Research Group of Pharmacology, Department of Pharmacy, University of Tromsø, Tromsø, Norway.
PLoS One. 2012;7(2):e31915. doi: 10.1371/journal.pone.0031915. Epub 2012 Feb 23.
Pax6 and Pax6(5a) are two isoforms of the evolutionary conserved Pax6 gene often co-expressed in specific stochiometric relationship in the brain and the eye during development. The Pax6(5a) protein differs from Pax6 by having a 14 amino acid insert in the paired domain, causing the two proteins to have different DNA binding specificities. Difference in functions during development is proven by the fact that mutations in the 14 amino acid insertion for Pax6(5a) give a slightly different eye phenotype than the one described for Pax6. Whereas quite many Pax6 target genes have been published during the last years, few Pax6(5a) specific target genes have been reported on. However, target genes identified by Pax6 knockout studies can probably be Pax6(5a) targets as well, since this isoform also will be affected by the knockout. In order to identify new Pax6 target genes, and to try to distinguish between genes regulated by Pax6 and Pax6(5a), we generated FlpIn-3T3 cell lines stably expressing Pax6 or Pax6(5a). RNA was harvested from these cell lines and used in gene expression microarrays where we identified a number of genes differentially regulated by Pax6 and Pax6(5a). A majority of these were associated with the extracellular region. By qPCR we verified that Ncam1, Ngef, Sphk1, Dkk3 and Crtap are Pax6(5a) specific target genes, while Tgfbi, Vegfa, EphB2, Klk8 and Edn1 were confirmed as Pax6 specific target genes. Nbl1, Ngfb and seven genes encoding different glycosyl transferases appeared to be regulated by both. Direct binding to the promoters of Crtap, Ctgf, Edn1, Dkk3, Pdgfb and Ngef was verified by ChIP. Furthermore, a change in morphology of the stably transfected Pax6 and Pax6(5a) cells was observed, and the Pax6 expressing cells were shown to have increased proliferation and migration capacities.
Pax6 和 Pax6(5a) 是进化上保守的 Pax6 基因的两种同工型，在发育过程中经常在大脑和眼睛中以特定的化学计量关系共同表达。Pax6(5a) 蛋白在配对结构域中有 14 个氨基酸插入，导致两种蛋白具有不同的 DNA 结合特异性。在发育过程中功能的差异是由以下事实证明的：Pax6(5a) 中 14 个氨基酸插入的突变导致的眼睛表型与描述的 Pax6 略有不同。尽管在过去几年中已经发表了相当多的 Pax6 靶基因，但报道的 Pax6(5a) 特异性靶基因很少。然而，通过 Pax6 敲除研究鉴定的靶基因也可能是 Pax6(5a) 的靶基因，因为这种同工型也会受到敲除的影响。为了鉴定新的 Pax6 靶基因，并试图区分由 Pax6 和 Pax6(5a) 调节的基因，我们生成了稳定表达 Pax6 或 Pax6(5a)的 FlpIn-3T3 细胞系。从这些细胞系中提取 RNA，并用于基因表达微阵列，在微阵列中我们鉴定了一些由 Pax6 和 Pax6(5a) 差异调节的基因。其中大多数与细胞外区域相关。通过 qPCR，我们验证了 Ncam1、Ngef、Sphk1、Dkk3 和 Crtap 是 Pax6(5a) 的特异性靶基因，而 Tgfbi、Vegfa、EphB2、Klk8 和 Edn1 被证实是 Pax6 的特异性靶基因。Nbl1、Ngfb 和七个编码不同糖基转移酶的基因似乎受到两者的调节。通过 ChIP 验证了 Crtap、Ctgf、Edn1、Dkk3、Pdgfb 和 Ngef 启动子的直接结合。此外，观察到稳定转染的 Pax6 和 Pax6(5a) 细胞形态发生变化，并且表达 Pax6 的细胞显示出增加的增殖和迁移能力。
