Understanding epigenetic remodeling in primordial germ cells

描述（由申请人提供）：在怀孕期间，三代DNA共存。妈妈的，婴儿的和婴儿的生殖系。在第一个和第二个三个月期间，来自婴儿的祖生殖系细胞（称为原始生殖细胞（PGCs））的DNA的大部分甲基化胞嘧啶被去除。这一行为对于消除在父母配子发生期间和/或在受精后婴儿早期发育期间获得的甲基化错误至关重要。如果胞嘧啶甲基化中的错误没有被去除，异常甲基化的等位基因有在下一代中作为疾病表观等位基因遗传的风险。考虑到环境可以稳定地影响基因组，包括子宫内婴儿PGC的基因组，有必要了解PGC中调节DNA去甲基化的机制，以制定防止疾病表观等位基因在后代中传播的策略。最近，我的小组发现，人类PGCs中的DNA去甲基化在两个阶段进行调节，但全球（第1阶段）和局部（第2阶段）的去甲基化机制尚不清楚。在这个项目中，我们的目标是发现新的细节，在人类PGCs的DNA去甲基化的动态和使用条件性删除小鼠PGCs在体内以及小鼠PGCs的分化，从胚胎干细胞在体外，以解决有关的特定机制，负责在哺乳动物生殖系去甲基化的假设。在目标1中，我们将首次在人类PGC中鉴定在碱基分辨率下胞嘧啶甲基化的动态去除，并解决蛋白质精氨酸甲基转移酶5（Prmt 5）抑制的含有PHD和RING指结构域的泛素样1（Uhrf 1）负责哺乳动物中1期DNA去甲基化的假设。在目标2中，使用小鼠PGC中的条件性缺失，我们将解决Dnmt 1在其主要辅因子Uhrf 1不存在的情况下维持PGC中离散位点的胞嘧啶甲基化的假设。在目标3中，我们转向2期去甲基化，以直接解决以下假设：通过泰特甲基胞嘧啶双加氧酶将5-甲基胞嘧啶转化为5-羟甲基胞嘧啶在PGCs中印迹控制中心的去甲基化中具有功能作用。总之，这项资助的结果将导致对调节生殖系表观遗传的机制的新见解，在未来的工作中，我们的目标将是防止表观等位基因被获得和传播。

DESCRIPTION (provided by applicant): During pregnancy three generations of DNA co-exist. Mum's, baby's and the germ line of baby. During the first and second trimester the majority of methylated cytosines from the DNA of baby's progenitor germ line cells, called primordial germ cells (PGCs) are removed. This act is essential to remove errors in methylation acquired during gametogenesis in the parents, and/or during early development of baby after fertilization. If errors in cytosine methylation are not removed, the abnormally methylated alleles have a risk of being inherited as disease epialleles in the following generation. Given that the environment can stably influence the genome, including the genome of baby's PGCs in utero, there is a need to understand the mechanisms that regulate DNA demethylation in PGCs in order to develop strategies to guard against the transmission of disease epialleles in future generations. Recently my group discovered that DNA demethylation in human PGCs is regulated in two phases however the mechanisms underlying demethylation globally (phase 1) and locally (phase 2) are unclear. In this project we aim to uncover new details on the dynamics of DNA demethylation in human PGCs and use conditional deletions of mouse PGCs in vivo as well as differentiation of mouse PGCs from embryonic stem cells in vitro to address hypotheses regarding the specific mechanisms responsible for demethylation in the mammalian germ line. In aim 1 we will identify the dynamic removal of cytosine methylation at base resolution for the very first time in human PGCs and address the hypothesis that Ubiquitin- like, containing PHD and RING finger domains, 1 (Uhrf1) repression by protein arginine methyltransferase 5 (Prmt5) is responsible for the phase 1 DNA demethylation in mammals. In aim 2 using a conditional deletion in mouse PGCs we will address the hypothesis that Dnmt1 maintains cytosine methylation at discreet loci in PGCs in the absence of its major cofactor Uhrf1. In aim 3, we turn to phase 2 demethylation to directly address the hypothesis that conversion of 5-methylcytosine to 5-hydroxymethylcytosine by Tet methylcytosine dioxygenases has a functional role in the demethylation of imprinting control centers in PGCs. Taken together, results from this grant will lead to new insights into the mechanisms that regulate germ line epigenetic inheritance, and in future work our goal will be to prevent epialleles from being acquired and transmitted.