53BP1 Tudor Domain Research Articles

HDGFRP3 interaction with 53BP1 promotes DNA double-strand break repair.

The 53BP1-dependent end-joining pathway plays a critical role in double-strand break (DSB) repair. However, the regulators of 53BP1 in chromatin remain incompletely characterized. In this study, we identified HDGFRP3 (hepatoma-derived growth factor related protein 3) as a 53BP1-interacting protein. The HDGFRP3-53BP1 interaction is mediated by the PWWP domain of HDGFRP3 and the Tudor domain of 53BP1. Importantly, we observed that the HDGFRP3-53BP1 complex co-localizes with 53BP1 or γH2AX at sites of DSB and participates in the response to DNA damage repair. Loss of HDGFRP3 impairs classical non-homologous end-joining repair (NHEJ), curtails the accumulation of 53BP1 at DSB sites, and enhances DNA end-resection. Moreover, the HDGFRP3-53BP1 interaction is required for cNHEJ repair, 53BP1 recruitment at DSB sites, and inhibition of DNA end resection. In addition, loss of HDGFRP3 renders BRCA1-deficient cells resistant to PARP inhibitors by facilitating end-resection in BRCA1 deficient cells. We also found that the interaction of HDGFRP3 with methylated H4K20 was dramatically decreased; in contrast, the 53BP1-methylated H4K20 interaction was increased after ionizing radiation, which is likely regulated by protein phosphorylation and dephosphorylation. Taken together, our data reveal a dynamic 53BP1-methylated H4K20-HDGFRP3 complex that regulates 53BP1 recruitment at DSB sites, providing new insights into our understanding of the regulation of 53BP1-mediated DNA repair pathway.

The nuclear kinesin KIF18B promotes 53BP1-mediated DNA double-strand break repair.

53BP1 is recruited to chromatin in the vicinity of DNA double-strand breaks (DSBs). We identify the nuclear kinesin, KIF18B, as a 53BP1-interacting protein and define its role in 53BP1-mediated DSB repair. KIF18B is a molecular motor protein involved in destabilizing astral microtubules during mitosis. It is primarily nuclear throughout the interphase and is constitutively chromatin bound. Our observations indicate a nuclear function during the interphase for a kinesin previously implicated in mitosis. We identify a central motif in KIF18B, which we term the Tudor-interacting motif (TIM), because of its interaction with the Tudor domain of 53BP1. TIM enhances the interaction between the 53BP1 Tudor domain and dimethylated lysine 20 of histone H4. TIM and the motor function of KIF18B are both required for efficient 53BP1 focal recruitment in response to damage and for fusion of dysfunctional telomeres. Our data suggest a role for KIF18B in efficient 53BP1-mediated end-joining of DSBs.

TIRR inhibits the 53BP1-p53 complex to alter cell-fate programs

53BP1 influences genome stability via two independent mechanisms: (1) regulating DNA double-strand break (DSB) repair and (2) enhancing p53 activity. We discovered a protein, Tudor-interacting repair regulator (TIRR), that associates with the 53BP1 Tudor domain and prevents its recruitment to DSBs. Here, we elucidate how TIRR affects 53BP1 function beyond its recruitment to DSBs and biochemically links the two distinct roles of 53BP1. Loss of TIRR causes an aberrant increase in the gene transactivation function of p53, affecting several p53-mediated cell-fate programs. TIRR inhibits the complex formation between the Tudor domain of 53BP1 and a dimethylated form of p53 (K382me2) that is poised for transcriptional activation of its target genes. TIRR mRNA expression levels negatively correlate with the expression of key p53 target genes in breast and prostate cancers. Further, TIRR loss is selectively not tolerated in p53-proficient tumors. Therefore, we establish that TIRR is an important inhibitor of the 53BP1-p53 complex.

Nudix Hydrolase NUDT16 Regulates 53BP1 Protein by Reversing 53BP1 ADP-Ribosylation.

