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. 1988 Apr 25;16(8):3239–3253. doi: 10.1093/nar/16.8.3239

In vivo functional analysis of in vitro protein binding sites in the immunoglobulin heavy chain enhancer.

B P Tsao 1, X F Wang 1, C L Peterson 1, K Calame 1
PMCID: PMC336491  PMID: 3131736

Abstract

We have systematically investigated the functional role of protein binding sites within the mouse immunoglobulin heavy chain enhancer which we previously identified by in vitro binding studies (1,2). Each binding site was deleted, mutant enhancers were cloned 3' of the chloramphenicol acetyl transferase gene in the vector pA10CAT2 and transfected into plasmacytoma cells. We demonstrate that the newly identified site E, located at 324-338 bp, is important for enhancer function; previously identified sites B(uE1), Cl(uE2), C2(uE3) and C3 were also shown to be important for enhancer activity. Sites A and D are not required for IgH enhancer function, as assayed by our methods. Thus, including the octamer site, six protein binding sites which bind at least six different proteins are important for enhancer function in vivo.

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Selected References

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  1. Augereau P., Chambon P. The mouse immunoglobulin heavy-chain enhancer: effect on transcription in vitro and binding of proteins present in HeLa and lymphoid B cell extracts. EMBO J. 1986 Aug;5(8):1791–1797. doi: 10.1002/j.1460-2075.1986.tb04428.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Borrelli E., Hen R., Wasylyk C., Wasylyk B., Chambon P. The immunoglobulin heavy chain enhancer is stimulated by the adenovirus type 2 E1A products in mouse fibroblasts. Proc Natl Acad Sci U S A. 1986 May;83(9):2846–2849. doi: 10.1073/pnas.83.9.2846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Calame K. L. Mechanisms that regulate immunoglobulin gene expression. Annu Rev Immunol. 1985;3:159–195. doi: 10.1146/annurev.iy.03.040185.001111. [DOI] [PubMed] [Google Scholar]
  5. Cockerill P. N., Garrard W. T. Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. Cell. 1986 Jan 31;44(2):273–282. doi: 10.1016/0092-8674(86)90761-0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Dougherty J. P., Augereau P., Chambon P. The mouse immunoglobulin heavy-chain gene enhancer contains sequences that inhibit transcription in vitro in HeLa cell extracts. Mol Cell Biol. 1986 Nov;6(11):4117–4121. doi: 10.1128/mcb.6.11.4117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ephrussi A., Church G. M., Tonegawa S., Gilbert W. B lineage--specific interactions of an immunoglobulin enhancer with cellular factors in vivo. Science. 1985 Jan 11;227(4683):134–140. doi: 10.1126/science.3917574. [DOI] [PubMed] [Google Scholar]
  9. Falkner F. G., Zachau H. G. Correct transcription of an immunoglobulin kappa gene requires an upstream fragment containing conserved sequence elements. Nature. 1984 Jul 5;310(5972):71–74. doi: 10.1038/310071a0. [DOI] [PubMed] [Google Scholar]
  10. Fletcher C., Heintz N., Roeder R. G. Purification and characterization of OTF-1, a transcription factor regulating cell cycle expression of a human histone H2b gene. Cell. 1987 Dec 4;51(5):773–781. doi: 10.1016/0092-8674(87)90100-0. [DOI] [PubMed] [Google Scholar]
  11. Gerster T., Matthias P., Thali M., Jiricny J., Schaffner W. Cell type-specificity elements of the immunoglobulin heavy chain gene enhancer. EMBO J. 1987 May;6(5):1323–1330. doi: 10.1002/j.1460-2075.1987.tb02371.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hall C. V., Jacob P. E., Ringold G. M., Lee F. Expression and regulation of Escherichia coli lacZ gene fusions in mammalian cells. J Mol Appl Genet. 1983;2(1):101–109. [PubMed] [Google Scholar]
