Yang Ying, Chauhan Bharesh K, Cveklova Kveta, Cvekl Ales
The Department of Ophthalmology, Albert Einstein College of Medicine, 909 Ullmann, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
J Mol Biol. 2004 Nov 19;344(2):351-68. doi: 10.1016/j.jmb.2004.07.102.
Mammalian alphaB-crystallin is highly expressed both in lens epithelium and lens fibers. In contrast, gammaF-crystallin is highly expressed in the lens fiber cells. Crystallin gene expression in lens is regulated at the level of transcription by a sparse number of specific DNA-binding transcription factors. Here, we report studies on transcriptional regulation of mouse alphaB- and gammaF-crystallin promoters by specific combinations of Pax6/Pax6(5a), large Mafs (MafA, MafB, c-Maf, and NRL), Sox1, Sox2, Six3, and RARbeta/RXRbeta. Two sets of these factors, co-expressed both in lens epithelium and in lens fibers, were tested in co-transfection assays using cultured lens and non-lens cells. Regulation of alphaB-crystallin was studied in the presence of lens epithelial-factors Pax6, MafB, and RARbeta/RXRbeta, and lens fiber-factors Pax6, MafA, c-Maf, and NRL. Pax6 proteins activated the alphaB-crystallin promoter (-162 to +45) with any combination of Mafs. Addition of RARbeta/RXRbeta further increased its promoter activity. Gel shift assays using lens nuclear extracts demonstrated interactions of Pax6, Maf, and retinoic acid nuclear receptor proteins with two lens-specific regions, the distal LSR1 (-147/-118) and proximal LSR2 (-78/-40), of the alphaB-crystallin promoter. In contrast, Pax6 proteins acted as repressors of gammaF-crystallin promoter activity elicited by a combination of large Mafs, Sox, and RARbeta/RXRbeta proteins in transiently transfected lens and non-lens cells. The results show that Pax6 conversely regulates these two lens crystallin promoters. We propose that the opposite roles of Pax6 in crystallin gene regulation are results of different promoter architectures of the alphaB- and gammaF-crystallin genes, developmentally regulated association of transcription factors with the corresponding cis-regulatory sites, and specific recruitment of transcriptional co-activators and co-repressors by Pax6.
哺乳动物的αB-晶状体蛋白在晶状体上皮细胞和晶状体纤维中均有高表达。相比之下，γF-晶状体蛋白在晶状体纤维细胞中高表达。晶状体中的晶状体蛋白基因表达在转录水平上受到少量特定DNA结合转录因子的调控。在此，我们报告了关于Pax6/Pax6(5a)、大Maf蛋白（MafA、MafB、c-Maf和NRL）、Sox1、Sox2、Six3以及RARβ/RXRβ的特定组合对小鼠αB-和γF-晶状体蛋白启动子转录调控的研究。在使用培养的晶状体和非晶状体细胞进行的共转染实验中，对两组在晶状体上皮细胞和晶状体纤维中均共表达的这些因子进行了测试。在存在晶状体上皮因子Pax6、MafB以及RARβ/RXRβ和晶状体纤维因子Pax6、MafA、c-Maf和NRL的情况下，研究了αB-晶状体蛋白的调控。Pax6蛋白与任何Maf组合均可激活αB-晶状体蛋白启动子（-162至+45）。添加RARβ/RXRβ可进一步增强其启动子活性。使用晶状体核提取物进行的凝胶迁移实验表明，Pax6、Maf和视黄酸核受体蛋白与αB-晶状体蛋白启动子的两个晶状体特异性区域，即远端LSR1（-147/-118）和近端LSR2（-78/-40）相互作用。相反，在瞬时转染的晶状体和非晶状体细胞中，Pax6蛋白可作为由大Maf蛋白、Sox蛋白和RARβ/RXRβ蛋白组合引发的γF-晶状体蛋白启动子活性的抑制因子。结果表明，Pax6对这两个晶状体晶状体蛋白启动子具有相反的调控作用。我们认为，Pax6在晶状体蛋白基因调控中的相反作用是αB-和γF-晶状体蛋白基因不同启动子结构、转录因子与相应顺式调控位点的发育调控关联以及Pax6对转录共激活因子和共抑制因子的特异性招募的结果。
