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. 1984 Dec 1;3(12):3013–3021. doi: 10.1002/j.1460-2075.1984.tb02248.x

Activation of immunoglobulin mu gene expression involves stepwise demethylation.

S Gerondakis, A Boyd, O Bernard, E Webb, J M Adams
PMCID: PMC557805  PMID: 6098461

Abstract

In an attempt to identify stages of mu gene activation subsequent to VHDHJH assembly, we investigated two pre-B cell lines (A1 and A8) that have both alleles of the JH locus rearranged but do not make mu polypeptides. The block in A1 reflects incorrect VHDHJH assembly on both alleles. In A8, although an out-of-phase VHDHJH-C mu allele is transcribed, the properly assembled allele is silent, despite having a normal VH promoter and mu enhancer. Thus, transcription can be restricted to a single mu allele. Low level mu transcription in both lines was associated with demethylation of the VHDHJH and enhancer regions but not of the C mu gene. Markedly elevated mu transcription ensued on lipopolysaccharide stimulation or fusion to a plasmacytoma, but only fusion induced C mu demethylation. Hence stepwise demethylation is implicated in mu gene regulation, but enhanced expression can also occur independently of, or prior to, demethylation.

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Selected References

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

  1. Adams J. M., Kemp D. J., Bernard O., Gough N., Webb E., Tyler B., Gerondakis S., Cory S. Organization and expression of murine immunoglobulin genes. Immunol Rev. 1981;59:5–32. doi: 10.1111/j.1600-065x.1981.tb00454.x. [DOI] [PubMed] [Google Scholar]
  2. Akira S., Sugiyama H., Sakaguchi N., Kishimoto T. Immunoglobulin gene expression and DNA methylation in murine pre-B cell lines. EMBO J. 1984 Mar;3(3):677–681. doi: 10.1002/j.1460-2075.1984.tb01866.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alt F. W., Bothwell A. L., Knapp M., Siden E., Mather E., Koshland M., Baltimore D. Synthesis of secreted and membrane-bound immunoglobulin mu heavy chains is directed by mRNAs that differ at their 3' ends. Cell. 1980 Jun;20(2):293–301. doi: 10.1016/0092-8674(80)90615-7. [DOI] [PubMed] [Google Scholar]
  4. Alt F. W., Rosenberg N., Casanova R. J., Thomas E., Baltimore D. Immunoglobulin heavy-chain expression and class switching in a murine leukaemia cell line. Nature. 1982 Mar 25;296(5855):325–331. doi: 10.1038/296325a0. [DOI] [PubMed] [Google Scholar]
  5. Alt F. W., Rosenberg N., Enea V., Siden E., Baltimore D. Multiple immunoglobulin heavy-chain gene transcripts in Abelson murine leukemia virus-transformed lymphoid cell lines. Mol Cell Biol. 1982 Apr;2(4):386–400. doi: 10.1128/mcb.2.4.386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Alt F., Rosenberg N., Lewis S., Thomas E., Baltimore D. Organization and reorganization of immunoglobulin genes in A-MULV-transformed cells: rearrangement of heavy but not light chain genes. Cell. 1981 Dec;27(2 Pt 1):381–390. doi: 10.1016/0092-8674(81)90421-9. [DOI] [PubMed] [Google Scholar]
  7. Alwine J. C., Kemp D. J., Stark G. R. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5350–5354. doi: 10.1073/pnas.74.12.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Baltimore D., Rosenberg N., Witte O. N. Transformation of immature lymphoid cells by Abelson murine leukemia virus. Immunol Rev. 1979;48:3–22. doi: 10.1111/j.1600-065x.1979.tb00296.x. [DOI] [PubMed] [Google Scholar]
  9. Banerji J., Olson L., Schaffner W. A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes. Cell. 1983 Jul;33(3):729–740. doi: 10.1016/0092-8674(83)90015-6. [DOI] [PubMed] [Google Scholar]
  10. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  11. Bernard O., Gough N. M., Adams J. M. Plasmacytomas with more than one immunoglobulin kappa mRNA: implications for allelic exclusion. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5812–5816. doi: 10.1073/pnas.78.9.5812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Bothwell A. L., Paskind M., Reth M., Imanishi-Kari T., Rajewsky K., Baltimore D. Heavy chain variable region contribution to the NPb family of antibodies: somatic mutation evident in a gamma 2a variable region. Cell. 1981 Jun;24(3):625–637. doi: 10.1016/0092-8674(81)90089-1. [DOI] [PubMed] [Google Scholar]
  13. Bourgois A., Fougereau M., De Preval C. Sequence of amino acids of the NH 2 -terminal region of a mouse-clonal immunoglobulin heavy chain. Eur J Biochem. 1972 Jan 21;24(3):446–455. doi: 10.1111/j.1432-1033.1972.tb19705.x. [DOI] [PubMed] [Google Scholar]
  14. Boyd A. W., Goding J. W., Schrader J. W. The regulation of growth and differentiation of a murine B cell lymphoma. I. Lipopolysaccharide-induced differentiation. J Immunol. 1981 Jun;126(6):2461–2465. [PubMed] [Google Scholar]
