Effect of CTCF-binding motif on regulation of PAX6 transcription.

PURPOSE Previous studies indicate that the CCCTC binding transcription factor (CTCF) regulates homeobox PAX6 gene transcription in corneal epithelial cells. In the present study, the effect was investigated of CTCF activity on PAX6 transcription through interaction with five essential motifs located in an 80-bp region upstream from the PAX6 P0 promoter. METHODS An electrophoretic mobility shift assay (EMSA) was used to determine the interaction between CTCF and DNA binding motifs. DNA mutagenesis was applied in identification of DNA motif functions. Immunohistochemistry and Western blot analyses were performed to detect the stress-induced effect on CTCF activity. RESULTS The five identified CTCF-binding motifs were mutated one by one or in different combinations. Interactions of CTCF with these mutated motifs were determined by EMSA and DNA-binding competitions. All five CCCTC motifs were functional for the CTCF binding and DNA-binding activity of CTCF was proportionally decreased after increases in mutations of motif numbers. In addition, ultraviolet (UV) irradiation and epidermal growth factor (EGF) induced suppression and activation of CTCF expression, respectively. Effects of UV and EGF induction were due to alterations in CTCF expression and activity resulting in changes in CTCF DNA binding activity to the PAX6 promoter region detected by EMSA. CONCLUSIONS These findings indicate that CTCF regulates PAX6 expression in response to stress-induced conditions and that the molecular base of CTCF controlling PAX6 expression is through five functional and specific motifs in the region upstream from the PAX6 P0 promoter in corneal epithelial cells.