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 is a transcription factor important for early embryo development. It is expressed in several cancer cell lines and tumors. In glioblastoma, PAX6 has been shown to function as a tumor suppressor. Dickkopf 3 (Dkk3) is well established as a tumor suppressor in several tumor types, but not much is known about the regulation of its expression. We have previously found that Pax6 and Pax6(5a) increase the expression of the Dkk3 gene in two stably transfected mouse fibroblast cell lines. In this study the molecular mechanism behind this regulation is looked at. Western blot and reverse transcriptase quantitative PCR (RT-qPCR) confirmed higher level of Dkk3 expression in both Pax6 and Pax6(5a) expressing cell lines compared to the control cell line. By the use of bioinformatics and electrophoretic mobility shift assay (EMSA) we have mapped a functional Pax6 binding site in the mouse Dkk3 promoter. The minimal Dkk3 promoter fragment required for transcriptional activation by Pax6 and Pax6(5a) was a 200 bp region just upstream of the transcriptional start site. Mutation of the evolutionary conserved binding site in this region abrogated transcriptional activation and binding of Pax6/Pax6(5a) to the mouse Dkk3 promoter. Since the identified Pax6 binding site in this promoter is conserved, RT-qPCR and Western blot were used to look for regulation of Dkk3/REIC expression in human cell lines. Six of eight cell lines tested showed changes in Dkk3/REIC expression after PAX6 siRNA knockdown. Interestingly, we observed that the Pax6/Pax6(5a) expressing mouse fibroblast cell lines were less responsive to canonical Wnt pathway stimulation than the control cell line when TOP/FOP activity and the levels of active β-catenin and GSK3-β Ser9 phosphorylation were measured after LiCl stimulation.

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