Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1993 Sep;12(9):3529–3537. doi: 10.1002/j.1460-2075.1993.tb06027.x

A selective defect in IgG2b switching as a result of targeted mutation of the I gamma 2b promoter and exon.

J Zhang 1, A Bottaro 1, S Li 1, V Stewart 1, F W Alt 1
PMCID: PMC413629  PMID: 8253079

Abstract

LPS stimulation of B lymphocytes induces germline transcription of and subsequent switching to the gamma 2b gene. Mature germline transcripts contain an I exon (non-coding) spliced to the C gamma 2b exons. To investigate the role of germline transcription and/or transcripts in heavy chain class switching, we have replaced the germline I gamma 2b promoter and I exon in ES cells with an expressed neomycin resistance gene. The mutated chromosome retains the downstream target sequence for switch recombination (S regions) and all sequences necessary for expression of a switched gamma 2b gene. Wild-type or mutant ES cells were injected into RAG-2 deficient blastocysts to generate somatic chimeras in which all lymphocytes were ES-cell derived. Chimeras derived from injection of heterozygous mutant ES cells had normal levels of serum IgG2b, but their splenic B cells showed a partial decrease in ability to switch to gamma 2b. Strikingly, B lymphocytes from chimeras derived by injection of homozygous mutant ES cells were deficient in IgG2b production both in vivo and in vitro, but normal with respect to production of other Ig heavy chain isotypes. Additional studies demonstrated that lack of ability to produce IgG2b by the mutant B cells correlated with lack of germline transcription and resulted from a specific defect in class-switch recombination to S gamma 2b. Together, these studies demonstrate that the I region is an important regulatory element for control of class-switch recombination.

Full text

PDF
3532

Images in this article

3530Image
on p.3530
3531Image
on p.3531
3532Image
on p.3532
3534Image
on p.3534
3535Image
on p.3535

