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. 1997 Jun 15;25(12):2303–2310. doi: 10.1093/nar/25.12.2303

V(D)J recombination frequency is affected by the sequence interposed between a pair of recombination signals: sequence comparison reveals a putative recombinational enhancer element.

F A Roch 1, R Hobi 1, M W Berchtold 1, C C Kuenzle 1
PMCID: PMC146747  PMID: 9235545

Abstract

The immunoglobulin heavy chain intron enhancer (Emu) not only stimulates transcription but also V(D)J recombination of chromosomally integrated recombination substrates. We aimed at reproducing this effect in recombination competent cells by transient transfection of extrachromosomal substrates. These we prepared by interposing between the recombination signal sequences (RSS) of the plasmid pBlueRec various fragments, including Emu, possibly affecting V(D)J recombination. Our work shows that sequences inserted between RSS 23 and RSS 12, with distances from their proximal ends of 26 and 284 bp respectively, can markedly affect the frequency of V(D)J recombination. We report that the entire Emu, the Emu core as well as its flanking 5' and 3' matrix associated regions (5' and 3' MARs) upregulate V(D)J recombination while the downstream section of the 3' MAR of Emu does not. Also, prokaryotic sequences markedly suppress V(D)J recombination. This confirms previous results obtained with chromosomally integrated substrates, except for the finding that the full length 3' MAR of Emu stimulates V(D)J recombination in an episomal but not in a chromosomal context. The fact that other MARs do not share this activity suggests that the effect is no mediated through attachment of the recombination substrate to a nuclear matrix-associated recombination complex but through cis-activation. The presence of a 26 bp A-T-rich sequence motif in the 5' and 3' MARs of Emu and in all of the other upregulating fragments investigated, leads us to propose that the motif represents a novel recombinational enhancer element distinct from those constituting the Emu core.

