Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Aug;17(8):4553–4561. doi: 10.1128/mcb.17.8.4553

Enhancer control of local accessibility to V(D)J recombinase.

M T McMurry 1, C Hernandez-Munain 1, P Lauzurica 1, M S Krangel 1
PMCID: PMC232309  PMID: 9234713

Abstract

We have studied the role of transcriptional enhancers in providing recombination signal sequence (RSS) accessibility to V(D)J recombinase by examining mice carrying a transgenic human T-cell receptor (TCR) delta gene minilocus. This transgene is composed of unrearranged variable (Vdelta and Vdelta2), diversity (Ddelta3), joining (Jdelta1 and Jdelta3), and constant (Cdelta) gene segments. Previous data indicated that with the TCR delta enhancer (Edelta) present in the Jdelta3-Cdelta intron, V(D)J recombination proceeds stepwise, first V to D and then VD to J. With the enhancer deleted or mutated, V-to-D rearrangement is intact, but VD-to-J rearrangement is inhibited. We proposed that Edelta is necessary for J segment but not D segment accessibility and that J segment inaccessibility in the enhancerless minilocus resulted in the observed V(D)J recombination phenotype. In this study, we tested this notion by using ligation-mediated PCR to assess the formation of recombination-activating gene (RAG)-dependent double-strand breaks (DSBs) at RSSs 3' of Ddelta3 and 5' of Jdelta1. In five lines of mice carrying multicopy integrants of constructs that either lacked Edelta or carried an inactivated Edelta, the frequency of DSBs 5' of Jdelta1 was dramatically reduced relative to that in the wild type, whereas the frequency of DSBs 3' of Ddelta3 was unaffected. We interpret these results to indicate that Edelta is required for Jdelta1 but not Ddelta3 accessibility within the minilocus, and we conclude that enhancers regulate V(D)J recombination by providing local accessibility to the recombinase. cis-acting elements other than Edelta must maintain Ddelta3 in an accessible state in the absence of Edelta. The analysis of DSB formation in a single-copy minilocus integrant indicates that efficient DSB formation at the accessible RSS 3' of Ddelta3 requires an accessible partner RSS, arguing that RSS synapsis is required for DSB formation in chromosomal substrates in vivo.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

