CTCF Mediates Cytokine– and Stress–Induced Alterations in Pax6 Transcription

Abstract

Abstract: : Purpose: Our previous study found that CTCF negatively controls Pax6 gene expression through its interaction with a repressor element located at its upstream promoter. The present work explored the molecular mechanism of this interaction. Methods:Human and rabbit corneal epithelial cells (HCE and RCE cells) were cultured in DMEM/F12 medium containing 10% FBS in 37 °C incubator gassed with 5% CO₂. Western analysis was used to determine target protein expressions. Pax6 transcriptional activity was measured with the luciferase reporter assay. The CTCF binding activity to the Pax6 promoter repressor was determined with the gel retardation assay for DNA–binding proteins. The gel retardation assay used normal DNA repressor fragment or its various mutants with CTCF binding sites. Results:UV irradiation reduced CTCF protein content in corneal epithelial cell, resulting in decrease in CTCF DNA binding activity. In contrast, EGF (0 ng/ml) stimulated CTCF expression and attenuated Pax6 repressor DNA–binding activity. The protein–binding ability of the Pax6 promoter repressor was dependent on its 5 binding sites (CCCTC sequence) to the CTCF protein. When the 5 CTCF–binding sites repeats were mutated, respectively with single, double, triple and quadruple site mutations, the protein–binding activity of the Pax6 promoter repressor was progressively reduced. When all 5 CTCF binding sites were mutated, its protein–binding activity was no longer detectable. The results clearly indicate that the Pax6 promoter repressor interacts with the CTCF protein. Additionally, the reporter assay showed that the transcriptional activity of the Pax6 promoter was undoubtedly increased when its CTCF binding sites in the repressor element were mutated, which indicates the functional significance of this physical interaction between the Pax6 promoter repressor and the CTCF protein. Conclusions: Our results reveal a regulatory mechanism that involves Pax6 transcription regulation. This suggests that CTCF protein regulation of Pax6 transcription plays a significant role in controlling corneal epithelial cell growth and fate in response to cytokine and stress stimulation.

Keywords: gene/expression • transcription factors • stress response