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Selected References

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  1. Akamatsu Y., Tsurushita N., Nagawa F., Matsuoka M., Okazaki K., Imai M., Sakano H. Essential residues in V(D)J recombination signals. J Immunol. 1994 Nov 15;153(10):4520–4529. [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. 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]
  5. Blunt T., Finnie N. J., Taccioli G. E., Smith G. C., Demengeot J., Gottlieb T. M., Mizuta R., Varghese A. J., Alt F. W., Jeggo P. A. Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation. Cell. 1995 Mar 10;80(5):813–823. doi: 10.1016/0092-8674(95)90360-7. [DOI] [PubMed] [Google Scholar]
  6. Bories J. C., Demengeot J., Davidson L., Alt F. W. Gene-targeted deletion and replacement mutations of the T-cell receptor beta-chain enhancer: the role of enhancer elements in controlling V(D)J recombination accessibility. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7871–7876. doi: 10.1073/pnas.93.15.7871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boubnov N. V., Hall K. T., Wills Z., Lee S. E., He D. M., Benjamin D. M., Pulaski C. R., Band H., Reeves W., Hendrickson E. A. Complementation of the ionizing radiation sensitivity, DNA end binding, and V(D)J recombination defects of double-strand break repair mutants by the p86 Ku autoantigen. Proc Natl Acad Sci U S A. 1995 Jan 31;92(3):890–894. doi: 10.1073/pnas.92.3.890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Boubnov N. V., Wills Z. P., Weaver D. T. Coding sequence composition flanking either signal element alters V(D)J recombination efficiency. Nucleic Acids Res. 1995 Mar 25;23(6):1060–1067. doi: 10.1093/nar/23.6.1060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bouvier G., Watrin F., Naspetti M., Verthuy C., Naquet P., Ferrier P. Deletion of the mouse T-cell receptor beta gene enhancer blocks alphabeta T-cell development. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7877–7881. doi: 10.1073/pnas.93.15.7877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Capone M., Watrin F., Fernex C., Horvat B., Krippl B., Wu L., Scollay R., Ferrier P. TCR beta and TCR alpha gene enhancers confer tissue- and stage-specificity on V(D)J recombination events. EMBO J. 1993 Nov;12(11):4335–4346. doi: 10.1002/j.1460-2075.1993.tb06118.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chant J., Stowers L. GTPase cascades choreographing cellular behavior: movement, morphogenesis, and more. Cell. 1995 Apr 7;81(1):1–4. doi: 10.1016/0092-8674(95)90363-1. [DOI] [PubMed] [Google Scholar]
  12. Chen J., Young F., Bottaro A., Stewart V., Smith R. K., Alt F. W. Mutations of the intronic IgH enhancer and its flanking sequences differentially affect accessibility of the JH locus. EMBO J. 1993 Dec;12(12):4635–4645. doi: 10.1002/j.1460-2075.1993.tb06152.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Difilippantonio M. J., McMahan C. J., Eastman Q. M., Spanopoulou E., Schatz D. G. RAG1 mediates signal sequence recognition and recruitment of RAG2 in V(D)J recombination. Cell. 1996 Oct 18;87(2):253–262. doi: 10.1016/s0092-8674(00)81343-4. [DOI] [PubMed] [Google Scholar]
  15. Döbbeling U., Hobi R., Berchtold M. W., Kuenzle C. C. V(D)J recombination is regulated similarly in RAG-transfected fibroblasts and pre-B cells. J Mol Biol. 1996 Aug 23;261(3):309–314. doi: 10.1006/jmbi.1996.0462. [DOI] [PubMed] [Google Scholar]
  16. Eastman Q. M., Leu T. M., Schatz D. G. Initiation of V(D)J recombination in vitro obeying the 12/23 rule. Nature. 1996 Mar 7;380(6569):85–88. doi: 10.1038/380085a0. [DOI] [PubMed] [Google Scholar]
  17. Ezekiel U. R., Engler P., Stern D., Storb U. Asymmetric processing of coding ends and the effect of coding end nucleotide composition on V(D)J recombination. Immunity. 1995 Apr;2(4):381–389. doi: 10.1016/1074-7613(95)90146-9. [DOI] [PubMed] [Google Scholar]
  18. Ferguson S. E., Accavitti M. A., Wang D. D., Chen C. L., Thompson C. B. Regulation of RAG-2 protein expression in avian thymocytes. Mol Cell Biol. 1994 Nov;14(11):7298–7305. doi: 10.1128/mcb.14.11.7298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fernex C., Caillol D., Capone M., Krippl B., Ferrier P. Sequences affecting the V(D)J recombinational activity of the IgH intronic enhancer in a transgenic substrate. Nucleic Acids Res. 1994 Mar 11;22(5):792–798. doi: 10.1093/nar/22.5.792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fernex C., Capone M., Ferrier P. The V(D)J recombinational and transcriptional activities of the immunoglobulin heavy-chain intronic enhancer can be mediated through distinct protein-binding sites in a transgenic substrate. Mol Cell Biol. 1995 Jun;15(6):3217–3226. doi: 10.1128/mcb.15.6.3217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ferradini L., Gu H., De Smet A., Rajewsky K., Reynaud C. A., Weill J. C. Rearrangement-enhancing element upstream of the mouse immunoglobulin kappa chain J cluster. Science. 1996 Mar 8;271(5254):1416–1420. doi: 10.1126/science.271.5254.1416. [DOI] [PubMed] [Google Scholar]
