Structural Studies and Functional Regulation of KLF4

KLF4的结构研究和功能调控

• 批准号: 7149716
• 负责人: Walden Ai
• 金额: $ 12.95万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7149716
• 关键词: SDS polyacrylamide gel electrophoresis; artificial chromosomes; aspartic endopeptidases; biological models; biological signal transduction; cell differentiation; cell proliferation; chromatin immunoprecipitation; colon neoplasms; enzyme inhibitors; gastrins; gastrointestinal epithelium; gene expression; gene induction /repression; genetic regulation; genetic transcription; goblet cells; laboratory mouse; metaplasia; model design /development; molecular genetics; protein localization; protein protein interaction; protein structure function; transcription factor; transfection

DESCRIPTION (provided by applicant): My long term goal is to understand the role of KLF4, a gut enriched transcriptional factor, in cancer development in the gastrinintestinal tract (Gl tract). KLF4 has potential use in the development of therapeutic strategies to treat cancer, especially colon cancer. Colorectal cancer is one of the most common cancers in Western countries and is the third most common cause of cancer-related deaths in the US. Approximately 50% of individuals develop a colorectal neoplasm by age 70 years. At the molecular level, in vitro systems are an irreplaceable method for studying the mechanism of KLF4 regulates the transcription of genes important for cancer development. Therefore I propose to pursue in-depth studies (Aim1 and Aim 2) using different cancer cell lines and human umbilical vein endothelial cells as in vitro systems to determine how KLF4 regulates transcription. These studies will identify the functional domains in KLF4 that are responsible for its proper localization; its interaction with another protein, Tip60; and its transcriptional activities. In addition, mouse models have been widely used to study human diseases. In Aim3, we will use a mouse model to study the in vivo regulation of KLF4 by Notch signaling pathway, inhibition of which causes goblet cell metaplasia. In an animal study, KLF4 has been shown to control goblet cell differentiation in colon. On the other hand, goblet cell differentiation is known to be controlled by Notch signal pathway, raising the possibility that KLF4 is under the control of Notch signaling pathway. Specifically, we will use a KLF4-GFP construct derived from a bacterial artificial chromosome (BAC) clone as the transgene to generate a transgenic mouse model (KLF4-GFP). Since GFP is under the control of native KLF4 promoter, this model will minimize the likelihood of misexpression and the insertion-position effects that are seen in transgenic studies using a small construct as the transgene. We will study the in vivo regulation of KLF4 by Notch signaling pathway using a gamma-secretase inhibitor and a mouse model overexpressing human progastrin, since in this model an increase of goblet cell number has been observed and KLF4 is upregulated by progastrin from our preliminary data.

描述（由申请人提供）： 我的长期目标是了解KLF 4（一种肠道富集的转录因子）在胃泌素肠道（GI道）癌症发展中的作用。KLF 4在开发治疗癌症，特别是结肠癌的治疗策略中具有潜在的用途。结直肠癌是西方国家最常见的癌症之一，也是美国癌症相关死亡的第三大常见原因。大约50%的人在70岁时发展为结直肠肿瘤。在分子水平上，体外系统是研究KLF 4调节癌症发展重要基因转录机制的不可替代的方法。因此，我建议进行深入的研究（Aim 1和Aim 2），使用不同的癌细胞系和人脐静脉内皮细胞作为体外系统，以确定KLF 4如何调节转录。这些研究将确定KLF 4中负责其正确定位的功能结构域;其与另一种蛋白质Tip 60的相互作用;以及其转录活性。此外，小鼠模型已被广泛用于研究人类疾病。在Aim 3中，我们将使用小鼠模型来研究Notch信号通路对KLF 4的体内调节，抑制Notch信号通路会导致杯状细胞化生。在一项动物研究中，KLF 4已被证明可以控制结肠中的杯状细胞分化。另一方面，已知杯状细胞分化受Notch信号通路控制，提高了KLF 4受Notch信号通路控制的可能性。具体而言，我们将使用来源于细菌人工染色体（BAC）克隆的KLF 4-GFP构建体作为转基因以产生转基因小鼠模型（KLF 4-GFP）。由于GFP是在天然KLF 4启动子的控制下，该模型将最大限度地减少在使用小构建体作为转基因的转基因研究中看到的错误表达和插入位置效应的可能性。我们将使用γ-分泌酶抑制剂和过表达人前胃泌素的小鼠模型研究Notch信号通路对KLF 4的体内调节，因为在该模型中观察到杯状细胞数量增加，并且根据我们的初步数据，KLF 4被前胃泌素上调。