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（由申请方提供）：NLRP 7或KHDC 3L（啮齿类动物中不存在的两种基因）的母体效应突变导致反复性双亲遗传性葡萄胎妊娠（BiHM），在通常在卵母细胞中获得甲基化的印迹种系差异甲基化区域（gDMR）处具有特征性DNA甲基化缺失。这表明NLRP 7和KHDC 3L是确定哪些gDMR需要在发育中的卵母细胞中重编程其印记标记和/或重编程过程本身所必需的。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。由于啮齿类动物没有NLRP 7和KHDC 3L基因，排除了小鼠模型的产生，我们建立了人胚胎干细胞（hESC）模型，使用H9（WA 09）系（NIH reg.0062）来研究它们的功能。我们发现，稳定的NLRP 7敲低hESC改变了许多CpG位点的DNA甲基化水平，并增强了BMP 4诱导的hESC向滋养层细胞的分化。我们还发现NLRP 7与KHDC 3L、YY 1、CTCF和CpG结合蛋白CFP 1结合。hESC中的初步基因表达和DNA甲基化分析数据表明，NLRP 7水平影响一组基因的表达和甲基化，这些基因包括在印迹中具有已知或推定作用的表观遗传调节因子。此外，NLRP 7和KHDC 3L在分裂细胞中上调，其中它们共同依赖地定位于有丝分裂纺锤体，这是一个有趣的发现，考虑到印记的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。这些新的发现使我们形成了我们的假设，即NLRP 7和KHDC 3L直接合作地在母体gDMR上建立或维持印记标记，并且它们对于识别需要获得DNA甲基化或在受精后维持它的一组定义的gDMR至关重要。我们提出了三个具体的目标来解决这个假设。具体目标1是通过产生KHDC 3L敲低hESC模型并将其细胞表型与现有NLRP 7敲低的细胞表型进行比较，研究KHDC 3L与NLRP 7在gDMR重编程中的协同功能。我们还将详细描述NLRP 7和KHDC 3L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KHDC 3L和NLRP 7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KHDC 3L和NLRP 7的影响。这将通过测试候选相互作用物，通过表征这些蛋白质在生殖系DMR重编程中发挥作用的复合物，以及通过选定相互作用物的ChIPseq来实现。具体目标3是进行基于高通量测序的分析，以研究NLRP 7或KHDC 3L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NLRP 7和KHDC 3L如何影响gDMR重编程。这可能导致人类基因组印记的基本机制的理解上的重大突破。