Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Mar;16(3):829–838. doi: 10.1128/mcb.16.3.829

A DNA-bending protein interacts with an essential upstream regulatory element of the human embryonic beta-like globin gene.

M A Dyer 1, R Naidoo 1, R J Hayes 1, C J Larson 1, G L Verdine 1, M H Baron 1
PMCID: PMC231063  PMID: 8622684

Abstract

The mammalian beta-like globin gene family has served as an important model system for analysis of tissue- and developmental state-specific gene regulation. Although the activities of a number of regulatory proteins have been implicated in the erythroid cell-specific transcription of globin genes, the mechanisms that restrict their expression to discrete stages of development are less well understood. We have previously identified a novel regulatory element (PRE II) upstream from the human embryonic beta-like globin gene (epsilon) that synergizes with other sequences to confer tissue- and stage-specific expression on a minimal epsilon-globin gene promoter in cultured embryonic erythroid cells. Binding of an erythroid nuclear protein (PRE II-binding factor [PRE-IIBF]) to the PRE II control element is required for promoter activation. Here we report on some of the biochemical properties of PREIIBF, including the characterization of its specificity and affinity for DNA. The embryonic and adult forms of PREIIBF recognize their cognate sequences with identical specificities, supporting our earlier conclusion that they are very similar proteins. PREIIBF binds DNA as a single polypeptide with an Mr of approximately 80,000 to 85,000 and introduces a bend into the target DNA molecule. These results suggest a mechanism by which PREIIBF may contribute to the regulation of the embryonic beta-like globin gene within the context of a complex locus.

Full Text

The Full Text of this article is available as a PDF (557.6 KB).

