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...

S-adenosylmethionine (SAM) is an important metabolite as a methyl-group donor in DNA and histone methylation, tuning regulation of gene expression. Appropriate intracellular SAM levels must be maintained, because methyltransferase reaction rates can be limited by SAM availability. In response to SAM depletion, MAT2A, which encodes a ubiquitous mammalian methionine adenosyltransferase isozyme, was upregulated through mRNA stabilization. SAM-depletion reduced N-methyladenosine (mA) in the 3' UTR of MAT2A. In vitro reactions using recombinant METTL16 revealed multiple, conserved methylation targets in the 3' UTR. Knockdown of METTL16 and the mA reader YTHDC1 abolished SAM-responsive regulation of MAT2A. Mutations of the target adenine sites of METTL16 within the 3' UTR revealed that these mAs were redundantly required for regulation. MAT2A mRNA methylation by METTL16 is read by YTHDC1, and we suggest that this allows cells to monitor and maintain intracellular SAM levels.

While all three coactivators ARA 70 , steroid receptor coactivator 1, and RAC3͞ACTR can enhance androgen receptor (AR) transcriptional activity at 1 nM dihydrotestosterone, we here demonstrate that only ARA 70 can induce AR transcriptional activity >30-fold in the presence of 10 nM 17␤-estradiol (E2), but not diethylstilbestrol. The significance of this newly described E2-induced AR transcriptional activity in DU145 human prostate cancer cells was further strengthened by finding patients with Reifenstein partial-androgen-insensitive syndrome that fail in the E2-AR-ARA 70 pathway. Together, our data suggest, for the first time, testosterone͞dihydrotestosterone may not be the only ligands for the AR. E2 represents another important natural ligand for AR that may play an essential role for the AR function and the development of the male reproductive system.

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.

The Mre11/Rad50/Xrs2 (MRX) complex is involved in DNA damage repair, DNA damage response, telomere control, and meiotic recombination. Here, we constructed and characterized novel mutant alleles of XRS2. The alleles with mutations in the C-terminal conserved domain of Xrs2 were grouped into the same class. Mutant Xrs2 in this class lacked Mre11 interaction ability. The second class, lacking a C-terminal end, showed defects only in telomere control. A previous study showed that this C-terminal end contains a Tel1-association domain. These results indicate that Xrs2 contains two functional domains, Mre11-and Tel1-binding domains. While the Mre11-binding domain is essential for Xrs2 function, the Tel1-binding domain may be essential only for Tel1 function in telomere maintenance. The third class, despite containing a large deletion in the N-terminal region, showed no defects in DNA damage repair. However, some mutants, which showed a reduced level of Xrs2 protein, were partially defective in formation of meiotic DSBs and telomere maintenance. These defects were suppressed by overexpression of the mutant Xrs2 protein. This result suggests that the total amount of Xrs2 protein is a critical determinant for the function of the MRX complex especially with regard to telomere maintenance and meiotic DSB formation.