Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Feb 15;322(Pt 1):199–206. doi: 10.1042/bj3220199

Regulation of the human alpha 2(1) procollagen gene by sequences adjacent to the CCAAT box.

M Collins 1, V D Leaner 1, M Madikizela 1, M I Parker 1
PMCID: PMC1218177  PMID: 9078262

Abstract

The human, rat, mouse and chicken alpha 2(1) procollagen promoters analysed to date all contain an inverted CCAAT box at -80. In this study we have examined the binding of nuclear proteins to the proximal promotor of the human alpha 2(1) procollagen gene, where an inverted CCAAT box is flanked by a downstream GGAGG sequence and its inverted counterpart (CCTCC) on the upstream end. Each of the GGAGG sequences is separated from the inverted CCAAT box by a single pyrimidine nucleotide (5'-CCTCCCATTGGTGGAGGCCCTTTT-3'). Electrophoretic mobility-shift assays (EMSAs) revealed that two distinct DNA-protein complexes formed on this DNA sequence. Methylation interference analysis and in vitro mutagenesis studies revealed that the integrity of the sequence 5'-CCTCCCATTGG-3' (the GGAGG/CCAAT-binding element or G/CBE) was important for the binding of the CCAAT-binding factor (CBF) (complex I). Competition studies showed that complex formation on the human G/CBE could be competed by mouse CBE and nuclear factor-Y (NF-Y) oligonucleotides, suggesting that mouse CBE and human G/CBE-binding proteins belong to the same family of CCAAT box binding proteins. Furthermore, antibodies to mouse CBF specifically supershifted the G/CBE complex (complex I) in EMSAs. The downstream GGAGG and 3'-flanking sequences (5'-GGAGGCCCTTTT-3') or collagen modulating element (CME), however, were important for the formation of a novel DNA protein complex (complex III). The formation of this complex was not competed out by CBE or NF-Y oligonucleotides, nor was DNA-protein complex formation affected by the anti-CBF antibody. Functional analysis of G/CBE and CME elements subjected to mutagenesis, using promoter-chloroamphenicol acetyl transferase constructs in transient transfection assays, showed that both these elements were essential for activity of the human promoter. These experiments identified a novel regulatory element in the human alpha 2(1) procollagen gene which is not present in the rodent gene.

