The role of Core Binding Factors (CBFs) in the periovulatory process

描述（由申请人提供）：排卵前促性腺激素激增启动卵泡成熟的最后过程，最终排卵为扩展的卵丘-卵母细胞复合体（COC）和黄体（CL）的形成。促性腺激素激增引起卵泡成功成熟的一个重要步骤是特异性转录因子的表达。然而，我们对LH诱导的关键转录因子的身份和调控作用的了解仍然非常有限。我们实验室和其他人最近的研究揭示了一个小家族的核转录因子，核心结合因子（CBF），作为一个关键的转录调控因子参与排卵期的过程。CBF由两个亚基组成：由Runx 1、Runx 2和Runx 3基因之一编码的DNA结合α亚基和非DNA结合β亚基CBF 2。为了发挥功能，RUNX蛋白需要与CBF 2二聚化。我们的初步数据显示，在排卵期卵泡中，LH峰快速诱导CBF组分（Runx 1，Runx 2和CBF 2）。使用体外模型，我们进一步证明了CBF（RUNX 1/CBF 2和RUNX 2/CBF 2）调节排卵期基因的表达，这些基因对COC扩增、排卵和黄体化至关重要。此外，我们的初步研究表明，抑制RUNX活性在体外阻断COC扩增。基于这些新发现，我们假设CBF是成功COC扩增、排卵和黄素化所必需的关键转录调节因子。 小鼠CBF组分基因的纯合无效突变导致早期致死，因此，无法确定CBF在卵巢中的功能。证明CBF在体内的生理重要性的另一个挑战是Runx 1和Runx 2的重叠表达及其在排卵期卵泡中的功能冗余。为了解决这个问题，我们建议建立一种新的转基因小鼠模型，其中CBFs在卵巢细胞中特异性失活。这将通过使用Cre-lox技术删除卵巢细胞中的CBF 2来实现。由于CBF 2是RUNX 1和RUNX 2的结合伴侣，因此CBF 2的靶向缺失消除了所有CBF（RUNX 1/CBF 2和RUNX 2/CBF 2）的活性。使用该转基因小鼠模型，我们将通过检查该突变小鼠的卵巢表型来检验CBF的靶向失活导致有缺陷的COC扩增、排卵和黄体化的假设（具体目标#1）。使用这种突变的小鼠卵巢，我们将确定在排卵期卵泡细胞中CBF的下游基因，并开始描绘排卵期卵泡发育的最后阶段所必需的转录调控途径（具体目标#2）。 这些研究将建立一个转基因动物模型，不仅要确定在体内的卵巢功能的CBFs，而且要确定的转录调控机制，控制排卵期的过程。从这个提议中获得的信息将提供新的见解COC扩张，排卵和CL形成的机制。对排卵期过程的细胞/分子机制的深入了解将有助于更好地诊断卵巢病理学，并有助于对这些过程进行生理学操作，以促进或抑制生育能力。 公共卫生相关性：拟议研究的总体目标是研究LH峰如何诱导成熟卵子从卵巢释放和黄体形成，这是女性生育的重要步骤。目前的建议集中在确定一个小家族的转录因子，核心结合因子（CBF）作为关键LH诱导的介质在这些过程中的功能。这些知识可以应用于促进和抑制卵巢生理学的这些关键方面，从而使我们能够更好地管理生育，不育和卵巢疾病。

DESCRIPTION (provided by applicant): The preovulatory gonadotropin surge initiates the final process of follicular maturation that culminates in ovulation of an expanded cumulus-oocyte complex (COC) and formation of the corpus luteum (CL). One essential step elicited by the gonadotropin surge required for successful follicular maturation is the expression of specific transcription factors. However, our knowledge of the identity and regulatory actions of LH-induced key transcription factors remains very limited. Recent studies from our laboratory and others shed light on a small family of nuclear transcription factor, Core binding factor (CBF), as a key transcriptional regulator involved in periovulatory processes. CBF is composed of two subunits; DNA binding alpha-subunit encoded by one of Runx1, Runx2, and Runx3 genes and non-DNA binding beta-subunit, CBF2. To be functional, RUNX proteins need to be dimerized with CBF2. Our preliminary data showed the rapid induction of CBF components (Runx1, Runx2 and CBF2) by the LH surge in periovulatory follicles. Using an in vitro model, we further demonstrated that CBFs (RUNX1/CBF2 and RUNX2/CBF2) regulate the expression of periovulatory genes that are known to be critical for COC expansion, ovulation, and luteinization. Moreover, our pilot study revealed that inhibition of RUNX activity blocked COC expansion in vitro. Based on these novel findings, we hypothesized that CBFs are key transcriptional regulators necessary for successful COC expansion, ovulation, and luteinization. Homozygous null mutation of genes for CBF components in mice results in early lethality and, consequently, fails to define the function of CBFs in the ovary. The additional challenge in demonstrating the physiological importance of CBFs in vivo is the overlapping expression of Runx1 and Runx2 and their functional redundancy in periovulatory follicles. To circumvent this problem, we propose to establish a novel transgenic mouse model in which CBFs are inactivated specifically in ovarian cells. This will be accomplished by deleting CBF2 in ovarian cells using Cre-lox technology. Since CBF2 is a binding partner for both RUNX1 and RUNX2, targeted deletion of CBF2 abrogates the activity of all CBFs (RUNX1/CBF2 and RUNX2/CBF2). Using this transgenic mouse model, we will test the hypothesis that targeted inactivation of CBFs results in defective COC expansion, ovulation, and luteinization by examining the ovarian phenotype of this mutant mouse (Specific Aim #1). Using this mutant mouse ovary, we will identify the genes downstream of CBFs in periovulatory follicular cells and begin to delineate the transcriptional regulatory pathways necessary for the final stage of periovulatory follicle development (Specific Aim #2). These studies will establish a genetically modified animal model not only to define the in vivo ovarian function of CBFs, but also to identify the transcriptional regulatory machinery that controls the periovulatory process. Information derived from this proposal will provide new insight into the mechanism(s) involved in COC expansion, ovulation, and CL formation. Gaining a thorough understanding of cellular/molecular mechanisms of the periovulatory process will lead to better diagnostic evaluation of ovarian pathology and facilitate the physiological manipulation of these processes to either promote or inhibit fertility. PUBLIC HEALTH RELEVANCE: The overall goal of the proposed study is to investigate how the LH surge induces the release of a mature egg from the ovary and corpus luteum formation, which are essential steps for female fertility. The current proposal focuses on determining the function of a small family of transcription factors, Core binding factors (CBFs) as key LH-induced mediators in these processes. Such knowledge can be applied for promoting and inhibiting these critical facets of ovarian physiology, thereby allowing us to better manage fertility, infertility, and ovarian-based disorders.