Selected References

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

  1. Affolter M., Percival-Smith A., Müller M., Leupin W., Gehring W. J. DNA binding properties of the purified Antennapedia homeodomain. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4093–4097. doi: 10.1073/pnas.87.11.4093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andrews N. C., Erdjument-Bromage H., Davidson M. B., Tempst P., Orkin S. H. Erythroid transcription factor NF-E2 is a haematopoietic-specific basic-leucine zipper protein. Nature. 1993 Apr 22;362(6422):722–728. doi: 10.1038/362722a0. [DOI] [PubMed] [Google Scholar]
  3. Avitahl N., Calame K. The C/EBP family of proteins distorts DNA upon binding but does not introduce a large directed bend. J Biol Chem. 1994 Sep 23;269(38):23553–23562. [PubMed] [Google Scholar]
  4. Baron M. H., Farrington S. M. Positive regulators of the lineage-specific transcription factor GATA-1 in differentiating erythroid cells. Mol Cell Biol. 1994 May;14(5):3108–3114. doi: 10.1128/mcb.14.5.3108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baron M. H., Maniatis T. Rapid reprogramming of globin gene expression in transient heterokaryons. Cell. 1986 Aug 15;46(4):591–602. doi: 10.1016/0092-8674(86)90885-8. [DOI] [PubMed] [Google Scholar]
  6. Baron M. H., Maniatis T. Regulated expression of human alpha- and beta-globin genes in transient heterokaryons. Mol Cell Biol. 1991 Mar;11(3):1239–1247. doi: 10.1128/mcb.11.3.1239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brown B. A., Padgett R. W., Hardies S. C., Hutchison C. A., 3rd, Edgell M. H. beta-globin transcript found in induced murine erythroleukemia cells is homologous to the beta h0 and beta h1 genes. Proc Natl Acad Sci U S A. 1982 May;79(9):2753–2757. doi: 10.1073/pnas.79.9.2753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brüggemeier U., Kalff M., Franke S., Scheidereit C., Beato M. Ubiquitous transcription factor OTF-1 mediates induction of the MMTV promoter through synergistic interaction with hormone receptors. Cell. 1991 Feb 8;64(3):565–572. doi: 10.1016/0092-8674(91)90240-y. [DOI] [PubMed] [Google Scholar]
  9. Cao S. X., Gutman P. D., Dave H. P., Schechter A. N. Identification of a transcriptional silencer in the 5'-flanking region of the human epsilon-globin gene. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5306–5309. doi: 10.1073/pnas.86.14.5306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chao M. V., Mellon P., Charnay P., Maniatis T., Axel R. The regulated expression of beta-globin genes introduced into mouse erythroleukemia cells. Cell. 1983 Feb;32(2):483–493. doi: 10.1016/0092-8674(83)90468-3. [DOI] [PubMed] [Google Scholar]
  11. Crothers D. M., Gartenberg M. R., Shrader T. E. DNA bending in protein-DNA complexes. Methods Enzymol. 1991;208:118–146. doi: 10.1016/0076-6879(91)08011-6. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Dillon N., Grosveld F. Transcriptional regulation of multigene loci: multilevel control. Trends Genet. 1993 Apr;9(4):134–137. doi: 10.1016/0168-9525(93)90208-y. [DOI] [PubMed] [Google Scholar]
  14. Du W., Thanos D., Maniatis T. Mechanisms of transcriptional synergism between distinct virus-inducible enhancer elements. Cell. 1993 Sep 10;74(5):887–898. doi: 10.1016/0092-8674(93)90468-6. [DOI] [PubMed] [Google Scholar]
  15. Engel J. D. Developmental regulation of human beta-globin gene transcription: a switch of loyalties? Trends Genet. 1993 Sep;9(9):304–309. doi: 10.1016/0168-9525(93)90248-g. [DOI] [PubMed] [Google Scholar]
  16. Fisher D. E., Parent L. A., Sharp P. A. Myc/Max and other helix-loop-helix/leucine zipper proteins bend DNA toward the minor groove. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11779–11783. doi: 10.1073/pnas.89.24.11779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fraser P., Pruzina S., Antoniou M., Grosveld F. Each hypersensitive site of the human beta-globin locus control region confers a different developmental pattern of expression on the globin genes. Genes Dev. 1993 Jan;7(1):106–113. doi: 10.1101/gad.7.1.106. [DOI] [PubMed] [Google Scholar]
  18. Gartenberg M. R., Ampe C., Steitz T. A., Crothers D. M. Molecular characterization of the GCN4-DNA complex. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6034–6038. doi: 10.1073/pnas.87.16.6034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Glover J. N., Harrison S. C. Crystal structure of the heterodimeric bZIP transcription factor c-Fos-c-Jun bound to DNA. Nature. 1995 Jan 19;373(6511):257–261. doi: 10.1038/373257a0. [DOI] [PubMed] [Google Scholar]
