The GAR Motif of 53BP1 is Arginine Methylated by PRMT1 and is Necessary for 53BP1 DNA Binding Activity

The p53-binding protein 1 (53BP1) is rapidly recruited to sites of DNA double-strand breaks and forms characteristics nuclear foci, demonstrating its role in the early events of detection, signaling and repair of damaged DNA. 53BP1 contains a glycine arginine rich (GAR) motif of unknown function within its kinetochore binding domain. Herein, we show that the GAR motif of 53BP1 is arginine methylated by protein arginine methyltransferase 1 (PRMT1), the same methyltransferase that methylates MRE11. 53BP1 contains asymmetric dimethylarginines (aDMA) within cells, as detected with methylarginine-specific antibodies. Amino acid substitution of the arginines within the GAR motif of 53BP1 abrogated binding to single and double-stranded DNA, demonstrating that the GAR motif is required for DNA binding activity of 53BP1. Fibroblast cells treated with methylase inhibitors failed to relocalize 53BP1 to sites of DNA damage and formed few ?-H2AX foci, consistent with our previous data that MRE11 fails to relocalize to DNA damage sites in cells treated with methylase inhibitors. Our findings identify the GAR motif as a region required for 53BP1 DNA binding activity and is the site of methylation by PRMT1.     

Methylation of EZH2 by PRMT1 regulates its stability and promotes breast cancer metastasis

Enhancer of zeste homolog 2 (EZH2), a key histone methyltransferase and EMT inducer, is overexpressed in diverse carcinomas, including breast cancer. However, the molecular mechanisms of EZH2 dysregulation in cancers are still largely unknown. Here, we discover that EZH2 is asymmetrically dimethylated at R342 (meR342-EZH2) by PRMT1. meR342-EZH2 was found to inhibit the CDK1-mediated phosphorylation of EZH2 at T345 and T487, thereby attenuating EZH2 ubiquitylation mediated by the E3 ligase TRAF6. We also demonstrate that meR342-EZH2 resulted in a decrease in EZH2 target gene expression, but an increase in breast cancer cell EMT, invasion and metastasis. Moreover, we confirm the positive correlations among PRMT1, meR342-EZH2 and EZH2 expression in the breast cancer tissues. Finally, we report that high expression levels of meR342-EZH2 predict a poor clinical outcome in breast cancer patients. Our findings may provide a novel diagnostic target and promising therapeutic target for breast cancer metastasis.Subject terms: Metastasis, Tumour biomarkers, Ubiquitylation, Gene regulation     

AHNAK controls 53BP1-mediated p53 response by restraining 53BP1 oligomerization and phase separation

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53BP1 is required for class switch recombination

53BP1 participates early in the DNA damage response and is involved in cell cycle checkpoint control. Moreover, the phenotype of mice and cells deficient in 53BP1 suggests a defect in DNA repair (Ward et al., 2003b). Therefore, we asked whether or not 53BP1 would be required for the efficient repair of DNA double strand breaks. Our data indicate that homologous recombination by gene conversion does not depend on 53BP1. Moreover, 53BP1-deficient mice support normal V(D)J recombination, indicating that 53BP1 is not required for "classic" nonhomologous end joining. However, class switch recombination is severely impaired in the absence of 53BP1, suggesting that 53BP1 facilitates DNA end joining in a way that is not required or redundant for the efficient closing of RAG-induced strand breaks. These findings are similar to those observed in mice or cells deficient in the tumor suppressors ATM and H2AX, further suggesting that the functions of ATM, H2AX, and 53BP1 are closely linked.     

The tandem BRCT domain of 53BP1 is not required for its repair function

53BP1 plays an important role in cellular response to DNA damage. It is thought to be the mammalian homologue of budding yeast Rad9 and/or fission yeast Crb2. Rad9/Crb2 are bona fide checkpoint proteins whose activation requires their corresponding C-terminal tandem BRCT (BRCA1 C-terminal) motifs, which mediate their oligomerization and phosphorylation at multiple sites following DNA damage. Here we show that the function of human 53BP1 similarly depends on its oligomerization and phosphorylation at multiple sites but in a BRCT domain-independent manner. Moreover, unlike its proposed yeast counterparts, human 53BP1 only has limited checkpoint functions but rather acts as an adaptor in the repair of DNA double strand breaks. This difference in function may reflect the higher complexity of the DNA damage response network in metazoa including the evolution of other BRCT domain-containing proteins that may have functions redundant or overlapping with those of 53BP1.     

