Mechanisms of Gastrointestional Growth & Transformation

胃肠生长机制

基本信息

批准号：
7895949
负责人：
JUANITA L. MERCHANT
金额：
$ 7.37万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-15 至 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7895949
关键词：
Acetylation Apoptosis Ataxia Telangiectasia Binding Binding Proteins Biological Assay Butyrates Cell Cycle Cell Cycle Arrest Cell Cycle Regulation Pathway Cell Fractionation Cells Chromatin Complex Confocal Microscopy DNA DNA Binding DNA Damage DNA-Binding Proteins EP300 gene Elements Event Exhibits Funding G1 Arrest GC Rich Sequence Gene Targeting Genes Genetic Goals Growth HDAC1 gene Histone Acetylation Histone Deacetylase Histone Deacetylase Inhibitor Histones Malignant neoplasm of gastrointestinal tract Mediating Molecular Mutate Mutation Neoplastic Cell Transformation Nuclear Protein Nuclear Proteins Phosphorylation Protein Binding Protein p53 Proteins Recruitment Activity Regulation Role Site Site-Directed Mutagenesis Spermatogenesis TP53 gene Testing Trichostatin A Tumor Suppressor Proteins Zinc Fingers ataxia telangiectasia mutated protein base cell growth chromatin remodeling gastrointestinal histone acetyltransferase prevent promoter protein protein interaction response transcription factor

项目摘要

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.

丁酸盐的基因特异性作用的分子基础仍然不明确。丁酸盐的主要已知功能涉及抑制组蛋白脱乙酰酶 (HDAC)，从而导致乙酰化增加。除了组蛋白乙酰化之外，现在已知 DNA 结合蛋白也会被乙酰化。乙酰化转录因子的拟议功能各不相同，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 配合，介导丁酸对 p21waf1 启动子的影响。然而，Sp1 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，它结合富含 GC 的位点并介导 p21waf1 的丁酸诱导。了解丁酸盐通过 ZBP-89 抑制生长的机制是这一竞争性更新的重点。 ZBP-89 是一种 89 kDa Kruppel 型锌指蛋白，由 794 个残基组成。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 期停滞。我们最近发现 ZBP-89 以丁酸盐特异性方式与肿瘤抑制蛋白共济失调毛细血管扩张突变 (ATM) 相互作用。 ATM 调节 DNA 损伤后 G1 和 G2 细胞停滞相关的因子。 ATM 通过 p53 Ser15 磷酸化介导细胞周期停滞。 p53 Ser15 磷酸化需要 ZBP-89。因此，该提案的具体目标是 1) 剖析 ZBP-89 与 ATM 响应丁酸盐的相互作用。 2) 剖析 ZBP-89 响应丁酸盐对 p53 激活的调节。 3) 剖析p300 HAT激活调节ZBP-89的机制。 4)确定ZBP-89是否具有抑癌功能。 ZBP-89 各个域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。将使用共聚焦显微镜、细胞分级分离和 ChIP 测定来研究 ZBP-89 与染色质的相互作用。通过这种方式，我们将进一步了解 HDAC 的丁酸盐抑制最终如何抑制细胞生长并防止肿瘤转化。