Derivation and Characterization of Germ Cells from Embryonic Stem Cells

描述(申请人提供)：这项建议的目标是从联邦批准的两种人类胚胎干细胞(HESCs)系UC01和UC06(HSF-1和HSF-6)中培养生殖细胞，以评估BLIMP1介导的转录抑制和组蛋白H4精氨酸3(H4R3me2)的二甲基化在人类生殖细胞形成中的作用。了解生殖细胞发育的分子调控对于改善人类健康非常重要，因为异常的生殖细胞可能导致：不孕不育，影响美国10%的生殖年龄人口；生殖细胞肿瘤，这是困扰15至35岁男性的最常见的癌症类型；以及出生缺陷，发生在父母生殖系发育异常的孩子中。在这项建议中，我们旨在评估人类胎儿生殖细胞形成初期转录抑制缺陷是否会导致生殖细胞发育受损。转录抑制的作用将从三个具体目标进行评估。具体目标1涉及确定人类胚胎妊娠6-9周的生殖细胞的转录特征。这将通过FACS从人胎儿性腺中分离的表达谱生殖细胞来实现。这一特定目的的结果将被用来进一步阐明通过hESC分化获得的生殖细胞形成的阶段，寻找能够区分hESCs和生殖细胞的其他细胞表面标记，并作为评估转录抑制因子在生殖细胞体外发育中的作用的基础。在特定的目标2中，我们将评估转录抑制因子BLIMP1在调节人类生殖细胞形成中的作用。已知BLIMP1对小鼠模型中生殖细胞的形成是必不可少的。在这个特定的目标中，我们的目标是在hESCs中敲除和过表达BLIMP1，并分析人类生殖细胞的形成。这项实验将确定在转基因对照ESCs中生殖细胞的产量是否会减少。我们还将通过流式细胞仪从分化的人类胚胎干细胞中分离人类生殖细胞，并评估生殖细胞特异性基因以及与体细胞分化相关的基因的转录。作为使用胚胎干细胞研究生殖细胞形成的积极对照和验证，我们将比较我们的结果与野生型和Blimp1缺失突变的小鼠胚胎干细胞在体外形成生殖系的能力。最后，在特定的目标3，将评估PRMT5和组蛋白H4R3me2在人类生殖细胞衍生中的功能，并将利用染色质免疫沉淀(CHIP)和芯片在全基因组水平上鉴定胚胎干细胞和生殖细胞中BLIMP1、PRMT5和组蛋白H4R3的二甲基化与启动子的结合位置。PRMT5是一种鱼精蛋白精氨酸甲基转移酶，它与BLIMP1相互作用，介导组蛋白H4R3的二甲基化，该基因座需要被抑制才能发生生殖细胞形成。具有相同BLIMP1、PRMT5和组蛋白H4R3me2启动子的基因将在未来的提案中进行分析。这一建议是评估对正常人类生殖细胞发育至关重要的其他下游目标的第一步。公共卫生相关性：这项提议的结果对于解开导致人类不孕不育的分子机制将是重要的。

DESCRIPTION (provided by applicant): The objective of this proposal is to generate germ cells from two federally approved lines of human embryonic stem cells (hESCs) called UC01 and UC06 (HSF-1 and HSF-6) in order to evaluate the role of transcriptional repression mediated by BLIMP1 and dimethylation of Histone H4 Arginine 3 (H4R3me2) on human germ cell formation. Understanding the molecular regulation of germ cell development is important to improving human health as abnormal germ cells can result in; infertility, which affects 10% of the reproductive age population in the United States, germ cell tumors, which are the most common cancer type to afflict males between the ages of 15 and 35, and birth defects, which occur in children born to parents with abnormal germ line development. In this proposal, we aim to evaluate whether defects in transcriptional repression during the initial stages of germ cell formation in the human fetus result in compromised germ cell development. The role of transcriptional repression will be evaluated in three Specific Aims. Specific Aim 1 involves determining the transcriptional signature of human germ cells from week 6-9 of human fetal gestation. This will be achieved by expression profiling germ cells isolated from human fetal gonads by FACS. Results from this Specific Aim will be used to further clarify the stage of germ cell formation acquired with hESC differentiation, to identify additional cell surface markers that can distinguish hESCs from germ cells, and as a foundation for evaluating the role of transcriptional repressors in germ cell development in vitro. In Specific Aim 2, we will evaluate the effect of a transcriptional repressor, BLIMP1 for a role in regulating human germ cell formation. It is known that BLIMP1 is essential for germ cell formation in murine models. In this Specific Aim, our goal is to knockdown and over express BLIMP1 in hESCs and assay human germ cell formation. This experiment will determine whether the yield of germ cells is reduced in genetically modified verses control ESCs. We will also FACS and isolate human germ cells from differentiating hESCs and evaluate transcription of germ cell specific genes, as well as genes associated with somatic cell differentiation. As a positive control and validation for the use of ESCs to study germ cell formation, we will compare our results to wild type and Blimp1 null mutant murine ESCs for their ability to form germ line in vitro. Finally in Specific Aim 3, the function of PRMT5 and Histone H4R3me2 in human germ cell derivation will be evaluated, and the binding sites of BLIMP1, PRMT5 and dimethylation of Histone H4R3 at promoters in ESCs and germ cells will be identified at a genome-wide level using Chromatin Immunoprecipitation (ChIP) followed by chip. PRMT5 is a protamine arginine methyltransferase that interacts with BLIMP1 to mediate dimethylation of histone H4R3 at loci that need to be repressed in order for germ cell formation to occur. Genes with promoters that share BLIMP1, PRMT5 and Histone H4R3me2 will be analyzed in future proposals. This proposal constitutes the first step from which additional downstream targets essential for normal human germ cell development can be evaluated. PUBLIC HEALTH RELEVANCE: Results from this proposal will be important for unraveling molecular mechanisms that lead specifically to human infertility.