried out as for Fig. 1. Note that K5Ac is also a strong inhibitor of NuA4 activity, though this effect is not modulated by methylation of arginine 3. The error bars indicate standard deviations.
FIG. 4.
FIG. 4.
Phosphorylation of histone H4 at serine 1 is regulated in vivo. (A) Yeast cultures were grown in the absence (control) or presence of the indicated drugs or substances, which induce cell cycle blockage or signaling response. Native chromatin was purified from yeast and analyzed by SDS-PAGE/Western blotting with the indicated antibodies. (B) Dot blots using 100 ng of the indicated peptides were probed with anti-S1P, anti-R3M, or anti-K5Ac antibody to test the effects of neighboring modifications on epitope recognition.
FIG. 5.
FIG. 5.
A histone H4 Ser1 kinase is found associated with the Sin3/Rpd3 histone deacetylase complex. (A) Strains bearing mutations in residue serine 1 or arginine 3 of histone H4 are not sensitive to MMS or hydroxyurea. Wild-type and the indicated mutant histone H4 strains with episomally expressed histone H3 and H4 in addition to normal yeast cells (chromosomally expressed histones; BY4741) were grown to log phase (OD600, 0.5 to 1). Fivefold serial dilutions starting from an OD600 of 0.02 were plated on YPD, YPD plus 0.03% MMS, and YPD plus 130 mM HU and grown for 3 to 4 days at 30°C. (B) The Rpd3 complex is associated with histone kinase activities, including one for H4 Ser1. Recombinant yeast histone H4 (rH4; lanes 3 and 6) and purified yeast histones from the strains in panel A (wild type, lanes 2 and 5; S1A, lanes 1 and 4) were incubated in the absence (lanes 4 to 6) and presence (lanes 1 to 3) of affinity-purified Rpd3 complexes with both cold ATP and [γ-32P]ATP, separated by SDS-PAGE, transferred to nitrocellulose, and exposed to film. A control reaction with Rpd3-TAP only is shown in lane 7. Kinase activity was found associated with Rpd3-TAP that targets both recombinant and purified histone H4 (lanes 2 and 3), but not when serine 1 was mutated (lane 1). (C) Reactions similar to those in panel B were performed with purified yeast histones in the absence (lanes 1 and 2) and presence of Sin3/Rpd3 HDAC complexes purified through Rpd3-TAP (lanes 3 and 4) or Sin3-TAP (lanes 6 and 7), separated by SDS-PAGE, blotted to nitrocellulose, and probed for the presence of H4 phospho-serine 1. While some basal levels of phospho-serine 1 were observed on endogenous yeast histone H4 (lane 2), this signal increased in the presence of Rpd3-TAP (lane 4) or Sin3-TAP (lane 7). (D) Purified CK2 phosphorylates histone H4 Ser1 in vitro. CK2 was affinity purified through Ckb2-TAP and was subjected to kinase assays with wild-type or mutant (S1A) purified yeast histones. As for Sin3/Rpd3, CK2 phosphorylates histone H3 and H4 in vitro, and the 32P-labeled H4 signal is lost on the S1A mutant (top, lane 1 versus lane 2). Western analysis also showed the strong H4 Ser1 kinase activity of CK2 (bottom).
FIG. 6.
FIG. 6.
Phosphorylation of H4 at serine 1 is linked to transcription and correlates with local H4 deacetylation in vivo. (A) Summary of the physical associations detected in proteomic studies between CK2 and transcription regulators. (B) ChIP analysis of H4 P-Ser1 on the HSP104 gene before and after heat shock. Cross-linked chromatin was prepared from cells grown in normal conditions or incubated for 10 min at 39°C. Immunoprecipitation was performed with anti-P-Ser1 and analyzed by real-time PCR (in triplicate). Ratios of IP to input signal (percent) are presented after subtraction of background signal (no antibody). Results from a control locus and two other genes are also presented (large intergenic region on chromosome V and coding regions of TAF2 and PMA1). Phosphorylation of H4 Ser1 specifically increases over the HSP104 gene during activation (“promoter” corresponds to the upstream activation sequence, while “coding” is 1.9 kb downstream from the transcription start site). (C) ChIP analysis of H4 acetylation at lysine 8 in the same conditions. In contrast to P-Ser1 in panel B, Ac-Lys8 signals are drastically decreased over the HSP104 gene upon transcription activation, while the control locus and other genes are not affected.
FIG. 7.
FIG. 7.
Nucleosomal histone H4 becomes phosphorylated at serine 1 near a DNA double-strand break in vivo. (A) Cross-linked chromatin was prepared from cells expressing the HO endonuclease under the control of the GAL promoter and incubated for the indicated length of time in galactose-containing medium. IPs were performed with anti-H2A P-Ser129 and analyzed by real-time PCR with primers for regions next to the single HO-induced double-strand break at the MAT locus. The numbers represent the change of IP ratio to the control locus (InterV) at the different time points compared to time zero (set to 1). In these conditions, the HO cutting site is cleaved more than 80% (data not shown). H2A S129 is phosphorylated to near-maximum levels near the HO break within 30 min. (B) ChIP analysis of H4 P-Ser1 in the same conditions. Histone H4 also becomes phosphorylated near a DNA double-strand break in vivo, but at later time points than H2A. (C) ChIP analysis of H4 Ac-Lys8 in the same conditions. Histone H4 becomes more acetylated in local chromatin upon double-strand break formation but drops at later time points, concomitant with the increase of P-Ser1.
FIG. 8.
FIG. 8.
Model for the interplay of histone phosphorylation events and the NuA4 HAT complex during the repair of DNA double-strand breaks. Appearance of a double-strand break is depicted in the context of chromatin. The proposed access, repair, and restore steps of chromatin modification during DNA repair are indicated. Phosphorylation of H2A(X), recruitment of NuA4, and chromatin acetylation are early events, while chromatin deacetylation and H4 phosphorylation are proposed to occur at the late stage of chromatin reassembly after DNA damage is repaired. In this situation, phosphorylation of H4 blocks local reacetylation by NuA4.
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