53BP1 controls two downstream subpathways, one mediated by PTIP and Artemis and the other by RIF1 and MAD2L2/Shieldin, to coordinate DNA repair pathway choices. However, the upstream regulator(s) of 53BP1 function in DNA repair remain unknown. We and others recently reported that TIRR associates with 53BP1 to stabilize it and prevents 53BP1 localization to DNA damage sites by blocking 53BP1 Tudor domain binding to H4K20me2 sites. Here, we report that the Nudix hydrolase NUDT16, a TIRR homolog, regulates 53BP1 stability. We identified a novel posttranslational modification of 53BP1 by ADP-ribosylation that is targeted by a PAR-binding E3 ubiquitin ligase, RNF146, leading to 53BP1 polyubiquitination and degradation. In response to DNA damage, ADP-ribosylated 53BP1 increased significantly, resulting in its ubiquitination and degradation. These data suggest that NUDT16 plays a major role in controlling 53BP1 levels under both normal growth conditions and during DNA damage. Notably, overexpression of a NUDT16 catalytically inactive mutant blocked 53BP1 localization to double-strand breaks because (i) the mutant binding to TIRR increased after IR; (ii) the mutant enhanced 53BP1 Tudor domain binding to TIRR, and (iii) the mutant impaired the interaction of 53BP1 Tudor domain with H4K20me2. Moreover, NUDT16's catalytic hydrolase activity was required for 53BP1 de-ADP-ribosylation, 53BP1 protein stability, and its function in cell survival. In summary, we demonstrate that NUDT16 regulates 53BP1 stability and 53BP1 recruitment at double-strand breaks, providing yet another mechanism of 53BP1 regulation.Significance: This study provides a novel mechanism of 53BP1 regulation by demonstrating that NUDT16 has hydrolase activities that remove ADP-ribosylation of 53BP1 to regulate 53BP1 stability and 53BP1 localization at DSBs.

Computational approaches to studying methylated H4K20 recognition by DNA repair factor 53BP1.

Histone lysine methylation regulates the recruitment of mammalian DNA repair factor 53BP1 to the histone H4 lysine 20 (H4K20), through specific recognition of the tandem Tudor domain of 53BP1. The di- and mono-methylated H4K20 bind to 53BP1 with high affinity, but the non- and tri-methylated H4K20 do not. Here, we develop a new approach to carry out computational study to unravel the binding mechanism of methylated H4K20 by 53BP1 and to compute relative binding affinities of different methylations of H4K20 by 53BP1. First, hot spots in 53BP1 were predicted by computational alanine scanning and aromatic cages formed by W1495, Y1500, Y1502, and Y1523 are found to provide the dominant binding to di- and mono-methylated H4K20 in addition to D1521. Secondly, a de-methylation method is proposed to predict relative binding free energies between 53BP1 and different methylated states of H4K20. Finally, the tri-methylated and non-methylated H4K20/53BP1 complexes are found to be dynamically unstable, explaining the experimental finding that neither can bind to 53BP1. The present work provides an important theoretical basis for our understanding of histone methylations of H4K20 and their recognition mechanism by DNA repair factor 53BP1.

PTEN Methylation by NSD2 Controls Cellular Sensitivity to DNA Damage.

The function of PTEN in the cytoplasm largely depends on its lipid-phosphatase activity, though which it antagonizes the PI3K-AKT oncogenic pathway. However, molecular mechanisms underlying the role of PTEN in the nucleus remain largely elusive. Here, we report that DNA double-strand breaks (DSB) promote PTEN interaction with MDC1 upon ATM-dependent phosphorylation of T/S398-PTEN. Importantly, DNA DSBs enhance NSD2 (MMSET/WHSC1)-mediated dimethylation of PTEN at K349, which is recognized by the tudor domain of 53BP1 to recruit PTEN to DNA-damage sites, governing efficient repair of DSBs partly through dephosphorylation of γH2AX. Of note, inhibiting NSD2-mediated methylation of PTEN, either through expressing methylation-deficient PTEN mutants or through inhibiting NSD2, sensitizes cancer cells to combinatorial treatment with a PI3K inhibitor and DNA-damaging agents in both cell culture and in vivo xenograft models. Therefore, our study provides a novel molecular mechanism for PTEN regulation of DSB repair in a methylation- and protein phosphatase-dependent manner. SIGNIFICANCE: NSD2-mediated dimethylation of PTEN is recognized by the 53BP1 tudor domain to facilitate PTEN recruitment into DNA-damage sites, governing efficient repair of DNA DSBs. Importantly, inhibiting PTEN methylation sensitizes cancer cells to combinatorial treatment with a PI3K inhibitor combined with DNA-damaging agents in both cell culture and in vivo xenograft models.This article is highlighted in the In This Issue feature, p. 1143.

