The surface cells of corneal epithelium are regularly shed off and replaced by new cells that are derived from limbal epithelial stem cells (LESC). LESC are believed to reside in the basal layer of the limbal epithelium and are characterized with high expression levels of ΔNp63, a transcription factor (TF) which is believed to play roles in the regulation of LESC proliferation. In this study, we examined the transcriptional regulation of ΔNp63 in limbal epithelial cell. We employed DNA pull down assay followed by LC/MS analysis and cDNA microarray analysis to identify the TFs that were capable of binding to ΔNp63 promoter or were expressed at higher levels in limbus over cornea. The TFs thus selected were further examined for their in vivo ΔNp63 promoter binding by chromatin immunoprecipitation assay. We identified six putative TFs (PAX6, EGR1, CEBPB, JUN, ATF3, and ARID5B) through the aforementioned approaches. Among them, PAX6 and EGR1 were shown to promote the transcription of ΔNp63 and led to increased cell proliferation. In contrast, CEBPB and ATF3 appeared to exert little or no effect on ΔNp63 expression, however, their silencing suppressed cell proliferation. Although JUN exhibited low promoter-binding specificity, however, it affected ΔNp63 expression and limbal epithelial cell proliferation in ways similar to that of PAX6 and EGR1. Intriguingly, ARID5B was highly expressed in the limbal epithelial cell, however, its silencing by siRNA did not obviously affect the expression of ΔNp63, nor did it reduce cell proliferation of the limbal epithelial cell.
Copyright © 2012 Wiley Periodicals, Inc.