  21. Grosschedl R., Giese K., Pagel J. HMG domain proteins: architectural elements in the assembly of nucleoprotein structures. Trends Genet. 1994 Mar;10(3):94–100. doi: 10.1016/0168-9525(94)90232-1. [DOI] [PubMed] [Google Scholar]
  22. Grosveld F., van Assendelft G. B., Greaves D. R., Kollias G. Position-independent, high-level expression of the human beta-globin gene in transgenic mice. Cell. 1987 Dec 24;51(6):975–985. doi: 10.1016/0092-8674(87)90584-8. [DOI] [PubMed] [Google Scholar]
  23. Gutman P. D., Cao S. X., Dave H. P., Mittelman M., Schechter A. N. Binding of erythroid and non-erythroid nuclear proteins to the silencer of the human epsilon-globin-encoding gene. Gene. 1992 Jan 15;110(2):197–203. doi: 10.1016/0378-1119(92)90648-9. [DOI] [PubMed] [Google Scholar]
  24. Kadonaga J. T. Purification of sequence-specific binding proteins by DNA affinity chromatography. Methods Enzymol. 1991;208:10–23. doi: 10.1016/0076-6879(91)08004-2. [DOI] [PubMed] [Google Scholar]
  25. Katahira M., Sugeta H., Kyogoku Y. A new model for the bending of DNAs containing the oligo(dA) tracts based on NMR observations. Nucleic Acids Res. 1990 Feb 11;18(3):613–618. doi: 10.1093/nar/18.3.613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kerppola T. K., Curran T. DNA bending by Fos and Jun: the flexible hinge model. Science. 1991 Nov 22;254(5035):1210–1214. doi: 10.1126/science.1957173. [DOI] [PubMed] [Google Scholar]
  27. Kerppola T. K., Curran T. Selective DNA bending by a variety of bZIP proteins. Mol Cell Biol. 1993 Sep;13(9):5479–5489. doi: 10.1128/mcb.13.9.5479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kim J., Zwieb C., Wu C., Adhya S. Bending of DNA by gene-regulatory proteins: construction and use of a DNA bending vector. Gene. 1989 Dec 21;85(1):15–23. doi: 10.1016/0378-1119(89)90459-9. [DOI] [PubMed] [Google Scholar]
  29. Kuprash D. V., Rice N. R., Nedospasov S. A. Homodimer of p50 (NF kappa B1) does not introduce a substantial directed bend into DNA according to three different experimental assays. Nucleic Acids Res. 1995 Feb 11;23(3):427–433. doi: 10.1093/nar/23.3.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Larson C. J., Verdine G. L. A high-capacity column for affinity purification of sequence-specific DNA-binding proteins. Nucleic Acids Res. 1992 Jul 11;20(13):3525–3525. doi: 10.1093/nar/20.13.3525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lister J., Forrester W. C., Baron M. H. Inhibition of an erythroid differentiation switch by the helix-loop-helix protein Id1. J Biol Chem. 1995 Jul 28;270(30):17939–17946. doi: 10.1074/jbc.270.30.17939. [DOI] [PubMed] [Google Scholar]
  32. Love J. J., Li X., Case D. A., Giese K., Grosschedl R., Wright P. E. Structural basis for DNA bending by the architectural transcription factor LEF-1. Nature. 1995 Aug 31;376(6543):791–795. doi: 10.1038/376791a0. [DOI] [PubMed] [Google Scholar]
  33. McCaffrey P. G., Luo C., Kerppola T. K., Jain J., Badalian T. M., Ho A. M., Burgeon E., Lane W. S., Lambert J. N., Curran T. Isolation of the cyclosporin-sensitive T cell transcription factor NFATp. Science. 1993 Oct 29;262(5134):750–754. doi: 10.1126/science.8235597. [DOI] [PubMed] [Google Scholar]
  34. Merika M., Orkin S. H. Functional synergy and physical interactions of the erythroid transcription factor GATA-1 with the Krüppel family proteins Sp1 and EKLF. Mol Cell Biol. 1995 May;15(5):2437–2447. doi: 10.1128/mcb.15.5.2437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Miller I. J., Bieker J. J. A novel, erythroid cell-specific murine transcription factor that binds to the CACCC element and is related to the Krüppel family of nuclear proteins. Mol Cell Biol. 1993 May;13(5):2776–2786. doi: 10.1128/mcb.13.5.2776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Murre C., McCaw P. S., Baltimore D. A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell. 1989 Mar 10;56(5):777–783. doi: 10.1016/0092-8674(89)90682-x. [DOI] [PubMed] [Google Scholar]
  37. Natesan S., Gilman M. Z. DNA bending and orientation-dependent function of YY1 in the c-fos promoter. Genes Dev. 1993 Dec;7(12B):2497–2509. doi: 10.1101/gad.7.12b.2497. [DOI] [PubMed] [Google Scholar]
  38. Nuez B., Michalovich D., Bygrave A., Ploemacher R., Grosveld F. Defective haematopoiesis in fetal liver resulting from inactivation of the EKLF gene. Nature. 1995 May 25;375(6529):316–318. doi: 10.1038/375316a0. [DOI] [PubMed] [Google Scholar]
