ROLE OF MATERNAL PAD16 IN EMBRYONIC DEVELOPMENT

描述（由申请人提供）：从最终分化的卵母细胞到全能受精卵的转变机制尚不清楚。在之前的资助期间，我们从小鼠卵子蛋白质组中克隆并鉴定了一个高度丰富的卵母细胞和胚胎限制基因，肽精氨酸脱亚胺酶6 (PADI6)，并发现它代表了一个酶家族（PADI）的新成员，该酶家族将蛋白精氨酸残基转化为瓜氨酸。我们对PADI6 -/-表型的持续分析表明，PADI6 -/-雌性由于在双细胞阶段胚胎发育停止而不育，从而表明PADI6代表了一种新的母体效应基因。在超微结构水平上，PADI6定位于卵和胚胎限制结构，即细胞骨架片（CSS），在许多哺乳动物中占细胞质的15%以上。虽然这种含有细胞角蛋白的结构的功能尚不清楚，但CSS在卵母细胞生长过程中形成，并在关键发育时间点经历戏剧性的重组；提示在胚胎重编程中的作用。引人注目的是，初步的超微结构分析显示PADI6 -/-卵母细胞和卵子的细胞骨架片完全分散，而大多数其他结构看起来正常。我们还发现PADI6 -/-双细胞胚胎的转录活性严重受损，这表明PADI6缺陷是由于未能激活胚胎转录而产生的。基于我们的初步发现，我们假设，在卵母细胞中，PADI6通过其n端结构域与细胞角蛋白相互作用，导致分散的CSS成分成核。在受精后，我们假设PADI6被激活（很可能是通过钙信号），并通过其c端使瓜氨酸化细胞角蛋白，导致CSS重组和胚胎基因组激活（EGA）所需的相关蛋白的阶段依赖性释放。本应用程序的具体目的是：1)测试假设，CSS成分成核需要PADI6进入卵母细胞内成熟的60 nm复合物。2)验证PADI6在基因组激活前早期胚胎细胞质-核信号事件中发挥作用的假设。3)验证细胞骨架片蛋白被PADI6瓜氨酸化是CSS吻合和早期发育所必需的假设。

DESCRIPTION (provided by applicant): The mechanisms governing the transition from a terminally differentiated oocyte to a totipotent zygote remain poorly understood. During the previous funding period, we cloned and characterized a highly-abundant oocyte and embryo-restricted gene, peptidylarginine deiminase 6 (PADI6), from the murine egg proteome and found that it represents a novel member of an enzyme family (PADI) that converts protein arginine residues to citrulline. Our ongoing analysis of the PADI6 -/- phenotype indicates that PADI6 -/- females are infertile due to an arrest in embryogenesis at the two-cell stage, thus demonstrating that PADI6 represents a novel maternal effect gene. At the ultrastructural level, PADI6 localizes to an egg and embryo-restricted structure, the cytoskeletal sheets (CSS), that occupies over 15% of the cytoplasm in many mammals. While the function of this cytokeratin-containing structure is unknown, the CSS form during oocyte growth and undergo dramatic reorganizations at critical developmental time points; suggestive of a role in embryonic reprogramming. Strikingly, preliminary ultrastructural analysis reveals a complete dispersal of the cytoskeletal sheets in PADI6 -/- oocytes and eggs while most other structures appear normal. We also found that transcriptional activity in PADI6 -/- two-cell embryos is severely compromised, suggesting that the PADI6 defect arises due to failure to activate embryonic transcription. Based on our preliminary findings, we hypothesize that, in the oocyte, PADI6 interacts with cytokeratin via its N-terminal domain leading to nucleation of the dispersed CSS components. Following fertilization, we hypothesize that PADI6 becomes activated (most likely by calcium signaling) and citrullinates cytokeratin via its C-terminus, leading to CSS reorganization and a stage-dependent release of associated proteins which are required for embryonic genome activation (EGA). The specific aims of this application are to: 1) Test the hypothesis that PADI6 is required for nucleation of CSS components into the mature 60 nm complex in the oocyte. 2) Test the hypothesis that PADI6 plays a role in cytoplasmic-to-nuclear signaling events in the early embryo prior to genome activation. 3) Test the hypothesis that citrullination of cytoskeletal sheet proteins by PADI6 is required for CSS anastomoses and for early development.