Full Text

The Full Text of this article is available as a PDF (530.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Boast S., Su M. W., Ramirez F., Sanchez M., Avvedimento E. V. Functional analysis of cis-acting DNA sequences controlling transcription of the human type I collagen genes. J Biol Chem. 1990 Aug 5;265(22):13351–13356. [PubMed] [Google Scholar]
  2. Bornstein P. Regulation of expression of the alpha 1 (I) collagen gene: a critical appraisal of the role of the first intron. Matrix Biol. 1996 Apr;15(1):3–10. doi: 10.1016/s0945-053x(96)90121-3. [DOI] [PubMed] [Google Scholar]
  3. Bornstein P., Sage H. Regulation of collagen gene expression. Prog Nucleic Acid Res Mol Biol. 1989;37:67–106. doi: 10.1016/s0079-6603(08)60695-9. [DOI] [PubMed] [Google Scholar]
  4. Bornstein P., Sage H. Structurally distinct collagen types. Annu Rev Biochem. 1980;49:957–1003. doi: 10.1146/annurev.bi.49.070180.004521. [DOI] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Chodosh L. A., Baldwin A. S., Carthew R. W., Sharp P. A. Human CCAAT-binding proteins have heterologous subunits. Cell. 1988 Apr 8;53(1):11–24. doi: 10.1016/0092-8674(88)90483-7. [DOI] [PubMed] [Google Scholar]
  7. De Crombrugghe B., Karsenty G., Maity S., Vuorio T., Rossi P., Ruteshouser E. C., McKinney S. H., Lozano G. Transcriptional mechanisms controlling types I and III collagen genes. Ann N Y Acad Sci. 1990;580:88–96. doi: 10.1111/j.1749-6632.1990.tb17921.x. [DOI] [PubMed] [Google Scholar]
  8. Dickson L. A., de Wet W., Di Liberto M., Weil D., Ramirez F. Analysis of the promoter region and the N-propeptide domain of the human pro alpha 2(I) collagen gene. Nucleic Acids Res. 1985 May 24;13(10):3427–3438. doi: 10.1093/nar/13.10.3427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Framson P., Bornstein P. A serum response element and a binding site for NF-Y mediate the serum response of the human thrombospondin 1 gene. J Biol Chem. 1993 Mar 5;268(7):4989–4996. [PubMed] [Google Scholar]
  11. Goldberg H., Helaakoski T., Garrett L. A., Karsenty G., Pellegrino A., Lozano G., Maity S., de Crombrugghe B. Tissue-specific expression of the mouse alpha 2(I) collagen promoter. Studies in transgenic mice and in tissue culture cells. J Biol Chem. 1992 Sep 25;267(27):19622–19630. [PubMed] [Google Scholar]
  12. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  13. Guenette D. K., Ritzenthaler J. D., Foley J., Jackson J. D., Smith B. D. DNA methylation inhibits transcription of procollagen alpha 2(I) promoters. Biochem J. 1992 May 1;283(Pt 3):699–703. doi: 10.1042/bj2830699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hatamochi A., Golumbek P. T., Van Schaftingen E., de Crombrugghe B. A CCAAT DNA binding factor consisting of two different components that are both required for DNA binding. J Biol Chem. 1988 Apr 25;263(12):5940–5947. [PubMed] [Google Scholar]
  15. Hatamochi A., Paterson B., de Crombrugghe B. Differential binding of a CCAAT DNA binding factor to the promoters of the mouse alpha 2(I) and alpha 1(III) collagen genes. J Biol Chem. 1986 Aug 25;261(24):11310–11314. [PubMed] [Google Scholar]
  16. Herbomel P., Bourachot B., Yaniv M. Two distinct enhancers with different cell specificities coexist in the regulatory region of polyoma. Cell. 1984 Dec;39(3 Pt 2):653–662. doi: 10.1016/0092-8674(84)90472-0. [DOI] [PubMed] [Google Scholar]
  17. Inagaki Y., Truter S., Ramirez F. Transforming growth factor-beta stimulates alpha 2(I) collagen gene expression through a cis-acting element that contains an Sp1-binding site. J Biol Chem. 1994 May 20;269(20):14828–14834. [PubMed] [Google Scholar]
  18. Karsenty G., Golumbek P., de Crombrugghe B. Point mutations and small substitution mutations in three different upstream elements inhibit the activity of the mouse alpha 2(I) collagen promoter. J Biol Chem. 1988 Sep 25;263(27):13909–13915. [PubMed] [Google Scholar]
  19. Karsenty G., de Crombrugghe B. Conservation of binding sites for regulatory factors in the coordinately expressed alpha 1 (I) and alpha 2 (I) collagen promoters. Biochem Biophys Res Commun. 1991 May 31;177(1):538–544. doi: 10.1016/0006-291x(91)92017-e. [DOI] [PubMed] [Google Scholar]
  20. Kim C. G., Sheffery M. Physical characterization of the purified CCAAT transcription factor, alpha-CP1. J Biol Chem. 1990 Aug 5;265(22):13362–13369. [PubMed] [Google Scholar]
  21. Lichtsteiner S., Schibler U. A glycosylated liver-specific transcription factor stimulates transcription of the albumin gene. Cell. 1989 Jun 30;57(7):1179–1187. doi: 10.1016/0092-8674(89)90055-x. [DOI] [PubMed] [Google Scholar]
  22. Maity S. N., Golumbek P. T., Karsenty G., de Crombrugghe B. Selective activation of transcription by a novel CCAAT binding factor. Science. 1988 Jul 29;241(4865):582–585. doi: 10.1126/science.3399893. [DOI] [PubMed] [Google Scholar]
  23. Maity S. N., de Crombrugghe B. Biochemical analysis of the B subunit of the heteromeric CCAAT-binding factor. A DNA-binding domain and a subunit interaction domain are specified by two separate segments. J Biol Chem. 1992 Apr 25;267(12):8286–8292. [PubMed] [Google Scholar]
  24. Maity S. N., de Crombrugghe B. Biochemical analysis of the B subunit of the heteromeric CCAAT-binding factor. A DNA-binding domain and a subunit interaction domain are specified by two separate segments. J Biol Chem. 1992 Apr 25;267(12):8286–8292. [PubMed] [Google Scholar]
