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终过程，最终导致扩大的卵丘复合物（COC）的排卵和黄体（CL）的形成。促性腺激素激增所引发的卵泡成功成熟的一个重要步骤是特定转录因子的表达。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核转录因子的一个小家族，核心结合因子（CBF），作为一个关键的转录调节因子参与排卵期过程。CBF由两个亚基组成；由Runx1、Runx2和Runx3基因之一编码的DNA结合α亚基和非DNA结合β亚基CBF2。为了发挥功能，RUNX蛋白需要与CBF2二聚化。我们的初步数据显示，排卵卵泡中LH激增可以快速诱导CBF成分（Runx1， Runx2和CBF2）。通过体外模型，我们进一步证明了CBFs （RUNX1/CBF2和RUNX2/CBF2）调节对COC扩增、排卵和黄体生成至关重要的排卵期基因的表达。此外，我们的初步研究表明，抑制RUNX活性可阻断体外COC扩增。基于这些新发现，我们假设CBFs是成功的COC扩增、排卵和黄体化所必需的关键转录调节因子。小鼠中CBF成分基因的纯合零突变导致早期死亡，因此不能确定CBF在卵巢中的功能。在体内证明CBFs生理重要性的另一个挑战是Runx1和Runx2的重叠表达及其在排卵卵泡中的功能冗余。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cbf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CBF2来实现。由于CBF2是RUNX1和RUNX2的结合伙伴，靶向删除CBF2将取消所有cbf （RUNX1/CBF2和RUNX2/CBF2）的活性。使用这种转基因小鼠模型，我们将通过检查这种突变小鼠的卵巢表型来检验CBFs的靶向失活导致COC扩增、排卵和黄体生成缺陷的假设（Specific Aim #1）。利用这一突变小鼠卵巢，我们将鉴定排卵周卵泡细胞中CBFs下游的基因，并开始描绘排卵周卵泡发育最后阶段所需的转录调控途径（Specific Aim #2）。这些研究将建立一种转基因动物模型，不仅可以确定CBFs的体内卵巢功能，还可以确定控制排卵期过程的转录调控机制。从这个建议中得到的信息将为COC扩张、排卵和CL形成的机制提供新的见解。全面了解排卵期过程的细胞/分子机制将有助于更好地诊断卵巢病理，并促进这些过程的生理操作，以促进或抑制生育。

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.