Understanding epigenetic remodeling in primordial germ cells

描述(申请人提供)：怀孕期间，三代DNA共存。妈妈的，婴儿的和婴儿的胚胎系。在怀孕的第一和第二个三个月，婴儿的祖先生殖细胞DNA中的大多数甲基化胞嘧啶被去除，称为原始生殖细胞(PGCs)。这一行为对于消除父母配子发生期间和/或受精后婴儿早期发育过程中获得的甲基化错误是必不可少的。如果不消除胞嘧啶甲基化的错误，异常甲基化的等位基因有可能在下一代遗传为疾病表观等位基因。鉴于环境可以稳定地影响基因组，包括婴儿宫内PGCs的基因组，有必要了解PGCs中DNA去甲基化的调节机制，以便制定策略，防止疾病表位基因在后代中传播。最近，我的研究小组发现，人PGCs的DNA去甲基化有两个阶段的调控，但其机制尚不清楚，包括整体(阶段1)和局部(阶段2)。在这个项目中，我们旨在揭示人类生殖细胞DNA去甲基化动力学的新细节，并利用体内条件缺失的小鼠PGCs以及体外从胚胎干细胞分化的小鼠PGCs来解决关于哺乳动物生殖系中负责去甲基化的特定机制的假说。在目标1中，我们将首次在人类PGCs中鉴定碱基分辨下胞嘧啶甲基化的动态去除，并解决这样的假设，即包含PHD和环指结构域的泛素样蛋白1(Uhrf1)被蛋白质精氨酸甲基转移酶5(PRMT5)抑制，负责哺乳动物DNA第一阶段的去甲基化。在目标2中，使用小鼠PGCs的条件缺失，我们将解决这样的假设，即Dnmt1在缺少其主要辅因子uhrf1的情况下，在PGCs中的谨慎位点维持胞嘧啶甲基化。在目标3中，我们转向第二阶段去甲基化，以直接解决这一假说，即四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PGC印迹控制中心的去甲基化中具有功能作用。综上所述，这项资助的结果将导致对胚系表观遗传调控机制的新见解，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.