Collagen α1(I) Gene (COL1A1) Is Repressed by RFX Family

Abstract

Collagen type I is composed of three polypeptide chains transcribed from two separate genes (COL1A1 and COL1A2) with different promoters requiring coordinate regulation. Our recent publications, centering on COL1A2 regulation, demonstrate that methylation in the first exon of COL1A2 at a regulatory factor for X box (RFX) site (at ؊1 Collagen, a major extracellular matrix component, plays a significant role influencing cell behavior and maintaining tissue structure (1). Type I collagen, the most abundant member of the collagen family, is composed of two identical ␣1(I) polypeptide chains and a similar, but distinct polypeptide chain designated ␣2(I). Collagen expression is controlled in a developmental and tissue-specific manner. In adult tissue, type I gene expression is largely inactivated, but stimulated after injury. The two type I collagen genes (COL1A1 and COL1A2) are within separate chromosomes with different regulatory sequences in their promoters, yet they need to be regulated coordinately to ensure proper function.toSynthesis of collagen by cells in culture is down-regulated upon oncogenic transformation with viruses or chemical carcinogens (2-5). We have previously demonstrated (5) that collagen ␣2(I) is not expressed in a tumorigenic cell line, W8, after treatment of the parental liver epithelial-like cell line, K16, with the carcinogen 2-N-(acetoxyacetyl)-aminofluorine. Analysis of genomic DNA isolated from W8 cells revealed that the promoter-5Ј region of the ␣2(I) gene, COL1A2, was methylated (6). Furthermore, reporter constructs containing the COL1A2 promoter region (218 bp), as well as the first exon (54 bp) of the COL1A2 gene, were inactivated by DNA methylation in transient transfection experiments and in vitro transcription assays (6, 7). The inhibition of reporter gene expression was attributable to CpG methylation within the first exon surrounding the transcription start site of the COL1A2 gene. Finally, the collagen transcription start site (Ϫ1 to ϩ20) contains a low affinity-binding site for the regulatory factor for X box (RFX) family (8, 9). The binding affinity of RFX1 is increased if the CpG site at ϩ7 is methylated or mutated to thymidine on the coding strand.RFX1 is a member of the RFX family of closely related proteins, , that can bind methylated DNA sequences with higher affinity within a sequence-specific 14-bp consensus sequence. Methylation-dependent binding sites have been located for RFX at the beginning of human genes such as hypoxanthine phosphoribosyltransferase, ␣-galactosidase A, human leukocyte antigens, and the apoferritin H gene (14) suggesting a role for this protein family in DNA methylation induced gene repression. Most importantly, the RFX proteins repress COL1A2 gene expression in transient transfection as well as in vitro transcription assays (8).Our data suggest that RFX5 complex and CIITA play an important role in repression of COL1A2 gene when fibroblast cells are treated with IFN-␥ 1 (15, 16). RFX5, a member of the RFX family binds to DNA in a sequence-specifi...

Peroxisome Proliferator-activated Receptor γ Interacts with CIITA·RFX5 Complex to Repress Type I Collagen Gene Expression

“…Therefore, we chose instead to use DNA competitors to demonstrate the specificity and recruitment of RFX binding in DNA affinity pulldown assays. It should be pointed out that this method has been exploited before and has satisfactorily served the same purpose in demonstrating the specificity and recruitment dependence or RFX5⅐CIITA complex to this site (22)(23)(24). Further experiments extending this line of investigation are currently underway and will eventually provide extra evidence to our hypothesis.…”

“…4 and 5). Combined with the finding that TGF-␤ attenuates CIITA expression (41,42), we propose that CIITA is a key protein, when facilitated by other factors such as RFX5 (22,24), which determines the ultimate consequences of collagen transcription in response to different signaling pathways. Therefore, we investigated whether PPAR␥ agonists alter the IFN-␥ response through the CIITA⅐RFX5 complex.…”

“…A larger col1a2-luciferase construct (pGL3-ColLuc) containing sequences from Ϫ357 to ϩ55 bp of mouse col1a2 promoter fused to the luciferase reporter gene was a gift from Dr. Jenny Ting (29). The human COL1A1 promoter plasmids were all cloned into a pGL2-luciferase vector from the Ϫ804hCOL1-luc plasmid described previously (24). Fulllength PPAR␥ construct was generated as previously described (30).…”

“…We have demonstrated that interferon-␥ (IFN-␥), an important inflammatory cytokine, represses collagen transcription, in part, through the induction of regulatory factor for X box 5 (RFX5) complex proteins and Class II transactivator (CIITA), and their recruitment to the collagen transcription start site (22)(23)(24). RFX5 and CIITA are important activators of the immune response through transcriptional activation of major histocompatibility complex II (MHC II).…”

Detection of COL1A1-PDGFB fusion transcripts and PDGFB/PDGFRB mRNA expression in dermatofibrosarcoma protuberans

“…It is known that the COL1A1 promoter activity is sensitive to DNA methylation and the COL1A1 gene is methylated in some human cancer cells. 28 Such epigenetic change could affect the transcription of the COL1A1-PDGFB fusion gene.…”