Mechanisms of Gastrointestional Growth & Transformation

丁酸盐的基因特异性作用的分子基础仍然不清楚。丁酸酯的主要已知功能涉及抑制组蛋白脱乙酰酶（HDAC），导致乙酰化增加。除了组蛋白乙酰化之外，现在已知DNA结合蛋白被乙酰化。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，丁酸的遗传靶点是结合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合作以介导丁酸对p21 waf 1启动子的作用。然而，Sp1不与p300复合，而是结合HDAC 1。我们以前已经表明，ZBP-89是另一种DNA结合蛋白，结合GC丰富的网站和介导丁酸诱导p21 waf 1。了解丁酸盐通过ZBP-89抑制生长的机制是这种竞争性更新的重点。ZBP-89是由794个残基组成的89 kDa Kruppel型锌指蛋白。在过去的资助期间，我们证明了ZBP-89与肿瘤抑制蛋白p53相互作用诱导G1期阻滞。我们最近发现，ZBP-89与肿瘤抑制蛋白共济失调毛细血管扩张症，突变（ATM）在丁酸特异性的方式相互作用。ATM调节DNA损伤后G1和G2期细胞停滞的相关因子。ATM通过p53在Ser 15的磷酸化介导细胞周期停滞。ZBP-89是p53丝氨酸15位磷酸化所必需的。因此，本提案的具体目的是1）剖析ZBP-89与ATM响应丁酸盐的相互作用。2)探讨丁酸盐对ZBP-89激活p53的调节作用。3)探讨p300 HAT激活在ZBP-89调控中的作用机制。4)确定ZBP-89是否具有肿瘤抑制功能。ZBP-89的各个结构域中的定点突变将用于剖析与这些细胞周期调节剂的相互作用。ZBP-89与染色质的相互作用将使用共聚焦显微镜、细胞分级分离和ChIP测定进行研究。通过这种方式，我们将进一步了解丁酸盐抑制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.