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

项目摘要 候选人。我是生殖内分泌学和不孕不育科的内科医生和讲师 贝丝以色列女执事医疗中心(BIDMC)。K08奖将为我提供必要的培训和 有成为生殖细胞(GC)生物学独立研究员的经验，专注于基因研究 人类配子发生的基础调控网络。 背景资料。发育中的人类和非人类灵长类胚胎中的一小部分细胞 获得形成生殖系的能力，并最终成熟为功能配子，同时保持其 多能性的潜力是生物学中最不被理解的问题之一。尤其是基因调控 网络和表观遗传重塑，在早期规范GC命运的能力 (原始)生殖细胞(PGC)在原肠周期仍不清楚。对基础的知识 机制将允许开发更有效的从多能干细胞分化为GC的方案 细胞(PSCs)体外培养。此外，它还将允许研究表观遗传失调之间的联系 在GC规范和严重的人类疾病期间，如妊娠丢失、先天性缺陷和常见的 晚发性疾病，如不孕不育、糖尿病、吸毒和癌症。 研究。本K08的目标是识别和验证关键基因调控元件(Gres)。 食蟹猴GC命运和原生殖细胞分化能力的诱导 原肠形成。我们的中心假设是，早期中胚层中关键GRE的表观遗传启动 多能状态下胚种谱系指定的发育能力。这项研究将 追求两个具体目标：(1)确定GC命运潜在的GRE 通过表观遗传足迹(2)确定这些GRE的子集作为GC的关键决定因素 应用大规模平行报告分析(MPRA)和CRISPR表观基因组研究能力和分化 编辑介导性功能丧失实验。在未来，这些成果将提供使用的平台 表观基因组编辑作为提高GC规范效率的工具，用于GC和GCs的转分化 体细胞，并研究突变对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的干细胞生物学专家 中心。我已经组建了一个跨哈佛网络的多学科指导团队：亚历克斯·迈斯纳博士 史蒂夫·麦卡罗尔博士(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，该计划包括动手实验室培训，如 以及生物信息学的正式和实践教学，以及对大型基因组数据集的分析 BIDMC坚定的机构承诺，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.