Foxa2 and Pet1 direct and indirect synergy drive serotonergic neuronal differentiation [ATAC-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. Overall design: ATAC-seq was used to characterize chromatin accessibility 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）实现的神经元编程，在再生医学的临床应用中具有广阔前景。然而，转录因子如何在基因组层面协同调控其活性，并控制不同神经元命运的具体机制，目前仍不明确。本研究借助胚胎干细胞的直接神经元重编程模型，解析了一系列转录因子对特定神经元调控程序的贡献。我们对已被证实可诱导生成5-羟色胺能神经元的Ascl1-Lmx1b-Foxa2-Pet1转录因子组合进行解构分析，发现向Ascl1中逐步添加其他转录因子，可将神经元命运定向引导为弥散性单胺能神经元命运。如同体内调控网络的运作模式，添加先锋转录因子（pioneer factor）Foxa2可抑制Phox2b的表达，从而诱导5-羟色胺能神经元命运。Foxa2与Pet1似乎可协同作用，上调5-羟色胺能神经元命运的相关程序。二者仅在少数基因组区域存在共结合，但大多结合于不同的调控位点。与其他重编程转录因子的组合结合模式不同，Pet1并不会严格遵循Foxa2先锋因子的结合规则。本研究结果凸显了一项核心挑战：即如何构建可推广的通用规则，以准确描述调控不同神经元亚型的转录因子组合的行为模式。实验整体设计：本研究在小鼠胚胎干细胞中分别表达Ascl1、Lmx1b、Pet1与Foxa2的不同组合，通过ATAC-seq技术表征各组的染色质开放状态。