The Y-box binding protein YB-1 suppresses collagen alpha 1(I) gene transcription via an evolutionarily conserved regulatory element in the proximal promoter
Jt. Norman et al., The Y-box binding protein YB-1 suppresses collagen alpha 1(I) gene transcription via an evolutionarily conserved regulatory element in the proximal promoter, J BIOL CHEM, 276(32), 2001, pp. 29880-29890
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.