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的母体效应突变，这是啮齿动物中不存在的两个基因，引起复发性双遗传遗传的摩尔妊娠（BIHM），具有特征性的DNA甲基化，在刻痕的生殖线下具有差异性生殖的特征性DNA甲基化（GDMRS）（GDMRS），它们正式获得了甲基化的甲基化甲基化。这表明NLRP7和KHDC3L是确定GDMR需要在发育中的卵母细胞和/或重新编程过程本身中重编程其印迹标记所需的。基因组烙印的这些基本方面仍然很少了解，尤其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，因此无法使用H9（WA09）线（NIHReg。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，并增加了BMP4诱导的hESC分化为滋养细胞。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水平会影响一个基因子集的表达和甲基化，包括包括表观遗传调节剂，在印记中具有已知或推定的作用。此外，NLRP7和KHDC3L在分裂细胞中被上调，它们共同依赖于有丝分裂的纺锤体，考虑到重新印记的重新编程是在减麻液中发生的，这是一种细胞分裂的形式，并且需要通过有丝分裂的细胞分裂来维持烙印，这是一个有趣的发现。这些新发现使我们提出了假设，即NLRP7和KHDC3L直接且合作地在母体GDMRS建立或维持印迹痕迹方面起作用，并且它们对于识别一组定义的GDMR至关重要，这些GDMR需要以获取DNA甲基化或维持IT后的IT后进行IT后的特征。我们提出了三个特定的目的来解决这一假设。具体目的1是通过生成KHDC3L敲低hESC模型来研究KHDC3L与NLRP7在重编程中的合作功能，并将其细胞表型与现有的NLRP7敲低的相比。我们还将详细描述NLRP7和KHDC3L的相互作用，并确定他们是否可以挽救彼此的损失。具体目的2是鉴定KHDC3L和NLRP7的蛋白结合伙伴，以及它们的功能，特别是DNA结合的功能如何受KHDC3L和NLRP7的影响。这将通过测试候选相互作用者，通过表征这些蛋白质在生殖线DMR重新编程中以及通过选定相互作用者的chipseq中发挥作用的复合物来实现。具体目的3是执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甲基。我们预计所有这些实验的结果的综合分析将揭示NLRP7和KHDC3L如何影响GDMR重新编程。这可能会导致理解人类基因组印记的基本机制的重大突破。