Department of Genetics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
J Biol Chem. 2011 Dec 16;286(50):43259-71. doi: 10.1074/jbc.M111.264580. Epub 2011 Oct 13.
Tissue morphogenesis requires intricate temporal and spatial control of gene expression that is executed through specific gene regulatory networks (GRNs). GRNs are comprised from individual subcircuits of different levels of complexity. An important question is to elucidate the mutual relationship between those genes encoding DNA-binding factors that trigger the subcircuit with those that play major "later" roles during terminal differentiation via expression of specific genes that constitute the phenotype of individual tissues. The ocular lens is a classical model system to study tissue morphogenesis. Pax6 is essential for both lens placode formation and subsequent stages of lens morphogenesis, whereas c-Maf controls terminal differentiation of lens fibers, including regulation of crystallins, key lens structural proteins required for its transparency and refraction. Here, we show that Pax6 directly regulates c-Maf expression during lens development. A 1.3-kb c-Maf promoter with a 1.6-kb upstream enhancer (CR1) recapitulated the endogenous c-Maf expression pattern in lens and retinal pigmented epithelium. ChIP assays revealed binding of Pax6 and c-Maf to multiple regions of the c-Maf locus in lens chromatin. To predict functional Pax6-binding sites, nine novel variants of Pax6 DNA-binding motifs were identified and characterized. Two of these motifs predicted a pair of Pax6-binding sites in the CR1. Mutagenesis of these Pax6-binding sites inactivated transgenic expression in the lens but not in retinal pigmented epithelium. These data establish a novel regulatory role for Pax6 during lens development, link together the Pax6/c-Maf/crystallin regulatory network, and suggest a novel type of GRN subcircuit that controls a major part of embryonic lens development.
组织形态发生需要精细的时空控制基因表达，这是通过特定的基因调控网络（GRN）来执行的。GRN 由不同复杂程度的单个子电路组成。一个重要的问题是阐明那些编码 DNA 结合因子的基因与那些在终末分化过程中通过表达构成个体组织表型的特定基因发挥主要“后期”作用的基因之间的相互关系。眼部晶状体是研究组织形态发生的经典模型系统。Pax6 对于晶状体基板的形成和随后的晶状体形态发生阶段都是必不可少的，而 c-Maf 则控制晶状体纤维的终末分化，包括晶状体蛋白的调节，晶状体蛋白是晶状体透明度和折射所必需的关键结构蛋白。在这里，我们表明 Pax6 在晶状体发育过程中直接调节 c-Maf 的表达。一个带有 1.6kb 上游增强子（CR1）的 1.3kb c-Maf 启动子在晶状体和视网膜色素上皮中重现了内源性 c-Maf 的表达模式。ChIP 分析显示 Pax6 和 c-Maf 结合到晶状体染色质中 c-Maf 基因座的多个区域。为了预测功能 Pax6 结合位点，鉴定并表征了九个新的 Pax6 DNA 结合基序变体。其中两个基序预测了 CR1 中一对 Pax6 结合位点。这些 Pax6 结合位点的突变使晶状体中的转基因表达失活，但在视网膜色素上皮中没有失活。这些数据确立了 Pax6 在晶状体发育过程中的新的调节作用，将 Pax6/c-Maf/crystallin 调控网络联系在一起，并提出了一种新的 GRN 子电路类型，该子电路控制了胚胎晶状体发育的主要部分。
