Abstract
The first intron of the human Proα1(I) collagen gene contains an orientation-dependent enhancer composed of both positive and negative cis-acting elements involved in the transcriptional regulation of this gene. Deletion of a 360 bp Sau 3A intronic fragment spanning nucleotide + 494 to + 854 (S360) resulted in dramatic down-regulation of pCOL-KT (Thompson et al., J Biol Chem 266: 2549–2556, 1991). Using a DNaseI protection assay, we demonstrate a single footprint located at + 590 to + 615 in the S360 fragment; nuclear extracts prepared from mesenchymal and nonmesenchymal cells exhibited similar binding characteristics. A double stranded oligonucleotide representing a consensus Ap-1 binding sequence competed with S360 for binding. In contrast to what occurred in response to S360 deletion which was always accompanied by reduced expression, the deletion of the Ap-1 binding site (+ 598 to + 604) caused either increased or decreased expression of the reporter gene depending on the target cell. Site-directed mutations in the Ap-1-like cis-element of Proα1(I) were also tested in transient expression assays. Consistent with the paradoxical results of Ap-1 deletion, we observed that the functional consequences of mutations in the Ap-1 site also varied in different cells. In A204 cells, one point mutation, which resulted in the loss of protein binding to S360, led to increased CAT activity while another point mutant, which retained binding of the Ap-1 like trans-acting factor(s), showed decreased CAT expression. The effects of these two mutations in the HFL-1 cells were exactly opposite of what was seen for A204 cells. Based on these observations, we postulate that the Ap-1 site plays a critical role in the transcriptional activity of the human Proα1(I) gene. The implications of an apparently dual mode of regulation through a single cis-regulatory element are discussed. (Mol Cell Biochem 118: 119–129, 1992)
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References
Raghow R, Thompson JP: Molecular mechanisms of collagen gene expression. Mol Cell Biochem 86: 5–18, 1989
Bornstein P, Sage H: Regulation of collagen gene expression. Prog Nucleic Acid Res Mol Biol 37: 67–106, 1989
Ramirez F, DiLiberto M: Complex and diversified regulatory programs control the expression of vertebrate collagen genes. FASEB J 4: 1616–1623, 1990
Vuorio E, de Crombrugghe B: The family of collagen genes. Ann Rev Biochem 59: 837–872, 1990
Rossouw CMS, Vergeer WP, du Plooy SJ, Bernard MP, Ramirez F, de Wet WJ: DNA sequences in the first intron of the human Proα1-(I) collagen gene enhance transcription. J Biol Chem 262: 15151–15157, 1987
Bornstein P, McKay J: The first intron of the α1(I) collagen gene contains several transcriptional regulatory elements. J Biol Chem 263: 1603–1606, 1988
Bornstein P, McKay J, Liska DJ, Apone S, Devarayalu S: Interactions between the promoter and first intron are involved in transcriptional control of α1(I) collagen gene expression. Mol Cell Biol 8: 4851–4857, 1988
Boast S, Su W, Ramirez F, Sanchez M, Avvedimento EV: Functional analysis of cis-acting DNA sequences controlling transcription of the human type I collagen gene. J Biol Chem 265: 13351–13356, 1990
Olsen A, Geddis AE, Prockop DJ: High levels of expression of a minigene version of the human Proα1(I) collagen gene in stably-transfected mouse fibroblasts: effects of deleting putative regulatory sequences in the first intron. J Biol Chem 266: 1117–1121, 1991
Slack JL, Liska DJ, Bornstein P: A upstream regulatory region mediates high level, tissue-specific expression of the human α1(I) collagen gene in transgenic mice. Mol Cell Biol 11: 2066–2074, 1991
