Characterization of the role of maternal effect gene Nlrp2 in reproduction

项目摘要 人NLRP 7的母体突变导致妊娠复发性葡萄胎妊娠， 印迹缺陷其高度同源的邻近基因NLRP 2的母体突变，导致多位点 在后代中表现为Beckwith-Wiedemann综合征的印迹障碍（MLID）。两者都有关联 母亲印记基因的异常甲基化啮齿动物有Nlrp 2基因，但没有Nlrp 7。我们 假设Nlrp 2可能结合了两种人类同源物的联合收割机功能，并产生了Nlrp 2突变小鼠 模型，以研究其母体失活导致观察到的后代和胎盘 异常我们发现NLRP 2蛋白是皮质下母性复合体（SCMC）的一个新成员， 在植入前胚胎中持续存在的卵母细胞胞质复合物，推测其在母体到 合子转换和合子基因组激活，这是胚胎从依赖转换的过程， 对母体贡献的转录本和蛋白质进行转录和翻译。我们发现一个 Nlrp 2的母源效应突变破坏了SCMC，导致Nlrp 2缺失的雌性产生更少更小的 有出生缺陷、生长异常和印记缺陷的后代。体外培养 这些Nlrp 2缺失雌性的胚胎具有更严重的早期卵裂阶段停滞的表型。这些 数据首次将SCMC与印记重编程联系起来，并表明Nlrp 2缺失小鼠是一种 很好的模型来研究这种相互作用的机制。他们支持这个项目的总体目标， 描述了NLRP 2（一种新的SCMC蛋白）的母体缺失如何改变后代的印记，以及这如何导致 一系列的生殖表型。为了实现这一目标，对于具体目标1，我们将表征细胞和 受精卵和植入前胚胎的发育事件是生殖和胚胎发育的起源， 印记表型对于具体目标2，我们将研究导致细胞凋亡的分子机制。 Nlrp 2的母体失活的生殖表型通过跟踪其他有趣的早期观察， 包括NLRP 2和DNA-甲基转移酶1（DNMT 1）共定位于植入前胚胎， Nlrp 2缺陷的卵母细胞基因表达谱改变。对于具体目标3，我们将在体内表征 由母体缺失Nlrp 2导致的胚胎和胎盘的可变后期发育异常。 这将增加对母体效应突变引起的生殖障碍的认识。所有表型 将通过转录组学、甲基化组学和DNMT 1结合分析来补充， 分子和表型数据。我们预测这项工作将揭示基因组学的重要新原理 在配子发生过程中和受精后的重编程，并将阐明SCMC如何影响这一过程。 对人类健康的长期益处将包括更好地了解多位点印迹障碍， 某些形式的不孕症和体外受精失败。

PROJECT SUMMARY Maternal mutations in human NLRP7 cause pregnancies recurrent hydatidiform molar pregnancies with imprinting defects. Maternal mutations in its highly homologous neighboring gene NLRP2, cause a multi-locus imprinting disorder (MLID) that manifests as Beckwith-Wiedemann syndrome in offspring. Both are associated with abnormal DNA methylation of maternally imprinted genes. Rodents have the Nlrp2 gene, but no Nlrp7. We hypothesized that Nlrp2 may combine functions of both human homologs and generated a Nlrp2-mutant mouse model to study the mechanisms by which its maternal inactivation causes the observed offspring and placental abnormalities. We found that NLRP2 protein is a new member of the subcortical maternal complex (SCMC), a cytoplasmic complex in oocytes that persists in preimplantation embryos with a presumed role in maternal-to- zygote transition and zygotic genome activation, which are the processes by which embryos switch from reliance on maternally contributed transcripts and proteins to their own transcription and translation. We found that a maternal-effect mutation in Nlrp2 disrupts the SCMC, causing Nlrp2-null females to produce fewer and smaller litters with offspring that have birth defects, growth abnormalities, and imprinting defects. In vitro cultured embryos of these Nlrp2-null females have a more severe phenotype with early cleavage-stage arrest. These data for the first time link the SCMC to imprinting reprogramming and indicate that Nlrp2-null mice are an excellent model to study the mechanisms of this interaction. They support the overarching goal of this project, to characterize how maternal loss of NLRP2, a new SCMC protein, alters imprinting in offspring, and how this leads to a range of reproductive phenotypes. Towards this goal, for specific aim 1, we will characterize the cellular and developmental events in the zygote and preimplantation embryo that are at the origin of the reproductive and imprinting phenotypes. For specific aim 2, we will investigate the molecular mechanisms that underlie the reproductive phenotypes of maternal inactivation of Nlrp2 by following up on other interesting early observations, which include that NLRP2 and DNA-methyltransferase 1 (DNMT1) co-localize in preimplantation embryos, and that Nlrp2-deficient oocytes have altered gene expression profiles. For specific aim 3 we will characterize in vivo the variable later developmental abnormalities of embryos and placentas resulting from maternal loss of Nlrp2. This will increase knowledge on the reproductive disorders caused by maternal effect mutations. All phenotyping will be complemented by transcriptomics, methylomics and profiling of DNMT1 binding to fully integrate molecular and phenotypic data. We predict that this work will uncover important new principles of genome reprogramming during gametogenesis and after fertilization and will clarify how the SCMC affects this process. The long-term benefit for human health will include better understanding of multi-locus imprinting disorders and some forms of infertility and in vitro fertilization failures.