  22. Gasser S. M., Laemmli U. K. Cohabitation of scaffold binding regions with upstream/enhancer elements of three developmentally regulated genes of D. melanogaster. Cell. 1986 Aug 15;46(4):521–530. doi: 10.1016/0092-8674(86)90877-9. [DOI] [PubMed] [Google Scholar]
  23. Gerstein R. M., Lieber M. R. Coding end sequence can markedly affect the initiation of V(D)J recombination. Genes Dev. 1993 Jul;7(7B):1459–1469. doi: 10.1101/gad.7.7b.1459. [DOI] [PubMed] [Google Scholar]
  24. Hesse J. E., Lieber M. R., Gellert M., Mizuuchi K. Extrachromosomal DNA substrates in pre-B cells undergo inversion or deletion at immunoglobulin V-(D)-J joining signals. Cell. 1987 Jun 19;49(6):775–783. doi: 10.1016/0092-8674(87)90615-5. [DOI] [PubMed] [Google Scholar]
  25. Hesse J. E., Lieber M. R., Mizuuchi K., Gellert M. V(D)J recombination: a functional definition of the joining signals. Genes Dev. 1989 Jul;3(7):1053–1061. doi: 10.1101/gad.3.7.1053. [DOI] [PubMed] [Google Scholar]
  26. Inoue H., Nojima H., Okayama H. High efficiency transformation of Escherichia coli with plasmids. Gene. 1990 Nov 30;96(1):23–28. doi: 10.1016/0378-1119(90)90336-p. [DOI] [PubMed] [Google Scholar]
  27. Kallenbach S., Goodhardt M., Rougeon F. A rapid test for V(D)J recombinase activity. Nucleic Acids Res. 1990 Nov 25;18(22):6730–6730. doi: 10.1093/nar/18.22.6730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kunze N., Klein M., Richter A., Knippers R. Structural characterization of the human DNA topoisomerase I gene promoter. Eur J Biochem. 1990 Dec 12;194(2):323–330. doi: 10.1111/j.1432-1033.1990.tb15620.x. [DOI] [PubMed] [Google Scholar]
  29. Lauster R., Reynaud C. A., Mårtensson I. L., Peter A., Bucchini D., Jami J., Weill J. C. Promoter, enhancer and silencer elements regulate rearrangement of an immunoglobulin transgene. EMBO J. 1993 Dec;12(12):4615–4623. doi: 10.1002/j.1460-2075.1993.tb06150.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Leu T. M., Schatz D. G. rag-1 and rag-2 are components of a high-molecular-weight complex, and association of rag-2 with this complex is rag-1 dependent. Mol Cell Biol. 1995 Oct;15(10):5657–5670. doi: 10.1128/mcb.15.10.5657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lieber M. R., Hesse J. E., Mizuuchi K., Gellert M. Developmental stage specificity of the lymphoid V(D)J recombination activity. Genes Dev. 1987 Oct;1(8):751–761. doi: 10.1101/gad.1.8.751. [DOI] [PubMed] [Google Scholar]
  32. Lin W. C., Desiderio S. Cell cycle regulation of V(D)J recombination-activating protein RAG-2. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2733–2737. doi: 10.1073/pnas.91.7.2733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lin W. C., Desiderio S. Regulation of V(D)J recombination activator protein RAG-2 by phosphorylation. Science. 1993 May 14;260(5110):953–959. doi: 10.1126/science.8493533. [DOI] [PubMed] [Google Scholar]
  34. Loc P. V., Strätling W. H. The matrix attachment regions of the chicken lysozyme gene co-map with the boundaries of the chromatin domain. EMBO J. 1988 Mar;7(3):655–664. doi: 10.1002/j.1460-2075.1988.tb02860.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. McBlane J. F., van Gent D. C., Ramsden D. A., Romeo C., Cuomo C. A., Gellert M., Oettinger M. A. Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps. Cell. 1995 Nov 3;83(3):387–395. doi: 10.1016/0092-8674(95)90116-7. [DOI] [PubMed] [Google Scholar]
  36. Menetski J. P., Gellert M. V(D)J recombination activity in lymphoid cell lines is increased by agents that elevate cAMP. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9324–9328. doi: 10.1073/pnas.87.23.9324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Neale G. A., Fitzgerald T. J., Goorha R. M. Expression of the V(D)J recombinase gene RAG-1 is tightly regulated and involves both transcriptional and post-transcriptional controls. Mol Immunol. 1992 Dec;29(12):1457–1466. doi: 10.1016/0161-5890(92)90219-n. [DOI] [PubMed] [Google Scholar]
