The Department of Genetics, Albert Einstein College of Medicine, Bronx, New York, United States of America.
PLoS One. 2013;8(1):e54507. doi: 10.1371/journal.pone.0054507. Epub 2013 Jan 14.
Pax6 encodes a specific DNA-binding transcription factor that regulates the development of multiple organs, including the eye, brain and pancreas. Previous studies have shown that Pax6 regulates the entire process of ocular lens development. In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific populations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification in telencephalon. In the pancreas, Pax6 is essential for the differentiation of α-, β- and δ-islet cells. To elucidate molecular roles of Pax6, chromatin immunoprecipitation experiments combined with high-density oligonucleotide array hybridizations (ChIP-chip) were performed using three distinct sources of chromatin (lens, forebrain and β-cells). ChIP-chip studies, performed as biological triplicates, identified a total of 5,260 promoters occupied by Pax6. 1,001 (133) of these promoter regions were shared between at least two (three) distinct chromatin sources, respectively. In lens chromatin, 2,335 promoters were bound by Pax6. RNA expression profiling from Pax6⁺/⁻ lenses combined with in vivo Pax6-binding data yielded 76 putative Pax6-direct targets, including the Gaa, Isl1, Kif1b, Mtmr2, Pcsk1n, and Snca genes. RNA and ChIP data were validated for all these genes. In lens cells, reporter assays established Kib1b and Snca as Pax6 activated and repressed genes, respectively. In situ hybridization revealed reduced expression of these genes in E14 cerebral cortex. Moreover, we examined differentially expressed transcripts between E9.5 wild type and Pax6⁻/⁻ lens placodes that suggested Efnb2, Fat4, Has2, Nav1, and Trpm3 as novel Pax6-direct targets. Collectively, the present studies, through the identification of Pax6-direct target genes, provide novel insights into the molecular mechanisms of Pax6 gene control during mouse embryonic development. In addition, the present data demonstrate that Pax6 interacts preferentially with promoter regions in a tissue-specific fashion. Nevertheless, nearly 20% of the regions identified are accessible to Pax6 in multiple tissues.
Pax6 编码一种特定的 DNA 结合转录因子，调节包括眼睛、大脑和胰腺在内的多种器官的发育。先前的研究表明，Pax6 调节晶状体发育的整个过程。在发育中的前脑，Pax6 在脑室区前体细胞和特定神经元群体中表达；Pax6 的缺失导致端脑细胞增殖和细胞命运特化紊乱。在胰腺中，Pax6 对于 α-、β-和 δ-胰岛细胞的分化是必需的。为了阐明 Pax6 的分子作用，使用三种不同来源的染色质（晶状体、前脑和 β 细胞）进行了染色质免疫沉淀实验（ChIP）与高密度寡核苷酸阵列杂交（ChIP-chip）的组合实验。ChIP-chip 研究作为生物学重复进行，共鉴定了 5260 个 Pax6 结合的启动子。其中 1001 个（133 个）启动子区域分别在至少两种（三种）不同的染色质来源之间共享。在晶状体染色质中，有 2335 个启动子被 Pax6 结合。将 Pax6⁺/⁻晶状体的 RNA 表达谱与体内 Pax6 结合数据相结合，产生了 76 个推定的 Pax6 直接靶标，包括 Gaa、Isl1、Kif1b、Mtmr2、Pcsk1n 和 Snca 基因。所有这些基因的 RNA 和 ChIP 数据都得到了验证。在晶状体细胞中，报告基因实验确定 Kib1b 和 Snca 分别为 Pax6 激活和抑制的基因。原位杂交显示这些基因在 E14 大脑皮层中的表达减少。此外，我们检查了 E9.5 野生型和 Pax6⁻/⁻晶状体基板之间差异表达的转录本，提示 Efnb2、Fat4、Has2、Nav1 和 Trpm3 为新的 Pax6 直接靶标。总的来说，本研究通过鉴定 Pax6 的直接靶基因，为 Pax6 基因在小鼠胚胎发育过程中的分子机制提供了新的见解。此外，本研究数据表明，Pax6 以组织特异性的方式优先与启动子区域相互作用。然而，鉴定出的区域中近 20%可在多种组织中被 Pax6 访问。
