Dissecting the function of Nemp1, a nuclear envelope protein critical for mammalian fertility

项目摘要/摘要 产生高质量的卵母细胞对女性的生育和健康以及健康的后代至关重要。核子 包膜蛋白1(Nemp1)是一种内核膜的跨膜蛋白。 在广泛的生物体中繁衍是必需的。小鼠Nemp1基因缺失导致近乎不育 雌性，与组成原始储备的卵母细胞的丧失有关。剩下的卵母细胞 发育能力差，染色体分离、染色质紧凑和 减数分裂完成。GWAS的研究表明，NEMP1的变异与早期绝经有关， 这表明Nemp1在生育中的作用对人类是保守的。我们的长期目标是了解 Nemp1在核膜上发挥作用，促进卵母细胞质量和人类生育能力。在这笔赠款中，我们将使用 建立小鼠模型以确定Nemp1基因缺失导致卵巢储备减少和发育不良的原因 潜力。我们将确定卵巢储备何时以及如何丧失，并确定涉及卵母细胞的途径 损失。我们将进行功能获得和功能丧失研究，以确定哪些细胞需要Nemp1 老鼠的卵巢。为了鉴定Nemp1相互作用的蛋白质，我们将利用新的小鼠品系WE 已经产生了，这使得我们可以从静息和 正在生长的卵母细胞。我们的初步研究表明，Nemp1积聚成极大的、规则的 聚焦在核膜上，揭示了正在生长的卵母细胞中一种新的核膜结构。蛋白质组学 分析将揭示定位于这些病灶(我们称之为NECS)的其他蛋白质。超分辨率 显微镜和电子显微镜将定义Nemp1团簇和邻近核的结构 信封。FRAP分析将阐明Nemp1簇和相关蛋白质的稳定性。我们的 初步数据表明，在Nemp1突变的卵母细胞中，染色质紧凑被破坏，并且 转录被解除管制。我们将使用RNAseq来定义Nemp1KO卵母细胞中基因表达的变化。 我们将使用CHIP-SEQ来定义突变卵母细胞中组蛋白修饰的变化，并确定这些变化 在使用Hi-C的3D染色质架构中。我们的初步数据表明，一个密切相关的基因Nemp2 在Nemp1KO卵母细胞中表达上调，因此我们将探讨Nemp2对Nemp1突变体的作用 表型。我们的初步数据表明，Nemp1的核质尾部可以与染色质相互作用 培养细胞：我们将使用转基因DamID阐明卵母细胞中Nemp1-染色质相互作用的区域 接近了。这些研究将共同阐明核膜的功能以及Nemp1的作用 支持创造健康的卵母细胞。

PROJECT SUMMARY/ABSTRACT Generating high quality oocytes is critical for a woman's fertility and health, and for healthy offspring. Nuclear envelope membrane protein 1 (Nemp1) is a transmembrane protein of the inner nuclear envelope that we found is needed for fertility in a wide range of organisms. Loss of Nemp1 in mice leads to near-sterility of females, associated with loss of the oocytes that compose the primordial reserve. The remaining oocytes have poor developmental competence, with defects in chromosome segregation, chromatin compaction and completion of meiosis. GWAS studies show that variants in NEMP1 are associated with early menopause, suggesting the role of Nemp1 in fertility is conserved to humans. Our long term goal is to understand how Nemp1 functions at the nuclear envelope to promote oocyte quality and human fertility. In this grant we will use mouse models to determine why loss of Nemp1 leads to reduced ovarian reserve and poor developmental potential. We will determine when and how the ovarian reserve is lost, and define pathways involved in oocyte loss. We will conduct gain of function and loss-of-function studies, to determine which cells require Nemp1 in the mouse ovary. To identify Nemp1-interacting proteins, we will take advantage of novel mouse lines we have generated, which allow us to conduct affinity-purification coupled mass spectrometry from resting and growing oocytes. Our preliminary studies have revealed that Nemp1 accumulates into extremely large, regular foci at the nuclear envelope, uncovering a novel nuclear envelope structure in growing oocytes. Proteomic analysis will reveal other proteins that localize to these foci (which we term NECs). Super-resolution microscopy and electron microscopy will define the structure of Nemp1 clusters and the adjacent nuclear envelope. FRAP analysis will clarify the stability of the Nemp1 clusters, and associated proteins. Our preliminary data indicate that chromatin compaction is disrupted in Nemp1 mutant oocytes, and that transcription is deregulated. We will use RNAseq to define change in gene expression in Nemp1KO oocytes. We will use ChIP-seq to define changes in histone modifications in mutant oocytes, and determine alterations in 3D chromatin architecture using Hi-C. Our preliminary data indicate that a closely related gene, Nemp2, is upregulated in Nemp1KO oocytes, so we will explore the contribution of Nemp2 to Nemp1 mutant phenotypes. Our preliminary data indicate that the nucleoplasmic tail of Nemp1 can interact with chromatin in cultured cells: we will clarify the regions of Nemp1-chromatin interactions in oocytes, using transgenic DamID approaches. Together these studies will illuminate the function of the nuclear envelope, and the role of Nemp1 in supporting the creation of healthy oocytes.