The RFX family interacts at the collagen (COL1A2) start site and represses transcription

Abstract

The transcription start site of the collagen α2(1) gene (COL1A2) has a sequence-specific binding site for a DNA methylation-responsive binding protein called regulatory factor for X-box 1 (RFX1) (Sengupta, P. K., Erhlich, M., and Smith, B. D. (1999) J. Biol. Chem. 274, 36649-36655). In this report, we demonstrate that RFX1 forms homodimers as well as heterodimers with RFX2 spanning the collagen transcription start site. Methylation at +7 on the coding strand increases RFX1 complex formation in gel shift assays. Methylation on the template strand, however, does not increase RFX1 complex formation. DNA from human fibroblasts contains minimal methylation on the coding strand (<4%) with variable methylation on the template strand. RFX1 acts as a repressor of collagen transcription as judged by in vitro transcription and co-transfection assays with an unmethylated collagen promoter-reporter construct. In addition, an RFX5 complex present in human fibroblasts interacts with the collagen RFX site, which is not sensitive to methylation. This is the first demonstration of RFX5 complex formation on a gene other than major histocompatibility complex (MHC) promoters. Also, RFX5 represses transcription of a collagen promoter-reporter construct in rat fibroblasts that have no detectable RFX5 complex formation or protein. RFX5 complex activates MHC II transcription by interacting with an interferon-γ (IFN-γ-inducible protein, major histocompatibility class II trans-activator (CIITA). Collagen transcription is repressed by IFN-γ in a dose-dependent manner in human but not in rat fibroblasts. IFN-γ enhances RFX5 binding activity, and CIITA is present in the RFX5 complex of IFN-γ-treated human fibroblasts. CIITA repressed collagen gene transcription more effectively in human fibroblasts than in rat fibroblasts, suggesting that the RFX5 complex may, in part, recruit CIITA protein to the collagen transcription start site. Thus the RFX family may be important repressors of collagen gene transcription through a RFX binding site spanning the transcription start site.