Table_2_Foxa2 and Pet1 Direct and Indirect Synergy Drive Serotonergic Neuronal Differentiation.XLSX

Neuronal programming by forced expression of transcription factors (TFs) holds promise for clinical applications of regenerative medicine. However, the mechanisms by which TFs coordinate their activities on the genome and control distinct neuronal fates remain obscure. Using direct neuronal programming of embryonic stem cells, we dissected the contribution of a series of TFs to specific neuronal regulatory programs. We deconstructed the Ascl1-Lmx1b-Foxa2-Pet1 TF combination that has been shown to generate serotonergic neurons and found that stepwise addition of TFs to Ascl1 canalizes the neuronal fate into a diffuse monoaminergic fate. The addition of pioneer factor Foxa2 represses Phox2b to induce serotonergic fate, similar to in vivo regulatory networks. Foxa2 and Pet1 appear to act synergistically to upregulate serotonergic fate. Foxa2 and Pet1 co-bind to a small fraction of genomic regions but mostly bind to different regulatory sites. In contrast to the combinatorial binding activities of other programming TFs, Pet1 does not strictly follow the Foxa2 pioneer. These findings highlight the challenges in formulating generalizable rules for describing the behavior of TF combinations that program distinct neuronal subtypes.

通过强制表达转录因子（TFs）进行神经元编程在再生医学的临床应用中具有广阔的前景。然而，TFs在基因组上协调其活动以及控制不同神经元命运的具体机制仍然扑朔迷离。利用胚胎干细胞的直接神经元编程，我们剖析了一系列TFs对特定神经元调控程序的贡献。我们解析了已被证明可生成5-羟色胺能神经元的Ascl1-Lmx1b-Foxa2-Pet1 TF组合，并发现逐步添加TFs至Ascl1将神经元命运引导至广泛的单胺能命运。先驱因子Foxa2的加入通过抑制Phox2b来诱导5-羟色胺能命运，与体内调控网络相似。Foxa2和Pet1似乎协同作用以上调5-羟色胺能命运。Foxa2和Pet1共同结合于基因组区域的一小部分，但主要结合于不同的调控位点。与其它编程TFs的组合结合活性不同，Pet1并不严格遵循Foxa2的先驱作用。这些发现突显了描述编程不同神经元亚型的TF组合行为时，制定普遍适用规则的挑战。