Characterization of the role of maternal effect gene Nlrp2 in reproduction

项目总结 人类NLRP7母体突变导致反复葡萄胎妊娠 印记缺陷。其高度同源的邻近基因NLRP2的母体突变导致多个基因座 印迹障碍(MLID)，在后代中表现为Beckwith-Wiedemann综合征。两者都是关联的 母体印记基因的DNA甲基化异常。啮齿动物有Nlrp2基因，但没有Nlrp7基因。我们 假设Nlrp2可能结合了两个人类同系物的功能，并产生了Nlrp2突变小鼠 研究其母体失活导致观察到的后代和胎盘的机制的模型 异常现象。我们发现NLRP2蛋白是皮层下母体复合体(SCMC)的一个新成员，一个 卵母细胞中的细胞质复合体，持续存在于植入前的胚胎中，推测在母体到母体之间的相互作用。 合子转换和合子基因组激活，这是胚胎从依赖状态转换的过程 关于母体提供的转录本和蛋白质来进行自己的转录和翻译。我们发现一个 Nlrp2的母性效应突变扰乱了SCMC，导致Nlrp2缺失的雌性后代越来越少 后代有先天缺陷、生长异常和印记缺陷的窝点。体外培养 这些Nlrp2缺失雌体的胚胎具有更严重的表型，并伴有早期卵裂停滞。这些 数据首次将SCMC与印记重新编程联系起来，并表明Nlrp2缺失的小鼠是一种 这是研究这种相互作用机制的极佳模型。他们支持这个项目的总体目标，以 描述母亲失去NLRP2，一种新的SCMC蛋白，如何改变后代的印记，以及这是如何导致 与一系列生殖表型有关。为了这个目标，对于特定的目标1，我们将描述细胞和 受精卵和着床前胚胎中的发育事件，这些事件是生殖和 印记表型。为了达到特定的目标2，我们将研究 Nlrp2母体失活的生殖表型通过跟踪其他有趣的早期观察， 包括NLRP2和DNA-甲基转移酶1(DNMT1)在植入前胚胎中共定位，以及 Nlrp2缺陷的卵母细胞改变了基因表达谱。对于特定的目标3，我们将在活体中表征 Nlrp2基因缺失导致的胚胎和胎盘的后期发育异常。 这将增加对母体效应突变引起的生殖障碍的了解。所有表型 将得到转录组学、甲基组学和DNMT1结合图谱的补充，以完全整合 分子和表型数据。我们预测，这项工作将发现基因组的重要新原理 在配子发生期间和受精后重新编程，并将阐明SCMC如何影响这一过程。 对人类健康的长期好处将包括更好地了解多位点印记疾病和 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.