Gaining insight into the mechanisms of communication between regulatory proteins/enzymes that modify or interact with the highly conserved histones is crucial to understanding all levels of DNA-related processes in the cell. It is becoming evident that the local environment of histone modifications is an important factor in dictating which interactions can and will occur. Inherent in this idea are the effects that the various modifications have on structure. Whether it is the general effect of loss of positive charge by acetylation causing loosening of the DNA or a specific local effect, such as phosphorylation of serine 10 on histone H3 affecting or being affected by lysine acetylation/methylation on the same tail, interrelatedness is a key. In addition, certain protein modules, such as bromodomains and chromodomains, have been shown to interact with acetylated lysines and methylated lysines, respectively, in a specific manner.
It was shown that serine 10 phosphorylation on histone H3 is associated with increased acetylation at lysine 14 and transcription activation of a subset of genes (6
). In contrast, for the histone H4 tail, our studies demonstrated that phosphorylation of serine 1 has a negative effect on acetylation by NuA4 in vitro. Interestingly, methyl-arginine 3 abrogates this phospho-serine 1 inhibition, though by itself this methylation has little effect on NuA4 acetylation in our peptide assays. On the other hand, methylation of the arginine 3 of histone H4 was previously shown to increase acetylation by human CBP in vitro (independently of other modifications) (53
). While PRMT1 was shown to be the enzyme responsible for methylating arginine 3 of histone H4 in mammalian cells, deletion of the yeast homolog Hmt1/Rmt1 does not cause loss of methylation on this residue (30
). This suggests that either another protein is responsible for Arg3 methylation in yeast or Hmt1/Rmt1 shares redundant functions with another methyltransferase. Nevertheless, several studies firmly linked mammalian PRMT1 to gene activation in the chromatin context (2
). In addition, yeast Hmt1/Rmt1 is recruited to specific genes in vivo during the beginning of the transcription elongation process (55
). Recently, an enzyme able to regulate H4 Me-Arg3 levels in vivo by deimination was identified and linked to transcription downregulation (10
Another point of note is that mammalian histone H2A has the same first five amino acids as H4 and can undergo the same modifications on serine 1, arginine 3, and lysine 5 (3
). The protein sequence of both the H2A.Z variant and the Tetrahymena
H4 tail has an alanine substituted for the usual serine at position 1. This substitution is accompanied by a loss of arginine 3, suggesting that in the absence of serine 1, there is no requirement for arginine 3 (16
). Taking this into account with our peptide data, it will be interesting to see whether the role of arginine 3 methylation is somehow related to phosphorylation of serine 1. A critical way to elucidate this relationship will be to obtain antibodies raised against the doubly modified H4 peptide (P-Ser1/diMe-Arg3).
In our analysis of native chromatin, we observed that several nuclear histone modifications, including phosphorylation, acetylation, and methylation of histone H4, are regulated throughout the cell cycle or upon exposure to drugs. After treatment with MMS, which introduces single- and double-strand breaks in DNA, phospho-serine 1 increases while acetylation of histone H4 decreases at lysines 5, 8, and 12 compared to control chromatin. This supports our theory that phospho-serine 1 is inhibitory to acetylation on the histone H4 tail. However, in the case of hydroxyurea-treated cells (blocked in S phase), isolated chromatin was found to have higher levels of phospho-serine 1 with no accompanying decrease in histone H4 acetylation. This discrepancy could be explained as follows. One possibility is that the remaining acetylation exists on different H4 tails than those with the phosphorylated serine. However, considering that modification of neighboring residues can greatly affect the function of an antibody by altering the epitope (Fig. ), as is the case for phospho-serine 1 and methyl-arginine 3, it is conceivable that methyl Arg3 is present but not detectable because of serine 1 phosphorylation on the same tail. Additional evidence that serine 1 phosphate is inhibitory to acetylation comes from a study employing mass spectrometry analysis of bulk histones from human cells (19
). In that report, the authors never detect phosphorylation and acetylation on the same histone H4 molecule. In fact, upon treatment with okadaic acid, a deacetylation event occurs before phosphorylation is observed, suggesting that the H4 tail must be deacetylated before the phosphate group can be added (19
). These data are clearly supported by our finding of an H4 Ser1 kinase associated with the Sin3/Rpd3 complex, the major histone deacetylase in yeast (Fig. ). Altogether, these results suggest that Sin3/Rpd3 complexes first deacetylate nucleosomal histone H4 tails, followed by Ser1 phosphorylation through associated CK2 activity. This phosphorylation event would ensure that any NuA4 or globally acting picNuA4 complexes (5
