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J. Biol. Chem., Vol. 277, Issue 28, 24926-24937, July 12, 2002
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From the Department of Biochemistry, Boston University
School of Medicine, Boston and the Boston Veterans Administration
Medical Center, Boston, Massachusetts 02118
The transcription start site of the collagen
2(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-
(IFN-)-inducible protein, major histocompatibility
class II trans-activator (CIITA). Collagen transcription is repressed
by IFN- in a dose-dependent manner in human but not in
rat fibroblasts. IFN- enhances RFX5 binding activity, and CIITA is
present in the RFX5 complex of IFN--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.
To whom correspondence should be addressed: Dept. of Biochemistry,
Boston University School of Medicine, 715 Albany St., Boston, MA 02118. Tel.: 617-638-4159; Fax: 617-638-5339; E-mail:
smith@biochem.bumc.bu.edu.
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