Recruitment of Ataxia-Telangiectasia Mutated to the p21^waf1 Promoter by ZBP-89 Plays a Role in Mucosal Protection

Background & Aims: Histone deacetylase inhibitors (HDACi) induce growth arrest, apoptosis, and differentiation, particularly in colon cancer cells where they are potential chemopreventive agents. HDACi induction of the cyclin-dependent kinase inhibitor p21^waf1 has been shown to require ataxia-telangiectasia mutated (ATM). Nevertheless, how ATM participates in p21^waf1 gene expression has not been defined. Methods: In vivo protein complexes forming in response to butyrate were studied using co-immunoprecipitation and mass spectroscopy. DNA elements in the p21^waf1 promoter were analyzed in vivo by chromatin immunoprecipitation and in vitro DNA affinity precipitation assays. The expression of p21^waf1 was analyzed by immunoblots and reporter assays. Results: Reduction of ZBP-89 or ATM with small interfering RNAs blocked HDACi-induced p21^waf1 expression. Chromatin immunoprecipitation and DNA affinity precipitation assays showed that both ZBP-89 and ATM are recruited to the GC-rich DNA elements of the p21^waf1 promoter with HDACi treatment. Co-immunoprecipitation revealed that ATM associates with ZBP-89 in an HDACi-dependent manner. Serial deletions revealed that ATM interacts with both the N-terminal and DNA binding domains of ZBP-89. Moreover, we found that immunodepletion of ZBP-89 prevented recruitment of ATM to the p21^waf1 promoter in vitro. Silencing of ZBP-89 expression blocked HDACi-induced phosphorylation of ATM^Ser1981 and p53^Ser15. ATM^Ser1981 phosphorylation in the colons of mutant mice expressing an N-terminally truncated form of ZBP-89 was not observed after ingestion of dextran sodium sulfate and correlated with exacerbation of the mucosal injury. Conclusions:ZBP-89 interacts with ATM in a butyrate-dependent manner and is essential for colonic homeostasis in the setting of acute mucosal injury.