Y-box-binding protein YB-1 mediates transcriptional repression of human α2(I) collagen gene expression by interferon-γ

Abstract

We have demonstrated previously that a proximal element within the human α2(I) collagen gene (COL1A2) promoter mediates transcriptional repression by interferon-γ (IFN-γ), and designated this region the IFN-γ response element (IgRE). Screening of a human fibroblast cDNA expression library with a radiolabeled IgRE probe exclusively yielded clones with a sequence identical to that of the transcription factor YB-1. Electrophoretic mobility shift assays (EMSA) using various IgRE-derived oligonucleotide probes containing serial two-base mutations showed that YB-1 protein was preferentially bound to the pyrimidine-rich sequence within the IgRE. This region is located immediately down-stream of and partly overlaps the previously reported Sp1/Sp3 binding site. Overexpression of YB-1 in human dermal fibroblasts decreased steady state levels of COL1A2 mRNA and repressed COL1A2 promoter activity in a dose-dependent manner. This inhibitory effect of YB-1 on COL1A2 expression was abolished by mutations of the IgRE shown to prevent YB-1 binding in EMSA. In addition, these mutations also abolished the inhibitory effect of IFN-γ, suggesting that YB-1 mediates the inhibitory action of IFN-γ on COL1A2 promoter through its binding to the IgRE. Also, overexpression of a deletion mutant YB-1, which lacks the carboxyl-terminal domain, abrogated the repression of COL1A2 transcription by IFN-γ. A functional correlation between IFN-γ and YB-1 was further supported by luciferase assays using four tandem repeats of the Y-box consensus oligonucleotide linked to a minimal promoter. EMSA and Western blot analysis using cytoplasmic and nuclear proteins implied that IFN-γ promotes the nuclear translocation of YB-1. Direct evidence for the nuclear translocation of YB-1 by IFN-γ was further provided by using a YB-1-green fluorescent protein expression plasmid transfected into human fibroblasts. Altogether, this study represents the definitive identification of the transcription factor responsible for IFN-γ-elicited inhibition of COL1A2 expression, namely YB-1.