Recruitment of ataxia-telangiectasia mutated to the p21waf1 promoter by ZBP-89 plays a role in mucosal protection
Recruitment of ataxia-telangiectasia mutated to the p21waf1 promoter by ZBP-89 plays a role in mucosal protection
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DOI：
10.1053/j.gastro.2006.06.014
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发表时间：
2006-09-01
期刊：
影响因子：
29.4
通讯作者：
Merchant, Juanita L.
中科院分区：
文献类型：
--
作者：
Bai, Longchuan;Kao, John Y.;Merchant, Juanita L.
背景与目的：组蛋白去乙酰化酶抑制剂（HDACi）可诱导生长停滞、细胞凋亡和分化，特别是在结肠癌细胞中，它们是潜在的化学预防药物。hdac诱导细胞周期蛋白依赖性激酶抑制剂p21（waf1）需要共济失调-毛细血管扩张突变（ATM）。然而，ATM如何参与p21（waf1）基因表达尚未明确。方法：采用免疫共沉淀法和质谱法研究丁酸盐在体内形成的蛋白质复合物。采用染色质免疫沉淀法和体外DNA亲和沉淀法分析p21（waf1）启动子中的DNA元件。采用免疫印迹法和报告基因法分析p21（waf1）的表达。结果：用小干扰rna减少ZBP-89或ATM可阻断hdac诱导的p21（waf1）表达。染色质免疫沉淀和DNA亲和沉淀实验表明，ZBP-89和ATM在HDACi处理下都被募集到p21（waf1）启动子富含gc的DNA元件上。共免疫沉淀显示ATM以hdac依赖的方式与ZBP-89结合。序列缺失表明，ATM与ZBP-89的n端和DNA结合域均有相互作用。此外，我们发现ZBP-89的免疫缺失阻止了ATM在体外募集p21（waf1）启动子。沉默ZBP-89表达可阻断hdaci诱导的ATM（Ser1981）和P53（Ser15）磷酸化。在摄入葡聚糖硫酸钠后，在表达n端截断形式ZBP-89的突变小鼠结肠中未观察到ATM（Ser1981）磷酸化，并与粘膜损伤加剧相关。结论：ZBP-89以丁酸盐依赖的方式与ATM相互作用，在急性粘膜损伤的情况下对结肠内稳态至关重要。
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.