  15. Boyd A. W., Schrader J. W. Mechanism of effector-cell blockade. I. Antigen-induced suppression of Ig synthesis in a hybridoma cell line, and correlation with cell-associated antigen. J Exp Med. 1980 Jun 1;151(6):1436–1451. doi: 10.1084/jem.151.6.1436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  17. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  18. Cory S., Adams J. M. Deletions are associated with somatic rearrangement of immunoglobulin heavy chain genes. Cell. 1980 Jan;19(1):37–51. doi: 10.1016/0092-8674(80)90386-4. [DOI] [PubMed] [Google Scholar]
  19. Cory S., Adams J. M., Kemp D. J. Somatic rearrangements forming active immunoglobulin mu genes in B and T lymphoid cell lines. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4943–4947. doi: 10.1073/pnas.77.8.4943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Cory S. Immunoglobulin variable region genes. Surv Synth Pathol Res. 1984;3(2):149–164. doi: 10.1159/000156923. [DOI] [PubMed] [Google Scholar]
  21. Durnam D. M., Hoffman J. S., Quaife C. J., Benditt E. P., Chen H. Y., Brinster R. L., Palmiter R. D. Induction of mouse metallothionein-I mRNA by bacterial endotoxin is independent of metals and glucocorticoid hormones. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1053–1056. doi: 10.1073/pnas.81.4.1053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Early P., Hood L. Allelic exclusion and nonproductive immunoglobulin gene rearrangements. Cell. 1981 Apr;24(1):1–3. doi: 10.1016/0092-8674(81)90492-x. [DOI] [PubMed] [Google Scholar]
  23. Early P., Nottenburg C., Weissman I., Hood L. Immunoglobulin gene rearrangements in normal mouse B cells. Mol Cell Biol. 1982 Jul;2(7):829–836. doi: 10.1128/mcb.2.7.829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Early P., Rogers J., Davis M., Calame K., Bond M., Wall R., Hood L. Two mRNAs can be produced from a single immunoglobulin mu gene by alternative RNA processing pathways. Cell. 1980 Jun;20(2):313–319. doi: 10.1016/0092-8674(80)90617-0. [DOI] [PubMed] [Google Scholar]
  25. Gearhart P. J., Johnson N. D., Douglas R., Hood L. IgG antibodies to phosphorylcholine exhibit more diversity than their IgM counterparts. Nature. 1981 May 7;291(5810):29–34. doi: 10.1038/291029a0. [DOI] [PubMed] [Google Scholar]
  26. Gillies S. D., Morrison S. L., Oi V. T., Tonegawa S. A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene. Cell. 1983 Jul;33(3):717–728. doi: 10.1016/0092-8674(83)90014-4. [DOI] [PubMed] [Google Scholar]
  27. Groudine M., Weintraub H. Propagation of globin DNAase I-hypersensitive sites in absence of factors required for induction: a possible mechanism for determination. Cell. 1982 Aug;30(1):131–139. doi: 10.1016/0092-8674(82)90019-8. [DOI] [PubMed] [Google Scholar]
  28. Hohn B. In vitro packaging of lambda and cosmid DNA. Methods Enzymol. 1979;68:299–309. doi: 10.1016/0076-6879(79)68021-7. [DOI] [PubMed] [Google Scholar]
  29. Kataoka T., Nikaido T., Miyata T., Moriwaki K., Honjo T. The nucleotide sequences of rearranged and germline immunoglobulin VH genes of a mouse myeloma MC101 and evolution of VH genes in mouse. J Biol Chem. 1982 Jan 10;257(1):277–285. [PubMed] [Google Scholar]
  30. Kemp D. J., Harris A. W., Adams J. M. Transcripts of the immunoglobulin C mu gene vary in structure and splicing during lymphoid development. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7400–7404. doi: 10.1073/pnas.77.12.7400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kemp D. J., Harris A. W., Cory S., Adams J. M. Expression of the immunoglobulin C mu gene in mouse T and B lymphoid and myeloid cell lines. Proc Natl Acad Sci U S A. 1980 May;77(5):2876–2880. doi: 10.1073/pnas.77.5.2876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kemp D. J., Morahan G., Cowman A. F., Harris A. W. Production of RNA for secreted immunoglobulin mu chains does not require transcriptional termination 5' to the microM exons. Nature. 1983 Jan 6;301(5895):84–86. doi: 10.1038/301084a0. [DOI] [PubMed] [Google Scholar]
  33. Kemp D. J., Tyler B., Bernard O., Gough N., Gerondakis S., Adams J. M., Cory S. Organization of genes and spacers within the mouse immunoglobulin VH locus. J Mol Appl Genet. 1981;1(3):245–261. [PubMed] [Google Scholar]
  34. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. doi: 10.1038/256495a0. [DOI] [PubMed] [Google Scholar]
