BACKGROUND Homeodomain (HD) transcription factor (TF) NKX2-1 critical for the regional specification of the medial ganglionic eminence (MGE) as well as promoting the GABAergic and cholinergic neuron fates via the induction of TFs such as LHX6 and LHX8. NKX2-1 defines MGE regional identity in large part through transcriptional repression, while specification and maturation of GABAergic and cholinergic fates is mediated in part by transcriptional activation via TFs such as LHX6 and LHX8. Here we analyze the signaling and TF pathways, downstream of NKX2-1, required for GABAergic and cholinergic neuron fate maturation. METHODS Differential ChIP-seq analysis was used to identify regulatory elements (REs) where chromatin state was sensitive to change in the Nkx2-1cKO MGE at embryonic day (E) 13.5. TF motifs in the REs were identified using RSAT. CRISPR-mediated genome editing was used to generate enhancer knockouts. Differential gene expression in these knockouts was analyzed through RT-qPCR and in situ hybridization. Functional analysis of motifs within hs623 was analyzed via site directed mutagenesis and reporter assays in primary MGE cultures. RESULTS We identified 4782 activating REs (aREs) and 6391 repressing REs (rREs) in the Nkx2-1 conditional knockout (Nkx2-1cKO) MGE. aREs are associated with basic-Helix-Loop-Helix (bHLH) TFs. Deletion of hs623, an intragenic Tcf12 aRE, caused a reduction of Tcf12 expression in the sub-ventricular zone (SVZ) and mantle zone (MZ) of the MGE. Mutation of LHX, SOX and octamers, within hs623, caused a reduction of hs623 activity in MGE primary cultures. CONCLUSIONS Tcf12 expression in the SVZ of the MGE is mediated through aRE hs623. The activity of hs623 is dependent on LHX6, SOX and octamers. Thus, maintaining the expression of Tcf12 in the SVZ involves on TF pathways parallel and genetically downstream of NKX2-1.

中文翻译：

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在MGE发育过程中指导细胞状态进程的转录网络的基因组分析。

背景同源域（HD）转录因子（TF）NKX2-1对于内侧神经节隆起（MGE）的区域规范以及通过诱导TF（例如LHX6和LHX8）促进GABA能和胆碱能神经元命运至关重要。NKX2-1很大程度上通过转录抑制来定义MGE区域同一性，而GABA能和胆碱能的命运的规范和成熟则部分地通过TF（例如LHX6和LHX8）的转录激活来介导。在这里，我们分析了GABA能和胆碱能神经元命运成熟所需的NKX2-1下游的信号传导和TF途径。方法采用差动ChIP-seq分析来鉴定调节元件（RE），在胚胎第13.5天，染色质状态对Nkx2-1cKO MGE的变化敏感。使用RSAT识别RE中的TF基序。CRISPR介导的基因组编辑用于产生增强子敲除。通过RT-qPCR和原位杂交分析了这些基因敲除中的差异基因表达。hs623内基序的功能分析通过原位MGE培养中的定点诱变和报告基因分析进行了分析。结果我们在Nkx2-1条件基因敲除（Nkx2-1cKO）MGE中鉴定了4782个激活RE（aRE）和6391个抑制RE（rRE）。aRE与基本螺旋-螺旋-螺旋（bHLH）TF相关联。内源性Tcf12 aRE hs623的缺失导致MGE的心室下区（SVZ）和地幔区（MZ）中Tcf12表达的减少。hs623内LHX，SOX和八聚体的突变导致MGE原代培养物中hs623活性降低。结论MGE SVZ中的Tcf12表达是通过aRE hs623介导的。hs623的活性取决于LHX6，SOX和八聚体。因此，维持Tcf12在SVZ中的表达涉及与NKX2-1平行且遗传下游的TF途径。