Selected References

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

  1. Berton M. T., Uhr J. W., Vitetta E. S. Synthesis of germ-line gamma 1 immunoglobulin heavy-chain transcripts in resting B cells: induction by interleukin 4 and inhibition by interferon gamma. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2829–2833. doi: 10.1073/pnas.86.8.2829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blackwell T. K., Alt F. W. Mechanism and developmental program of immunoglobulin gene rearrangement in mammals. Annu Rev Genet. 1989;23:605–636. doi: 10.1146/annurev.ge.23.120189.003133. [DOI] [PubMed] [Google Scholar]
  3. Burstein H. J., Tepper R. I., Leder P., Abbas A. K. Humoral immune functions in IL-4 transgenic mice. J Immunol. 1991 Nov 1;147(9):2950–2956. [PubMed] [Google Scholar]
  4. Coffman R. L., Lebman D. A., Shrader B. Transforming growth factor beta specifically enhances IgA production by lipopolysaccharide-stimulated murine B lymphocytes. J Exp Med. 1989 Sep 1;170(3):1039–1044. doi: 10.1084/jem.170.3.1039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Coffman R. L., Ohara J., Bond M. W., Carty J., Zlotnik A., Paul W. E. B cell stimulatory factor-1 enhances the IgE response of lipopolysaccharide-activated B cells. J Immunol. 1986 Jun 15;136(12):4538–4541. [PubMed] [Google Scholar]
  6. Cory S., Jackson J., Adams J. M. Deletions in the constant region locus can account for switches in immunoglobulin heavy chain expression. Nature. 1980 Jun 12;285(5765):450–456. doi: 10.1038/285450a0. [DOI] [PubMed] [Google Scholar]
  7. Davis M. M., Kim S. K., Hood L. E. DNA sequences mediating class switching in alpha-immunoglobulins. Science. 1980 Sep 19;209(4463):1360–1365. doi: 10.1126/science.6774415. [DOI] [PubMed] [Google Scholar]
  8. Dunnick W., Rabbitts T. H., Milstein C. An immunoglobulin deletion mutant with implications for the heavy-chain switch and RNA splicing. Nature. 1980 Aug 14;286(5774):669–675. doi: 10.1038/286669a0. [DOI] [PubMed] [Google Scholar]
  9. Esser C., Radbruch A. Immunoglobulin class switching: molecular and cellular analysis. Annu Rev Immunol. 1990;8:717–735. doi: 10.1146/annurev.iy.08.040190.003441. [DOI] [PubMed] [Google Scholar]
  10. Esser C., Radbruch A. Rapid induction of transcription of unrearranged S gamma 1 switch regions in activated murine B cells by interleukin 4. EMBO J. 1989 Feb;8(2):483–488. doi: 10.1002/j.1460-2075.1989.tb03401.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Field S. J., Johnson R. S., Mortensen R. M., Papaioannou V. E., Spiegelman B. M., Greenberg M. E. Growth and differentiation of embryonic stem cells that lack an intact c-fos gene. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9306–9310. doi: 10.1073/pnas.89.19.9306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gauchat J. F., Lebman D. A., Coffman R. L., Gascan H., de Vries J. E. Structure and expression of germline epsilon transcripts in human B cells induced by interleukin 4 to switch to IgE production. J Exp Med. 1990 Aug 1;172(2):463–473. doi: 10.1084/jem.172.2.463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gerondakis S. Structure and expression of murine germ-line immunoglobulin epsilon heavy chain transcripts induced by interleukin 4. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1581–1585. doi: 10.1073/pnas.87.4.1581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Iwasato T., Shimizu A., Honjo T., Yamagishi H. Circular DNA is excised by immunoglobulin class switch recombination. Cell. 1990 Jul 13;62(1):143–149. doi: 10.1016/0092-8674(90)90248-d. [DOI] [PubMed] [Google Scholar]
  15. Jung S., Rajewsky K., Radbruch A. Shutdown of class switch recombination by deletion of a switch region control element. Science. 1993 Feb 12;259(5097):984–987. doi: 10.1126/science.8438159. [DOI] [PubMed] [Google Scholar]
  16. Kataoka T., Kawakami T., Takahashi N., Honjo T. Rearrangement of immunoglobulin gamma 1-chain gene and mechanism for heavy-chain class switch. Proc Natl Acad Sci U S A. 1980 Feb;77(2):919–923. doi: 10.1073/pnas.77.2.919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kataoka T., Miyata T., Honjo T. Repetitive sequences in class-switch recombination regions of immunoglobulin heavy chain genes. Cell. 1981 Feb;23(2):357–368. doi: 10.1016/0092-8674(81)90131-8. [DOI] [PubMed] [Google Scholar]
  18. Kearney J. F., Cooper M. D., Lawton A. R. B cell differentiation induced by lipopolysaccharide. IV. Development of immunoglobulin class restriction in precursors of IgG-synthesizing cells. J Immunol. 1976 Nov;117(5 Pt 1):1567–1572. [PubMed] [Google Scholar]
  19. Kohl N. E., Legouy E., DePinho R. A., Nisen P. D., Smith R. K., Gee C. E., Alt F. W. Human N-myc is closely related in organization and nucleotide sequence to c-myc. Nature. 1986 Jan 2;319(6048):73–77. doi: 10.1038/319073a0. [DOI] [PubMed] [Google Scholar]
  20. Layton J. E., Vitetta E. S., Uhr J. W., Krammer P. H. Clonal analysis of B cells induced to secrete IgG by T cell-derived lymphokine(s). J Exp Med. 1984 Dec 1;160(6):1850–1863. doi: 10.1084/jem.160.6.1850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lebman D. A., Nomura D. Y., Coffman R. L., Lee F. D. Molecular characterization of germ-line immunoglobulin A transcripts produced during transforming growth factor type beta-induced isotype switching. Proc Natl Acad Sci U S A. 1990 May;87(10):3962–3966. doi: 10.1073/pnas.87.10.3962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lennon G. G., Perry R. P. C mu-containing transcripts initiate heterogeneously within the IgH enhancer region and contain a novel 5'-nontranslatable exon. Nature. 1985 Dec 5;318(6045):475–478. doi: 10.1038/318475a0. [DOI] [PubMed] [Google Scholar]
