ZNF506-dependent positive feedback loop regulates H2AX signaling after DNA damage

ARTICLE DOI: 10.1038/s41467-018-05161-0 OPEN ZNF506-dependent positive feedback loop regulates H2AX signaling after DNA damage 1234567890():,; Somaira Nowsheen 1,2, Khaled Aziz1, Kuntian Luo3, Min Deng3, Bo Qin3, Jian Yuan2,4, Karthik B. Jeganathan5, Jia Yu2, Henan Zhang6, Wei Ding6, Jan M. van Deursen5 & Zhenkun Lou 2,3 Cells respond to cytotoxic DNA double-strand breaks by recruiting repair proteins to the damaged site. Phosphorylation of the histone variant H2AX at S139 and Y142 modulate its interaction with downstream DNA repair proteins and their recruitment to DNA lesions. Here we report ATM-dependent ZNF506 localization to the lesion through MDC1 following DNA damage. ZNF506, in turn, recruits the protein phosphatase EYA, resulting in dephosphorylation of H2AX at Y142, which further facilitates the recruitment of MDC1 and other downstream repair factors. Thus, ZNF506 regulates the early dynamic signaling in the DNA damage response (DDR) pathway and controls progressive downstream signal amplification. Cells lacking ZNF506 or harboring mutations found in cancer patient samples are more sensitive to radiation, offering a potential new therapeutic option for cancers with mutations in this pathway. Taken together, these results demonstrate how the DDR pathway is orchestrated by ZNF506 to maintain genomic integrity. 1 Mayo Clinic Medical Scientist Training Program, Mayo Clinic School of Medicine and Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic, Rochester, MN 55905, USA. 2 Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA. 3 Department of Oncology, Mayo Clinic, Rochester, MN 55905, USA. 4 Research Center for Translational Medicine, Key Laboratory of Arrhythmias of the Ministry of Education of China, East Hospital, Tongji University School of Medicine, Shanghai 200120, China. 5 Department of Pediatrics and Adolescent Medicine, Mayo Clinic, Rochester, MN 55905, USA. 6 Department of Hematology, Mayo Clinic, Rochester, MN 55905, USA. Correspondence and requests for materials should be addressed to Z.L. (email: ) NATURE COMMUNICATIONS | (2018)9:2736 | DOI: 10.1038/s41467-018-05161-0 | www.nature.com/naturecommunications 1 ARTICLE O NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05161-0 ur genome is under constant stress, both from exogenous and endogenous agents that can lead to various forms of DNA damage. Of the various types of DNA damage, DNA double-strand breaks are the most lethal. Since one unrepaired DNA double-strand break can potentially be lethal to the cell, cells have evolved an intricate system called the DNA damage response system to combat this threat and maintain genomic integrity1–3. This response to cytotoxic DNA double-strand breaks involves accrual of DNA repair proteins to the damaged site. This complex response system starts with the protein kinase ATM sensing the damaged DNA and phosphorylating the histone variant H2AX at its serine 139 (Ser139) residue, thereby forming γ-H2AX4. γ-H2AX is required for subsequent interaction with downstream DNA repair proteins5,6. The mediator protein MDC1 interacts with γ-H2AX using its BRCT domain7,8. However, before H2AX can recruit MDC1 via its phosphorylated S139 site, it has to be dephosphorylated at Y142 by the phosphatase EYA5,6. After MDC1 recruitment, MDC1 in turn recruits the E3 ligase RNF8, which ubiquitylates the polycomb group-like protein L3MBTL29. Next, another E3 ligase, RNF168, recognizes the ubiquitylated L3MBTL2 and localizes to the sites of DNA damage9. This protein, RNF168, ubiquitylates lysine residues on histones H2A and H2AX (K13 and K15 residues) and further amplifies the response10. These ubiquitylated histone residues are recognized by DNA repair proteins such as 53BP1 and RAP80, which localize to the damage site to amplify and promote DNA double-strand break repair11–16. As mentioned above, DNA repair is very complex with significant cross-talk between the various pathways. The DNA double-strand break response can be simplified to two major repair pathways: homologous recombination and nonhomologous end joining. Homologous recombination-mediated DNA double-strand break repair is restricted to the S/G2 phase of the cell cycle since it requires the sister chromatid as a template for high-fidelity repair. Among others, BRCA1, BRCA2, and Rad51 are critical for efficient homologous recombinationmediated DNA double-strand break repair17–19. In contrast, non-homologous end-joining-mediated DNA double-strand break repair can occur throughout the cell cycle and is error prone. Key players in this pathway include proteins such as DNAPK/Ku complex, 53BP1, Artemis, and Ligase IV. Regulators such as Rev7, TIRR, UHRF1, and Rif1 dictate the choice of DNA repair pathway20–24. As DNA double-strand break repair pathways are often aberrant in cancers such as leukemia, identifying key regulators of this pathway is important for ascertaining novel therapeutic targets. In order to identify new regulators of this pathway, we analyzed the sequencing data of patients with a rare type of leukemia called T-cell prolymphocytic leukemia (T-PLL) in the Mayo Clinic patient database. We identified ZNF506 as one of the frequently mutated genes in this patient population. To our knowledge, there are no reports on ZNF506 in any biological context; hence, we proceeded to identify the function of this protein. In this study, we reveal that ZNF506 regulates gradual H2AX dephosphorylation at Y142. We report that DNA damageinduced ATM-dependent phosphorylation of ZNF506 localizes it to the DNA damage site through its interaction with MDC1. ZNF506, in turn, facilitates recruitment of the protein phosphatase EYA to the DNA lesion, dephosphorylating H2AX at Y142 and leading to recruitment of MDC1 and other downstream repair factors. In this way, ZNF506 regulates the dynamics at an early point in the DNA damage response pathway and controls progressive downstream signal amplification. Overexpression of ZNF506 confers resistance to radiation. Conversely, cells lacking ZNF506 or harboring mutations found in patient samples are 2 more sensitive to radiation and DNA-damaging agents, offering a potential new therapeutic option for cancers with mutations in this pathway. Collectively, these results identify ZNF506 as a key target of ATM following DNA damage and establish the function of ZNF506 in maintaining genomic integrity through its role in the DNA damage response pathway. Results ZNF506 localizes to DNA lesions in an MDC1-dependent manner. Since the gene was identified in T-PLL, a disease characterized by mutations in DNA double-strand break repair proteins such as ATM25–27, we tested whether ZNF506 plays a role in DNA double-strand break repair. We knocked down ZNF506 in U2OS cells using short hairpin RNAs (shRNAs) and assessed radiation-induced γ-H2AX foci, a marker for DNA double-strand breaks4. As shown in Fig. 1a, b, downregulation of ZNF506 res (...truncated)