THE CHICK AND HUMAN COLLAGEN ALPHA-1(XII) GENE PROMOTER - ACTIVITY OFHIGHLY CONSERVED REGIONS AROUND THE FIRST EXON AND IN THE FIRST INTRON

A single gene encodes collagen XII, an extracellular matrix protein wi th three large fibronectin-related subunits connected via a short coll agen triple helix. Since collagen XII is a component of a specific sub set of collagen fibrils in tissues bearing high tensile stress, we are interested to know how its restricted expression is regulated. To thi s aim, we have isolated the region around the first exon of both the c hick and human collagen alpha 1(XII) gene. The upstream sequences of t he two genes share common features but are not related. Strong similar ity starts about 100 bp 5' of the first exon and ends 100 bp into the first intron. In addition, two large conserved regions (56-63% similar ity) were found in the first intron. A single major and two clusters o f minor transcription start sites were identified in both the chick an d human gene. To test for promoter activity, conserved fragments from the chick gene were cloned into reporter plasmids for transient transf ection of fibroblasts. A 70-bp stretch containing a conserved nuclear factor-1 binding sequence just upstream of the first transcription sta rt site was found to work as a basal promoter. An adjacent, but nonove rlapping short segment including the more downstream start sites and a conserved TATTAA sequence exhibited independent promoter activity. GC -rich sequences just 5' and 3' of the minimal promoter fragments were required for full activity. In contrast, inclusion of more upstream se quences (up to 2.4 kb) had no effect. The two conserved regions in the first intron showed no promoter activity on their own but modulated a ctivity when linked to autologous or heterologous promoters. Specifica lly, one of these intronic regions might contain enhancer element(s) t hat respond to mechanical stress acting on the fibroblasts. We conclud e that the collagen XII gene is driven by a basal promoter with two ha lves that can act independently; conserved control regions are located around the first exon and in the first intron.