δEF1 binds to a far upstream sequence of the mouse pro-α1(I) collagen gene and represses its expression in osteoblasts

The transcription of type I collagen genes is tightly regulated, but few cis-acting elements have been identified that can modulate the levels of expression of these genes. Generation of transgenic mice harboring various segments of the mouse pro-alpha1(I) collagen promoter led us to suspect that a repressor element was located between -10.5 and -17 kilobase pairs. Stable and transient transfection experiments in ROS17/2.8 osteoblastic cells confirmed the existence of such a repressor element at about -14 kilobase pairs and showed that it consisted in an almost perfect three-time repeat of a 41-base pair sequence. This element, which we named COIN-1, contains three E2-boxes, and a point mutation in at least two of them completely abolished its repressor effect. In gel shift assays, COIN-1 bound a DNA-binding protein named delta EF1/ZEB-1, and mutations that abolished the repressor effect of COIN-1 also suppressed the binding of delta EF1. We also showed that the repressor effect of COIN-1 was not mediated by chromatin compaction. Furthermore, overexpression of delta EF1 in ROS17/2.8 osteoblastic cells enhanced the inhibitory effect of COIN-1 in a dose-dependent manner and repressed the expression of the pro-alpha1(I) collagen gene. Thus, delta EF1 appears to repress the expression of the mouse pro-alpha1(I) collagen gene, through its binding to COIN-1.