Structural Studies and Functional Regulation of KLF4

描述(由申请人提供)： 我的长期目标是了解KLF4，一种肠道丰富的转录因子，在胃泌素肠道(Gl道)癌症发展中的作用。KLF4在开发治疗癌症，特别是结肠癌的治疗策略方面具有潜在的用途。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岁之前会患上结直肠肿瘤。在分子水平上，体外系统是研究KLF4调控肿瘤发生的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(Aim1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KLF4如何调控转录。这些研究将确定KLF4中负责其正确定位的功能结构域；它与另一种蛋白质Tip60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Aim3中，我们将使用一个小鼠模型来研究KLF4在体内通过抑制Notch信号通路引起杯状细胞化生的调节。在一项动物研究中，KLF4已被证明可以控制结肠中的杯状细胞分化。另一方面，已知杯状细胞的分化受Notch信号通路控制，这增加了KLF4受Notch信号通路控制的可能性。具体地说，我们将使用来自细菌人工染色体(BAC)克隆的KLF4-GFP构造作为转基因来产生转基因小鼠模型(KLF4-GFP)。由于GFP受天然KLF4启动子的控制，该模型将最大限度地减少错误表达的可能性和在使用小构建物作为转基因的转基因研究中看到的插入-定位效应。我们将使用一种伽马分泌酶抑制剂和一个高表达人孕激素的小鼠模型来研究Notch信号通路对KLF4的体内调节，因为在这个模型中观察到杯状细胞数量的增加，从我们的初步数据来看，孕激素上调了KLF4的表达。

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.