Chromatin reorganization plays an important role in DNA repair, apoptosis, and cell cycle checkpoints. Among proteins involved in chromatin reorganization, TIP60 histone acetyltransferase has been shown to play a role in DNA repair and apoptosis. However, how TIP60 regulates chromatin reorganization in the response of human cells to DNA damage is largely unknown. Here, we show that ionizing irradiation induces TIP60 acetylation of histone H2AX, a variant form of H2A known to be phosphorylated following DNA damage. Furthermore, TIP60 regulates the ubiquitination of H2AX via the ubiquitin-conjugating enzyme UBC13, which is induced by DNA damage. This ubiquitination of H2AX requires its prior acetylation. We also demonstrate that acetylation-dependent ubiquitination by the TIP60-UBC13 complex leads to the release of H2AX from damaged chromatin. We conclude that the sequential acetylation and ubiquitination of H2AX by TIP60-UBC13 promote enhanced histone dynamics, which in turn stimulate a DNA damage response.Chromatin reorganization by histone modification and mobilization plays a crucial role in DNA metabolism, including replication, transcription, and repair. It appears that histone modification and mobilization can reorganize chromatin to allow DNA repair machinery to access damaged chromosomal DNA (11,29,52,56,57).H2AX is a histone variant that differs from H2A at various amino acid residues along the entire protein and in its Cterminal extensions. H2AX is phosphorylated after the induction of DNA double-strand breaks (DSBs), and the phosphorylated H2AX (␥-H2AX) participates in focus formation at sites of DNA damage. After induction of DSBs, the MRN complex (MRE11, RAD50, and NBS1) binds to broken DNA ends and recruits active ATM, ATR, and/or DNA protein kinase, resulting in the initial phosphorylation of H2AX (32,38,40). MDC1 then associates with ␥-H2AX and recruits additional activated ATM to the sites of DSBs (23,46). This positive feedback loop leads to the expansion of the ␥-H2AX region surrounding DSBs and provides docking sites for many DNA damage and repair proteins, including the MRN complex, 53BP1, and BRCA1 (5, 6, 46). ␥-H2AX plays a role in the accumulation but not in the initial recruitment of repair factors such as the MRN complex, 53BP1, and BRCA1 (10, 63). Therefore, modifications of H2AX other than phosphorylation could play a role in the initial step of the DNA damage response.Until recently, the biological significance of ubiquitination in the DNA damage response has been unclear. H2B ubiquitination regulates the damage checkpoint response (15). H2A is ubiquitinated during the response to UV-induced DNA damage (8). UV-induced DNA damage also causes the ubiquitination of histones H3 and H4, resulting in their release from chromatin (60). Interestingly, ubiquitin-conjugated proteins appear to be accumulated at sites of DSBs, forming nuclear foci like ␥-H2AX (34). These findings raise the possibility that histone ubiquitination is also involved in the reorganization of chromatin in response to D...

The transcription repressor Bach1 is a sensor and effector of heme that regulates the expression of heme oxygenase 1 and globin genes. Heme binds to Bach1, inhibiting its DNA binding activity and inducing its nuclear export. We found that hemin further induced the degradation of endogenous Bach1 in NIH 3T3 cells, murine embryonic fibroblasts, and murine erythroleukemia cells. In contrast, succinylacetone, an inhibitor of heme synthesis, caused accumulation of Bach1 in murine embryonic fibroblasts, indicating that physiological levels of heme regulated the Bach1 turnover. Polyubiquitination and rapid degradation of overexpressed Bach1 were induced by hemin treatment. HOIL-1, an ubiquitin-protein ligase which recognizes heme-bound, oxidized iron regulatory protein 2, was found to bind with Bach1 when both were overexpressed in NIH 3T3 cells. HOIL-1 stimulated the polyubiquitination of Bach1 in a purified in vitro ubiquitination system depending on the intact heme binding motifs of Bach1. Expression of dominant-negative HOIL-1 in murine erythroleukemia cells resulted in higher stability of endogenous Bach1, raising the possibility that the heme-regulated degradation involved HOIL-1 in murine erythroleukemia cells. These results suggest that heme within a cell regulates the polyubiquitination and degradation of Bach1.