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

  1. Alt F. W., Blackwell T. K., Yancopoulos G. D. Development of the primary antibody repertoire. Science. 1987 Nov 20;238(4830):1079–1087. doi: 10.1126/science.3317825. [DOI] [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. 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]
  4. 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]
  5. 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]
  6. 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]
  7. Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Chien Y. H., Iwashima M., Wettstein D. A., Kaplan K. B., Elliott J. F., Born W., Davis M. M. T-cell receptor delta gene rearrangements in early thymocytes. Nature. 1987 Dec 24;330(6150):722–727. doi: 10.1038/330722a0. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Elliott J. F., Rock E. P., Patten P. A., Davis M. M., Chien Y. H. The adult T-cell receptor delta-chain is diverse and distinct from that of fetal thymocytes. Nature. 1988 Feb 18;331(6157):627–631. doi: 10.1038/331627a0. [DOI] [PubMed] [Google Scholar]
  12. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Forrester W. C., van Genderen C., Jenuwein T., Grosschedl R. Dependence of enhancer-mediated transcription of the immunoglobulin mu gene on nuclear matrix attachment regions. Science. 1994 Aug 26;265(5176):1221–1225. doi: 10.1126/science.8066460. [DOI] [PubMed] [Google Scholar]
  15. Gellert M. Molecular analysis of V(D)J recombination. Annu Rev Genet. 1992;26:425–446. doi: 10.1146/annurev.ge.26.120192.002233. [DOI] [PubMed] [Google Scholar]
  16. Gorman J. R., van der Stoep N., Monroe R., Cogne M., Davidson L., Alt F. W. The Ig(kappa) enhancer influences the ratio of Ig(kappa) versus Ig(lambda) B lymphocytes. Immunity. 1996 Sep;5(3):241–252. doi: 10.1016/s1074-7613(00)80319-2. [DOI] [PubMed] [Google Scholar]
  17. Han S., Zheng B., Schatz D. G., Spanopoulou E., Kelsoe G. Neoteny in lymphocytes: Rag1 and Rag2 expression in germinal center B cells. Science. 1996 Dec 20;274(5295):2094–2097. doi: 10.1126/science.274.5295.2094. [DOI] [PubMed] [Google Scholar]
  18. Hendrickson E. A., Liu V. F., Weaver D. T. Strand breaks without DNA rearrangement in V (D)J recombination. Mol Cell Biol. 1991 Jun;11(6):3155–3162. doi: 10.1128/mcb.11.6.3155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hernández-Munain C., Lauzurica P., Krangel M. S. Regulation of T cell receptor delta gene rearrangement by c-Myb. J Exp Med. 1996 Jan 1;183(1):289–293. doi: 10.1084/jem.183.1.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hikida M., Mori M., Takai T., Tomochika K., Hamatani K., Ohmori H. Reexpression of RAG-1 and RAG-2 genes in activated mature mouse B cells. Science. 1996 Dec 20;274(5295):2092–2094. doi: 10.1126/science.274.5295.2092. [DOI] [PubMed] [Google Scholar]
  21. Jackson S. P., Jeggo P. A. DNA double-strand break repair and V(D)J recombination: involvement of DNA-PK. Trends Biochem Sci. 1995 Oct;20(10):412–415. doi: 10.1016/s0968-0004(00)89090-8. [DOI] [PubMed] [Google Scholar]
  22. Jenuwein T., Forrester W. C., Qiu R. G., Grosschedl R. The immunoglobulin mu enhancer core establishes local factor access in nuclear chromatin independent of transcriptional stimulation. Genes Dev. 1993 Oct;7(10):2016–2032. doi: 10.1101/gad.7.10.2016. [DOI] [PubMed] [Google Scholar]
  23. Kirchgessner C. U., Patil C. K., Evans J. W., Cuomo C. A., Fried L. M., Carter T., Oettinger M. A., Brown J. M. DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect. Science. 1995 Feb 24;267(5201):1178–1183. doi: 10.1126/science.7855601. [DOI] [PubMed] [Google Scholar]
  24. Krangel M. S., Yssel H., Brocklehurst C., Spits H. A distinct wave of human T cell receptor gamma/delta lymphocytes in the early fetal thymus: evidence for controlled gene rearrangement and cytokine production. J Exp Med. 1990 Sep 1;172(3):847–859. doi: 10.1084/jem.172.3.847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Lauzurica P., Krangel M. S. Enhancer-dependent and -independent steps in the rearrangement of a human T cell receptor delta transgene. J Exp Med. 1994 Jan 1;179(1):43–55. doi: 10.1084/jem.179.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lauzurica P., Krangel M. S. Temporal and lineage-specific control of T cell receptor alpha/delta gene rearrangement by T cell receptor alpha and delta enhancers. J Exp Med. 1994 Jun 1;179(6):1913–1921. doi: 10.1084/jem.179.6.1913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lauzurica P., Zhong X. P., Krangel M. S., Roberts J. L. Regulation of T cell receptor delta gene rearrangement by CBF/PEBP2. J Exp Med. 1997 Apr 7;185(7):1193–1201. doi: 10.1084/jem.185.7.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lewis S. M., Hesse J. E. Cutting and closing without recombination in V(D)J joining. EMBO J. 1991 Dec;10(12):3631–3639. doi: 10.1002/j.1460-2075.1991.tb04929.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lewis S. M. The mechanism of V(D)J joining: lessons from molecular, immunological, and comparative analyses. Adv Immunol. 1994;56:27–150. doi: 10.1016/s0065-2776(08)60450-2. [DOI] [PubMed] [Google Scholar]
