Chauhan Bharesh K, Yang Ying, Cveklová Kveta, Cvekl Ales
Department of Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Nucleic Acids Res. 2004 Mar 12;32(5):1696-709. doi: 10.1093/nar/gkh334. Print 2004.
Pax6 is essential for development of the eye, olfactory system, brain and pancreas. Haploinsufficiency of Pax6 causes abnormal eye development. Two forms of Pax6 protein, PAX6 and PAX6(5a), differ in a 14 amino acid insertion encoded by an alternatively spliced exon 5a in the N-terminal DNA-binding paired domain (PD), and they are simultaneously expressed. Here, we show that PAX6 and PAX6(5a) together synergistically activate transcription from promoters recognized by Pax6 PD and PD5a, but not by their homeodomain. This synergism promotes activation of transcription by c-Maf and MafA on the alphaB-crystallin promoter, and is required for transcriptional co-activation by RARbeta/RXRbeta and PAX6/PAX6(5a) on the gammaF-crystallin promoter. To determine the role of this synergism in haploinsufficiency, we tested four human missense (G18W, R26G, G64V and R128C) and one nonsense (R317X) mutants, with reporters driven by Pax6 PD consensus binding sites and the alphaB-crystallin promoter. The simultaneous activity of Pax6 proteins [PAX6, mutated PAX6, PAX6(5a) and mutated PAX6(5a)] modeling haploinsufficiency yielded results not predicted by properties of individual PAX6 or PAX6(5a). Taken together, these results indicate that complex ocular phenotypes due to Pax6 haploinsufficiency originate, at least partially, from functional interactions between alternatively spliced PAX6 and PAX6(5a) variants and other factors, e.g. MafA/c-Maf.
Pax6对于眼睛、嗅觉系统、大脑和胰腺的发育至关重要。Pax6单倍剂量不足会导致眼睛发育异常。两种形式的Pax6蛋白，即PAX6和PAX6(5a)，在由选择性剪接的外显子5a编码的N端DNA结合配对结构域（PD）中有14个氨基酸的插入差异，且它们同时表达。在此，我们表明PAX6和PAX6(5a)共同协同激活由Pax6 PD和PD5a识别的启动子的转录，但不由它们的同源结构域识别的启动子激活转录。这种协同作用促进了c-Maf和MafA对αB-晶状体蛋白启动子的转录激活，并且是RARβ/RXRβ和PAX6/PAX6(5a)对γF-晶状体蛋白启动子进行转录共激活所必需的。为了确定这种协同作用在单倍剂量不足中的作用，我们用由Pax6 PD共有结合位点和αB-晶状体蛋白启动子驱动的报告基因测试了四个人类错义突变体（G18W、R26G、G64V和R128C）和一个无义突变体（R317X）。模拟单倍剂量不足的Pax6蛋白[PAX6、突变的PAX6、PAX6(5a)和突变的PAX6(5a)]的同时活性产生了单独的PAX6或PAX6(5a)特性无法预测的结果。综上所述，这些结果表明，由于Pax6单倍剂量不足导致的复杂眼部表型至少部分源于选择性剪接的PAX6和PAX6(5a)变体与其他因子（如MafA/c-Maf）之间的功能相互作用。
