Identification and characterization of conserved cis-regulatory elements in the human keratocan gene promoter

Keratocan. along with lumican and mimecan, represent the keratan sulfate-co ntaining proteoglycans of the vertebrate cornea that play a key role in dev elopment and maintenance of corneal transparency. In this study, we cloned 4.1 kb of the human Kera 5'-flanking, region and characterized the promoter structure. Using primer extension and ribonuclease protection assay, we id entify two major transcriptional start sites in the first exon. Using lucif erase reporter gene transfection analysis of 5'-deletion and mutation const ructs, we demonstrate positive and negative regulatory elements within a 1. 3 kb upstream sequence. Comparison of human and bovine 5'-flanking sequence s reveals three highly conserved regions: a 450 bp region in the first exon , a 92 bp promoter proximal conserved regulatory region identified as an en hancer in the natural context, and a 223 bp promoter distal conserved regul atory region identified as a silencer both in the natural context and in a heterologous promoter system. In addition, a conserved CArG-box residing 85 1 bp upstream of the first transcription start site also can lead to the re pression of Kera expression in cultured corneal keratocytes. DNaseI footpri nting and electrophoretic mobility shift assay demonstrate that cell type-s pecific factors bind to regulatory elements located in the conserved region s. Competition experiments indicate that the CTC factor and a protein that binds to the CAGA motif are likely to be among the multiple factors involve d in the transcriptional regulation of the human Kera gene. (C) 2000 Elsevi er Science B.V. All rights reserved.