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PMID: 8065328 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

DNA methylation represses the murine alpha 1(I) collagen promoter by an indirect mechanism.

Molecular and cellular biology ·Vol. 14 ·No. 9 ·1994-09-00 ·Pages 5950-60

Rhodes K, Rippe RA, Umezawa A, Nehls M, Brenner DA, Breindl M

Abstract

Several lines of evidence indicate that DNA methylation plays a role in the transcriptional regulation of the murine alpha 1(I) collagen gene. To study the molecular mechanisms involved, a reporter gene construct containing the alpha 1(I) promoter and part of the first exon linked to the luciferase gene (Col3luc) was methylated in vitro and transfected into murine fibroblasts and embryonal carcinoma cells. Methylation resulted in repression of the alpha 1(I) promoter in both cell types, although it was less pronounced in embryonal carcinoma cells than in fibroblasts. The extent of repression depended on the density of methylation. DNase footprint and mobility shift assays indicated that the trans-acting factors binding to the alpha 1(I) promoter and first exon are ubiquitous factors and that their DNA binding is not inhibited by methylation. Transfection of Col3luc into Drosophila SL2 cells together with expression vectors for the transcription factors Sp1 and NF-1 showed that DNA methylation also inhibits the alpha 1(I) promoter in nonvertebrate cells, although to a much lesser extent than in murine cells. However, Sp1 and NF-1 transactivated the unmethylated and methylated reporter gene in SL2 cells equally well, confirming that these factors can bind and transactivate methylated DNA and indicating that DNA methylation represses the alpha 1(I) promoter by an indirect mechanism. This was further confirmed by cotransfection experiments with unspecific methylated competitor DNA which partially restored the activity of the methylated alpha 1(I) promoter. Our results suggest that DNA methylation can inhibit promoter activity by an indirect mechanism independent of methyl-C-binding proteins and that in vertebrate cells, chromatin structure and methyl-C-binding proteins cooperatively mediate the transcriptional inhibitory effect of DNA methylation.

