In this study, we show that Rvb1 is required for the activity of the Tip60 complex and that the complex has a specific role in decreasing phospho-H2AX in vivo after DNA damage. The early detection of phospho-H2AX in Rvb1-depleted cells suggests that the latent period of the phospho-H2AX signal in wild-type cells is due to the constitutive activity of Rvb1 on phospho-H2AX-containing nucleosomes even early in the cell's response. At later time points, the Rvb1 deficit manifests itself as the continued accumulation of phospho-H2AX beyond a point when normal cells have started downregulating the phosphorylation. The identification of Rvb1 in various chromatin-remodeling complexes involved in the DNA damage response by us as well as others had suggested a role of the protein in the DNA damage response. The increase in phospho-H2AX levels in Rvb1-depleted cells subjected to DNA damage is in agreement with this possibility.
The overexpression of histone H4 that cannot be acetylated by Tip60 results in increased levels of chromatin-bound phospho-H2AX, consistent with a role of the histone H4 acetyltransferase, Tip60, in the dephosphorylation of phospho-H2AX. We cannot formally rule out that the increase in phospho-H2AX that we observed in cells was secondary to a transcriptional role of Tip60. Despite this caveat, we prefer the hypothesis that the Rvb1-Tip60 chromatin-remodeling factor is necessary at sites of DNA damage for the dephosphorylation of phospho-H2AX because the Tip60 complex associates with the sites of damaged DNA and is known to promote the opening of the chromatin by the posttranslational modification of the histone tails (this study and references 31
phospho-H2AX is exchanged from the chromatin before dephosphorylation by PPH3 (23
), and chromatin remodeling by the Drosophila
Tip60 complex is important for the in vitro dephosphorylation of a related histone, phospho-H2AV (27
). Although PP2A is known to remove the phosphate directly from phospho-H2AX in mammalian cells (9
), a chromatin-remodeling step upstream of the dephosphorylation may be necessary. Our results show that the removal of Rvb1 or of Tip60 results in the accumulation of phospho-H2AX on the chromatin but that the removal of PP2A leads to the accumulation of the phospho-H2AX in the soluble fraction and the chromatin. The abundance of PP2A and the interaction of PP2A with H2AX are independent of a functional Rvb1-Tip60 complex (Fig. and data not shown). These results are consistent with the possibility that chromatin remodeling by the Rvb1-Tip60 complex at sites of DNA damage is required before phospho-H2AX can be dephosphorylated by PP2A.
The hIno80 and SRCAP complexes are mammalian homologs of the Ino80 and Swr1 complexes, two chromatin-remodeling complexes in yeast known to contain Rvb1. Both the yeast proteins are recruited to double-strand breaks, and Ino80 is required for the eviction of phospho-H2A nucleosomes from the sites of DNA damage (34
). Decreasing hIno80 (or SRCAP) to 10% of the wild-type levels did not impair phospho-H2AX dephosphorylation in our experiments with mammalian cells, while similar decreases of Rvb1 or Tip60 impaired phospho-H2AX dephosphorylation. These results suggest that in mammalian cells, the Tip60 complex is of greater importance than the Ino80 complex in the dephosphorylation of phospho-H2AX.
Our results agree with those in a very recent paper saying that Tip60 associates with and is required for the mobilization of H2AX in the first 5 min after the induction of double-strand breaks with ionizing radiation (19
). Ikura et al., however, suggest a different mechanism, in which Tip60 acetylates H2AX on K5 and promotes UBC13-mediated polyubiquitination of H2AX on K119 prior to the release of H2AX from the chromatin (19
). We have failed to see any acetylation of K5 on H2AX after UV irradiation (data not shown) and have instead noted acetylation on H4 associated with H2AX and have mimicked the phenotype of Tip60 depletion by overexpressing nonacetylable H4 (Fig. ). The differences noted may be due to differences in the agents used to induce double-stranded DNA breaks: ionizing radiation breaks the DNA directly, while UV or chemicals induce breaks mostly after DNA replication across the sites of DNA damage. We prefer the explanation that both H4 and H2AX are important substrates of Tip60, with H2AX acetylation and ubiquitinylation being more important very early in the DNA damage response (within minutes) while H4 acetylation is more important on a longer time scale for curtailing the phospho-H2AX response.
The Tip60 complex has been implicated not only in DNA damage repair but also in the activation of checkpoint pathways (40
). The Tip60 complex acetylates ATM after DNA damage, and this acetylation is required for the activation of ATM. As Rvb1 regulates the acetyltransferase activity of the Tip60 complex, a decrease in the specific activation of ATR/ATM may explain why phospho-Chk1 and phospho-Chk2 are not hyperactivated after Rvb1 depletion, despite the increase in phospho-H2AX. In addition, because other substrates of ATR or ATM are not hyperphosphorylated, we do not think that Rvb1 depletion increases the phosphorylation of H2AX by increasing the activity of ATR or ATM. Instead, we favor the hypothesis that the Tip60 complex is required for the dephosphorylation of phospho-H2AX and that it is the loss of this function that leads to the increased phosphorylation of H2AX after Tip60 or Rvb1 depletion. The chemical inhibition of Tip60 and Rvb1 is expected to prolong the phospho-H2AX response while maintaining the normal kinetics of activation and inactivation of the downstream Chk1 and Chk2 kinases that arrest the cell cycle. By delinking the kinetics of phospho-H2AX activation from Chk1 or Chk2 activation, inhibitors of the Rvb1-Tip60 complex may be useful as sensitizers for chemo- or radiotherapy of cancers.
A role for Rvb1 in the activity of the Tip60 complex links the activity of Rvb1 to that of critical tumor-suppressive and oncogenic transcription factors that are regulated by Tip60. Tip60 is required for the optimum function of p53 on specific p53-driven promoters (43
), so that our results suggest that Rvb1 will also be required for p53-mediated pathways of apoptosis after DNA damage. Tip60 is also required for gene expression from Myc-driven promoters (15
). Therefore, a role for Rvb1 in Tip60 activity may explain why Rvb1 is required for cell transformation by Myc (47
). Similarly, a role for Rvb1 in the action of chromatin-remodeling complexes that interact with beta-catenin may explain the role of Rvb1 in the transmission of the growth-suppressive signal from the adenomatous polyposis coli tumor suppressor protein (4
The only molecular function attributed to Rvb1 until now has been in the recruitment of Arp5 to the yeast Ino80 complex (22
). The discovery reported here expands the role of Rvb1 in chromatin remodeling and highlights the importance of Rvb1 in the DNA damage response of mammalian cells. Future studies will investigate how the absence of Rvb1 impairs the HAT activity of the Tip60 complex and the consequences of Rvb1 depletion for the sensitivity of cells to DNA damage.