A Methylation-responsive MDBP/RFX Site Is in the First Exon of the Collagen α2(I) Promoter

Abstract

DNA methylation inhibits transcription driven by the collagen ␣2(I) promoter and the 5 end of the gene in transient transfection and in vitro transcription assays. DNA-binding proteins in a unique family of ubiquitously expressed proteins, methylated DNA-binding protein (MDBP)/regulatory factor for X box (RFX), form specific complexes with a sequence overlapping the transcription start site of the collagen ␣2(I) gene. Complex formation increased when the CpG site at ؉7 base pairs from the transcription start site was methylated. The identity of the protein was demonstrated by co-migration and cross-competition for a characteristic slowly migrating doublet complex formed on MDBP/RFX recognition sequences and the collagen sequences by band shift assays. A RFX1-specific antibody supershifted the collagen DNA-protein complexes. Furthermore, in vitro translated RFX1 protein formed a specific complex with the collagen sequence that was also supershifted with the RFX1 antibody. MDBP/RFX displayed a higher affinity binding to the collagen sequence if the CpG at ؉7 was mutated in a manner similar to TpG. This same mutation within reporter constructs inhibited transcription in transfection and in vitro transcription assay. These results support the hypothesis that DNA methylationinduced inactivation of collagen ␣2(I) gene transcription is mediated, in part, by increased binding of MDBP/ RFX to the first exon in response to methylation in this region.Type I collagen, the most abundant collagen molecule within the collagen family, normally consists of a heterotrimer of two ␣1(I) chains and one ␣2(I) chain. Synthesis of these genes is often down-regulated upon oncogenic transformation of cells in culture (1-4). We have previously demonstrated down-regulation of the collagen ␣2(I) gene, encoding one of the subunits of Type I collagen in an epithelial-like cell line from rat liver, K16 cells upon their conversion to a tumorigenic line, W8, after treatment with the carcinogen 2-N-(acetoxyacetyl)-aminofluorine (3). The promoter-5Ј region of the ␣2 gene was methylated in the nonexpressing W8 cells and not in the expressing K16 cells (5). Furthermore, reporter gene expression downstream of the 218-bp 1 promoter and the 54-bp 5Ј region of the rat and human collagen ␣2(I) genes was inactivated by in vitro DNA methylation in transient transfection experiments, whereas an analogous expression plasmid with the SV40 early promoter/ enhancer driving expression was not (6). We also demonstrated that a minimal collagen ␣2(I) promoter containing the preinitiation region (Ϫ41 to ϩ54) driving expression of the luciferase reporter gene was inactivated by DNA methylation (7). The inhibition of reporter gene expression was attributable to CpG methylation, specifically of collagen ␣2(I) sequences. However, all the methylation sites were located in the first exon, not in the promoter. DNA methylation in the promoter and 5Ј region of genes often correlates with decreased transcription of vertebrate genes, and many studies indicate that this methyla...

Interferon γ Repression of Collagen (COL1A2) Transcription Is Mediated by the RFX5 Complex

“…Our previous data demonstrate that several members of the RFX family interact with the collagen start site and repress transcription (30,31). The RFX5 complex in human fibroblast nuclear extracts interacts with the collagen transcription start site as judged by gel shift migration patterns and blocking of binding by RFX5 and RFXB antibodies.…”

“…Since the RFX5 complex contains three proteins, we continued these experiments by examining the role of the two additional subunits of the RFX5 complex, RFXB, and RFXAP, on collagen gene transcription. Rat fibroblasts were used for these experiments because these fibroblasts do not exhibit an RFX5 binding activity or demonstrable RFX5 protein by Western analysis (30).…”

“…Previously we have demonstrated that the RFX family of transcription factors can bind to the ␣2(I) collagen gene (COL1A2) at the transcription start site (29,30). In addition, RFX5 and CIITA repress collagen gene expression in fibroblasts (31).…”

“…This protein is considered a master regulator of MHC-II transcription and its mRNA levels are greatly stimulated by IFN-␥ in many cells, including fibroblasts (14 -16). CIITA is a histone acetyltransferase capable of acetylating histones and activating transcription through its interactions with the general transcription factors (TBP, TAF II 30, TAF II 70, TF II B) (17)(18)(19) and other histone acetyltransferases (p300/CBP, pCAF) (20 -22). It has been suggested that CIITA could repress collagen through interaction with p300/CBP (23).…”

Major Histocompatibility Class II Transactivator (CIITA) Mediates Repression of Collagen (COL1A2) Transcription by Interferon γ (IFN-γ)

“…DNA Affinity Pull-down Assay-The collagen sequence (COL1A2 Ϫ25/ϩ30, Genbank TM accession number AF004877) with a HindIII overhang was synthesized as complementary strands and annealed as previously described (40). Double-stranded collagen DNA was biotinlabeled by incubating with Klenow fragment (NE Biolabs) and biotin-14-dATP (Invitrogen) supplemented with regular dCTP, dTTP, and dGTP at room temperature for 30 min.…”