MeSH Terms
Animals Base Sequence Cell Line Chromatin/ultrastructure Collagen/genetics DNA/metabolism DNA-Binding Proteins/metabolism Gene Expression Methylation Mice Molecular Sequence Data Oligodeoxyribonucleotides/chemistry Promoter Regions, Genetic Protein Binding RNA, Messenger/genetics Transcription Factors/metabolism Transcription, Genetic
Chemicals
Chromatin DNA-Binding Proteins Oligodeoxyribonucleotides RNA, Messenger Transcription Factors Collagen DNA
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Rhodes K
Department of Biology, San Diego State University, California 92182.
Rippe R A
Umezawa A
Nehls M
Brenner D A
Breindl M
References (64)
64 references, click to expand
  1. CpG methylation inhibits proenkephalin gene expression and binding of the transcription factor AP-2.
    Nucleic Acids Res. 1990 Jul 11;18(13):3975-82 PMID: 1695733
  2. Inactive chromatin spreads from a focus of methylation.
    Mol Cell Biol. 1993 Dec;13(12):7372-9 PMID: 7504173
  3. Retrovirus-induced interference with collagen I gene expression in Mov13 fibroblasts is maintained in the absence of DNA methylation.
    Mol Cell Biol. 1991 Jan;11(1):47-54 PMID: 1702514
  4. Methylation-sensitive sequence-specific DNA binding by the c-Myc basic region.
    Science. 1991 Jan 11;251(4990):186-9 PMID: 1987636
  5. 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):2549-56 PMID: 1990005
  6. Sequence-specific antirepression of histone H1-mediated inhibition of basal RNA polymerase II transcription.
    Science. 1991 Feb 8;251(4994):643-9 PMID: 1899487
  7. DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein.
    Cell. 1991 Mar 22;64(6):1123-34 PMID: 2004419
  8. An avian 40 KDa nucleoprotein binds preferentially to a promoter sequence containing one single pair of methylated CpG.
    Nucleic Acids Res. 1991 Mar 11;19(5):1029-34 PMID: 2020543
  9. Transcription factors nuclear factor I and Sp1 interact with the murine collagen alpha 1 (I) promoter.
    Mol Cell Biol. 1991 Aug;11(8):4065-73 PMID: 2072909
  10. Inhibition of promoter activity by methylation: possible involvement of protein mediators.
    Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6515-8 PMID: 1650472
  11. Estradiol down regulates the binding activity of an avian vitellogenin gene repressor (MDBP-2) and triggers a gradual demethylation of the mCpG pair of its DNA binding site.
    Nucleic Acids Res. 1991 Oct 25;19(20):5771-5 PMID: 1945854
  12. DNA CpG methylation inhibits binding of NF-kappa B proteins to the HIV-1 long terminal repeat cognate DNA motifs.
    New Biol. 1991 Oct;3(10):969-76 PMID: 1768651
  13. A dictionary of transcription control sequences.
    DNA Seq. 1990;1(1):3-11 PMID: 2132957
  14. Repression of genes by DNA methylation depends on CpG density and promoter strength: evidence for involvement of a methyl-CpG binding protein.
    EMBO J. 1992 Jan;11(1):327-33 PMID: 1310933
  15. Activation of collagen IV gene expression in F9 teratocarcinoma cells by 3-deazaadenosine analogs. Indirect inhibitors of methylation.
    J Biol Chem. 1992 Mar 5;267(7):4988-91 PMID: 1537874
  16. DNA methylation inhibits transcription of procollagen alpha 2(I) promoters.
    Biochem J. 1992 May 1;283 ( Pt 3):699-703 PMID: 1590760
  17. CpG methylation of an endogenous retroviral enhancer inhibits transcription factor binding and activity.
    Gene Expr. 1991;1(3):185-96 PMID: 1820217
  18. Developmental changes in the methylation status of regulatory elements in the murine alpha 1(I) collagen gene.
    Gene Expr. 1992;2(1):59-69 PMID: 1617303
  19. The essentials of DNA methylation.
    Cell. 1992 Jul 10;70(1):5-8 PMID: 1377983
  20. Vector methylation inhibits transcription from the SV40 early promoter.
    FEBS Lett. 1992 Aug 31;309(1):97-102 PMID: 1324853
  21. NF-I/Sp1 switch elements regulate collagen alpha 1(I) gene expression.
    DNA Cell Biol. 1992 Jul-Aug;11(6):443-52 PMID: 1524678
  22. Characterization of MeCP2, a vertebrate DNA binding protein with affinity for methylated DNA.
    Nucleic Acids Res. 1992 Oct 11;20(19):5085-92 PMID: 1408825
  23. The repressor MDBP-2 is a member of the histone H1 family that binds preferentially in vitro and in vivo to methylated nonspecific DNA sequences.
    Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9499-503 PMID: 1409659
  24. Methylation in the preinitiation domain suppresses gene transcription by an indirect mechanism.
    Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10119-23 PMID: 1332030
  25. Methylation-sensitive DNA binding by v-myb and c-myb proteins.
    Oncogene. 1993 Jan;8(1):111-5 PMID: 8423989
  26. Characterization of DBPm, a plant protein that binds to DNA containing 5-methylcytosine.
    Biochim Biophys Acta. 1993 Feb 20;1172(1-2):108-16 PMID: 8439549
  27. CpG methylation inactivates the promoter activity of the human retinoblastoma tumor-suppressor gene.
    Oncogene. 1993 Apr;8(4):1063-7 PMID: 8455933
  28. 5-methylcytosine is localized in nucleosomes that contain histone H1.
    Proc Natl Acad Sci U S A. 1983 Sep;80(18):5490-4 PMID: 6577440
  29. The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter.
    Cell. 1983 Nov;35(1):79-87 PMID: 6313230
  30. Complete DNA methylation does not prevent polyoma and simian virus 40 virus early gene expression.
    Proc Natl Acad Sci U S A. 1983 Nov;80(21):6470-4 PMID: 6314328
  31. A protein from human placental nuclei binds preferentially to 5-methylcytosine-rich DNA.
