Small molecules modulate chromatin accessibility to promote NEUROG2-mediated fibroblast-to-neuron reprogramming [ChIP-Seq]

Pro-neural transcription factors and small molecules can induce the transdifferentiation of fibroblasts into functional neurons; however, a molecular mechanism detailing the immediate-early events that catalyze this conversion has not been well defined. We previously demonstrated that NEUROG2, forskolin (F), and dorsomorphin (D) can induce functional neurons with high-efficiency. Here, we used this model to define the genetic and epigenetic events that initiate an acquisition of neuronal identity. We demonstrate that NEUROG2 is a pioneer factor, FD enhances both genome-wide NEUROG2 chromatin occupancy and H3K27 acetylation, and synergistic transcription by these factors is essential to successful reprogramming. CREB1, activated by FD, promotes neuron survival and acts with NEUROG2 to upregulate SOX4, which co-activates NEUROD1 and NEUROD4. In addition to this hierarchical function, SOX4 targets SWI/SNF subunits and SOX4 knockdown results in extensive loss of open chromatin and abolishes reprogramming. Applying these insights, adult human glioblastoma and skin fibroblast reprogramming was improved using SOX4, SMARCA4, and chromatin modifying chemicals. Overall design: Histone 3 lysine 27 acetylation (3 input datasets, 6 immunoprecipitation datasets), histone 3 lysine 27 methylation (3 input datasets, 6 immunoprecipitation datasets), NEUROG2 (6 input datasets, 12 immunoprecipitation datasets), and SOX4 (1 input dataset, 3 immunoprecipitation datasets)

前神经转录因子与小分子化合物可诱导成纤维细胞转分化为功能神经元，但介导该转化的早期分子事件机制尚未被充分阐明。我们此前已证实，神经源性分化因子2（NEUROG2）、福司柯林（forskolin, F）与多马司芬（dorsomorphin, D）可高效诱导生成功能神经元。本研究以此模型为基础，解析了启动神经元身份获得的遗传与表观遗传事件。我们证实NEUROG2为先锋转录因子，FD可同时增强全基因组范围内NEUROG2的染色质结合与组蛋白H3赖氨酸27乙酰化（H3K27 acetylation）水平，且二者的协同转录对于重编程成功至关重要。经FD激活的环腺苷酸应答元件结合蛋白1（CREB1）可促进神经元存活，并与NEUROG2协同上调SOX4的表达，而SOX4可共同激活NEUROD1与NEUROD4。除上述层级调控功能外，SOX4可靶向SWI/SNF复合物亚基；敲低SOX4会导致开放染色质大面积丢失，并完全阻断重编程过程。基于上述发现，我们使用SOX4、SMARCA4与染色质修饰化合物，优化了成人胶质母细胞瘤细胞与皮肤成纤维细胞的重编程效率。实验整体设计：组蛋白H3赖氨酸27乙酰化（3份输入数据集、6份免疫沉淀数据集）、组蛋白H3赖氨酸27甲基化（3份输入数据集、6份免疫沉淀数据集）、NEUROG2（6份输入数据集、12份免疫沉淀数据集）与SOX4（1份输入数据集、3份免疫沉淀数据集）的相关检测。