Recruitment of ataxia-telangiectasia mutated to the p21(waf1) promoter by ZBP-89 plays a role in mucosal protection.
OBJECTIVE
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.
Bai
Longchuan
L
Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA.
Kao
John Y
JY
Law
David J
DJ
Merchant
Juanita L
JL
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D003092:Colitis / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D016264:Dextran Sulfate / Q000633:toxicity; D015870:Gene Expression; D006655:Histone Deacetylases / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012313:RNA / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C110951:ZNF148 protein, human; D012313:RNA; D016264:Dextran Sulfate; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases; D006655:Histone Deacetylases