The molecular basis for the gene specific effects of butyrate remains poorly defined. Butyrate's major known function involves inhibition of histone deacetylases (HDACs) resulting in increased acetylation. In addition to histone acetylation, it is now known that DNA binding proteins become acetylated. The proposed function of acetylated transcription factors varies and includes increased or decreased DNA binding as well as protein stability. In many instances, the genetic targets of butyrate are GC-rich sequences that bind Sp1 and Sp3. Recruitment of the histone acetyltransferase (HAT) p300 cooperates with Sp1 and Sp3 to mediate the effects of butyrate to the p21waf1 promoter. However, Sp1 does not complex with p300, but instead binds HDAC1. We have shown previously that ZBP-89 is another DNA binding protein that binds GC-rich sites and mediates butyrate induction of p21waf1. Understanding the mechanisms by which butyrate suppresses growth through ZBP-89 is the focus of this competing renewal. ZBP-89 is an 89 kDa Kruppel-type zinc finger protein composed of 794 residues. During the past funding period, we demonstrated that ZBP-89 interacts with the tumor suppressor protein p53 to induce G1 arrest. We have recently found that ZBP-89 interacts with the tumor suppressor protein ataxia telangiectasia, mutated (ATM) in a butyrate specific manner. ATM modulates factors involved in both G1 and G2 cell arrest after DNA damage. ATM mediates cell cycle arrest through phosphorylation of p53 at Ser15. ZBP-89 is required for phosphorylation of p53 at Ser15. Therefore the specific aims of this proposal are 1) To dissect the interaction of ZBP-89 with ATM in response to butyrate. 2) To dissect the regulation of p53 activation by ZBP-89 in response to butyrate. 3) To dissect the mechanisms of p300 HAT activation in the regulation of ZBP-89. 4) To determine whether ZBP-89 exhibits tumor suppressor function. Site-direct mutations in various domains of ZBP-89 will be used to dissect the interactions with these cell cycle regulators. ZBP-89 interactions with chromatin will be studied using confocal microscopy, cell fractionation and ChIP assays. In this way, we will further the understanding of how butyrate inhibition of HDACs ultimately suppresses cell growth and prevents neoplastic transformation.
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