Plaza S, Dozier C, Langlois M C, Saule S
Laboratoire de différenciation cellulaire et moléculaire, Centre National de la Recherche Scientifique EP 56. Institut Pasteur, Lille, France.
Mol Cell Biol. 1995 Feb;15(2):892-903. doi: 10.1128/MCB.15.2.892.
Using nuclear run-on assays, we showed that the tissue-specific expression of quail Pax-6 (Pax-QNR) P0-initiated mRNAs is due in part to regulation of the gene at the transcriptional level. Regulatory sequences governing neuroretina-specific expression of the P0-initiated mRNAs were investigated. By using reporter-based expression assays, we characterized a region within the Pax-QNR gene, located 7.5 kbp downstream from the P0 promoter, that functions as an enhancer in neuroretina cells but not in nonexpressing P0-initiated mRNA cells (quail embryo cells and quail retinal pigment epithelial cells). This enhancer element functioned in a position- and orientation-independent manner both on the Pax-QNR P0 promoter and the heterologous thymidine kinase promoter. Moreover, this enhancer element exhibited a developmental stage-specific activity during embryonic neuroretina development: in contrast to activity at day E7, the enhancer activity was very weak at day E5. This paralleled the level of expression of P0-initiated mRNAs observed at the same stages. Using footprinting, gel retardation, and Southwestern (DNA-protein) analysis, we demonstrated the existence of four neuroretina-specific nuclear protein-binding sites, involving multiple unknown factors. In addition we showed that the quail enhancer element is structurally and functionally conserved in mice. All of these results strongly suggest that this enhancer element may contribute to the neuroretina-specific transcriptional regulation of the Pax-6 gene in vivo.
通过核转录分析，我们发现鹌鹑Pax-6（Pax-QNR）由P0起始的mRNA的组织特异性表达部分归因于该基因在转录水平的调控。我们研究了调控P0起始的mRNA在神经视网膜特异性表达的调控序列。通过基于报告基因的表达分析，我们鉴定了Pax-QNR基因内一个位于P0启动子下游7.5kbp处的区域，该区域在神经视网膜细胞中起增强子作用，但在不表达P0起始mRNA的细胞（鹌鹑胚胎细胞和鹌鹑视网膜色素上皮细胞）中不起作用。该增强子元件在Pax-QNR P0启动子和异源胸苷激酶启动子上均以位置和方向独立的方式发挥作用。此外，该增强子元件在胚胎神经视网膜发育过程中表现出特定发育阶段的活性：与E7时的活性相比，E5时的增强子活性非常弱。这与在相同阶段观察到的P0起始mRNA的表达水平相一致。通过足迹法、凝胶阻滞和西南（DNA-蛋白质）分析，我们证明了存在四个神经视网膜特异性核蛋白结合位点，涉及多种未知因子。此外，我们还表明鹌鹑增强子元件在结构和功能上在小鼠中是保守的。所有这些结果强烈表明，该增强子元件可能在体内对Pax-6基因的神经视网膜特异性转录调控起作用。