  25. McKeon C., Ohkubo H., Pastan I., de Crombrugghe B. Unusual methylation pattern of the alpha 2 (l) collagen gene. Cell. 1982 May;29(1):203–210. doi: 10.1016/0092-8674(82)90104-0. [DOI] [PubMed] [Google Scholar]
  26. Namba M., Nishitani K., Kimoto T. Characteristics of WI-38 cells (WI-38 CT-1) transformed by treatment with Co-60 gamma rays. Gan. 1980 Jun;71(3):300–307. [PubMed] [Google Scholar]
  27. Niederreither K., D'Souza R. N., de Crombrugghe B. Minimal DNA sequences that control the cell lineage-specific expression of the pro alpha 2(I) collagen promoter in transgenic mice. J Cell Biol. 1992 Dec;119(5):1361–1370. doi: 10.1083/jcb.119.5.1361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Oikarinen J., Hatamochi A., de Crombrugghe B. Separate binding sites for nuclear factor 1 and a CCAAT DNA binding factor in the mouse alpha 2(I) collagen promoter. J Biol Chem. 1987 Aug 15;262(23):11064–11070. [PubMed] [Google Scholar]
  29. Parker M. I., Smith A. A., Gevers W. Absence of alpha 2(1) procollagen synthesis in a clone of SV40-transformed WI-38 human fibroblasts. J Biol Chem. 1989 May 5;264(13):7147–7152. [PubMed] [Google Scholar]
  30. Parker M. I., Smith A. A., Mundell K., Collins M., Boast S., Ramirez F. The abolition of collagen gene expression in SV40-transformed fibroblasts is associated with trans-acting factor switching. Nucleic Acids Res. 1992 Nov 11;20(21):5825–5830. doi: 10.1093/nar/20.21.5825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Parker M. I., de Haan J. B., Gevers W. DNA hypermethylation in sodium butyrate-treated WI-38 fibroblasts. J Biol Chem. 1986 Feb 25;261(6):2786–2790. [PubMed] [Google Scholar]
  32. Pogulis R. J., Freytag S. O. Contribution of specific cis-acting elements to activity of the mouse pro-alpha 2(I) collagen enhancer. J Biol Chem. 1993 Feb 5;268(4):2493–2499. [PubMed] [Google Scholar]
  33. Prockop D. J., Kivirikko K. I., Tuderman L., Guzman N. A. The biosynthesis of collagen and its disorders (first of two parts). N Engl J Med. 1979 Jul 5;301(1):13–23. doi: 10.1056/NEJM197907053010104. [DOI] [PubMed] [Google Scholar]
  34. Raghow R., Thompson J. P. Molecular mechanisms of collagen gene expression. Mol Cell Biochem. 1989 Mar 16;86(1):5–18. doi: 10.1007/BF00231686. [DOI] [PubMed] [Google Scholar]
  35. Rossi P., Karsenty G., Roberts A. B., Roche N. S., Sporn M. B., de Crombrugghe B. A nuclear factor 1 binding site mediates the transcriptional activation of a type I collagen promoter by transforming growth factor-beta. Cell. 1988 Feb 12;52(3):405–414. doi: 10.1016/s0092-8674(88)80033-3. [DOI] [PubMed] [Google Scholar]
  36. Rossi P., de Crombrugghe B. Identification of a cell-specific transcriptional enhancer in the first intron of the mouse alpha 2 (type I) collagen gene. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5590–5594. doi: 10.1073/pnas.84.16.5590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sayers J. R., Schmidt W., Eckstein F. 5'-3' exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res. 1988 Feb 11;16(3):791–802. doi: 10.1093/nar/16.3.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Seed B., Sheen J. Y. A simple phase-extraction assay for chloramphenicol acyltransferase activity. Gene. 1988 Jul 30;67(2):271–277. doi: 10.1016/0378-1119(88)90403-9. [DOI] [PubMed] [Google Scholar]
  39. Slack J. L., Liska D. J., Bornstein P. Regulation of expression of the type I collagen genes. Am J Med Genet. 1993 Jan 15;45(2):140–151. doi: 10.1002/ajmg.1320450203. [DOI] [PubMed] [Google Scholar]
  40. Smith B. D., Marsilio E. Methylation of the alpha 2(I) collagen gene in chemically transformed rat liver epithelial cells. Biochem J. 1988 Jul 1;253(1):269–273. doi: 10.1042/bj2530269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Thompson J. P., Simkevich C. P., Holness M. A., Kang A. H., 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. 1991 Feb 5;266(4):2549–2556. [PubMed] [Google Scholar]
  42. Tronche F., Rollier A., Bach I., Weiss M. C., Yaniv M. The rat albumin promoter: cooperation with upstream elements is required when binding of APF/HNF1 to the proximal element is partially impaired by mutation or bacterial methylation. Mol Cell Biol. 1989 Nov;9(11):4759–4766. doi: 10.1128/mcb.9.11.4759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Vuust J., Sobel M. E., Martin G. R. Regulation of type I collagen synthesis. Total pro alpha 1(I) and pro alpha 2(I) mRNAs are maintained in a 2:1 ratio under varying rates of collagen synthesis. Eur J Biochem. 1985 Sep 16;151(3):449–453. doi: 10.1111/j.1432-1033.1985.tb09122.x. [DOI] [PubMed] [Google Scholar]
  44. Yoshioka H., Greenwel P., Inoguchi K., Truter S., Inagaki Y., Ninomiya Y., Ramirez F. Structural and functional analysis of the promoter of the human alpha 1(XI) collagen gene. J Biol Chem. 1995 Jan 6;270(1):418–424. doi: 10.1074/jbc.270.1.418. [DOI] [PubMed] [Google Scholar]
  45. de Haan J. B., Gevers W., Parker M. I. Effects of sodium butyrate on the synthesis and methylation of DNA in normal cells and their transformed counterparts. Cancer Res. 1986 Feb;46(2):713–716. [PubMed] [Google Scholar]
  46. van der Rest M., Garrone R. Collagen family of proteins. FASEB J. 1991 Oct;5(13):2814–2823. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES