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. 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）与同源结构域（homeodomain, HD）构成。在发育中的前脑内，Pax6表达于脑室区前体细胞以及特定神经元亚群中；Pax6缺失会导致细胞增殖紊乱与细胞命运特化异常。Pax6同时调控整个晶状体发育程序。 为重构Pax6依赖型基因调控网络（gene regulatory networks, GRNs），研究团队利用小鼠晶状体与前脑的染色质开展了染色质免疫共沉淀测序（ChIP-seq）实验。最终分别鉴定出3723个（前脑）与3514个（晶状体）含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"共有结合峰"。 对Pax6结合峰的分析显示，其基序与已证实的Pax6-PD、Pax6-PD/HD及Pax6-HD结合序列高度相似。 对H3K4me1、H3K4me3、H3K27ac、H3K27me3及RNA聚合酶II（Pol II）的图谱分析揭示了Pax6结合的组织特异性增强子的不同亚型。 Pax6通过激活（如Dmrta1与Ngn2）及抑制（如Ascl1、Fezf2与Gsx2）转录因子，直接调控皮层神经发生过程。在晶状体内，Pax6则通过调控成纤维细胞生长因子（fibroblast growth factor, FGF）信号通路（包括Fgfr2、Ccnd1与Prox1）及Wnt信号通路（包括Dkk3、Wnt7a、Lrp6、Bcl9l与Ccnd1）的相关组分，直接控制细胞周期退出。 综上，本研究完成了晶状体与前脑内Pax6依赖型基因调控网络的全基因组分析，阐明了Pax6在器官发生中的全新功能。 本研究对小鼠胚胎前脑与新生晶状体中的Pax6进行了检测。研究人员分别对小鼠E12.5胚胎前脑及新生晶状体开展了ChIP-seq实验，获取了Pax6、H3K4me1、H3K4me3、H3K27ac、H3K27me3及Pol II的全基因组结合位点数据。同时，研究团队还对小鼠E12.5胚胎前脑以及新生晶状体上皮细胞与纤维细胞开展了RNA测序（RNA-seq）实验。