Liska DJ, Slack JL, Bornstein P: A highly conserved intronic sequence is involved in transcriptional regulation of the α1(I) collagen gene. Gene Reg. 1: 487–498, 1990
Thompson JP, Simkevich CP, Hotness MA, Kang AH, Raghow R: In vitro methylation of the promoter and enhancer of Pro alpha 1(I) collagen gene leads to its transcriptional inactivation. J Biol Chem 266: 2549–2556, 1991
Reue K, Leff T, Breslow JL: Human apolipoprotein CIII gene expression is regulated by positive and negative cis-acting elements and tissue-specific protein factors. J Biol Chem 263: 6852–6864, 1988
Briggs MR, Kadonaga JT, Bell SP, Tjian R: Purification and biochemical characterization of the promoter-specific transcription factor, Sp1. Science 234: 47–52, 1986
Harrington MA, Jones PA, Imagawa M, Karin M: Cytosine methylation does not affect binding of transcription factor Sp-1. Proc Natl Acad Sci USA 85: 2066–2070, 1988
Revzin A: Gel electrophoresis assay for DNA protein interactions. Biotechniques 7: 346–355, 1989
Kunkel TA: Rapid and efficient site-specific mutagenesis without phenotype selection. Proc Natl Acad Sci USA 82: 488–492, 1985
Graham F, van der Eb A: A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52: 456–457, 1973
Gorman CM, Moffat LF, Howard BH: Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol 2: 1044–1051, 1982
Herbomel P, Bourachot B, Yaniv M: Two distinct enhancers with different cell specificities coexist in the regulatory region of polyoma. Cell 39: 653–655, 1984
Simkevich CP, Thompson JP, Poppleton H, Raghow R: Transcriptional tissue specificity of the human Proα1(I) collagen gene is determined by a negative cis-regulatory element in the promoter. Biochem J 286: 179–185, 1992
Mitchell PJ, Tjian R: Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 245: 371–378, 1989
Imagawa M, Chiu R, Karin M: Transcription factor AP-2 mediates induction by two different signal-transduction pathways: protein kinase C and cAMP. Cell 51: 251–260, 1987
Lee W, Haslinger A, Karin M, Tjian R: Activation of transcription by two factors that bind promoter and enhancer sequences of the human metallothionein gene and SV40. Nature 325: 368–372, 1987
Mermod N, Trevor TJ, Tjian R: Enhancer binding factors AP-4 and AP-1 act in concert to activate SV40 late transcription in vitro. Nature 332: 557–561, 1988
Angel P, Karin M: The role of jun, fos and the Ap-1 complex in cell proliferation and transformation. Biochem Biophys Acta 1072: 129–157, 1991
Curran T, Franza R: Fos and jun: the Ap-1 connection. Cell 55: 395–397, 1988
Kuroki Y, Shinozawa S, Sugimoto T, Fujita T: Constitutive expression of c-fos gene inhibits type I collagen synthesis in transformed ostcoblasts. Biochem Biophys Res Comm 182: 1389–1394, 1992
Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson D: Control of gene expression. In: Molecular Biology of the Cell. Garland Publishers, New York and London, 1989, pp 551–612
Atchison ML: Enhancers: mechanism of action and cell specificity. Ann Rev Cell Biol 4: 127–153, 1988
Maniatis T, Goodbourn S, Fischer J: Regulation of inducible and tissue-specific gene expression. Science 236: 1237–1245, 1987
Serfling E, Jasin M, Schaffner W: Enhancers and eukaryotic gene transcription. Trends Genet 1: 224–230, 1985
Sherwood AL, Bornstein P: Transcriptional control of the α1(I) collagen gene involves orientation and position specific intronic sequences. Biochem J 265: 895–897, 1990
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Katai, H., Stephenson, J.D., Simkevich, C.P. et al. An AP-1-like motif in the first intron of human Proα1(I) collagen gene is a critical determinant of its transcriptional activity. Mol Cell Biochem 116, 119–129 (1992). https://doi.org/10.1007/BF00299391
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DOI: https://doi.org/10.1007/BF00299391