  38. Oltz E. M., Alt F. W., Lin W. C., Chen J., Taccioli G., Desiderio S., Rathbun G. A V(D)J recombinase-inducible B-cell line: role of transcriptional enhancer elements in directing V(D)J recombination. Mol Cell Biol. 1993 Oct;13(10):6223–6230. doi: 10.1128/mcb.13.10.6223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ramsden D. A., McBlane J. F., van Gent D. C., Gellert M. Distinct DNA sequence and structure requirements for the two steps of V(D)J recombination signal cleavage. EMBO J. 1996 Jun 17;15(12):3197–3206. [PMC free article] [PubMed] [Google Scholar]
  40. Roman C. A., Baltimore D. Genetic evidence that the RAG1 protein directly participates in V(D)J recombination through substrate recognition. Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2333–2338. doi: 10.1073/pnas.93.6.2333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Romig H., Fackelmayer F. O., Renz A., Ramsperger U., Richter A. Characterization of SAF-A, a novel nuclear DNA binding protein from HeLa cells with high affinity for nuclear matrix/scaffold attachment DNA elements. EMBO J. 1992 Sep;11(9):3431–3440. doi: 10.1002/j.1460-2075.1992.tb05422.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Romig H., Ruff J., Fackelmayer F. O., Patil M. S., Richter A. Characterisation of two intronic nuclear-matrix-attachment regions in the human DNA topoisomerase I gene. Eur J Biochem. 1994 Apr 1;221(1):411–419. doi: 10.1111/j.1432-1033.1994.tb18753.x. [DOI] [PubMed] [Google Scholar]
  43. Roth D. B., Menetski J. P., Nakajima P. B., Bosma M. J., Gellert M. V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes. Cell. 1992 Sep 18;70(6):983–991. doi: 10.1016/0092-8674(92)90248-b. [DOI] [PubMed] [Google Scholar]
  44. Schatz D. G., Oettinger M. A., Schlissel M. S. V(D)J recombination: molecular biology and regulation. Annu Rev Immunol. 1992;10:359–383. doi: 10.1146/annurev.iy.10.040192.002043. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Smider V., Rathmell W. K., Lieber M. R., Chu G. Restoration of X-ray resistance and V(D)J recombination in mutant cells by Ku cDNA. Science. 1994 Oct 14;266(5183):288–291. doi: 10.1126/science.7939667. [DOI] [PubMed] [Google Scholar]
  47. Spanopoulou E., Zaitseva F., Wang F. H., Santagata S., Baltimore D., Panayotou G. The homeodomain region of Rag-1 reveals the parallel mechanisms of bacterial and V(D)J recombination. Cell. 1996 Oct 18;87(2):263–276. doi: 10.1016/s0092-8674(00)81344-6. [DOI] [PubMed] [Google Scholar]
  48. Spitzner J. R., Chung I. K., Muller M. T. Eukaryotic topoisomerase II preferentially cleaves alternating purine-pyrimidine repeats. Nucleic Acids Res. 1990 Jan 11;18(1):1–11. doi: 10.1093/nar/18.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. Stief A., Winter D. M., Strätling W. H., Sippel A. E. A nuclear DNA attachment element mediates elevated and position-independent gene activity. Nature. 1989 Sep 28;341(6240):343–345. doi: 10.1038/341343a0. [DOI] [PubMed] [Google Scholar]
  51. Taccioli G. E., Gottlieb T. M., Blunt T., Priestley A., Demengeot J., Mizuta R., Lehmann A. R., Alt F. W., Jackson S. P., Jeggo P. A. Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination. Science. 1994 Sep 2;265(5177):1442–1445. doi: 10.1126/science.8073286. [DOI] [PubMed] [Google Scholar]
  52. Takahama Y., Singer A. Post-transcriptional regulation of early T cell development by T cell receptor signals. Science. 1992 Nov 27;258(5087):1456–1462. doi: 10.1126/science.1439838. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. Tonegawa S. Somatic generation of antibody diversity. Nature. 1983 Apr 14;302(5909):575–581. doi: 10.1038/302575a0. [DOI] [PubMed] [Google Scholar]
  55. Xu Y., Davidson L., Alt F. W., Baltimore D. Deletion of the Ig kappa light chain intronic enhancer/matrix attachment region impairs but does not abolish V kappa J kappa rearrangement. Immunity. 1996 Apr;4(4):377–385. doi: 10.1016/s1074-7613(00)80251-4. [DOI] [PubMed] [Google Scholar]
  56. van Gent D. C., Mizuuchi K., Gellert M. Similarities between initiation of V(D)J recombination and retroviral integration. Science. 1996 Mar 15;271(5255):1592–1594. doi: 10.1126/science.271.5255.1592. [DOI] [PubMed] [Google Scholar]
  57. van Gent D. C., Ramsden D. A., Gellert M. The RAG1 and RAG2 proteins establish the 12/23 rule in V(D)J recombination. Cell. 1996 Apr 5;85(1):107–113. doi: 10.1016/s0092-8674(00)81086-7. [DOI] [PubMed] [Google Scholar]

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