Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1996 Dec 15;24(24):5034–5044. doi: 10.1093/nar/24.24.5034

An enhancer LEF-1/TCF-1 site is essential for insertion site-independent transgene expression in thymus.

T L Haynes 1, M B Thomas 1, M R Dusing 1, M T Valerius 1, S S Potter 1, D A Wiginton 1
PMCID: PMC146351  PMID: 9016677

Abstract

Transcriptional activation of eukaryotic genes involves assembly of specific multiprotein complexes on the promoters and enhancers of the genes. Recently, it has been proposed that the role of some of the proteins in the complex may be architectural, involving DNA bending, orchestration of protein-protein interaction and modulation of nucleosome structure. This role has been proposed for the HMG proteins LEF-1 and TCF-1. We examined the role of a LEF-1/TCF-1 binding site in the human adenosine deaminase (ADA) thymic enhancer. Mutational analysis demonstrated that a functional LEF-1/TCF-1 binding site is not required for enhancer-mediated transcriptional activation in transient transfection studies, but is essential for enhancer function in the in vivo chromatin context of transgenic mice. Mutation of the LEF-1/TCF-1 site destroyed the ability of the ADA enhancer/locus control region to specify high level, insertion site-independent transgene expression in thymus. DNase I and DpnII accessibility experiments indicated dramatic changes in the chromatin organization of the ADA enhancer in transgenic mice with a mutated LEF-1/TCF-1 site. This supports the hypothesis that factors binding the LEF-1/TCF-1 site play an architectural role during the in vivo activation of the ADA enhancer, possibly involving chromatin modification.

Full Text

The Full Text of this article is available as a PDF (535.2 KB).

