Mechanisms of Gastrointestional Growth & Transformation

丁酸盐基因特异性作用的分子基础仍不明确。丁酸盐已知的主要功能包括抑制组蛋白去乙酰化酶（hdac），导致乙酰化增加。除了组蛋白乙酰化，现在已知DNA结合蛋白也会乙酰化。乙酰化转录因子的功能各不相同，包括增加或减少DNA结合以及蛋白质稳定性。在许多情况下，丁酸盐的遗传靶标是结合Sp1和Sp3的富含gc的序列。组蛋白乙酰转移酶（HAT） p300的募集与Sp1和Sp3协同介导丁酸盐对p21waf1启动子的影响。但是Sp1并不与p300复合，而是与HDAC1结合。我们之前已经证明ZBP-89是另一种DNA结合蛋白，可以结合富含gc的位点并介导丁酸诱导p21waf1。了解丁酸盐通过ZBP-89抑制生长的机制是这一竞争更新的重点。ZBP-89是由794个残基组成的89 kDa的kruppel型锌指蛋白。在过去的资助期内，我们证明了ZBP-89与肿瘤抑制蛋白p53相互作用诱导G1阻滞。我们最近发现ZBP-89以丁酸盐特异性的方式与肿瘤抑制蛋白ataxia毛细血管扩张突变（ATM）相互作用。ATM可调节DNA损伤后参与G1和G2细胞阻滞的因子。ATM通过p53的Ser15位点磷酸化介导细胞周期阻滞。ZBP-89是p53丝氨酸15位点磷酸化所必需的。因此，本提案的具体目的是：1)分析ZBP-89与ATM对丁酸盐的相互作用。2)分析ZBP-89对丁酸盐对p53激活的调控作用。3)探讨p300 HAT活化调控ZBP-89的机制。4)确定ZBP-89是否具有抑瘤功能。ZBP-89不同结构域的位点直接突变将用于剖析与这些细胞周期调节因子的相互作用。ZBP-89与染色质的相互作用将通过共聚焦显微镜、细胞分离和ChIP分析进行研究。通过这种方式，我们将进一步了解丁酸抑制hdac最终如何抑制细胞生长并防止肿瘤转化。

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.