  23. Lutzker S., Alt F. W. Structure and expression of germ line immunoglobulin gamma 2b transcripts. Mol Cell Biol. 1988 Apr;8(4):1849–1852. doi: 10.1128/mcb.8.4.1849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lutzker S., Rothman P., Pollock R., Coffman R., Alt F. W. Mitogen- and IL-4-regulated expression of germ-line Ig gamma 2b transcripts: evidence for directed heavy chain class switching. Cell. 1988 Apr 22;53(2):177–184. doi: 10.1016/0092-8674(88)90379-0. [DOI] [PubMed] [Google Scholar]
  25. Mansour S. L., Thomas K. R., Capecchi M. R. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature. 1988 Nov 24;336(6197):348–352. doi: 10.1038/336348a0. [DOI] [PubMed] [Google Scholar]
  26. Matsuoka M., Yoshida K., Maeda T., Usuda S., Sakano H. Switch circular DNA formed in cytokine-treated mouse splenocytes: evidence for intramolecular DNA deletion in immunoglobulin class switching. Cell. 1990 Jul 13;62(1):135–142. doi: 10.1016/0092-8674(90)90247-c. [DOI] [PubMed] [Google Scholar]
  27. Mortensen R. M., Conner D. A., Chao S., Geisterfer-Lowrance A. A., Seidman J. G. Production of homozygous mutant ES cells with a single targeting construct. Mol Cell Biol. 1992 May;12(5):2391–2395. doi: 10.1128/mcb.12.5.2391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nikaido T., Yamawaki-Kataoka Y., Honjo T. Nucleotide sequences of switch regions of immunoglobulin C epsilon and C gamma genes and their comparison. J Biol Chem. 1982 Jul 10;257(13):7322–7329. [PubMed] [Google Scholar]
  29. Nilsson L., Islam K. B., Olafsson O., Zalcberg I., Samakovlis C., Hammarström L., Smith C. I., Sideras P. Structure of TGF-beta 1-induced human immunoglobulin C alpha 1 and C alpha 2 germ-line transcripts. Int Immunol. 1991 Nov;3(11):1107–1115. doi: 10.1093/intimm/3.11.1107. [DOI] [PubMed] [Google Scholar]
  30. Rabbitts T. H., Forster A., Dunnick W., Bentley D. L. The role of gene deletion in the immunoglobulin heavy chain switch. Nature. 1980 Jan 24;283(5745):351–356. doi: 10.1038/283351a0. [DOI] [PubMed] [Google Scholar]
  31. Radcliffe G., Lin Y. C., Julius M., Marcu K. B., Stavnezer J. Structure of germ line immunoglobulin alpha heavy-chain RNA and its location on polysomes. Mol Cell Biol. 1990 Jan;10(1):382–386. doi: 10.1128/mcb.10.1.382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rothman P., Chen Y. Y., Lutzker S., Li S. C., Stewart V., Coffman R., Alt F. W. Structure and expression of germ line immunoglobulin heavy-chain epsilon transcripts: interleukin-4 plus lipopolysaccharide-directed switching to C epsilon. Mol Cell Biol. 1990 Apr;10(4):1672–1679. doi: 10.1128/mcb.10.4.1672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rothman P., Li S. C., Alt F. W. The molecular events in heavy chain class-switching. Semin Immunol. 1989 Sep;1(1):65–77. [PubMed] [Google Scholar]
  34. Rothman P., Lutzker S., Gorham B., Stewart V., Coffman R., Alt F. W. Structure and expression of germline immunoglobulin gamma 3 heavy chain gene transcripts: implications for mitogen and lymphokine directed class-switching. Int Immunol. 1990;2(7):621–627. doi: 10.1093/intimm/2.7.621. [DOI] [PubMed] [Google Scholar]
  35. Shinkai Y., Rathbun G., Lam K. P., Oltz E. M., Stewart V., Mendelsohn M., Charron J., Datta M., Young F., Stall A. M. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell. 1992 Mar 6;68(5):855–867. doi: 10.1016/0092-8674(92)90029-c. [DOI] [PubMed] [Google Scholar]
  36. Sideras P., Mizuta T. R., Kanamori H., Suzuki N., Okamoto M., Kuze K., Ohno H., Doi S., Fukuhara S., Hassan M. S. Production of sterile transcripts of C gamma genes in an IgM-producing human neoplastic B cell line that switches to IgG-producing cells. Int Immunol. 1989;1(6):631–642. doi: 10.1093/intimm/1.6.631. [DOI] [PubMed] [Google Scholar]
  37. Snapper C. M., Paul W. E. Interferon-gamma and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science. 1987 May 22;236(4804):944–947. doi: 10.1126/science.3107127. [DOI] [PubMed] [Google Scholar]
  38. Snapper C. M., Peschel C., Paul W. E. IFN-gamma stimulates IgG2a secretion by murine B cells stimulated with bacterial lipopolysaccharide. J Immunol. 1988 Apr 1;140(7):2121–2127. [PubMed] [Google Scholar]
  39. Stavnezer-Nordgren J., Sirlin S. Specificity of immunoglobulin heavy chain switch correlates with activity of germline heavy chain genes prior to switching. EMBO J. 1986 Jan;5(1):95–102. doi: 10.1002/j.1460-2075.1986.tb04182.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stavnezer J., Radcliffe G., Lin Y. C., Nietupski J., Berggren L., Sitia R., Severinson E. Immunoglobulin heavy-chain switching may be directed by prior induction of transcripts from constant-region genes. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7704–7708. doi: 10.1073/pnas.85.20.7704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Thomas B. J., Rothstein R. Elevated recombination rates in transcriptionally active DNA. Cell. 1989 Feb 24;56(4):619–630. doi: 10.1016/0092-8674(89)90584-9. [DOI] [PubMed] [Google Scholar]
  42. Yancopoulos G. D., DePinho R. A., Zimmerman K. A., Lutzker S. G., Rosenberg N., Alt F. W. Secondary genomic rearrangement events in pre-B cells: VHDJH replacement by a LINE-1 sequence and directed class switching. EMBO J. 1986 Dec 1;5(12):3259–3266. doi: 10.1002/j.1460-2075.1986.tb04637.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. von Schwedler U., Jäck H. M., Wabl M. Circular DNA is a product of the immunoglobulin class switch rearrangement. Nature. 1990 May 31;345(6274):452–456. doi: 10.1038/345452a0. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES