Dissecting neural differentiation regulatory networks through epigenetic footprinting

Human pluripotent stem cell derived models that accurately recapitulate neural development in vitro and allow for the generation of specific neuronal subtypes are of major interest to the stem cell and biomedical community. Notch signaling, particularly through the Notch effector HES5, is a major pathway critical for the onset and maintenance of neural progenitor cells (NPCs) in the embryonic and adult nervous system1-3. Use of a HES5 reporter enables the isolation distinct populations of human embryonic stem (ES) cell derived NPCs that represent building blocks of cortical development in vitro4. Here, we report the transcriptional and epigenomic analysis of six consecutive stages of human ES cell differentiation along the neural lineage aimed at modeling key cell fate decisions including specification, expansion and patterning during the ontogeny of neural stem and progenitor cells. In order to dissect the regulatory mechanisms that orchestrate the stage-specific differentiation process we developed a computational framework to infer key regulators of each cell state transition based on the progressive remodeling of the epigenetic landscape and then validated these through a pooled shRNA screen. We were also able to refine our previous observations on epigenetic priming at transcription factor binding sites and show here that they are mediated by combinations of core and stage-specific factors. Taken together, we demonstrate the utility of our reference maps and outline a general framework, not limited to the context of the neural lineage, to dissect regulatory circuits of differentiation.

能够在体外精准复刻神经发育过程、并可诱导生成特定神经元亚型的人类多能干细胞（human pluripotent stem cell）衍生模型，一直备受干细胞与生物医学领域的广泛关注。Notch信号通路（Notch signaling），尤其是通过其效应分子HES5，是调控胚胎及成年神经系统中神经祖细胞（neural progenitor cells, NPCs）起始与维持的关键通路1-3。借助HES5报告基因系统，可分离得到不同群体的人类胚胎干细胞（human embryonic stem cell, ES细胞）衍生的神经祖细胞，这类细胞可作为体外模拟皮层发育的基本构建单元4。本研究对人类ES细胞沿神经谱系分化的六个连续阶段开展了转录组与表观基因组（epigenomic）分析，旨在模拟神经干细胞与神经祖细胞个体发育过程中关键的细胞命运决定事件，包括特化、扩增与模式化。为解析调控该阶段特异性分化过程的核心机制，本研究基于表观基因组的动态重塑，开发了一套计算框架以推断每个细胞状态转换过程中的关键调控因子，并通过混合shRNA筛选对这些调控因子进行了验证。本研究还优化了此前关于转录因子结合位点表观遗传预激活（epigenetic priming）的研究结果，并证实这类预激活过程由核心因子与阶段特异性因子共同介导。综上，本研究验证了所构建参考图谱的应用价值，并提出了一套不限于神经谱系的通用分析框架，用于解析分化过程中的调控环路。