Plaza S, Turque N, Dozier C, Bailly M, Saule S
Laboratoire de Differenciation Cellulaire et Moléculaire, CNRS EP56 Institut Pasteur, Lille, France.
Oncogene. 1995 Jan 19;10(2):329-40.
To understand the regulation of the Pax-6 gene, which plays an important role in eye development, we have characterized the promoter region of the quail Pax-6(Pax-QNR) gene. In addition to TATA and CAAT boxes, sequence analysis revealed several putative cis-regulatory elements among which three myb-responsive elements (MRE). C-myb encodes a nuclear, DNA-binding phosphoprotein that functions as transcriptional regulator. Co-transfection in quail embryo cells of the Pax-QNR/pax-6 promoter with a vector expressing the 75 kDa c-myb protein resulted in an increase in Pax-QNR promoter activity. By footprinting experiments we identified multiple binding sites for the myb protein within the promoter region. Protein containing the myb DNA-binding domain fused to the VP16-transactivation domain was fully efficient in Pax-QNR promoter transactivation, demonstrating that myb can transactivate through a direct binding on DNA. However, a myb truncated protein devoid of DNA-binding domain was also able to transactivate the Pax-QNR promoter. These results show that this promoter can be transactivated by the myb protein directly as well as indirectly. Finally we show by in situ hybridization that c-myb is strongly expressed in the developing neuroretina, simultaneously with Pax-QNR. These observations suggest that the c-myb protein may be a regulator of Pax-QNR/pax-6.
为了解在眼睛发育中起重要作用的Pax-6基因的调控机制，我们对鹌鹑Pax-6（Pax-QNR）基因的启动子区域进行了特征分析。除了TATA盒和CAAT盒外，序列分析还揭示了几个假定的顺式调控元件，其中包括三个myb反应元件（MRE）。C-myb编码一种核内DNA结合磷蛋白，其作为转录调节因子发挥作用。将Pax-QNR/pax-6启动子与表达75 kDa c-myb蛋白的载体共转染到鹌鹑胚胎细胞中，导致Pax-QNR启动子活性增加。通过足迹实验，我们在启动子区域内鉴定出了多个myb蛋白结合位点。含有与VP16反式激活结构域融合的myb DNA结合结构域的蛋白在Pax-QNR启动子反式激活中完全有效，表明myb可以通过直接结合DNA进行反式激活。然而，一种缺乏DNA结合结构域的myb截短蛋白也能够反式激活Pax-QNR启动子。这些结果表明，该启动子可以被myb蛋白直接或间接反式激活。最后，我们通过原位杂交表明，c-myb与Pax-QNR同时在发育中的神经视网膜中强烈表达。这些观察结果表明，c-myb蛋白可能是Pax-QNR/pax-6的调节因子。
