Epigenetic determinants of competence for germ line specification during human embryonic development

项目摘要 候选人我是一名医生和讲师，在生殖内分泌和不孕不育科， 贝斯以色列女执事医疗中心（BIDMC）。K08奖将为我提供必要的培训， 经验，成为生殖细胞（GC）生物学的独立研究者，重点是基因 人类配子发生的调控网络。 背景人类和非人类灵长类动物胚胎中的一小部分细胞 获得形成生殖系的能力，并最终成熟为功能性配子，同时保持其 多能性的潜力是生物学中最不了解的问题之一。特别是，基因调控 网络和表观遗传重塑，这决定了GC的命运，在规范早期 （原始）生殖细胞（PGCs）在原肠胚形成期，仍然未知。知识的基础 这些机制将允许开发更有效的从多能干细胞分化GCs的方案。 细胞（PSC）。此外，它将允许调查表观遗传失调之间的联系， 在GC规范和严重的人类疾病，如流产，先天性缺陷和常见的 迟发性疾病，如不孕症、糖尿病、吸毒和癌症。 RESEARCH.本K08的目的是识别和验证关键基因调控元件（GRES） 在食蟹猴中控制GC命运和PGCs分化能力的诱导 原肠胚形成我们的中心假设是，早期中胚层中关键GRES的表观遗传启动介导了 发育能力在生殖谱系规范从多能状态。这项研究将 追求两个具体的目标：（1）确定推定的GRES潜在的发展能力的GC的命运 通过表观遗传足迹法（2）确定这些GRES的子集作为GC的关键决定因素 使用大规模平行报告基因测定（MPRA）和CRISPR表观基因组的感受态和分化 编辑介导的功能丧失实验。在未来，这些成果将提供平台， 表观基因组编辑作为提高GC特化效率的工具， 研究表突变对GC和胚胎发育结果的影响， 动物模型 辅导。我的主要导师凯文·埃根博士是哈佛干细胞研究所的干细胞生物学专家 中心我在哈佛网络中组建了一个多学科的指导团队：亚历克斯·迈斯纳博士 (an表观遗传学和表观基因组学专家），史蒂夫·麦卡罗博士（基因组学和生物信息学的领导者）。 训练研究目标得到了培训计划的支持，其中包括动手实验室培训， 以及生物信息学的正式和实践教学法，以及大型基因组数据集的分析， BIDMC的强大机构承诺以及通过哈佛催化剂的赠款审查和支持计划。

Project Summary CANDIDATE. I am a physician and Instructor in the Division of Reproductive Endocrinology and Infertility at Beth Israel Deaconess Medical Center (BIDMC). A K08 Award will provide me with the necessary training and experience to become an independent investigator in germ cell (GC) biology, with a focus on the gene regulatory networks underlying human gametogenesis. BACKGROUND. How a small subset of cells in the developing human and non-human primate embryo acquires competency to form the germ line and ultimately mature into functional gametes while maintaining its potential for pluripotency is among the least understood questions in biology. In particular, the gene regulatory networks and epigenetic remodeling, which govern competence for the GC fate during specification of early (primordial) germ cells (PGCs) in the peri-gastrulating period, remain unknown. Knowledge of the underlying mechanisms will allow the development of more efficient differentiation protocols for GCs from pluripotent stem cells (PSCs) in vitro. In addition, it will allow the investigation of the link between epigenetic dysregulation during GC specification and critical human disorders such as pregnancy loss, congenital defects and common late-onset diseases such as infertility, diabetes, drug addiction and cancer. RESEARCH. The objective for this K08 is to identify and validate the key gene regulatory elements (GREs) governing induction of competence for the GC fate and the differentiation of PGCs during cynomolgus gastrulation. Our central hypothesis is that epigenetic priming of key GREs in incipient mesoderm mediates developmental competence during germ lineage specification from the pluripotent state. This research will pursue two specific aims: (1) to identify putative GREs underlying developmental competence for the GC fate through epigenetic footprinting (2) to identify the subset of these GREs functioning as key determinants of GC competence and differentiation using massively parallel reporter assays (MPRA) and CRISPR epigenome editing mediated loss of function experiments. In the future, these results will provide the platform to use epigenome editing as a tool to improve the efficiency of GC specification, for trans-differentiation of GCs from somatic cells and to study the effect of epimutations on developmental outcomes in GCs and embryos using animal models. MENTORING. My primary mentor, Dr. Kevin Eggan, is an expert in stem cell biology at the Harvard Stem Cell Center. I have assembled a multi-disciplinary mentoring team across the Harvard network: Dr. Alex Meissner (an expert in epigenetics and epigenomics), Dr. Steve McCarroll (a leader in genomics and bioinformatics). TRAINING. The research objectives are supported by a training plan that includes hands-on lab training as well as formal and hands-on didactics in bioinformatics and the analysis of large genomic datasets alongside a strong institutional commitment at BIDMC and the grant review and support program through Harvard Catalyst.