  15. Imler J. L., Lemaire C., Wasylyk C., Wasylyk B. Negative regulation contributes to tissue specificity of the immunoglobulin heavy-chain enhancer. Mol Cell Biol. 1987 Jul;7(7):2558–2567. doi: 10.1128/mcb.7.7.2558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Junker S., Pedersen S. Time course of arrest of immunoglobulin expression in heterokaryons and early hybrids of human lymphoma cells and mouse fibroblasts. A study of transcriptional and translational events. Exp Cell Res. 1985 Jun;158(2):349–359. doi: 10.1016/0014-4827(85)90460-4. [DOI] [PubMed] [Google Scholar]
  17. Kadesch T., Zervos P., Ruezinsky D. Functional analysis of the murine IgH enhancer: evidence for negative control of cell-type specificity. Nucleic Acids Res. 1986 Oct 24;14(20):8209–8221. doi: 10.1093/nar/14.20.8209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Landolfi N. F., Capra J. D., Tucker P. W. Interaction of cell-type-specific nuclear proteins with immunoglobulin VH promoter region sequences. Nature. 1986 Oct 9;323(6088):548–551. doi: 10.1038/323548a0. [DOI] [PubMed] [Google Scholar]
  19. Lenardo M., Pierce J. W., Baltimore D. Protein-binding sites in Ig gene enhancers determine transcriptional activity and inducibility. Science. 1987 Jun 19;236(4808):1573–1577. doi: 10.1126/science.3109035. [DOI] [PubMed] [Google Scholar]
  20. Mattaj I. W., Lienhard S., Jiricny J., De Robertis E. M. An enhancer-like sequence within the Xenopus U2 gene promoter facilitates the formation of stable transcription complexes. Nature. 1985 Jul 11;316(6024):163–167. doi: 10.1038/316163a0. [DOI] [PubMed] [Google Scholar]
  21. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  22. Mercola M., Goverman J., Mirell C., Calame K. Immunoglobulin heavy-chain enhancer requires one or more tissue-specific factors. Science. 1985 Jan 18;227(4684):266–270. doi: 10.1126/science.3917575. [DOI] [PubMed] [Google Scholar]
  23. Neuberger M. S. Expression and regulation of immunoglobulin heavy chain gene transfected into lymphoid cells. EMBO J. 1983;2(8):1373–1378. doi: 10.1002/j.1460-2075.1983.tb01594.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nielsen D. A., Chou J., MacKrell A. J., Casadaban M. J., Steiner D. F. Expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5198–5202. doi: 10.1073/pnas.80.17.5198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Oi V. T., Morrison S. L., Herzenberg L. A., Berg P. Immunoglobulin gene expression in transformed lymphoid cells. Proc Natl Acad Sci U S A. 1983 Feb;80(3):825–829. doi: 10.1073/pnas.80.3.825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ornitz D. M., Hammer R. E., Davison B. L., Brinster R. L., Palmiter R. D. Promoter and enhancer elements from the rat elastase I gene function independently of each other and of heterologous enhancers. Mol Cell Biol. 1987 Oct;7(10):3466–3472. doi: 10.1128/mcb.7.10.3466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Parslow T. G., Blair D. L., Murphy W. J., Granner D. K. Structure of the 5' ends of immunoglobulin genes: a novel conserved sequence. Proc Natl Acad Sci U S A. 1984 May;81(9):2650–2654. doi: 10.1073/pnas.81.9.2650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Parslow T. G., Jones S. D., Bond B., Yamamoto K. R. The immunoglobulin octanucleotide: independent activity and selective interaction with enhancers. Science. 1987 Mar 20;235(4795):1498–1501. doi: 10.1126/science.3029871. [DOI] [PubMed] [Google Scholar]
