ROLE OF MATERNAL PAD16 IN EMBRYONIC DEVELOPMENT

描述（由申请人提供）：从终末分化的卵母细胞到全能合子的转变机制仍然知之甚少。在上一个资助期间，我们从小鼠卵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性基因肽基精氨酸脱亚胺酶6（PADI 6），并发现它代表了将蛋白质精氨酸残基转化为瓜氨酸的酶家族（PADI）的新成员。我们正在进行的PADI 6-/-表型的分析表明，PADI 6-/-女性是不育的，由于在两个细胞阶段的胚胎发生的逮捕，从而证明，PADI 6代表一种新的母体效应基因。在超微结构水平，PADI 6定位于卵和胚胎限制性结构，细胞骨架片（CSS），在许多哺乳动物中占据超过15%的细胞质。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，并在关键的发育时间点进行戏剧性的重组;暗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揭示了PADI 6-/-卵母细胞和卵子中细胞骨架片的完全分散，而大多数其他结构似乎正常。我们还发现，PADI 6-/-两细胞胚胎的转录活性受到严重损害，这表明PADI 6缺陷是由于未能激活胚胎转录而产生的。基于我们的初步研究结果，我们假设，在卵母细胞中，PADI 6通过其N-末端结构域与细胞角蛋白相互作用，导致分散的CSS组分成核。受精后，我们假设PADI 6被激活（最有可能是通过钙信号传导），瓜氨酸通过其C-末端使细胞角蛋白化，导致CSS重组和胚胎基因组激活（EGA）所需的相关蛋白质的阶段依赖性释放。本申请的具体目的是：1）测试PADI 6是CSS组分成核进入卵母细胞中成熟的60 nm复合物所需的假设。2)测试PADI 6在基因组激活之前的早期胚胎中的细胞质到核信号传导事件中起作用的假设。3)验证以下假设：通过PADI 6将瓜氨酸转化为细胞骨架片层蛋白是CSS类糖尿病和早期发育所必需的。

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.