Selected References

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

  1. Almouzni G., Clark D. J., Méchali M., Wolffe A. P. Chromatin assembly on replicating DNA in vitro. Nucleic Acids Res. 1990 Oct 11;18(19):5767–5774. doi: 10.1093/nar/18.19.5767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Archer T. K., Cordingley M. G., Wolford R. G., Hager G. L. Transcription factor access is mediated by accurately positioned nucleosomes on the mouse mammary tumor virus promoter. Mol Cell Biol. 1991 Feb;11(2):688–698. doi: 10.1128/mcb.11.2.688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Archer T. K., Lefebvre P., Wolford R. G., Hager G. L. Transcription factor loading on the MMTV promoter: a bimodal mechanism for promoter activation. Science. 1992 Mar 20;255(5051):1573–1576. doi: 10.1126/science.1347958. [DOI] [PubMed] [Google Scholar]
  4. Aronow B. J., Ebert C. A., Valerius M. T., Potter S. S., Wiginton D. A., Witte D. P., Hutton J. J. Dissecting a locus control region: facilitation of enhancer function by extended enhancer-flanking sequences. Mol Cell Biol. 1995 Feb;15(2):1123–1135. doi: 10.1128/mcb.15.2.1123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Aronow B. J., Silbiger R. N., Dusing M. R., Stock J. L., Yager K. L., Potter S. S., Hutton J. J., Wiginton D. A. Functional analysis of the human adenosine deaminase gene thymic regulatory region and its ability to generate position-independent transgene expression. Mol Cell Biol. 1992 Sep;12(9):4170–4185. doi: 10.1128/mcb.12.9.4170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Aronow B., Lattier D., Silbiger R., Dusing M., Hutton J., Jones G., Stock J., McNeish J., Potter S., Witte D. Evidence for a complex regulatory array in the first intron of the human adenosine deaminase gene. Genes Dev. 1989 Sep;3(9):1384–1400. doi: 10.1101/gad.3.9.1384. [DOI] [PubMed] [Google Scholar]
  7. Brickner A. G., Gossage D. L., Dusing M. R., Wiginton D. A. Identification of a murine homolog of the human adenosine deaminase thymic enhancer. Gene. 1995 Dec 29;167(1-2):261–266. doi: 10.1016/0378-1119(95)00673-7. [DOI] [PubMed] [Google Scholar]
  8. Carlsson P., Waterman M. L., Jones K. A. The hLEF/TCF-1 alpha HMG protein contains a context-dependent transcriptional activation domain that induces the TCR alpha enhancer in T cells. Genes Dev. 1993 Dec;7(12A):2418–2430. doi: 10.1101/gad.7.12a.2418. [DOI] [PubMed] [Google Scholar]
  9. Clevers H. C., Oosterwegel M. A., Georgopoulos K. Transcription factors in early T-cell development. Immunol Today. 1993 Dec;14(12):591–596. doi: 10.1016/0167-5699(93)90198-T. [DOI] [PubMed] [Google Scholar]
  10. Clevers H., Lonberg N., Dunlap S., Lacy E., Terhorst C. An enhancer located in a CpG-island 3' to the TCR/CD3-epsilon gene confers T lymphocyte-specificity to its promoter. EMBO J. 1989 Sep;8(9):2527–2535. doi: 10.1002/j.1460-2075.1989.tb08390.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ess K. C., Whitaker T. L., Cost G. J., Witte D. P., Hutton J. J., Aronow B. J. A central role for a single c-Myb binding site in a thymic locus control region. Mol Cell Biol. 1995 Oct;15(10):5707–5715. doi: 10.1128/mcb.15.10.5707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ferrari S., Harley V. R., Pontiggia A., Goodfellow P. N., Lovell-Badge R., Bianchi M. E. SRY, like HMG1, recognizes sharp angles in DNA. EMBO J. 1992 Dec;11(12):4497–4506. doi: 10.1002/j.1460-2075.1992.tb05551.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gastrop J., Hoevenagel R., Young J. R., Clevers H. C. A common ancestor of the mammalian transcription factors TCF-1 and TCF-1 alpha/LEF-1 expressed in chicken T cells. Eur J Immunol. 1992 May;22(5):1327–1330. doi: 10.1002/eji.1830220531. [DOI] [PubMed] [Google Scholar]
  15. Giese K., Amsterdam A., Grosschedl R. DNA-binding properties of the HMG domain of the lymphoid-specific transcriptional regulator LEF-1. Genes Dev. 1991 Dec;5(12B):2567–2578. doi: 10.1101/gad.5.12b.2567. [DOI] [PubMed] [Google Scholar]
  16. Giese K., Cox J., Grosschedl R. The HMG domain of lymphoid enhancer factor 1 bends DNA and facilitates assembly of functional nucleoprotein structures. Cell. 1992 Apr 3;69(1):185–195. doi: 10.1016/0092-8674(92)90129-z. [DOI] [PubMed] [Google Scholar]
  17. Giese K., Grosschedl R. LEF-1 contains an activation domain that stimulates transcription only in a specific context of factor-binding sites. EMBO J. 1993 Dec;12(12):4667–4676. doi: 10.1002/j.1460-2075.1993.tb06155.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Giese K., Kingsley C., Kirshner J. R., Grosschedl R. Assembly and function of a TCR alpha enhancer complex is dependent on LEF-1-induced DNA bending and multiple protein-protein interactions. Genes Dev. 1995 Apr 15;9(8):995–1008. doi: 10.1101/gad.9.8.995. [DOI] [PubMed] [Google Scholar]
  19. Gottschalk L. R., Leiden J. M. Identification and functional characterization of the human T-cell receptor beta gene transcriptional enhancer: common nuclear proteins interact with the transcriptional regulatory elements of the T-cell receptor alpha and beta genes. Mol Cell Biol. 1990 Oct;10(10):5486–5495. doi: 10.1128/mcb.10.10.5486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Harper M. E., Marselle L. M., Gallo R. C., Wong-Staal F. Detection of lymphocytes expressing human T-lymphotropic virus type III in lymph nodes and peripheral blood from infected individuals by in situ hybridization. Proc Natl Acad Sci U S A. 1986 Feb;83(3):772–776. doi: 10.1073/pnas.83.3.772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hayashi S., Gillam I. C., Delaney A. D., Tener G. M. Acetylation of chromosome squashes of Drosophila melanogaster decreases the background in autoradiographs from hybridization with [125I]-labeled RNA. J Histochem Cytochem. 1978 Aug;26(8):677–679. doi: 10.1177/26.8.99471. [DOI] [PubMed] [Google Scholar]
