Using PGCLCs as a model system for early germ cell development to characterize the roles of histone marks and their readers in DNA methylation homeostasis and transcriptional regulation

在基本层面上，表观遗传学领域涉及对DNA上的化学标记以及与DNA相关的蛋白质的研究，这些蛋白质要么促进或抑制DNA编码基因的开启或关闭。其中一种在该领域被广泛研究的标记被称为“甲基化”。我们的兴趣是以小鼠为模型系统，研究DNA甲基化和与DNA相互作用的蛋白质甲基化之间的相互作用，特别是在产生精子和卵子的细胞中。所谓的“原始生殖细胞”，是精子和卵子的前体，在早期胚胎中出现，当时这些表观遗传标记是相当动态的。我们将以胚胎干细胞为起点，以一种模拟这一关键发育时间窗口的新型细胞培养体系为起点，具体研究通过基因工程破坏DNA相关蛋白质上的甲基化，是否会导致目标生殖细胞基因、基因和/或寄生元素的异常打开和关闭，而这些基因通常富含在基因组中的这些标记。这些实验将揭示表观遗传标记在原始生殖细胞发育中的基本作用，以及“跨代遗传”的分子基础，即DNA上的标记或与DNA相关的蛋白质的变化与DNA本身在一代中的序列变化无关，从而导致下一代可遗传的变化。虽然这种可遗传变化的分子基础在很大程度上仍未确定，但它们可能依赖于在配子中建立的表观遗传标记，如这里正在研究的那些。由于这种表观遗传现象的机制研究在活体动物中很难研究，因为这些细胞只存在于少数几个细胞中，因此开发和应用生殖细胞发育的组织培养模型是这一基础研究的关键一步。

At a fundamental level, the field of epigenetics involves the study of chemical marks on DNA and the proteins associated with DNA that either promote or inhibit the turning on or off of genes encoded in DNA. One of these marks, which is widely studied in the field, is called "methylation". Our interest is in studying the interplay between the methylation on DNA and the methylation on proteins that interact with DNA, specifically in the cells that give rise to sperm and egg, using the mouse as a model system. So-called "primordial germ cells", the precursors to sperm and egg, emerge in the early embryo, when these epigenetic marks are quite dynamic. Using embryonic stem cells as a starting point and a novel cell culture system that mimics this essential developmental time window, we will specifically study whether disrupting the methylation on DNA-associated proteins via genetic engineering of the enzymes that deposit this mark, or a related set of proteins that recognize such marks in the cell, leads to the aberrant "turning on" of target germ cell genes, and/or parasitic elements, that are normally enriched for these marks in the genome and "turned off". These experiments will shed light on the fundamental role of epigenetic marks in primordial germ cell development as well as the molecular basis of "transgenerational inheritance", a phenomenon whereby changes in the marks on DNA or the proteins associated with DNA unrelated to changes in the sequence of the DNA itself in one generation lead to heritable alterations in the next generation. While the molecular basis of such heritable changes remain largely uncharacterized, they likely are dependent on epigenetic marks, established in the gametes, such as those under study here. As mechanistic studies of such epigenetic phenomena are difficult to study in living animals, given that these cells are present in only small numbers, development and application of a tissue culture model of germ cell development is an essential step in this line of basic research.