Foxa2 and Pet1 direct and indirect synergy drive serotonergic neuronal differentiation [ChIP-Seq]

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. ChIP-seq was used to characterize the genomic binding of induced transcription factors when various combinations of Ascl1, Lmx1b, Pet1, and Foxa2 were expressed in mouse embryonic stem cells.

通过强制表达转录因子（transcription factors, TFs）实现神经元编程，在再生医学的临床应用中展现出巨大潜力。然而，转录因子如何协同调控基因组活性、并控制特定神经元命运的机制仍有待阐明。本研究借助胚胎干细胞的直接神经元编程模型，解析了一系列转录因子对特定神经元调控程序的贡献。我们解构了此前被证实可生成血清素能神经元的Ascl1-Lmx1b-Foxa2-Pet1转录因子组合，发现逐步向Ascl1中添加其余转录因子，会将神经元命运导向弥散性单胺能神经元命运。先锋因子Foxa2可通过抑制Phox2b以诱导血清素能神经元命运，这与体内调控网络的特征相符。Foxa2与Pet1似乎可协同上调血清素能神经元命运的相关程序。Foxa2与Pet1仅在极小部分基因组区域存在共同结合，二者大多结合于不同的调控位点。与其他编程转录因子的组合结合模式不同，Pet1并未严格遵循Foxa2的先锋因子结合规律。上述研究结果凸显了构建可推广规则以描述不同神经元亚型编程相关转录因子组合行为的挑战。本研究在小鼠胚胎干细胞中表达Ascl1、Lmx1b、Pet1与Foxa2的不同组合，利用染色质免疫共沉淀测序（ChIP-seq）对诱导型转录因子的基因组结合特征进行了表征。