  22. Ho I. C., Leiden J. M. Regulation of the human T-cell receptor alpha gene enhancer: multiple ubiquitous and T-cell-specific nuclear proteins interact with four hypomethylated enhancer elements. Mol Cell Biol. 1990 Sep;10(9):4720–4727. doi: 10.1128/mcb.10.9.4720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ho I. C., Yang L. H., Morle G., Leiden J. M. A T-cell-specific transcriptional enhancer element 3' of C alpha in the human T-cell receptor alpha locus. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6714–6718. doi: 10.1073/pnas.86.17.6714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jones K. A., Peterlin B. M. Control of RNA initiation and elongation at the HIV-1 promoter. Annu Rev Biochem. 1994;63:717–743. doi: 10.1146/annurev.bi.63.070194.003441. [DOI] [PubMed] [Google Scholar]
  25. Kim J. Y., Gonzalez-Scarano F., Zeichner S. L., Alwine J. C. Replication of type 1 human immunodeficiency viruses containing linker substitution mutations in the -201 to -130 region of the long terminal repeat. J Virol. 1993 Mar;67(3):1658–1662. doi: 10.1128/jvi.67.3.1658-1662.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Leiden J. M. Transcriptional regulation of T cell receptor genes. Annu Rev Immunol. 1993;11:539–570. doi: 10.1146/annurev.iy.11.040193.002543. [DOI] [PubMed] [Google Scholar]
  27. Meyer K. B., Ireland J. Activation of the immunoglobulin kappa 3' enhancer in pre-B cells correlates with the suppression of a nuclear factor binding to a sequence flanking the active core. Nucleic Acids Res. 1994 May 11;22(9):1576–1582. doi: 10.1093/nar/22.9.1576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mitchell P. J., Tjian R. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science. 1989 Jul 28;245(4916):371–378. doi: 10.1126/science.2667136. [DOI] [PubMed] [Google Scholar]
  29. Oosterwegel M., van de Wetering M., Dooijes D., Klomp L., Winoto A., Georgopoulos K., Meijlink F., Clevers H. Cloning of murine TCF-1, a T cell-specific transcription factor interacting with functional motifs in the CD3-epsilon and T cell receptor alpha enhancers. J Exp Med. 1991 May 1;173(5):1133–1142. doi: 10.1084/jem.173.5.1133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Oosterwegel M., van de Wetering M., Timmerman J., Kruisbeek A., Destree O., Meijlink F., Clevers H. Differential expression of the HMG box factors TCF-1 and LEF-1 during murine embryogenesis. Development. 1993 Jun;118(2):439–448. doi: 10.1242/dev.118.2.439. [DOI] [PubMed] [Google Scholar]
  31. Redondo J. M., Hata S., Brocklehurst C., Krangel M. S. A T cell-specific transcriptional enhancer within the human T cell receptor delta locus. Science. 1990 Mar 9;247(4947):1225–1229. doi: 10.1126/science.2156339. [DOI] [PubMed] [Google Scholar]
  32. Sawada S., Littman D. R. Identification and characterization of a T-cell-specific enhancer adjacent to the murine CD4 gene. Mol Cell Biol. 1991 Nov;11(11):5506–5515. doi: 10.1128/mcb.11.11.5506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sheridan P. L., Sheline C. T., Cannon K., Voz M. L., Pazin M. J., Kadonaga J. T., Jones K. A. Activation of the HIV-1 enhancer by the LEF-1 HMG protein on nucleosome-assembled DNA in vitro. Genes Dev. 1995 Sep 1;9(17):2090–2104. doi: 10.1101/gad.9.17.2090. [DOI] [PubMed] [Google Scholar]
  34. Simpson R. T. Nucleosome positioning can affect the function of a cis-acting DNA element in vivo. Nature. 1990 Jan 25;343(6256):387–389. doi: 10.1038/343387a0. [DOI] [PubMed] [Google Scholar]
  35. Tjian R., Maniatis T. Transcriptional activation: a complex puzzle with few easy pieces. Cell. 1994 Apr 8;77(1):5–8. doi: 10.1016/0092-8674(94)90227-5. [DOI] [PubMed] [Google Scholar]
  36. Travis A., Amsterdam A., Belanger C., Grosschedl R. LEF-1, a gene encoding a lymphoid-specific protein with an HMG domain, regulates T-cell receptor alpha enhancer function [corrected]. Genes Dev. 1991 May;5(5):880–894. doi: 10.1101/gad.5.5.880. [DOI] [PubMed] [Google Scholar]
  37. Verbeek S., Izon D., Hofhuis F., Robanus-Maandag E., te Riele H., van de Wetering M., Oosterwegel M., Wilson A., MacDonald H. R., Clevers H. An HMG-box-containing T-cell factor required for thymocyte differentiation. Nature. 1995 Mar 2;374(6517):70–74. doi: 10.1038/374070a0. [DOI] [PubMed] [Google Scholar]
  38. Waterman M. L., Fischer W. H., Jones K. A. A thymus-specific member of the HMG protein family regulates the human T cell receptor C alpha enhancer. Genes Dev. 1991 Apr;5(4):656–669. doi: 10.1101/gad.5.4.656. [DOI] [PubMed] [Google Scholar]
  39. Waterman M. L., Jones K. A. Purification of TCF-1 alpha, a T-cell-specific transcription factor that activates the T-cell receptor C alpha gene enhancer in a context-dependent manner. New Biol. 1990 Jul;2(7):621–636. [PubMed] [Google Scholar]
  40. Zeichner S. L., Kim J. Y., Alwine J. C. Linker-scanning mutational analysis of the transcriptional activity of the human immunodeficiency virus type 1 long terminal repeat. J Virol. 1991 May;65(5):2436–2444. doi: 10.1128/jvi.65.5.2436-2444.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. van Genderen C., Okamura R. M., Fariñas I., Quo R. G., Parslow T. G., Bruhn L., Grosschedl R. Development of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient mice. Genes Dev. 1994 Nov 15;8(22):2691–2703. doi: 10.1101/gad.8.22.2691. [DOI] [PubMed] [Google Scholar]
  42. van de Wetering M., Clevers H. Sequence-specific interaction of the HMG box proteins TCF-1 and SRY occurs within the minor groove of a Watson-Crick double helix. EMBO J. 1992 Aug;11(8):3039–3044. doi: 10.1002/j.1460-2075.1992.tb05374.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES