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. 2001 Oct 1;29(19):4070-8.
doi: 10.1093/nar/29.19.4070.

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

J B Zheng  1 , Y H Zhou, T Maity, W S Liao, G F Saunders
Affiliations

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

J B Zheng et al. Nucleic Acids Res. .

Abstract

PAX6 is a transcription factor that plays a major role in ocular morphogenesis. PAX6 is expressed in the eye, central nervous system and pancreas. Two alternative promoters, P0 and P1, which are differentially regulated during development, drive PAX6 transcription. We identified a 57 bp cis-regulatory element in exon 1 of the human PAX6 gene exon 1 enhancer (EIE). EIE enhances P1-driven PAX6 expression. Three regions in E1E (E1E-1, E1E-2 and E1E-3) have sequence similarities with binding sites of transcription factors ARP-1, Isl-1 and SEF, respectively. As shown by electrophoretic mobility shift assays, E1E-3, but not E1E-1 or E1E-2, bound to proteins in nuclear extracts of human glioma cells and transcription factor SEF bound to E1E-3. As shown by transient transfection experiments, deletion or site-specific mutations in E1E-3 dramatically decreased P1 promoter activity. Mutations in E1E-2, however, did not affect function of the P1 promoter. Co-transfection of SEF and PAX6 promoter-reporter constructs showed that SEF up-regulates PAX6 gene expression through the P1 promoter. Two Sp1 sites in the E1E region were also shown to be important by transient co-transfection assays. Data from immunoprecipitation and transient transfection assays demonstrated that SEF and Sp1 interacted in vitro and may act together in vivo to regulate PAX6 expression.

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Figures

Figure 1
Figure 1
Nucleotide sequence of the P1 promoter of the human PAX6 gene. The transcriptional start site (+1) is marked with the bent arrow. The cis element E1E is indicated in bold. The TATA-like box, CCAAT boxes and potential cis-regulatory elements in the proximal promoter region mapped by Xu and Saunders (13) and Okladnova et al. (20) are either in boxes or underlined and labeled. Essential restriction sites used in subsequent subcloning are underlined and labeled above the sequence. Intron 1 is in lower case and labeled. The exon 2 region is labeled.
Figure 2
Figure 2
EMSA of potential protein binding sites in E1E. 32P-labeled probes for E1E-1, E1E-2, mE1E2, E1E-3, m1E1E3, m2E1E3 and m3E1E3 (Table 2) sites were incubated with equal amounts of protein in nuclear extracts from U251 cells.
Figure 3
Figure 3
DNA binding specificity of SEF to E1E-3. (A) EMSA with nuclear extracts. Double-stranded oligonucleotides of E1E-3 in the PAX6 P1 promoter and the SEF binding site (SEF) from the serum amyloid A3 promoter were 32P-labeled and incubated with nuclear extracts from LN229 cells. DNA–protein complexes were competed with wild-type or mutant E1E-3 or SEF oligonucleotides (lanes 2–5 and 7–10). (B) The reaction was incubated with preimmune or anti-SEF antibodies to supershift the DNA–protein complex (lanes 1–5 and 6–10) and the amounts of anti-SEF antibodies were increased to supershift the DNA–protein complex.
Figure 4
Figure 4
DNA binding properties of SEF protein to E1E-3. In an experiment similar to that in Figure 3, 32P-labeled E1E-3 or SEF oligonucleotides were incubated with SEF protein. The competitors used were the wild-type or mutant oligonucleotides, E1E-3 or SEF (lanes 2–5 and 7–10) or preimmune serum or anti-SEF antibodies to abolish the DNA–protein complex (lanes 12–15 and 17–20). In lanes 13–15 and 18–20 the amounts of anti-SEF antibodies were increased to abolish the DNA–protein complex.
Figure 5
Figure 5
Luciferase activity of the PAX6 P1 promoter with various mutations. The 346pGL3B construct was prepared by inserting the 346 bp SmaI–NaeI region of the human PAX6 P1 promoter into a luciferase basic vector (pGL3B). Each binding site was mutated (as described in Materials and Methods). U251 and LN229 cells were transfected with 0.5 µg of the constructs shown. The results were normalized to the activity of the control pGL3 basic transfected cells, to which a value of 100 was assigned.
Figure 6
Figure 6
Co-immunoprecipitation analysis of the association of SEF and Sp1. (A) Nuclear extract from LN229 cells was subjected to immunoprecipitation with anti-SEF antibody, anti-Sp1 antibody or anti-PAX6 antibody (as a control). In vitro synthesized Sp1 protein was used to indicate the position of the Sp1 band (lane 5) and a sample of in vitro synthesized SEF protein was used as a negative control for immunoblotting (lane 1). (B) In vitro synthesized SEF and Sp1 were immunoprecipitated together with anti-SEF antibody or anti-Sp1 antibody (lanes 6 and 9) or immuniprecipitated separately with anti-SEF antibody or anti-Sp1 antibody (lanes 7, 8, 10 and 11). Anti-Sp1 antibody was used to probe the membrane.
Figure 7
Figure 7
Transactivation of the PAX6 promoter by SEF and Sp1 expression constructs. (A) LN229 cells were co-transfected with 50 ng of 346pGL3B and increasing amounts of SEF expression plasmid DNA. The luciferase activities were normalized to the activity of 346pGL3B with co-transfection of the empty vector, to which a value of 1.0 was assigned (column 1). (B) LN229 cells were co-transfected with 50 ng of 346pGL3B and increasing amounts of the Sp1 expression plasmid DNA. The data were calculated from nine independent experiments repeated three or four times.
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