  29. Peterson C. L., Calame K. L. Complex protein binding within the mouse immunoglobulin heavy-chain enhancer. Mol Cell Biol. 1987 Dec;7(12):4194–4203. doi: 10.1128/mcb.7.12.4194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Peterson C. L., Orth K., Calame K. L. Binding in vitro of multiple cellular proteins to immunoglobulin heavy-chain enhancer DNA. Mol Cell Biol. 1986 Dec;6(12):4168–4178. doi: 10.1128/mcb.6.12.4168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rosales R., Vigneron M., Macchi M., Davidson I., Xiao J. H., Chambon P. In vitro binding of cell-specific and ubiquitous nuclear proteins to the octamer motif of the SV40 enhancer and related motifs present in other promoters and enhancers. EMBO J. 1987 Oct;6(10):3015–3025. doi: 10.1002/j.1460-2075.1987.tb02607.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Scheidereit C., Heguy A., Roeder R. G. Identification and purification of a human lymphoid-specific octamer-binding protein (OTF-2) that activates transcription of an immunoglobulin promoter in vitro. Cell. 1987 Dec 4;51(5):783–793. doi: 10.1016/0092-8674(87)90101-2. [DOI] [PubMed] [Google Scholar]
  33. Schlokat U., Bohmann D., Schöler H., Gruss P. Nuclear factors binding specific sequences within the immunoglobulin enhancer interact differentially with other enhancer elements. EMBO J. 1986 Dec 1;5(12):3251–3258. doi: 10.1002/j.1460-2075.1986.tb04636.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schöler H. R., Gruss P. Cell type-specific transcriptional enhancement in vitro requires the presence of trans-acting factors. EMBO J. 1985 Nov;4(11):3005–3013. doi: 10.1002/j.1460-2075.1985.tb04036.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sen R., Baltimore D. In vitro transcription of immunoglobulin genes in a B-cell extract: effects of enhancer and promoter sequences. Mol Cell Biol. 1987 May;7(5):1989–1994. doi: 10.1128/mcb.7.5.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sen R., Baltimore D. Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell. 1986 Aug 29;46(5):705–716. doi: 10.1016/0092-8674(86)90346-6. [DOI] [PubMed] [Google Scholar]
  37. Sive H. L., Heintz N., Roeder R. G. Multiple sequence elements are required for maximal in vitro transcription of a human histone H2B gene. Mol Cell Biol. 1986 Oct;6(10):3329–3340. doi: 10.1128/mcb.6.10.3329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Staudt L. M., Singh H., Sen R., Wirth T., Sharp P. A., Baltimore D. A lymphoid-specific protein binding to the octamer motif of immunoglobulin genes. Nature. 1986 Oct 16;323(6089):640–643. doi: 10.1038/323640a0. [DOI] [PubMed] [Google Scholar]
  39. Wang X. F., Calame K. The endogenous immunoglobulin heavy chain enhancer can activate tandem VH promoters separated by a large distance. Cell. 1985 Dec;43(3 Pt 2):659–665. doi: 10.1016/0092-8674(85)90238-7. [DOI] [PubMed] [Google Scholar]
  40. Wasylyk C., Wasylyk B. The immunoglobulin heavy-chain B-lymphocyte enhancer efficiently stimulates transcription in non-lymphoid cells. EMBO J. 1986 Mar;5(3):553–560. doi: 10.1002/j.1460-2075.1986.tb04246.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Weinberger J., Baltimore D., Sharp P. A. Distinct factors bind to apparently homologous sequences in the immunoglobulin heavy-chain enhancer. 1986 Aug 28-Sep 3Nature. 322(6082):846–848. doi: 10.1038/322846a0. [DOI] [PubMed] [Google Scholar]
  42. Zenke M., Grundström T., Matthes H., Wintzerith M., Schatz C., Wildeman A., Chambon P. Multiple sequence motifs are involved in SV40 enhancer function. EMBO J. 1986 Feb;5(2):387–397. doi: 10.1002/j.1460-2075.1986.tb04224.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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