) could not reacetylate the H4 tail, establishing a more stable deacetylated state of the local chromatin. Accordingly, H4 and H2A P-Ser1 were recently analyzed during early murine development and described as stable “epigenetic” marks in contrast to the more dynamic and reversible acetylation and methylation at arginine 3 (46
). Another recent report detected increased H4 and H2A Ser1 phosphorylation in higher eukaryotes during S phase (3
), agreeing with the results we obtained in yeast chromatin (Fig. ) and early work linking P-Ser1 to newly synthesized histone H4 (45
). Cheung et al. also independently identified CK2 as a DNA damage-regulated kinase of histone H4 serine 1 (7
). This work clearly demonstrates that MMS-induced H4 P-Ser1 signal is lost in cka1
(Ts) double-mutant cells (CK2 has two kinase subunits in yeast, Cka1 and Cka2, and single mutants do not affect H4 P-Ser1 signals) (data not shown and reference 7
). Interestingly CK2 was also reported to phosphorylate human histone deacetylases 2 and 3, a modification that promotes both enzymatic activities, again linking the kinase to histone H4 deacetylation (51
Histone H4 serine 1 and arginine 3 mutants display no obvious growth phenotype and no sensitivity to the DNA-damaging agent MMS (Fig. ), indicating that these modifications are not by themselves required for cell survival or DNA damage response. Similar results were obtained in a recently published report (7
). Interestingly, data presented in this work suggests that phosphorylation of H4 serine 1 slightly affects the efficiency of DNA double-strand break repair by nonhomologous end joining. In any case, the overall weakness or absence of the phenotype of H4 serine 1/arginine 3 mutants suggests that these marks are redundant with other histone modifications, e.g., on the H2A tail. Nevertheless, we clearly demonstrate that H4 P-Ser1 is locally regulated during transcription and DNA double-strand break repair in vivo and its increase correlates with histone H4 deacetylation (Fig. and ). Importantly, it has been shown that large transcribed regions of many human and yeast genes were maintained at low levels of histone acetylation compared to the 5′/start site/promoter regions, even though these regions are read by elongating polymerases (33
). This accords with the fact that CK2 is found associated with many factors involved in transcription elongation (Fig. ). Our ChIP analysis of the HSP104
gene during activation also correlates histone H4 phosphorylation with deacetylation and transcription. Thus, we hypothesize that the role of H4 phosphorylation/deacetylation during transcription elongation may be linked to nucleosome stabilization after the passage of RNA polymerase II.
Maintenance of genome integrity is another critical nuclear process. The fact that H4 P-Ser1 is directly involved at sites of DNA double-strand breaks in addition to its role in gene transcription indicates that this specific chromatin modification is important in diverse nuclear functions. In a separate study, we previously showed that NuA4 binds to histone H2A phosphorylated on Ser129 near a double-strand break, allowing acetylation of the surrounding chromatin (12
). We have now found that histone H4 is phosphorylated at a later stage, after DNA break formation and induction of the damage response (Fig. ). P-Ser1 appearance correlates again with a decrease of H4 acetylation, and it is reasonable to think that it could be linked to chromatin restoration after DNA repair is complete (41
) (a suggested model is shown in Fig. ). Accordingly, a recent report showed that the Sin3/Rpd3 HDAC complex facilitates double-strand break repair and that histone H4 is deacetylated 4 h after the induction of the HO break (23
). The local recruitment of the Sin3/Rpd3 complex (presumably with CK2) between 2 and 4 h postbreak could explain our finding of increased H4 P-Ser1 and a drop in H4 acetylation during that period (Fig. ). Altogether, these findings indicate that NuA4 function/activity is differentially regulated by two distinct phospho-histone marks (H2A P-Ser129 and H4 P-Ser1) in a stepwise fashion during the process of DNA double-strand break repair (Fig. ).
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 (more ...)
In our previous work on the NuA4 HAT complex, we were surprised to find no major variation in its abundance and specific activity during the cell cycle and under other growth conditions (N. Lacoste and J. Côté, unpublished data). In this report, we demonstrate a new efficient way to regulate NuA4 acetyltransferase activity, i.e., through other posttranslational covalent modifications of its substrate, the histone H4 N-terminal domain. These findings add an important new circuitry to the cross talk that occurs between different histone modifications in chromatin and their diverse functional consequences. It also shows how NuA4-dependent acetylation is tightly regulated in vivo, at sites of both gene transcription and DNA repair.