  35. Mather E. L., Perry R. P. Methylation status and DNase I sensitivity of immunoglobulin genes: changes associated with rearrangement. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4689–4693. doi: 10.1073/pnas.80.15.4689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nelson K. J., Haimovich J., Perry R. P. Characterization of productive and sterile transcripts from the immunoglobulin heavy-chain locus: processing of micron and muS mRNA. Mol Cell Biol. 1983 Jul;3(7):1317–1332. doi: 10.1128/mcb.3.7.1317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nelson K. J., Mather E. L., Perry R. P. Lipopolysaccharide-induced transcription of the kappa immunoglobulin locus occurs on both alleles and is independent of methylation status. Nucleic Acids Res. 1984 Feb 24;12(4):1911–1923. doi: 10.1093/nar/12.4.1911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Paige C. J., Kincade P. W., Ralph P. Independent control of immunoglobulin heavy and light chain expression in a murine pre-B-cell line. Nature. 1981 Aug 13;292(5824):631–633. doi: 10.1038/292631a0. [DOI] [PubMed] [Google Scholar]
  41. Perry R. P., Kelley D. E., Coleclough C., Seidman J. G., Leder P., Tonegawa S., Matthyssens G., Weigert M. Transcription of mouse kappa chain genes: implications for allelic exclusion. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1937–1941. doi: 10.1073/pnas.77.4.1937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Premkumar E., Potter M., Singer P. A., Sklar M. D. Synthesis, surface deposition, and secretion of immunoglobulins by Abelson virus-transformed lymphosarcoma cell lines. Cell. 1975 Oct;6(2):149–159. doi: 10.1016/0092-8674(75)90005-7. [DOI] [PubMed] [Google Scholar]
  43. Riggs A. D., Jones P. A. 5-methylcytosine, gene regulation, and cancer. Adv Cancer Res. 1983;40:1–30. doi: 10.1016/s0065-230x(08)60678-8. [DOI] [PubMed] [Google Scholar]
  44. Rogers J., Wall R. Immunoglobulin heavy chain genes: demethylation accompanies class switching. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7497–7501. doi: 10.1073/pnas.78.12.7497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sakano H., Kurosawa Y., Weigert M., Tonegawa S. Identification and nucleotide sequence of a diversity DNA segment (D) of immunoglobulin heavy-chain genes. Nature. 1981 Apr 16;290(5807):562–565. doi: 10.1038/290562a0. [DOI] [PubMed] [Google Scholar]
  46. Sakano H., Maki R., Kurosawa Y., Roeder W., Tonegawa S. Two types of somatic recombination are necessary for the generation of complete immunoglobulin heavy-chain genes. Nature. 1980 Aug 14;286(5774):676–683. doi: 10.1038/286676a0. [DOI] [PubMed] [Google Scholar]
  47. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  48. Schrader J. W. Bone marrow differentiation in vitro. Crit Rev Immunol. 1983;4(3):197–277. [PubMed] [Google Scholar]
  49. Sigal N. H., Pickard A. R., Metcalf E. S., Gearhart P. J., Klinman N. R. Expression of phosphorylcholine-specific B cells during murine development. J Exp Med. 1977 Oct 1;146(4):933–948. doi: 10.1084/jem.146.4.933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Storb U., Arp B. Methylation patterns of immunoglobulin genes in lymphoid cells: correlation of expression and differentiation with undermethylation. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6642–6646. doi: 10.1073/pnas.80.21.6642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tonegawa S. Somatic generation of antibody diversity. Nature. 1983 Apr 14;302(5909):575–581. doi: 10.1038/302575a0. [DOI] [PubMed] [Google Scholar]
  52. Wabl M. R., Burrows P. D. Expression of immunoglobulin heavy chain at a high level in the absence of a proposed immunoglobulin enhancer element in cis. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2452–2455. doi: 10.1073/pnas.81.8.2452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Walker I. D., Harris A. W. Immunoglobulin Cmu RNA in T lymphoma cells is not translated. Nature. 1980 Nov 20;288(5788):290–293. doi: 10.1038/288290a0. [DOI] [PubMed] [Google Scholar]
  54. Warner N. L., Daley M. J., Richey J., Spellman C. Flow cytometry analysis of murine B cell lymphoma differentiation. Immunol Rev. 1979;48:197–243. doi: 10.1111/j.1600-065x.1979.tb00304.x. [DOI] [PubMed] [Google Scholar]
  55. Wilks A. F., Cozens P. J., Mattaj I. W., Jost J. P. Estrogen induces a demethylation at the 5' end region of the chicken vitellogenin gene. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4252–4255. doi: 10.1073/pnas.79.14.4252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wilks A., Seldran M., Jost J. P. An estrogen-dependent demethylation at the 5' end of the chicken vitellogenin gene is independent of DNA synthesis. Nucleic Acids Res. 1984 Jan 25;12(2):1163–1177. doi: 10.1093/nar/12.2.1163. [DOI] [PMC free article] [PubMed] [Google Scholar]

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