  31. Loh E. Y., Cwirla S., Serafini A. T., Phillips J. H., Lanier L. L. Human T-cell-receptor delta chain: genomic organization, diversity, and expression in populations of cells. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9714–9718. doi: 10.1073/pnas.85.24.9714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Mueller P. R., Wold B. In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science. 1989 Nov 10;246(4931):780–786. doi: 10.1126/science.2814500. [DOI] [PubMed] [Google Scholar]
  35. Nussenzweig A., Chen C., da Costa Soares V., Sanchez M., Sokol K., Nussenzweig M. C., Li G. C. Requirement for Ku80 in growth and immunoglobulin V(D)J recombination. Nature. 1996 Aug 8;382(6591):551–555. doi: 10.1038/382551a0. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Okada A., Mendelsohn M., Alt F. Differential activation of transcription versus recombination of transgenic T cell receptor beta variable region gene segments in B and T lineage cells. J Exp Med. 1994 Jul 1;180(1):261–272. doi: 10.1084/jem.180.1.261. [DOI] [PMC free article] [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. Pikaart M., Feng J., Villeponteau B. The polyomavirus enhancer activates chromatin accessibility on integration into the HPRT gene. Mol Cell Biol. 1992 Dec;12(12):5785–5792. doi: 10.1128/mcb.12.12.5785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Roberts J. L., Lauzurica P., Krangel M. S. Developmental regulation of VDJ recombination by the core fragment of the T cell receptor alpha enhancer. J Exp Med. 1997 Jan 6;185(1):131–140. doi: 10.1084/jem.185.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Roth D. B., Nakajima P. B., Menetski J. P., Bosma M. J., Gellert M. V(D)J recombination in mouse thymocytes: double-strand breaks near T cell receptor delta rearrangement signals. Cell. 1992 Apr 3;69(1):41–53. doi: 10.1016/0092-8674(92)90117-u. [DOI] [PubMed] [Google Scholar]
  43. Roth D. B., Zhu C., Gellert M. Characterization of broken DNA molecules associated with V(D)J recombination. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10788–10792. doi: 10.1073/pnas.90.22.10788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. Schlissel M., Constantinescu A., Morrow T., Baxter M., Peng A. Double-strand signal sequence breaks in V(D)J recombination are blunt, 5'-phosphorylated, RAG-dependent, and cell cycle regulated. Genes Dev. 1993 Dec;7(12B):2520–2532. doi: 10.1101/gad.7.12b.2520. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Sheehan K. M., Lieber M. R. V(D)J recombination: signal and coding joint resolution are uncoupled and depend on parallel synapsis of the sites. Mol Cell Biol. 1993 Mar;13(3):1363–1370. doi: 10.1128/mcb.13.3.1363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. Sleckman B. P., Gorman J. R., Alt F. W. Accessibility control of antigen-receptor variable-region gene assembly: role of cis-acting elements. Annu Rev Immunol. 1996;14:459–481. doi: 10.1146/annurev.immunol.14.1.459. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. Stanhope-Baker P., Hudson K. M., Shaffer A. L., Constantinescu A., Schlissel M. S. Cell type-specific chromatin structure determines the targeting of V(D)J recombinase activity in vitro. Cell. 1996 Jun 14;85(6):887–897. doi: 10.1016/s0092-8674(00)81272-6. [DOI] [PubMed] [Google Scholar]
  54. 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]
  55. 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]
  56. Villey I., Caillol D., Selz F., Ferrier P., de Villartay J. P. Defect in rearrangement of the most 5' TCR-J alpha following targeted deletion of T early alpha (TEA): implications for TCR alpha locus accessibility. Immunity. 1996 Oct;5(4):331–342. doi: 10.1016/s1074-7613(00)80259-9. [DOI] [PubMed] [Google Scholar]
  57. Weaver D. T. What to do at an end: DNA double-strand-break repair. Trends Genet. 1995 Oct;11(10):388–392. doi: 10.1016/s0168-9525(00)89121-0. [DOI] [PubMed] [Google Scholar]
  58. 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]
  59. Zhong X. P., Krangel M. S. An enhancer-blocking element between alpha and delta gene segments within the human T cell receptor alpha/delta locus. Proc Natl Acad Sci U S A. 1997 May 13;94(10):5219–5224. doi: 10.1073/pnas.94.10.5219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zhu C., Bogue M. A., Lim D. S., Hasty P., Roth D. B. Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates. Cell. 1996 Aug 9;86(3):379–389. doi: 10.1016/s0092-8674(00)80111-7. [DOI] [PubMed] [Google Scholar]
  61. 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]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES