The Role of NLRP7 and KHDC3L in Germline Imprinting and Embryonic Reprogramming

NLRP7 和 KHDC3L 在种系印记和胚胎重编程中的作用

• 批准号: 8814028
• 负责人: IGNATIA B VAN DEN VEYVER
• 金额: $ 34.74万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8814028
• 关键词: Address; Affect; BMP4; Binding; Binding Proteins; Cell Culture Techniques; Cell division; Cell model; Cells; ChIP-seq; Characteristics; Chromatin; Complex; DNA Binding; DNA Methylation; Data; Data Analyses; Defect; Disease; Embryo; Epigenetic Process; Fertilization; Fetus; Gene Expression; Gene Expression Profile; Generations; Genes; Genetic; Genetic Epistasis; Genomic Imprinting; Germ Cells; High-Throughput Nucleotide Sequencing; Human; Hydatidiform Mole; In Vitro; Inherited; Lead; Maintenance; Mass Spectrum Analysis; Massive Parallel Sequencing; Meiosis; Methylation; Mitotic; Mitotic spindle; Modeling; Molecular Profiling; Mono-S; Mus; Mutation; Oocytes; Parents; Pathogenesis; Pathway interactions; Phenotype; Placenta; Pregnancy; Primates; Process; Protein Binding; Proteins; RNA; Recurrence; Regulation; Reproduction; Rodent; Role; SETDB1 gene; Site; Tertiary Protein Structure; Testing; United States National Institutes of Health; Validation; WA09 Cell Line; base; bisulfite sequencing; cell type; demethylation; embryonic stem cell; genome wide methylation; genome-wide; human disease; human embryonic stem cell; imprint; loss of function; loss of function mutation; methylome; mouse model; overexpression; protein complex; protein function; public health relevance; research study; rodent genome; trophoblast

DESCRIPTION (provided by applicant): Maternal effect mutations of NLRP7 or KHDC3L, two genes that are not present in rodents, cause recurrent biparentally inherited molar pregnancies (BiHM) with characteristic absent DNA methylation at imprinted germline differentially-methylated regions (gDMRs) that normally gain methylation in oocytes. This indicates that NLRP7 and KHDC3L are required for the determination of which gDMRs need to have their imprinting marks reprogrammed in the developing oocyte, and/or for the reprogramming process itself. These fundamental aspects of genomic imprinting are still poorly understood, especially in humans and other primates. Because rodents do not have NLRP7 and KHDC3L genes, precluding generation of mouse models, we established a human embryonic stem cell (hESC) model, using the H9 (WA09) line (NIH reg. 0062) to study their function. We discovered that stable NLRP7 knockdown in hESCs changed DNA methylation levels at many CpG sites and augmented BMP4-induced differentiation of hESCs into trophoblast. We also found that NLRP7 binds to KHDC3L, YY1, CTCF and the CpG-binding protein CFP1. Preliminary gene-expression and DNA methylation profiling data in hESC indicate that NLRP7 levels influence expression and methylation of a subset of genes that include epigenetic regulators with known or putative roles in imprinting. Furthermore, NLRP7 and KHDC3L are upregulated in dividing cells where they co-dependently localize to the mitotic spindle, an intriguing discovery considering that reprogramming of imprinting occurs during meiosis, a form of cell division, and needs to be maintained through mitotic cell divisions. These new findings led us to formulate our hypothesis, that NLRP7 and KHDC3L directly and cooperatively act in establishment or maintenance of imprinting marks at maternal gDMRs and that they are critical for recognition of a defined set of gDMRs that need to acquire DNA methylation or maintain it post-fertilization. We propose three specific aims to address this hypothesis. Specific aim 1 is to investigate the cooperative function of KHDC3L with NLRP7 in reprogramming of gDMRs by generating a KHDC3L knockdown hESC model and compare its cellular phenotype to that of the existing NLRP7 knockdown. We will also characterize in detail how NLRP7 and KHDC3L interact, and determine if they can rescue each other's loss. Specific Aim 2 is to identify the protein binding partners of KHDC3L and NLRP7, and how their functions, specifically DNA-binding, are affected by KHDC3L and NLRP7. This will be accomplished by testing candidate interactors, by characterizing the complexes in which these proteins carry out their role in germline DMR repogramming, and by ChIPseq of selected interactors. Specific aim 3 is to perform high-throughput sequencing-based profiling to study how NLRP7 or KHDC3L affect the transcriptome and methylome. We anticipate that the integrated analysis of results from all these experiments will reveal how NLRP7 and KHDC3L affect gDMR reprogramming. This may result in a significant breakthrough in the understanding basic mechanisms of human genomic imprinting.

