NBS1 deficiency abolished the interaction between UFL1 and MRN complex (Fig.?1g), suggesting NBS1 might be important for UFL1 recruitment to DSB. Article is available as a Supplementary Information file. All data supporting the findings of this study are available from the corresponding author on affordable request. Abstract The ataxia-telangiectasia mutated (ATM) kinase, an upstream kinase of the DNA damage response (DDR), is usually rapidly activated following DNA damage, and phosphorylates its downstream targets to launch DDR signaling. However, the mechanism of ATM activation is still not completely comprehended. Here we report that Rabbit polyclonal to HAtag UFM1 specific ligase 1 (UFL1), an ufmylation E3 ligase, is usually important for ATM activation. UFL1 is usually recruited to double strand breaks by the MRE11/RAD50/NBS1 complex, and monoufmylates histone H4 following DNA damage. Monoufmylated histone H4 is usually important for Suv39h1 and Tip60 recruitment. Furthermore, ATM phosphorylates UFL1 at serine 462, enhancing UFL1 E3 ligase activity and promoting ATM I-191 activation in a positive feedback loop. These findings reveal that ufmylation of histone H4 by UFL1 is an important step for amplification of ATM activation and maintenance of genomic integrity. Introduction When DNA double-strand break (DSB) occurs, rapid DNA damage response (DDR) and DNA repair are required to preserve genome integrity1. The protein kinase ataxia-telangiectasia mutated (ATM) functions as I-191 an apical activator for the whole process, and controls signaling and the DNA repair network2,3. Germline mutations of the gene tend to destabilize ATM protein and cause ataxia-telangiectasia (AT) syndrome/LouisCBar syndrome. AT is usually a rare, neurodegenerative, and autosomal recessive disease-causing severe disability. AT patients display immunodeficiency, radiosensitivity, progressive cerebellar ataxia, and cancer susceptibility and commonly develop neurodegenerative disease, metabolic syndrome and cancer4,5. The MRE11CRAD50CNBS1 (MRN) complex is important for activation of ATM kinase6C9. Activated ATM phosphorylates histone H2AX at Ser139 (H2AX) close to DNA damage sites, and then recruits MDC1, which serves as a platform for binding more MRN complexes and other DNA repair proteins to amplify DDR signaling and promote DNA repair10C15. In addition, ATM activation is also dependent on the acetyltransferase Tip60. Tip60 is recruited to the sites of DNA damage by binding to H3K9me3, and in turn acetylates ATM at lysine 3016 and boosts ATM autophosphorylation and activation16,17. Tip60 itself is phosphorylated by c-Abl, which increases Tip60 activity and reinforces ATM activity18. However, early I-191 chromatin context leading to full ATM activation remains unclear. Post-translational modification is critical for ATM activation. In addition to phosphorylation, acetylation and methylation, ubiquitination is also important for ATM activation. Skp2 mediated NBS1 ubiquitination enhances the interaction between NBS1 and ATM and promotes ATM activation19. CHFR and RNF8 are also found to synergistically regulate histone H2B ubiquitination and chromatin I-191 relaxation, and promote ATM activation20. In ubiquitination reaction, three classes of enzymes work orchestrally to add ubiquitin to the substrate. The first enzyme, E1, consecutively thioesterificates and activates the C terminus of ubiquitin. E1 then transfers ubiquitin to the cysteine side chain in the second enzyme E2. At last, the E2 and E3 enzymes together transfer the ubiquitin (Ub) from the E2 enzyme to the substrate21. Conversely, ubiquitin conjugation could be removed by ubiquitin protease in a reaction called deubiquitination. In addition to ubiquitin, other Ub like polypeptides such as SUMO and NEDD8 could be conjugated to target proteins through E1-E2-E3 catalyzed reactions. Recently, ubiquitin-fold modifier1 (UFM1) C a new ubiquitin-like protein was identified22. UFM1 conjugation system is a ubiquitin-like modification system including UBA5 (E1-like), UFC1 (E2-like), and UFL1 (E3-like)22,23. Thus far, UFL1 is the only known E3 ligase that has been discovered to conjugate UFM1 to its substrates23. Similar to deubiquitination, UFM1 can be removed from I-191 substrates by UFM1-specific proteases (UFSP). Till now, only one functional UFSP protein called UFSP2 has been identified in human24. The ufmylation system is less explored. So far, it has only been discovered in animals and plants and only two substrates have been determined- UFBP1 and ASC122,25. Previous studies have demonstrated the important roles of ufmylation in erythroid development, breast cancer development, and protecting pancreatic beta cells from ER stress-induced apoptosis25C28. Here we reveal a role of ufmylation in the DDR and identify histone H4 as a substrate of the ufmylation system. We find that MRN.
- The structural hallmarks of AD neurodegeneration, neurofibrillary tangles and neuritic plaques, were prominently immunolabeled with Cdc25A antibodies
- Wash the cells once briefly with PBS, then incubate for 15 minutes in blocking solution made up of 4% normal goat serum and 0
- When gated about viable immune cells in the tumor, the annexin V+ single-positive cells are distinct from caspase-3/7+ or apoptotic cells (Figure 1B)
- Miller 1995b)
- Each dot represents one patient, and bars indicate mean with SEM