    Nature. 1984 Mar 15-21;308(5956):293-5 PMID: 6538260
  32. Histone-H1-dependent chromatin superstructures and the suppression of gene activity.
    Cell. 1984 Aug;38(1):17-27 PMID: 6467367
  33. Retrovirus-induced lethal mutation in collagen I gene of mice is associated with an altered chromatin structure.
    Cell. 1984 Aug;38(1):9-16 PMID: 6088079
  34. Retrovirus-induced de novo methylation of flanking host sequences correlates with gene inactivity.
    Nature. 1985 Jun 13-19;315(6020):594-7 PMID: 2989695
  35. DNA methylation affects the formation of active chromatin.
    Cell. 1986 Feb 28;44(4):535-43 PMID: 3456276
  36. Retrovirus insertion inactivates mouse alpha 1(I) collagen gene by blocking initiation of transcription.
    Nature. 1986 Mar 27-Apr 2;320(6060):365-7 PMID: 3960120
  37. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
    Nucleic Acids Res. 1983 Mar 11;11(5):1475-89 PMID: 6828386
  38. Chromatin structure is required to block transcription of the methylated herpes simplex virus thymidine kinase gene.
    Proc Natl Acad Sci U S A. 1987 Mar;84(5):1177-81 PMID: 3029768
  39. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor.
    Cell. 1987 Jun 19;49(6):729-39 PMID: 3034432
  40. Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development.
    Development. 1987 Mar;99(3):371-82 PMID: 3653008
  41. Determination of trace amounts of 5-methylcytosine in DNA by reverse-phase high-performance liquid chromatography.
    Anal Biochem. 1987 Jul;164(1):164-9 PMID: 2960248
  42. Human CCAAT-binding proteins have heterologous subunits.
    Cell. 1988 Apr 8;53(1):11-24 PMID: 3349524
  43. DNA methylation and gene activity.
    Cell. 1988 Apr 8;53(1):3-4 PMID: 3280142
  44. A high-efficiency HeLa cell nuclear transcription extract.
    DNA. 1988 Jan-Feb;7(1):47-55 PMID: 3349904
  45. Cytosine methylation does not affect binding of transcription factor Sp1.
    Proc Natl Acad Sci U S A. 1988 Apr;85(7):2066-70 PMID: 3281160
  46. Methylation of the alpha 2(I) collagen gene in chemically transformed rat liver epithelial cells.
    Biochem J. 1988 Jul 1;253(1):269-73 PMID: 3421946
  47. Genomic imprinting.
    Genes Dev. 1988 Aug;2(8):921-5 PMID: 3049238
  48. Analysis of Sp1 in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif.
    Cell. 1988 Dec 2;55(5):887-98 PMID: 3142690
  49. Sp1 transcription factor binds DNA and activates transcription even when the binding site is CpG methylated.
    Genes Dev. 1988 Sep;2(9):1127-35 PMID: 3056778
  50. Cytosine methylation prevents binding to DNA of a HeLa cell transcription factor required for optimal expression of the adenovirus major late promoter.
    Genes Dev. 1988 Sep;2(9):1136-43 PMID: 3192075
  51. Cloning and expression of AP-2, a cell-type-specific transcription factor that activates inducible enhancer elements.
    Genes Dev. 1988 Dec;2(12A):1557-69 PMID: 3063603
  52. Synergistic activation and repression of transcription by Drosophila homeobox proteins.
    Cell. 1989 Feb 24;56(4):573-83 PMID: 2563673
  53. CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation.
    Genes Dev. 1989 May;3(5):612-9 PMID: 2545524
  54. Regulatory elements in the 5'-flanking region and the first intron contribute to transcriptional control of the mouse alpha 1 type I collagen gene.
    Mol Cell Biol. 1989 May;9(5):2224-7 PMID: 2747648
  55. Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs.
    Cell. 1989 Aug 11;58(3):499-507 PMID: 2758464
  56. Analysis of the collagen alpha 1(I) promoter.
    Nucleic Acids Res. 1989 Aug 11;17(15):6055-64 PMID: 2771638
  57. Regulation of collagen gene expression.
    Prog Nucleic Acid Res Mol Biol. 1989;37:67-106 PMID: 2672111
  58. Cytosine methylation in CTF and Sp1 recognition sites of an HSV tk promoter: effects on transcription in vivo and on factor binding in vitro.
    Nucleic Acids Res. 1989 Dec 25;17(24):10179-90 PMID: 2557588
  59. DNA-mediated gene transfer into adult rat hepatocytes in primary culture.
    Mol Cell Biol. 1990 Feb;10(2):689-95 PMID: 2105458
  60. DNA methylation and collagen IV gene expression in F9 teratocarcinoma cells.
    J Biol Chem. 1990 Mar 25;265(9):4839-43 PMID: 2156857
  61. Two different negative and one positive regulatory factors interact with a short promoter segment of the alpha 1 (I) collagen gene.
    J Biol Chem. 1990 Jun 15;265(17):9934-42 PMID: 2351683
  62. Identification of two novel mouse nuclear proteins that bind selectively to a methylated c-Myc recognizing sequence.
    Nucleic Acids Res. 1993 May 11;21(9):2125-30 PMID: 8502552
  63. Histone H1-mediated inhibition of transcription initiation of methylated templates in vitro.
    J Biol Chem. 1993 Oct 15;268(29):21754-9 PMID: 8408030
  64. Binding of the transcription factor EBP-80 mediates the methylation response of an intracisternal A-particle long terminal repeat promoter.
    Mol Cell Biol. 1991 Jan;11(1):117-25 PMID: 1898760
Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.14.9.5950-5960.1994
Published
1994-09-00
Pages
5950-60
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC359121
Subset
IM
Grants
NCI NIH HHS · CA50528 · United States
NIGMS NIH HHS · GM41804 · United States
Databases
GENBANK
X54876
External Links
PubMed source DOI: 10.1128/mcb.14.9.5950-5960.1994 PMC Full Text View journal details
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