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

剖析 Nemp1（一种对哺乳动物生育能力至关重要的核膜蛋白）的功能

• 批准号: 10586929
• 负责人: Helen McNeill
• 金额: $ 58.3万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-15 至 2027-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10586929
• 关键词: 3-Dimensional; Adult; Affect; Affinity Chromatography; Alleles; Aneuploidy; Animals; Appearance; Architecture; Biological Assay; Birth; Cardiovascular Diseases; Cells; Cessation of life; ChIP-seq; Chimeric Proteins; Chromatin; Chromosome Pairing; Chromosome Segregation; Competence; Congenital Abnormality; Coupled; Cultured Cells; DNA Damage; Data; Defect; Development; Diagnosis; Electron Microscopy; Embryonic Development; Female; Female infertility; Female sterility; Fertility; Fertility Disorders; Fishes; Gene Expression; Genes; Genetic; Genetic Transcription; Genetic study; Germ Cells; Goals; Grant; Health; Hi-C; Histones; Human; Human Genome; Image; Infertility; Integral Membrane Protein; Link; Mammals; Mass Spectrum Analysis; Meiosis; Membrane Proteins; Menopause; Mothers; Mus; Muscular Atrophy; Mutation; Neurologic Dysfunctions; Nuclear Envelope; Nuclear Structure; Nucleoplasm; Oocytes; Oogenesis; Organism; Osteoporosis; Ovarian Follicle; Ovary; Ovulation; Pathway interactions; Phenotype; Premature Menopause; Proteins; Proteomics; Public Health; Publishing; Rest; Risk; Role; Sterility; Structure; Tail; Testing; Time; Transcript; Transgenic Organisms; Variant; Woman; Women' s Health; cardiovascular disorder risk; early onset; egg; env Gene Products; experimental study; female fertility; fly; gain of function; genome wide association study; granulosa cell; histone modification; in vivo; insight; loss of function; mouse model; mutant; novel; offspring; oocyte quality; ovarian reserve; superresolution microscopy; transcriptome sequencing; zygote

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.

项目总结/摘要 产生高质量的卵母细胞对于女性的生育力和健康以及健康的后代至关重要。核 包膜蛋白1（Envelope membrane protein 1，Nemp 1）是一种位于核内膜的跨膜蛋白， 在许多生物体中，都需要这种蛋白质。小鼠Nemp 1的缺失导致小鼠几乎不育， 女性，与组成原始储备的卵母细胞的损失有关。剩下的卵母细胞 发育能力差，染色体分离、染色质致密化和 完成减数分裂。GWAS研究表明NEMP 1的变异与早期绝经有关， 这表明Nemp 1在生育中的作用对人类是保守的。我们的长期目标是了解 Nemp 1在核膜中发挥作用，促进卵母细胞质量和人类生育能力。在这份补助金中，我们将使用 小鼠模型，以确定为什么Nemp 1的缺失导致卵巢储备减少和发育不良 潜力我们将确定何时以及如何卵巢储备功能丧失，并确定参与卵母细胞 损失我们将进行功能获得和功能丧失的研究，以确定哪些细胞需要Nemp 1。 小鼠卵巢。为了鉴定Nemp 1相互作用蛋白，我们将利用新的小鼠品系， 已经产生，这使我们能够进行亲和纯化耦合质谱从休息和 卵母细胞生长我们的初步研究表明，Nemp 1积累成非常大的，规则的， 在核膜的焦点，揭示了一个新的核膜结构在生长的卵母细胞。蛋白组 分析将揭示定位于这些病灶的其他蛋白质（我们称之为NEC）。超分辨率 显微镜和电子显微镜将确定Nemp 1簇的结构和相邻的核 信封. FRAP分析将阐明Nemp 1簇和相关蛋白的稳定性。我们 初步数据表明，Nemp 1突变卵母细胞的染色质致密化被破坏， 转录被解除管制。我们将使用RNAseq来定义Nemp 1 KO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， 在3D染色质结构中使用Hi-C。我们的初步数据表明，一个密切相关的基因，Nemp 2， 在Nemp 1 KO卵母细胞中上调，因此我们将探索Nemp 2对Nemp 1突变体的贡献 表型我们的初步数据表明，Nemp 1的核质尾可以与染色质相互作用， 培养的细胞：我们将阐明卵母细胞中Nemp 1-染色质相互作用的区域，使用转基因DamID 接近。总之，这些研究将阐明核膜的功能，以及Nemp 1的作用。 支持健康卵母细胞的产生。