In Vivo Trapping of Genes Involved in Ovulation

描述（由申请人提供）：排卵是一个精确定时的事件，成熟的卵母细胞从卵泡中释放出来。排卵过程与许多基因的转录调控有关，包括CCAAT/增强子结合蛋白（Cebpb）的上调，CCAAT/增强子结合蛋白是一种核受体转录因子，在排卵期颗粒细胞内。Cebpb在排卵过程中的重要作用已被基因靶向证实，它完全阻断了卵泡破裂。本实验的长期目标是在排卵期颗粒细胞中确定Cebpb调控基因，这是完成排卵过程所必需的。最近，我们的实验室采用了一种称为染色质免疫沉淀（CHIP）的新方法，该方法使我们能够识别染色质结构的区域变化，并将这些变化与基因活性（转录）的增加联系起来。此外，这种方法允许在体内捕获转录因子到多个基因的目标调控元件同时。我们将采用这种新方法来鉴定卵巢颗粒细胞内由Cebpb直接调节的未知基因。这项研究的基本原理是，随着基因的确定，机制将被阐明，以更好地了解排卵过程。PI在生殖生物学实验和分子技术方面具有专业知识，并使用该方法确定参与排卵过程的其他基因。合作者有广泛的背景研究Cebpb转录调控。她还培育出了缺乏Cebpb的小鼠。因此，项目负责人和合作者结合独特的才能，提供必要的资源和技能，以完成当前提案中的研究目标。这些研究将首次确定Cebpb转录因子促进排卵的机制和细胞信号通路。

DESCRIPTION (provided by applicant): Ovulation is a precisely timed event in which a mature oocyte is released from an ovarian follicle. The ovulatory process is associated with the transcriptional regulation of numerous genes, including the upregulation of CCAAT/enhancer binding protein ¿ (Cebpb), a nuclear receptor transcription factor, within the periovulatory granulosa cell. The essential role of Cebpb in the ovulatory process has been demonstrated by gene targeting which completely blocked follicular rupture in nullizygous females. The long-term objective of the experiments outlined in this proposal are to identify the Cebpb regulated gene(s) in periovulatory granulosa cells that are essential for completion of the ovulatory process. Recently, our laboratory utilized a new methodology referred to as chromatin immunoprecipitation (CHIP) that allows us to identify regional changes in chromatin structure and correlate those changes with increased gene activity (transcription). Additionally, this methodology allows the in vivo trapping of transcription factors to their target regulatory elements in multiple genes simultaneously. We will adapt this new methodology to identify unknown genes that are regulated directly by Cebpb within ovarian granulosa cells. The rationale that underlies the proposed research is that as genes are identified mechanisms will be elucidated to gain a better understanding of the process of ovulation. The PI has expertise in reproductive biology experiments and molecular techniques and has used the methodology to identify other genes involved in the process of ovulation. The collaborator has an extensive background examining Cebpb transcriptional regulation. She also has generated mice deficient in Cebpb. Therefore, the PI and collaborator combine unique talents that provide the necessary resources and skills to complete the research goals in the current proposal. These studies will for the first time determine the mechanism and cell signaling pathways by which the Cebpb transcription factor promotes ovulation.