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

描述(申请人提供)：NLRP7或KHDC3L的母系效应突变，这两个基因在啮齿类动物中不存在，会导致反复发生的双亲遗传性磨牙妊娠(BiHM)，其特征是在印记种系差异甲基化区域(GDMR)缺乏DNA甲基化，而这些区域通常会在卵母细胞中获得甲基化。这表明NLRP7和KHDC3L是确定哪些gDMR需要在发育中的卵母细胞中重新编程其印记标记和/或重新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。由于啮齿动物不具有NLRP7和KHDC3L基因，排除了小鼠模型的产生，我们利用H9(WA09)系(NIH reg.0062)来研究它们的功能。我们发现hESCs中稳定的NLRP7基因敲除改变了许多CpG位点的DNA甲基化水平，并增强了BMP4诱导的hESCs向滋养层细胞的分化。我们还发现NLRP7与KHDC3L、YY1、CTCF和CpG结合蛋白CFP1结合。HESC的初步基因表达和DNA甲基化图谱数据表明，NLRP7水平影响一组基因的表达和甲基化，这些基因包括已知或推测在印记中起作用的表观遗传调节因子。此外，NLRP7和KHDC3L在细胞分裂中上调，在那里它们共同依赖于有丝分裂纺锤体，这是一个有趣的发现，考虑到印记的重新编程发生在减数分裂期间，这是一种细胞分裂形式，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NLRP7和KHDC3L直接和协同地在母体gDMR上建立或维护印记标记，并且它们对于识别一组定义的gDMR至关重要，这些gDMR需要在受精后获得DNA甲基化或维持它。我们提出了三个具体目标来解决这一假设。具体目的1是通过建立KHDC3L基因敲除的hESC模型，研究KHDC3L与NLRP7在gDMRS重编程中的协同作用，并将其细胞表型与现有的NLRP7基因敲除的细胞表型进行比较。我们还将详细描述NLRP7和KHDC3L是如何相互作用的，并确定它们是否可以挽救彼此的损失。具体目的2是确定KHDC3L和NLRP7的蛋白质结合伙伴，以及KHDC3L和NLRP7如何影响它们的功能，特别是DNA结合。这将通过测试候选的交互作用因子，通过表征这些蛋白质在胚系DMR重编程序中执行其作用的复合体，以及通过选定的交互作用因子的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NLRP7或KHDC3L如何影响转录组和甲基组。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重新编程。这可能会在理解人类基因组印记的基本机制方面取得重大突破。

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.