Y-box-binding protein YB-1 mediates transcriptional repression of human alpha 2(I) collagen gene expression by interferon-gamma.
We have demonstrated previously that a proximal element within the human alpha2(I) collagen gene (COL1A2) promoter mediates transcriptional repression by interferon-gamma (IFN-gamma), and designated this region the IFN-gamma 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 downstream 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-gamma, suggesting that YB-1 mediates the inhibitory action of IFN-gamma 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-gamma. A functional correlation between IFN-gamma 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-gamma promotes the nuclear translocation of YB-1. Direct evidence for the nuclear translocation of YB-1 by IFN-gamma 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-gamma-elicited inhibition of COL1A2 expression, namely YB-1.
Higashi
Kiyoshi
K
Environmental Health Science Laboratory, Sumitomo Chemical Company, Ltd., Konohana-ku, Osaka 554-8558, Japan. higashik2@sc.sumitomo-chem.co.jp
Inagaki
Yutaka
Y
Suzuki
Noriyuki
N
Mitsui
Shinichi
S
Mauviel
Alain
A
Kaneko
Hideo
H
Nakatsuka
Iwao
I
D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics*; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics*; D004268:DNA-Binding Proteins; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000187:drug effects; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; D024042:Collagen Type I; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D007371:Interferon-gamma