Identification of two repressor elements in the mouse alpha 2(I) collagen promoter

We recently identified three areas of Sp1 binding located between -568 and -453 of the 5' flanking region of the murine alpha2(I) collagen promoter which are necessary for optimal activity. We now identify two additional regions of Sp1 binding located at -371 to -351 (region 4) and at -690 to -613 (region 5), which when mutated increased promoter activity in transfected rat hepatic stellate cells indicating they contain negative regulatory elements. AP-2 bound to region 4 while YY1 bound most strongly to region 5. AP-2 decreased Sp1 binding to region 4 and had a dual effect on Sp1 binding to region 5 decreasing and increasing Sp1 binding at low and high concentrations of AP-2, respectively. YY1 enhanced Sp1 binding to both regions. AP-2 inhibited or enhanced the stimulatory effect of a transfected Sp1 expression vector on the alpha2(I) collagen promoter in Drosophila cells at low or high AP-2 expression, respectively. YY1 enhanced or inhibited the activation of the promoter by low or high Sp1 expression, respectively. This study identifies two negative regulatory elements in the murine alpha2(I) collagen promoter and shows that AP-2 and YY1 interact with Sp1 at these sites and can inhibit the activating action of Sp1.