Activation of the human PAX6 gene through the exon 1 enhancer by transcription factors SEF and Sp1

PAX6 is a transcription factor that plays a major role in ocular morphogene sis. PAX6 is expressed in the eye, central nervous system and pancreas. Two alternative promoters, P0 and P1, which are differentially regulated durin g development, drive PAX6 transcription. We identified a 57 bp cis-regulato ry element in exon 1 of the human PAX6 gene exon 1 enhancer (EIE). EIE enha nces PI-driven PAX6 expression. Three regions In E1E (E1E-1, E1E-2 and E1E- 3) have sequence similarities with binding sites of transcription factors A RP-1, IsI-1 and SEF, respectively. As shown by electrophoretic mobility shi ft assays, E1E-3, but not E1E-1 or E1E-2, bound to proteins in nuclear extr acts of human glioma cells and transcription factor SEF bound to E1E-3. As shown by transient transfection experiments, deletion or site-specific muta tions in E1E-3 dramatically decreased P1 promoter activity. Mutations in E1 E-2, however, did not affect function of the P1 promoter. Co-transfection o f SEF and PAX6 promoter-reporter constructs showed that SEF up-regulates PA X6 gene expression through the P1 promoter. Two Spl sites in the El E regio n were also shown to be important by transient co-transfection assays. Data from immunoprecipitation and transient transfection assays demonstrated th at SEF and Sp1 interacted in vitro and may act together in vivo to regulate PAX6 expression.