A balanced deoxyribonucleotide (dNTP) supply is essential for DNA repair. Here, we found that ribonucleotide reductase (RNR) subunits RRM1 and RRM2 accumulated very rapidly at damage sites. RRM1 bound physically to Tip60. Chromatin immunoprecipitation analyses of cells with an I-SceI cassette revealed that RRM1 bound to a damage site in a Tip60-dependent manner. Active RRM1 mutants lacking Tip60 binding failed to rescue an impaired DNA repair in RRM1-depleted G1-phase cells. Inhibition of RNR recruitment by an RRM1 C-terminal fragment sensitized cells to DNA damage. We propose that Tip60-dependent recruitment of RNR plays an essential role in dNTP supply for DNA repair.Supplemental material is available at http://www.genesdev.org.

Mutations of the Glu76 residue of canonical histone H2B are frequently found in cancer cells. However, it is quite mysterious how a single amino acid substitution in one of the multiple H2B genes affects cell fate. Here we found that the H2B E76K mutation, in which Glu76 is replaced by Lys (E76K), distorted the interface between H2B and H4 in the nucleosome, as revealed by the crystal structure and induced nucleosome instability in vivo and in vitro. Exogenous production of the H2B E76K mutant robustly enhanced the colony formation ability of the expressing cells, indicating that the H2B E76K mutant has the potential to promote oncogenic transformation in the presence of wild-type H2B. We found that other cancer-associated mutations of histones, H3.1 E97K and H2A.Z.1 R80C, also induced nucleosome instability. Interestingly, like the H2B E76K mutant, the H3.1 E97K mutant was minimally incorporated into chromatin in cells, but it enhanced the colony formation ability. In contrast, the H2A.Z.1 R80C mutant was incorporated into chromatin in cells, and had minor effects on the colony formation ability of the cells. These characteristics of histones with cancer-associated mutations may provide important information toward understanding how the mutations promote cancer progression.

Cytolethal distending toxin (CDT), produced by Actinobacillus actinomycetemcomitans, is a putative virulence factor in the pathogenesis of periodontal diseases. It is a cell cycle specific inhibitor at the G 2 /M transition. CDTB, one of the subunits of the CDT holotoxin, is implicated in a genotoxic role after entering the target cells, whereby chromosomal damage induces checkpoint phosphorylation cascades. CDTB microinjected into the cytoplasm was shown to localize in the nucleus and induce chromatin collapse. To investigate the molecular mechanism involved in nuclear transport of CDTB, we used transient expression and microinjection of a CDTB-green fluorescent protein (GFP) fusion protein. After microinjection, His-tagged CDTB-GFP entered the nucleus in 3-4 h. Leptomycin B did not increase the speed of entry of the fusion protein, suggesting that the relatively slow entry of the fusion protein is not due to the CRM1-dependent nuclear export of the protein. Nuclear localization of the CDTB-GFP was temperature-dependent. An in vitro transport assay demonstrated that the nuclear localization of CDTB is mediated by active transport. An assay using transient expression of a series of truncated CDTB-GFP fusion proteins revealed that residues 48 -124 constitute the minimum region involved in nuclear transport of CDTB. A domain swapping experiment of the region involved in nuclear transport of CDTB with an SV40 T nuclear localization signal indicated that CDTB is composed of two domains, an N-terminal domain for nuclear transport and a C-terminal active domain. Our results strongly suggest that nuclear localization of CDTB is required for the holotoxin to induce cytodistension and cell cycle block. This is the first demonstration that a bacterial toxin possessing a unique domain for nuclear transport is transferred to the animal cell nucleus by active transport.Cytolethal distending toxin (CDT) 1 is a unique bacterial toxin that induces cell cycle arrest of cultured cells in the G 2 phase. It has been identified in several pathogenic bacteria including Campylobacter spp., Escherichia coli, Shigella dysenteriae, Haemophilus ducreyi, Helicobacter hepaticus, and Actinobacillus actinomycetemcomitans. The cells intoxicated with CDT show a cytopathic effect and distension in cell size, and eventually they die, which has been shown to be a common consequence of CDT treated cells (1-4). CDT holotoxin is composed of CDTA, -B, and -C, encoded by the cdtA, cdtB, and cdtC genes tandemly located on the cdt locus (1, 5-7). The CDTinduced G 2 arrest has been ascribed to the inactivation of the Cdc2-cyclin B complex, which is a key molecule for the progression of the cell cycle. In normal cells, dephosphorylation of the Thr-14 and Tyr-15 in Cdc2 triggers G 2 /M transition in the cell cycle. CDT-treated cells were found to maintain Cdc2 with these residues phosphorylated in the Cdc2-cyclin B complex (8). This is because of the recruitment of Cdc25C, a Cdc2-specific phosphatase, from the nucleus to cytoplasm, which prevents dephosphor...