Identification of in vivo DNA-binding mechanisms of Pax6 and reconstruction of Pax6-dependent gene regulatory networks during lens and forebrain development

The transcription factor Pax6 is comprised of the paired domain (PD) and homeodomain (HD). In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific subpopulations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification. Pax6 also regulates the entire lens developmental program. To reconstruct Pax6-dependent gene regulatory networks (GRNs), ChIP-seq studies were performed using lens and forebrain chromatin from mice. A total of 3,723 (forebrain) and 3,514 (lens) Pax6-containing peaks were identified, with ~70% of them found in both tissues and thereafter called “common” peaks. Analysis of Pax6-bound peaks identified motifs that closely resemble Pax6-PD, -PD/HD and -HD established binding sequences. Mapping of H3K4me1, H3K4me3, H3K27ac, H3K27me3 and RNA polymerase II revealed distinct types of tissue-specific enhancers bound by Pax6. Pax6 directly regulates cortical neurogenesis through activation (e.g. Dmrta1 and Ngn2) and repression (e.g. Ascl1, Fezf2, and Gsx2) of transcription factors. In lens, Pax6 directly regulates cell cycle exit control via components of FGF (Fgfr2, Ccnd1, and Prox1) and Wnt (Dkk3, Wnt7a, Lrp6, Bcl9l, and Ccnd1) signaling pathways. Collectively, these studies provide genome-wide analysis of Pax6-dependent GRNs in lens and forebrain and establish novel roles of Pax6 in organogenesis. Overall design: Examination of Pax6 in mouse embryonic forebrain and newborn lens. We performed ChIP-seq on mouse E12.5 embryonic forebrain and newborn lens. Genome-wide binding sites of Pax6, H3K4me1, H3K4me3, H3K27ac, H3K27me3, and Pol2 were generated. We also performed RNA-seq on mouse E12.5 embryonic forebrain and newborn lens epithelial cells and fibers.

转录因子Pax6由配对结构域（paired domain, PD）和同源结构域（homeodomain, HD）组成。在发育中的前脑中，Pax6表达于脑室区前体细胞与特定神经元亚群；Pax6缺失会导致细胞增殖紊乱及细胞命运特化异常。Pax6同时调控晶状体的完整发育程序。为重构依赖于Pax6的基因调控网络（gene regulatory network, GRN），研究人员使用小鼠晶状体和前脑的染色质开展了染色质免疫沉淀测序（ChIP-seq）实验。最终分别鉴定得到3723个（前脑）和3514个（晶状体）含有Pax6的结合峰，其中约70%的结合峰在两种组织中均存在，后续将其称为“共有”峰。对Pax6结合峰的分析发现了与已验证的Pax6-PD、-PD/HD及-HD结合序列高度相似的DNA基序。通过对H3K4me1、H3K4me3、H3K27ac、H3K27me3以及RNA聚合酶II的染色质图谱分析，揭示了Pax6结合的不同类型组织特异性增强子。Pax6通过激活（如Dmrta1和Ngn2）以及抑制（如Ascl1、Fezf2和Gsx2）转录因子，直接调控皮层神经发生过程。在晶状体中，Pax6通过调控成纤维细胞生长因子（FGF）信号通路组分（包括Fgfr2、Ccnd1和Prox1）与Wnt信号通路组分（包括Dkk3、Wnt7a、Lrp6、Bcl9l和Ccnd1），直接调控细胞周期退出过程。综上，本系列研究实现了晶状体和前脑中依赖Pax6的基因调控网络的全基因组分析，并阐明了Pax6在器官发生中的全新功能。整体实验设计：针对小鼠胚胎前脑与新生晶状体中的Pax6开展检测。研究人员对小鼠E12.5胚胎前脑及新生晶状体进行了ChIP-seq实验，获取了Pax6、H3K4me1、H3K4me3、H3K27ac、H3K27me3以及Pol2的全基因组结合位点图谱。同时，研究人员还对小鼠E12.5胚胎前脑以及新生晶状体上皮细胞和纤维细胞开展了RNA测序（RNA-seq）实验。