TRIP12 and UBR5 suppress spreading of chromatin ubiquitylation at damaged chromosomes.

Histone ubiquitylation is a prominent response to DNA double-strand breaks (DSBs), but how these modifications are confined to DNA lesions is not understood. Here, we show that TRIP12 and UBR5, two HECT domain ubiquitin E3 ligases, control accumulation of RNF168, a rate-limiting component of a pathway that ubiquitylates histones after DNA breakage. We find that RNF168 can be saturated by increasing amounts of DSBs. Depletion of TRIP12 and UBR5 allows accumulation of RNF168 to supraphysiological levels, followed by massive spreading of ubiquitin conjugates and hyperaccumulation of ubiquitin-regulated genome caretakers such as 53BP1 and BRCA1. Thus, regulatory and proteolytic ubiquitylations are wired in a self-limiting circuit that promotes histone ubiquitylation near the DNA lesions but at the same time counteracts its excessive spreading to undamaged chromosomes. We provide evidence that this mechanism is vital for the homeostasis of ubiquitin-controlled events after DNA breakage and can be subverted during tumorigenesis.

Micronucleus frequency in Danish schoolchildren and their mothers from the DEMOCOPHES population.

Micronucleus (MN) frequency is a biomarker for early genetic effects which is often used in human biomonitoring studies. Increased frequency of micronuclei has been associated with high levels of traffic exposure. Further high MN frequency was found predictive for cancer development in several studies of adults. In the present study, the MN frequency in blood samples from the Danish participants of the European pilot project DEMOCOPHES was analysed and related to the area of residence, self-reported and calculated exposure to road traffic as well as to mercury in hair and blood concentrations of persistent organic pollutants and dioxin-like activity measured in the same participants. The MN frequency analysis was performed with the cytokinesis-block micronucleus (CBMN) assay and included 100 children and 119 mothers. We found a significant correlation between mothers and children in the levels of micronuclei in 1000 binucleated T lymphocytes (‰MNBN) and in the proliferation index. Further the levels of ‰MNBN were significantly higher in mothers compared with their children. No significant associations were found for ‰MNBN for traffic related exposure in neither children nor their mothers. In children, a 2.5 times higher micronuclei in mononuclear T lymphocytes were found in children living within 50 m of a busy road, however, this was not found in mothers or in MNBN and the effect of exposure to road traffic on MN frequency needs further investigation. No significant associations were found between MN frequencies and the other biomarkers measured in the same participants.

Positive Predictive Value of Non-Traumatic Bleeding Diagnoses in the Danish National Patient Register.

Purpose: The majority of bleeding diagnoses in the Danish National Patient Registry have not been validated despite extensive use in epidemiological research. Therefore, we examined the positive predictive value (PPV) of non-traumatic bleeding diagnoses in the Danish National Patient Registry. Study Design: Population-based validation study. Patients and Methods: Based on a manual review of electronic medical records, we estimated the PPV of diagnostic coding (International Classification of Diseases, Tenth Revision (ICD-10)) for non-traumatic bleeding for all patients ≥65 years of age with any hospital contact in the North Denmark Region during March-December 2019 as registered in the Danish National Patient Registry. We calculated PPVs and associated 95% confidence intervals (CI) for non-traumatic bleeding diagnoses overall and stratified according to primary or secondary diagnosis, and according to major anatomical sites. Results: A total of 907 electronic medical records were available for review. The population mean age was 79.33 years (standard deviation (SD)=7.73) and 57.6% were males. Primary bleeding diagnoses accounted for 766 of the records and 141 were secondary bleeding diagnoses. The overall PPV for bleeding diagnoses was 94.0% (95% CI: 92.3-95.4). The PPV was 98.7% (95% CI: 97.6-99.3) for the primary diagnoses and 68.8% (95% CI: 60.7-75.9) for the secondary diagnoses. When stratified according to subgroups of major anatomical sites, the PPVs ranged between 94.1% and 100% for the primary diagnoses, and between 53.8% and 100% for secondary diagnoses. Conclusion: The overall validity of non-traumatic bleeding diagnoses in the Danish National Patient Registry is high and considered acceptable for epidemiological research. However, PPVs were substantially higher for primary than for secondary diagnosis.

