The Y-box binding protein YB-1 suppresses collagen alpha 1(I) gene transcription via an evolutionarily conserved regulatory element in the proximal promoter

Appropriate expression of collagen type I, a major component of connective tissue matrices, is dependent on tight transcriptional control and a number of transactivating and repressing factors have been characterized. Here we identify the Y-box binding protein-1 (YB-1) as a novel repressor of the co llagen type alpha1(I) (COL1A1) gene. Collagen type I mRNA and protein level s decreased upon overexpression of YB-1 by transfection in NRK fibroblasts. The human, rat, and mouse COL1A1 promoter -220/+115 contains three putativ e Y-boxes, one of these sites, designated collagen Y-box element (CYE), inc ludes a Y-box. plus an adjacent 3 ' inverted repeat. DNase-I footprinting a nd Southwestern blotting with fibroblast nuclear extract, demonstrated bind ing of several nuclear proteins across the CYE, one of which was identified as YB-1. Recombinant YB-1 bound the CYE sequence in gel shift assays with a preference for single-stranded templates. The entire sequence (-88/-48) w as required for high affinity binding. Complex formation of endogenous YB-1 with the CYE was established by supershift studies. COL1A1 promoter-report er constructs were suppressed up to 80% by cotransfection with YB-1 in a va riety of cell types. In addition, CYE conferred YB-1 responsiveness on two heterologous promoters further demonstrating the importance of this repress or region. Mung bean nuclease sensitivity analysis suggested that repressio n is most likely exerted through changes in DNA conformation.