53BP1 is a Positive Regulator of the BRCA1 Promoter

Germline mutations in the BRCA1 tumor suppressor gene contribute to familial breast and ovarian tumor formation. Sporadic breast and ovarian cancer, however, which accounts for more than 90% of total cases and virtually lacks BRCA1 mutations, exhibits reduced expression of the BRCA1 gene. The magnitude of this reduction correlates with disease progression. In this report we have identified an imperfect palindrome sequence for binding of the 53BP1-containing complex, -40TTCCGTGG CAACGGAA-25, within the BRCA1 minimal promoter. Overexpression of 53BP1 activates a luciferase reporter driven by the wild type BRCA1 minimal promoter, but not by the BRCA1 minimal promoter with mutated palindrome sequence. Depletion of endogenous 53BP1 by siRNA suppresses activity of the BRCA1 minimal promoter. In vitro and in vivo DNA-protein interaction studies demonstrate that this palindrome sequence binds to the 53BP1-containing complex. These findings establish a positive regulation of the BRCA1 promoter by 53BP1. Disruption of this regulation in cancer cells may provide a molecular mechanistic basis for sporadic breast and ovarian tumor formation.     

The direct interaction between 53BP1 and MDC1 is required for the recruitment of 53BP1 to sites of damage

The DNA damage response mediators, 53BP1 and MDC1, play a central role in checkpoint activation and DNA repair. Here we establish that human 53BP1 and MDC1 interact directly through the tandem BRCT domain of MDC1 and residues 1288-1409 of 53BP1. Following induction of DNA double strand breaks the interaction is reduced, probably due to competition between gamma-H2AX and 53BP1 for the binding of the tandem BRCT domain of MDC1. Furthermore, the MDC1 binding region of 53BP1 is required for focus formation by 53BP1. During mitosis the interaction between 53BP1 and MDC1 is enhanced. The interaction is augmented in a phospho-dependent manner, and the MDC1 binding region of 53BP1 is phosphorylated in vivo in mitotic cells; therefore, it is probably modulated by cell cycle-regulated kinases. Our results demonstrate that the 53BP1-MDC1 interaction per se is required for the recruitment of 53BP1 to sites of DNA breaks, which is known to be crucial for an efficient activation of the DNA damage response. Moreover, the results presented here suggest that the interaction between 53BP1 and MDC1 plays a role in the regulation of mitosis.     

Deep Protein Methylation Profiling by Combined Chemical and Immunoaffinity Approaches Reveals Novel PRMT1 Targets

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Highlights

• •Enrichment of methyl peptides using two orthogonal techniques.
• •Knockdown of PRMT1 leads to substantial changes in protein arginine “methylome”.
• •Discrimination of ADMA and SDMA using characteristic neutral losses.
• •Identification of PRMT1 targets and substrate scavenged by other PRMTs in the absence of PRMT1 activity.

     

OXR1A,a Coactivator of PRMT5 Regulating Histone Arginine Methylation

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Proapoptotic p53-interacting protein 53BP2 is induced by UV irradiation but suppressed by p53

p53 is an important mediator of the cellular stress response with roles in cell cycle control, DNA repair, and apoptosis. 53BP2, a p53-interacting protein, enhances p53 transactivation, impedes cell cycle progression, and promotes apoptosis through unknown mechanisms. We now demonstrate that endogenous 53BP2 levels increase following UV irradiation induced DNA damage in a p53-independent manner. In contrast, we found that the presence of a wild-type (but not mutant) p53 gene suppressed 53BP2 steady-state levels in cell lines with defined p53 genotypes. Likewise, expression of a tetracycline-regulated wild-type p53 cDNA in p53-null fibroblasts caused a reduction in 53BP2 protein levels. However, 53BP2 levels were not reduced if the tetracycline-regulated p53 cDNA was expressed after UV damage in these cells. This suggests that UV damage activates cellular factors that can relieve the p53-mediated suppression of 53BP2 protein. To address the physiologic significance of 53BP2 induction, we utilized stable cell lines with a ponasterone A-regulated 53BP2 cDNA. Conditional expression of 53BP2 cDNA lowered the apoptotic threshold and decreased clonogenic survival following UV irradiation. Conversely, attenuation of endogenous 53BP2 induction with an antisense oligonucleotide resulted in enhanced clonogenic survival following UV irradiation. These results demonstrate that 53BP2 is a DNA damage-inducible protein that promotes DNA damage-induced apoptosis. Furthermore, 53BP2 expression is highly regulated and involves both p53-dependent and p53-independent mechanisms. Our data provide new insight into 53BP2 function and open new avenues for investigation into the cellular response to genotoxic stress.     

