Epithelium specific Ets-2 (ESE-2), an epithelium-specific ETS-domain transcription factor, is highly expressed in differentiated keratinocytes. To understand the molecular mechanisms that govern the cell-type and differentiation-specific expression of ESE-2 in keratinocytes, we have focused our studies on the identification and characterization of its cis-regulatory elements. We first performed DNase I hypersensitive site mapping and demonstrated that the promoter region of ESE-2 is in open chromatin conformation in differentiated keratinocytes. Next, we performed transient transfection assays with several 5' serially deleted constructs containing segments of the ESE-2 promoter. These experiments have led to the identification of a short fragment that shows remarkable sequence conservation between several species and harbors most of the transcriptional activity. Interestingly, a high level of transcriptional activity was only observed when the transfected keratinocytes were induced to differentiate by increasing the calcium concentration in the cell-culture medium. To identify the factors that mediate the transcriptional activity, we analyzed this segment by mutational and electrophoretic mobility shift assays (EMSA) experiments. Our studies have identified a critical stretch of nucleotides that is important for both basal as well as calcium responsive reporter activity and that binds to a nuclear factor, keratinocyte restricted factor (KRF). KRF is a novel transcription factor that is restricted to nuclear extracts isolated from keratinocytes and that binds to unique DNA sequences, which do not resemble any known consensus binding motif for transcription factors. Our preliminary experiments shed light on the biochemical nature of KRF and set the stage for future studies in identification of KRF and testing its role in governing ESE-2 gene expression in vivo.
2005 Wiley-Liss, Inc.