Human RNF169 is a negative regulator of the ubiquitin-dependent response to DNA double-strand breaks.

Nonproteolytic ubiquitylation of chromatin surrounding deoxyribonucleic acid double-strand breaks (DSBs), mediated by the RNF8/RNF168 ubiquitin ligases, plays a key role in recruiting repair factors, including 53BP1 and BRCA1, to reestablish genome integrity. In this paper, we show that human RNF169, an uncharacterized E3 ubiquitin ligase paralogous to RNF168, accumulated in DSB repair foci through recognition of RNF168-catalyzed ubiquitylation products by its motif interacting with ubiquitin domain. Unexpectedly, RNF169 was dispensable for chromatin ubiquitylation and ubiquitin-dependent accumulation of repair factors at DSB sites. Instead, RNF169 functionally competed with 53BP1 and RAP80-BRCA1 for association with RNF168-modified chromatin independent of its catalytic activity, limiting the magnitude of their recruitment to DSB sites. By delaying accumulation of 53BP1 and RAP80 at damaged chromatin, RNF169 stimulated homologous recombination and restrained nonhomologous end joining, affecting cell survival after DSB infliction. Our results show that RNF169 functions in a noncanonical fashion to harness RNF168-mediated protein recruitment to DSB-containing chromatin, thereby contributing to regulation of DSB repair pathway utilization.

Epidermal growth factor receptor ligands as new extracellular targets for the metastasis-promoting S100A4 protein.

The function of S100A4, a member of the calcium-binding S100 protein family, has been associated with tumor invasion and metastasis. Although an essential pro-metastatic role of extracellular S100A4 in tumor progression has been demonstrated, the identification of the precise underlying mechanisms and protein partners (receptors) has remained elusive. To identify putative targets for extracellular S100A4, we screened a phage display peptide library using S100A4 as bait. We identified three independent peptide motifs with varying affinities for the S100A4 protein. Sequence analyses indicated that the most abundant peptide mimicked the F/YCC motif present in the epidermal growth factor domain of ErbB receptor ligands. S100A4 selectively interacted with a number of epidermal growth factor receptor (EGFR) ligands, demonstrating highest affinity for amphiregulin. Importantly, we found that S100A4 stimulated EGFR/ErbB2 receptor signaling and enhanced the amphiregulin-mediated proliferation of mouse embryonic fibroblasts. S100A4-neutralizing antibodies, as well as EGFR- and ErbB2 receptor-specific tyrosine kinase inhibitors, blocked these effects. The present results suggest that extracellular S100A4 regulates tumor progression by interacting with EGFR ligands, thereby enhancing EGFR/ErbB2 receptor signaling and cell proliferation. Structured digital abstract: * MINT-7256556: EGF (uniprotkb:P01133) binds (MI:0407) to S100A4 (uniprotkb:P26447) by far western blotting (MI:0047) * MINT-7256512: BC (uniprotkb:P35070) binds (MI:0407) to S100A4 (uniprotkb:P26447) by far western blotting (MI:0047) * MINT-7256485, MINT-7256618, MINT-7256636: AR (uniprotkb:P15514) binds (MI:0407) to S100A4 (uniprotkb:P26447) by far western blotting (MI:0047) * MINT-7256494: HB-EGF (uniprotkb:Q99075) binds (MI:0407) to S100A4 (uniprotkb:P26447) by far western blotting (MI:0047) * MINT-7256502: P53 (uniprotkb:P04637) binds (MI:0407) to S100A4 (uniprotkb:P26447) by far western blotting (MI:0047) * MINT-7256654: S100A2 (uniprotkb:P29034) binds (MI:0407) to AR (uniprotkb:P15514) by far western blotting (MI:0047) * MINT-7256693: S100A5 (uniprotkb:P33763) binds (MI:0407) to AR (uniprotkb:P15514) by far western blotting (MI:0047) * MINT-7256593: S100A4 (uniprotkb:P26447) binds (MI:0407) to BC (uniprotkb:P35070) by pull down (MI:0096) * MINT-7256567: S100A4 (uniprotkb:P26447) binds (MI:0407) to AR (uniprotkb:P15514) by pull down (MI:0096).