Identification of a fragment-like small molecule ligand for the methyl-lysine binding protein, 53BP1.

Improving our understanding of the role of chromatin regulators in the initiation, development, and suppression of cancer and other devastating diseases is critical, as they are integral players in regulating DNA integrity and gene expression. Developing small molecule inhibitors for this target class with cellular activity is a crucial step toward elucidating their specific functions. We specifically targeted the DNA damage response protein, 53BP1, which uses its tandem tudor domain to recognize histone H4 dimethylated on lysine 20 (H4K20me2), a modification related to double-strand DNA breaks. Through a cross-screening approach, we identified UNC2170 (1) as a micromolar ligand of 53BP1, which demonstrates at least 17-fold selectivity for 53BP1 as compared to other methyl-lysine (Kme) binding proteins tested. Structural studies revealed that the tert-butyl amine of UNC2170 anchors the compound in the methyl-lysine (Kme) binding pocket of 53BP1, making it competitive with endogenous Kme substrates. X-ray crystallography also demonstrated that UNC2170 binds at the interface of two tudor domains of a 53BP1 dimer. Importantly, this compound functions as a 53BP1 antagonist in cellular lysates and shows cellular activity by suppressing class switch recombination, a process which requires a functional 53BP1 tudor domain. These results demonstrate that UNC2170 is a functionally active, fragment-like ligand for 53BP1.

Structural Plasticity of Methyllysine Recognition by the Tandem Tudor Domain of 53BP1

p53 is dynamically regulated through various posttranslational modifications (PTMs), which differentially modulate its function and stability. The dimethylated marks p53K370me2 and p53K382me2 are associated with p53 activation or stabilization and both are recognized by the tandem Tudor domain (TTD) of 53BP1, a p53 cofactor. Here we detail the molecular mechanisms for the recognition of p53K370me2 and p53K382me2 by 53BP1. The solution structures of TTD in complex with the p53K370me2 and p53K382me2 peptides show a remarkable plasticity of 53BP1 in accommodating these diverse dimethyllysine-containing sequences. We demonstrate that dimeric TTDs are capable of interacting with the two PTMs on a single p53K370me2K382me2 peptide, greatly strengthening the 53BP1-p53 interaction. Analysis of binding affinities of TTD toward methylated p53 and histones reveals strong preference of 53BP1 for p53K382me2, H4K20me2, and H3K36me2 and suggests a possible role of multivalent contacts of 53BP1 in p53 targeting to and accumulation at the sites of DNA damage.

Structural Plasticity of Methyllysine Recognition by the Tandem Tudor Domain of 53BP1

Structural Plasticity of Methyllysine Recognition by the Tandem Tudor Domain of 53BP1

Acetylation limits 53BP1 association with damaged chromatin to promote homologous recombination

The pathogenic sequelae of BRCA1 mutation in human and mouse cells are mitigated by concomitant deletion of 53BP1, which binds histone H4 dimethylated at Lys20 (H4K20me2) to promote nonhomologous end-joining, suggesting a balance between BRCA1 and 53BP1 regulates DNA double-strand break (DSB) repair mechanism choice. Here, we document that acetylation is a key determinant of this balance. TIP60 acetyltransferase deficiency reduced BRCA1 at DSB chromatin with commensurate increases in 53BP1, while HDAC inhibition yielded the opposite effect. TIP60 -dependent H4 acetylation diminished 53BP1 binding to H4K20me2 in part through disruption of a salt bridge between H4K16 and Glu1551 in the 53BP1 Tudor domain. Moreover, TIP60 deficiency impaired HR and conferred sensitivity to PARP inhibition in a 53BP1-dependent manner. These findings demonstrate that acetylation in cis to H4K20me2 regulates relative BRCA1 and 53BP1 DSB chromatin occupancy to direct DNA repair mechanism.