描述（由申请人提供）：NLRP7 或 KHDC3L（啮齿动物中不存在的两个基因）的母体效应突变会导致复发性双亲遗传性葡萄胎妊娠 (BiHM)，其特征是印记种系差异甲基化区域 (gDMR) 缺乏 DNA 甲基化，而正常情况下，在卵母细胞中获得甲基化。这表明需要 NLRP7 和 KHDC3L 来确定哪些 gDMR 需要在发育中的卵母细胞中重编程其印记标记，和/或重编程过程本身。基因组印记的这些基本方面仍然知之甚少，特别是在人类和其他灵长类动物中。由于啮齿动物没有 NLRP7 和 KHDC3L 基因，无法产生小鼠模型，因此我们建立了人胚胎干细胞 (hESC) 模型，使用 H9 (WA09) 系（NIH reg. 0062）来研究其功能。我们发现 hESC 中稳定的 NLRP7 敲低改变了许多 CpG 位点的 DNA 甲基化水平，并增强了 BMP4 诱导的 hESC 向滋养层的分化。我们还发现 NLRP7 与 KHDC3L、YY1、CTCF 和 CpG 结合蛋白 CFP1 结合。 hESC 中的初步基因表达和 DNA 甲基化分析数据表明，NLRP7 水平影响基因子集的表达和甲基化，其中包括在印记中具有已知或推定作用的表观遗传调节因子。此外，NLRP7 和 KHDC3L 在分裂细胞中上调，它们共同依赖地定位于有丝分裂纺锤体，考虑到印记重编程发生在减数分裂（细胞分裂的一种形式）期间，并且需要通过有丝分裂细胞分裂来维持，这是一个有趣的发现。这些新发现使我们提出了我们的假设，即 NLRP7 和 KHDC3L 直接协同作用，在母体 gDMR 上建立或维持印记标记，并且它们对于识别一组需要获得 DNA 甲基化或维持 DNA 甲基化的定义的 gDMR 至关重要。受精后。我们提出了三个具体目标来解决这一假设。具体目标1是通过生成KHDC3L敲低hESC模型来研究KHDC3L与NLRP7在gDMR重编程中的协同功能，并将其细胞表型与现有NLRP7敲低的细胞表型进行比较。我们还将详细描述 NLRP7 和 KHDC3L 如何相互作用，并确定它们是否可以挽救彼此的损失。具体目标 2 是确定 KHDC3L 和 NLRP7 的蛋白质结合伴侣，以及它们的功能（特别是 DNA 结合）如何受到 KHDC3L 和 NLRP7 的影响。这将通过测试候选相互作用蛋白、表征这些蛋白质在种系 DMR 重编程中发挥作用的复合物以及通过所选相互作用蛋白的 ChIPseq 来完成。具体目标 3 是进行基于高通量测序的分析，以研究 NLRP7 或 KHDC3L 如何影响转录组和甲基化组。我们预计对所有这些实验结果的综合分析将揭示 NLRP7 和 KHDC3L 如何影响 gDMR 重编程。这可能会在理解人类基因组印记的基本机制方面带来重大突破。