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. 1993 Dec;12(12):4635–4645. doi: 10.1002/j.1460-2075.1993.tb06152.x

Mutations of the intronic IgH enhancer and its flanking sequences differentially affect accessibility of the JH locus.

J Chen 1, F Young 1, A Bottaro 1, V Stewart 1, R K Smith 1, F W Alt 1
PMCID: PMC413901  PMID: 8223473

Abstract

To investigate the role of intronic immunoglobulin heavy chain (IgH) enhancer (E mu) in generating accessibility of the JH locus for VDJ recombination, we generated ES cells in which E mu or its flanking sequences were mutated by replacement with or insertion of an expressed neor gene. Heterozygous mutant ES cells were used to generate chimeric mice from which pre-B cell lines were derived by transformation of bone marrow cells with Abelson murine leukemia virus (A-MuLV). Comparison of the rearrangement status of the normal and mutated alleles in individual pre-B cell lines allowed us to assay for cis-acting effects of the mutations. Replacement of a 700 bp region immediately downstream from the core E mu [which includes part of the 3' matrix associated region (MAR) and the I mu exon] had no obvious effect on rearrangement of the targeted allele, indicating that insertion of a transcribed neor gene into the JH-C mu intron does not affect JH accessibility. In contrast, replacement of an overlapping 1 kb DNA fragment that contains the E mu resulted in a dramatic cis-acting inhibition of rearrangement, demethylation and germline transcription of the associated JH locus. Surprisingly, insertion of the neor gene into the 5' MAR sequence approximately 100 bp upstream of the core E mu also dramatically decreased recombination of the linked JH locus; but, in many lines, did not prevent demethylation of this locus. We conclude that integrity of the E mu and upstream flanking sequences is required for efficient rearrangement of the JH locus and that demethylation of this locus, per se, does not necessarily make it a good substrate for VDJ recombination.