Oligomerization is required for p53 to be efficiently ubiquitinated by MDM2.

Wild-type p53 is degraded in part through the ubiquitin proteolysis pathway. Recent studies indicate that MDM2 can bind p53 and promote its rapid degradation although the molecular basis for this degradation has not been clarified. This report demonstrates that MDM2 can promote the ubiquitination of wild-type p53 and cancer-derived p53 mutants in transiently transfected cells. Deletion mutants that disrupted the oligomerization domain of p53 displayed low binding affinity for MDM2 and were poor substrates for ubiquitination. However, efficient MDM2 binding and ubiquitination were restored when an oligomerization-deficient p53 mutant was fused to the dimerization domain from another protein. These results indicate that oligomerization is required for p53 to efficiently bind and be ubiquitinated by MDM2. p53 ubiquitination was inhibited in cells exposed to UV radiation, and this inhibition coincided with a decrease in MDM2 protein levels and p53.MDM2 complex formation. In contrast, p53 dimerization was unaffected following UV treatment. These results suggest that UV radiation may stabilize p53 by blocking the ubiquitination and degradation of p53 mediated by MDM2.     

Oligomerization of oncoprotein p53

Cellular phosphoprotein p53, which seems to be a multifunctional protein, may be assigned to different structural subclasses. Recently established immortalized or transformed cell lines that overexpress p53 allowed us to perform a detailed analysis of the quaternary structure of p53. By means of sucrose density gradient centrifugation, we found in simian virus 40-transformed cells that overexpress p53, in addition to high-molecular-weight T-p53 complexes, low-molecular-weight forms. The level of T-p53 complexes within simian virus 40-transformed cells seemed to be determined by the intracellular concentration of p53. However, the presence of uncomplexed T antigen and p53 indicated that an appropriate modification of at least one of the two proteins appears to be necessary for complex formation. Using different monoclonal antibodies that distinguish between (i) p53 associated with T antigen or heat shock proteins and (ii) p53 in apparently free form, we found p53 from transformed cells always in high-molecular-weight forms. p53 from normal and immortalized cells, however, was found mainly in low-molecular-weight forms. Pulse-labeling experiments revealed that oligomerization of p53 is a very rapid process. Monomeric forms of p53 which could be detected only by 2 min of pulse-labeling were rapidly converted to stable, high-molecular-weight oligomers. Furthermore, our data indicate a correlation between the occurrence of p53 in high-molecular-weight forms and the transformation state of the cell.     

Impact of Histone H4 Lysine 20 Methylation on 53BP1 Responses to Chromosomal Double Strand Breaks

Recruitment of 53BP1 to chromatin flanking double strand breaks (DSBs) requires γH2AX/MDC1/RNF8-dependent ubiquitination of chromatin and interaction of 53BP1 with histone H4 methylated on lysine 20 (H4K20me). Several histone methyltransferases have been implicated in 53BP1 recruitment, but their quantitative contributions to the 53BP1 response are unclear. We have developed a multi-photon laser (MPL) system to target DSBs to subfemtoliter nuclear volumes and used this to mathematically model DSB response kinetics of MDC1 and of 53BP1. In contrast to MDC1, which revealed first order kinetics, the 53BP1 MPL-DSB response is best fitted by a Gompertz growth function. The 53BP1 MPL response shows the expected dependency on MDC1 and RNF8. We determined the impact of altered H4K20 methylation on 53BP1 MPL response kinetics in mouse embryonic fibroblasts (MEFs) lacking key H4K20 histone methyltransferases. This revealed no major requirement for the known H4K20 dimethylases Suv4-20h1 and Suv4-20h2 in 53BP1 recruitment or DSB repair function, but a key role for the H4K20 monomethylase, PR-SET7. The histone methyltransferase MMSET/WHSC1 has recently been implicated in 53BP1 DSB recruitment. We found that WHSC1 homozygous mutant MEFs reveal an alteration in balance of H4K20 methylation patterns; however, 53BP1 DSB responses in these cells appear normal.     

DSB repair pathway choice is regulated by recruitment of 53BP1 through cell cycle-dependent regulation of Sp1

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