Functional Outcomes of Cdk6 and Eya2 Interaction

描述（由申请人提供）：本提案旨在继续研究cdk 6分化功能的原始资助的工作。该领域的最新证据表明cdk 6是多能性的早期靶点，这表明它既促进自我更新又阻止分化，这是多能细胞和癌症干细胞的特征。我们的实验室已经确定了cdk 6和发育重要的转录因子Eya 2之间的一种新的相互作用。我们有证据表明cdk 6抑制Eya 2驱动的转录激活，我们已经开发出一种创新的方法来研究cdk 6使用卵巢癌组织培养细胞作为模型系统。这项工作的目标是研究cdk 6在分化和肿瘤发生中的新功能。对这个提议很重要的一点是，cdk 6癌基因正在成为多能性和自我更新的主要调控因子的早期靶点之一。它已被证明是多能性SOX 2、NANOG和OCT 4的三种主要调节剂的直接靶标（Bonini et al. 1998; Zhang et al. 2009）。因此，对cdk 6和Eya 2之间相互作用的理解可能会影响我们对癌症干细胞和发育生物学的理解。该提议的相关模型系统是卵巢，其中Eya 2在果蝇突变体（Bonini et al. 1998）和卵巢癌细胞系中具有已证实的作用，其中Eya 2升高（Zhang et al. 2009）。Eya 2升高与卵巢癌的不良结局相关（Zhang et al. 2009）。我们设想了一个模型，其中cdk 6和Eya 2相互作用，影响细胞周期和分化的时间，影响发展和转化。这里提出的目的化妆一个试点研究项目，探讨这种新的相互作用的生物相关性。这些目的包括：1）确认卵巢癌细胞系中cdk 6和Eya 2的相互作用。确认cdk 6影响Eya 2的转录激活功能，并确定这种影响需要cdk 6的哪些功能。为了进行带移、ChIP和ChIP-seq研究，以检查Eya 2/Six 4异二聚体的DNA结合和启动子占用。目的2）分离cdk 6突变体，该突变体破坏cdk 6与Eya 2和在酵母双杂交筛选中鉴定的其它蛋白的结合。该遗传筛选将利用cdk 6突变体库来搜索破坏或增强与Eya 2结合的突变体。目的3）通过免疫荧光和激酶/磷酸酶分析等多种方法，研究cdk 6 Eya 2相互作用的功能性后果。确定Eya 2、cdk 6和这些蛋白的突变体的过度表达对卵巢癌细胞的增殖、迁移和肿瘤发生的影响。这里提出的目的解决的假设，cdk 6和Eya 2相互作用，影响细胞增殖和分化的过程中重要的发展和癌症，通过检查的生物学相关性的新的相互作用的cdk 6和Eya 2，这是在我们的实验室确定。 公共卫生相关性：在这项建议中概述的工作的目标是调查的细胞周期调控蛋白，cdk 6和发育重要的转录因子，Eya 2的相互作用。了解这些蛋白质如何影响分化和细胞增殖对癌症干细胞和发育的生物学至关重要，并可能促进我们对卵巢癌的理解。

DESCRIPTION (provided by applicant): This proposal aims to continue the work of the original grant that investigated differentiation functions of cdk6. Recent evidence in the field suggests that cdk6 is an early target of pluripotency, suggesting it both promotes self-renewal and blocks differentiation, characteristics of pluripotent cells and cancer stem cells. Our lab has identified a novel interaction between cdk6 and the developmentally important transcription factor, Eya2. We have evidence that cdk6 squelches Eya2-driven transcriptional activation and we have developed an innovative approach to studying cdk6 using ovarian cancer tissue culture cells as a model system. The goal of the work outlined in this proposal is to investigate novel functions of cdk6 that contribute to the regulation of differentiation and oncogenesis. Important to this proposal is the fact that the cdk6 oncogene is emerging as one of the early targets of the major regulators of pluripotency and self-renewal. It has been shown to be a direct target of the three major regulators of pluripotency SOX2, NANOG and OCT4 (Bonini et al. 1998; Zhang et al. 2009). Thus, an understanding of the interaction between cdk6 and Eya2 could impact our understanding of the biology of both cancer stem cells and development. The relevant model system for this proposal is the ovary, where Eya2 has a demonstrated effect in Drosophila mutants (Bonini et al. 1998) and ovarian cancer cell lines, where Eya2 is elevated (Zhang et al. 2009). Elevated Eya2 has been associated with poor outcomes in ovarian cancer (Zhang et al. 2009). We envision a model in which cdk6 and Eya2 interact to affect the timing of cell cycle and differentiation that impacts development and transformation. Aims proposed here makeup a pilot research project that examines the biological relevance of this novel interaction. These Aims include 1) To confirm the interaction of cdk6 and Eya2 in an ovarian cancer cell line. To confirm that cdk6 influences the transcriptional activation function of Eya2 and determine what functions of cdk6 are required for this affect. To perform bandshift, ChIP and ChIP-seq studies to examine the DNA-binding and promoter occupancy of the Eya2/Six4 heterodimer. Aim 2) To isolate cdk6 mutants that disrupt binding of cdk6 to Eya2 and other proteins identified in a yeast two-hybrid screen. This genetic screen will utilize a pool of cdk6 mutants to search for mutants that disrupt or enhance binding to Eya2. Aim 3) To determine the functional consequences of the cdk6 Eya2 interaction using a variety of assays including immunofluorescence and kinase/phosphatase assays. To determine the effect of over-expression of Eya2, cdk6, and mutant versions of these proteins, on proliferation, migration, and tumorigenesis of ovarian cancer cells. The Aims presented here address the hypothesis that cdk6 and Eya2 interact to influence cell proliferation and differentiation-processes important in development and cancer-by examining the biological relevance of the novel interaction of cdk6 and Eya2, which was identified in our laboratory. PUBLIC HEALTH RELEVANCE: The goal of the work outlined in this proposal is to investigate the interaction of the cell cycle regulatory protein, cdk6 and the developmentally important transcription factor, Eya2. An understanding of how these proteins influence differentiation and cell proliferation is crucial to the biology of cancer stem cells and development and may advance our understanding ovarian cancer.