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Selected References

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

  1. Adra C. N., Boer P. H., McBurney M. W. Cloning and expression of the mouse pgk-1 gene and the nucleotide sequence of its promoter. Gene. 1987;60(1):65–74. doi: 10.1016/0378-1119(87)90214-9. [DOI] [PubMed] [Google Scholar]
  2. Alessandrini A., Desiderio S. V. Coordination of immunoglobulin DJH transcription and D-to-JH rearrangement by promoter-enhancer approximation. Mol Cell Biol. 1991 Apr;11(4):2096–2107. doi: 10.1128/mcb.11.4.2096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alt F. W., Oltz E. M., Young F., Gorman J., Taccioli G., Chen J. VDJ recombination. Immunol Today. 1992 Aug;13(8):306–314. doi: 10.1016/0167-5699(92)90043-7. [DOI] [PubMed] [Google Scholar]
  4. Alt F. W., Yancopoulos G. D., Blackwell T. K., Wood C., Thomas E., Boss M., Coffman R., Rosenberg N., Tonegawa S., Baltimore D. Ordered rearrangement of immunoglobulin heavy chain variable region segments. EMBO J. 1984 Jun;3(6):1209–1219. doi: 10.1002/j.1460-2075.1984.tb01955.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Anderson S. J., Abraham K. M., Nakayama T., Singer A., Perlmutter R. M. Inhibition of T-cell receptor beta-chain gene rearrangement by overexpression of the non-receptor protein tyrosine kinase p56lck. EMBO J. 1992 Dec;11(13):4877–4886. doi: 10.1002/j.1460-2075.1992.tb05594.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Bird A. The essentials of DNA methylation. Cell. 1992 Jul 10;70(1):5–8. doi: 10.1016/0092-8674(92)90526-i. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Cedar H. DNA methylation and gene activity. Cell. 1988 Apr 8;53(1):3–4. doi: 10.1016/0092-8674(88)90479-5. [DOI] [PubMed] [Google Scholar]
  11. Cockerill P. N., Yuen M. H., Garrard W. T. The enhancer of the immunoglobulin heavy chain locus is flanked by presumptive chromosomal loop anchorage elements. J Biol Chem. 1987 Apr 15;262(11):5394–5397. [PubMed] [Google Scholar]
  12. Davis M. M., Bjorkman P. J. T-cell antigen receptor genes and T-cell recognition. Nature. 1988 Aug 4;334(6181):395–402. doi: 10.1038/334395a0. [DOI] [PubMed] [Google Scholar]
  13. Dickinson L. A., Joh T., Kohwi Y., Kohwi-Shigematsu T. A tissue-specific MAR/SAR DNA-binding protein with unusual binding site recognition. Cell. 1992 Aug 21;70(4):631–645. doi: 10.1016/0092-8674(92)90432-c. [DOI] [PubMed] [Google Scholar]
  14. Early P., Huang H., Davis M., Calame K., Hood L. An immunoglobulin heavy chain variable region gene is generated from three segments of DNA: VH, D and JH. Cell. 1980 Apr;19(4):981–992. doi: 10.1016/0092-8674(80)90089-6. [DOI] [PubMed] [Google Scholar]
  15. Engler P., Haasch D., Pinkert C. A., Doglio L., Glymour M., Brinster R., Storb U. A strain-specific modifier on mouse chromosome 4 controls the methylation of independent transgene loci. Cell. 1991 Jun 14;65(6):939–947. doi: 10.1016/0092-8674(91)90546-b. [DOI] [PubMed] [Google Scholar]
  16. Ferrier P., Krippl B., Blackwell T. K., Furley A. J., Suh H., Winoto A., Cook W. D., Hood L., Costantini F., Alt F. W. Separate elements control DJ and VDJ rearrangement in a transgenic recombination substrate. EMBO J. 1990 Jan;9(1):117–125. doi: 10.1002/j.1460-2075.1990.tb08087.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gay D., Saunders T., Camper S., Weigert M. Receptor editing: an approach by autoreactive B cells to escape tolerance. J Exp Med. 1993 Apr 1;177(4):999–1008. doi: 10.1084/jem.177.4.999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Hsieh C. L., Lieber M. R. CpG methylated minichromosomes become inaccessible for V(D)J recombination after undergoing replication. EMBO J. 1992 Jan;11(1):315–325. doi: 10.1002/j.1460-2075.1992.tb05054.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Mombaerts P., Iacomini J., Johnson R. S., Herrup K., Tonegawa S., Papaioannou V. E. RAG-1-deficient mice have no mature B and T lymphocytes. Cell. 1992 Mar 6;68(5):869–877. doi: 10.1016/0092-8674(92)90030-g. [DOI] [PubMed] [Google Scholar]
  23. Nelsen B., Sen R. Regulation of immunoglobulin gene transcription. Int Rev Cytol. 1992;133:121–149. doi: 10.1016/s0074-7696(08)61859-8. [DOI] [PubMed] [Google Scholar]
  24. Oettinger M. A., Schatz D. G., Gorka C., Baltimore D. RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science. 1990 Jun 22;248(4962):1517–1523. doi: 10.1126/science.2360047. [DOI] [PubMed] [Google Scholar]
  25. Reth M. G., Jackson S., Alt F. W. VHDJH formation and DJH replacement during pre-B differentiation: non-random usage of gene segments. EMBO J. 1986 Sep;5(9):2131–2138. doi: 10.1002/j.1460-2075.1986.tb04476.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rosenberg N., Baltimore D. A quantitative assay for transformation of bone marrow cells by Abelson murine leukemia virus. J Exp Med. 1976 Jun 1;143(6):1453–1463. doi: 10.1084/jem.143.6.1453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Schatz D. G., Oettinger M. A., Baltimore D. The V(D)J recombination activating gene, RAG-1. Cell. 1989 Dec 22;59(6):1035–1048. doi: 10.1016/0092-8674(89)90760-5. [DOI] [PubMed] [Google Scholar]
  29. Schlissel M. S., Baltimore D. Activation of immunoglobulin kappa gene rearrangement correlates with induction of germline kappa gene transcription. Cell. 1989 Sep 8;58(5):1001–1007. doi: 10.1016/0092-8674(89)90951-3. [DOI] [PubMed] [Google Scholar]
  30. Schlissel M. S., Corcoran L. M., Baltimore D. Virus-transformed pre-B cells show ordered activation but not inactivation of immunoglobulin gene rearrangement and transcription. J Exp Med. 1991 Mar 1;173(3):711–720. doi: 10.1084/jem.173.3.711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Serwe M., Sablitzky F. V(D)J recombination in B cells is impaired but not blocked by targeted deletion of the immunoglobulin heavy chain intron enhancer. EMBO J. 1993 Jun;12(6):2321–2327. doi: 10.1002/j.1460-2075.1993.tb05886.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Smith R. K., Zimmerman K., Yancopoulos G. D., Ma A., Alt F. W. Transcriptional down-regulation of N-myc expression during B-cell development. Mol Cell Biol. 1992 Apr;12(4):1578–1584. doi: 10.1128/mcb.12.4.1578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Staudt L. M., Lenardo M. J. Immunoglobulin gene transcription. Annu Rev Immunol. 1991;9:373–398. doi: 10.1146/annurev.iy.09.040191.002105. [DOI] [PubMed] [Google Scholar]
  35. Su L. K., Kadesch T. The immunoglobulin heavy-chain enhancer functions as the promoter for I mu sterile transcription. Mol Cell Biol. 1990 Jun;10(6):2619–2624. doi: 10.1128/mcb.10.6.2619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Takeda S., Zou Y. R., Bluethmann H., Kitamura D., Muller U., Rajewsky K. Deletion of the immunoglobulin kappa chain intron enhancer abolishes kappa chain gene rearrangement in cis but not lambda chain gene rearrangement in trans. EMBO J. 1993 Jun;12(6):2329–2336. doi: 10.1002/j.1460-2075.1993.tb05887.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tiegs S. L., Russell D. M., Nemazee D. Receptor editing in self-reactive bone marrow B cells. J Exp Med. 1993 Apr 1;177(4):1009–1020. doi: 10.1084/jem.177.4.1009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tonegawa S. Somatic generation of antibody diversity. Nature. 1983 Apr 14;302(5909):575–581. doi: 10.1038/302575a0. [DOI] [PubMed] [Google Scholar]
  39. Tybulewicz V. L., Crawford C. E., Jackson P. K., Bronson R. T., Mulligan R. C. Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. Cell. 1991 Jun 28;65(7):1153–1163. doi: 10.1016/0092-8674(91)90011-m. [DOI] [PubMed] [Google Scholar]
  40. Yancopoulos G. D., Alt F. W. Developmentally controlled and tissue-specific expression of unrearranged VH gene segments. Cell. 1985 Feb;40(2):271–281. doi: 10.1016/0092-8674(85)90141-2. [DOI] [PubMed] [Google Scholar]
  41. Yancopoulos G. D., Blackwell T. K., Suh H., Hood L., Alt F. W. Introduced T cell receptor variable region gene segments recombine in pre-B cells: evidence that B and T cells use a common recombinase. Cell. 1986 Jan 31;44(2):251–259. doi: 10.1016/0092-8674(86)90759-2. [DOI] [PubMed] [Google Scholar]

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