Structural Insight into p53 Recognition by the 53BP1 Tandem Tudor Domain

The tumor suppressor p53 and the DNA repair factor 53BP1 (p53 binding protein 1) regulate gene transcription and responses to genotoxic stresses. Upon DNA damage, p53 undergoes dimethylation at Lys382 (p53K382me2), and this posttranslational modification is recognized by 53BP1. The molecular mechanism of nonhistone methyl-lysine mark recognition remains unknown. Here we report a 1. 6-Å-resolution crystal structure of the tandem Tudor domain of human 53BP1 bound to a p53K382me2 peptide. In the complex, dimethylated Lys382 is restrained by a set of hydrophobic and cation–π interactions in a cage formed by four aromatic residues and an aspartate of 53BP1. The signature HKKme2 motif of p53, which defines specificity, is identified through a combination of NMR resonance perturbations, mutagenesis, measurements of binding affinities and docking simulations, and analysis of the crystal structures of 53BP1 bound to p53 peptides containing other dimethyl-lysine marks, p53K370me2 (p53 dimethylated at Lys370) and p53K372me2 (p53 dimethylated at Lys372). Binding of the 53BP1 Tudor domain to p53K382me2 may facilitate p53 accumulation at DNA damage sites and promote DNA repair as suggested by chromatin immunoprecipitation and DNA repair assays. Together, our data detail the molecular mechanism of p53–53BP1 association and provide the basis for deciphering the role of this interaction in the regulation of p53 and 53BP1 functions.

An Oligomerized 53BP1 Tudor Domain Suffices for Recognition of DNA Double-Strand Breaks

53BP1, the vertebrate ortholog of the budding yeast Rad9 and fission yeast Crb2/Rhp9 checkpoint proteins, is recruited rapidly to sites of DNA double-strand breaks (DSBs). A tandem tudor domain in human 53BP1 that recognizes methylated residues in the histone core is necessary, but not sufficient, for efficient recruitment. By analysis of deletion mutants, we identify here additional elements in 53BP1 that facilitate recognition of DNA DSBs. The first element corresponds to an independently folding oligomerization domain. Replacement of this domain with heterologous tetramerization domains preserves the ability of 53BP1 to recognize DNA DSBs. A second element is only about 15 amino acids long and appears to be a C-terminal extension of the tudor domain, rather than an independently functioning domain. Recruitment of 53BP1 to sites of DNA DSBs is facilitated by histone H2AX phosphorylation and ubiquitination. However, none of the 53BP1 domains/elements important for recruitment are known to bind phosphopeptides or ubiquitin, suggesting that histone phosphorylation and ubiquitination regulate 53BP1 recruitment to sites of DNA DSBs indirectly.

Role for 53BP1 Tudor Domain Recognition of p53 Dimethylated at Lysine 382 in DNA Damage Signaling

Modification of histone proteins by lysine methylation is a principal chromatin regulatory mechanism (Shi, Y., and Whetstine, J. R. (2007) Mol. Cell 25, 1-14). Recently, lysine methylation has been shown also to play a role in regulating non-histone proteins, including the tumor suppressor protein p53 (Huang, J., and Berger, S. L. (2008) Curr. Opin. Genet. Dev. 18, 152-158). Here, we identify a novel p53 species that is dimethylated at lysine 382 (p53K382me2) and show that the tandem Tudor domain of the DNA damage response mediator 53BP1 acts as an "effector" for this mark. We demonstrate that the 53BP1 tandem Tudor domain recognizes p53K382me2 with a selectivity relative to several other protein lysine methylation sites and saturation states. p53K382me2 levels increase with DNA damage, and recognition of this modification by 53BP1 facilitates an interaction between p53 and 53BP1. The generation of p53K382me2 promotes the accumulation of p53 protein that occurs upon DNA damage, and this increase in p53 levels requires 53BP1. Taken together, our study identifies a novel p53 modification, demonstrates a new effector function for the 53BP1 tandem Tudor domain, and provides insight into how DNA damage signals are transduced to stabilize p53.