  39. Perkins A. C., Sharpe A. H., Orkin S. H. Lethal beta-thalassaemia in mice lacking the erythroid CACCC-transcription factor EKLF. Nature. 1995 May 25;375(6529):318–322. doi: 10.1038/375318a0. [DOI] [PubMed] [Google Scholar]
  40. Pierrou S., Hellqvist M., Samuelsson L., Enerbäck S., Carlsson P. Cloning and characterization of seven human forkhead proteins: binding site specificity and DNA bending. EMBO J. 1994 Oct 17;13(20):5002–5012. doi: 10.1002/j.1460-2075.1994.tb06827.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pil P. M., Lippard S. J. Specific binding of chromosomal protein HMG1 to DNA damaged by the anticancer drug cisplatin. Science. 1992 Apr 10;256(5054):234–237. doi: 10.1126/science.1566071. [DOI] [PubMed] [Google Scholar]
  42. Potter D. A., Larson C. J., Eckes P., Schmid R. M., Nabel G. J., Verdine G. L., Sharp P. A. Purification of the major histocompatibility complex class I transcription factor H2TF1. The full-length product of the nfkb2 gene. J Biol Chem. 1993 Sep 5;268(25):18882–18890. [PMC free article] [PubMed] [Google Scholar]
  43. Prentki P., Pham M. H., Galas D. J. Plasmid permutation vectors to monitor DNA bending. Nucleic Acids Res. 1987 Dec 10;15(23):10060–10060. doi: 10.1093/nar/15.23.10060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Raich N., Papayannopoulou T., Stamatoyannopoulos G., Enver T. Demonstration of a human epsilon-globin gene silencer with studies in transgenic mice. Blood. 1992 Feb 15;79(4):861–864. [PubMed] [Google Scholar]
  45. Riggs A. D., Suzuki H., Bourgeois S. Lac repressor-operator interaction. I. Equilibrium studies. J Mol Biol. 1970 Feb 28;48(1):67–83. doi: 10.1016/0022-2836(70)90219-6. [DOI] [PubMed] [Google Scholar]
  46. Rowley P. T., Ohlsson-Wilhelm B. M., Wisniewski L., Lozzio C. B., Lozzio B. B. K562 human leukemia cell passages differ in embryonic globin gene expression. Leuk Res. 1984;8(1):45–54. doi: 10.1016/0145-2126(84)90030-4. [DOI] [PubMed] [Google Scholar]
  47. Sauer R. T., Smith D. L., Johnson A. D. Flexibility of the yeast alpha 2 repressor enables it to occupy the ends of its operator, leaving the center free. Genes Dev. 1988 Jul;2(7):807–816. doi: 10.1101/gad.2.7.807. [DOI] [PubMed] [Google Scholar]
  48. Shen B., Kim J., Dorsett D. The enhancer-blocking suppressor of Hairy-wing zinc finger protein of Drosophila melanogaster alters DNA structure. Mol Cell Biol. 1994 Sep;14(9):5645–5652. doi: 10.1128/mcb.14.9.5645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Shirakata M., Hüppi K., Usuda S., Okazaki K., Yoshida K., Sakano H. HMG1-related DNA-binding protein isolated with V-(D)-J recombination signal probes. Mol Cell Biol. 1991 Sep;11(9):4528–4536. doi: 10.1128/mcb.11.9.4528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Shykind B. M., Kim J., Sharp P. A. Activation of the TFIID-TFIIA complex with HMG-2. Genes Dev. 1995 Jun 1;9(11):1354–1365. doi: 10.1101/gad.9.11.1354. [DOI] [PubMed] [Google Scholar]
  51. Stein B., Cogswell P. C., Baldwin A. S., Jr Functional and physical associations between NF-kappa B and C/EBP family members: a Rel domain-bZIP interaction. Mol Cell Biol. 1993 Jul;13(7):3964–3974. doi: 10.1128/mcb.13.7.3964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Stuvé L. L., Myers R. M. Identification and characterization of a beta-globin promoter-binding factor from murine erythroleukemia cells. Mol Cell Biol. 1993 Jul;13(7):4311–4322. doi: 10.1128/mcb.13.7.4311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Thompson J. F., Landy A. Empirical estimation of protein-induced DNA bending angles: applications to lambda site-specific recombination complexes. Nucleic Acids Res. 1988 Oct 25;16(20):9687–9705. doi: 10.1093/nar/16.20.9687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. Trepicchio W. L., Dyer M. A., Baron M. H. A novel developmental regulatory motif required for stage-specific activation of the epsilon-globin gene and nuclear factor binding in embryonic erythroid cells. Mol Cell Biol. 1994 Jun;14(6):3763–3771. doi: 10.1128/mcb.14.6.3763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Trepicchio W. L., Dyer M. A., Baron M. H. Developmental regulation of the human embryonic beta-like globin gene is mediated by synergistic interactions among multiple tissue- and stage-specific elements. Mol Cell Biol. 1993 Dec;13(12):7457–7468. doi: 10.1128/mcb.13.12.7457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Trepicchio W. L., Dyer M. A., Hardison R. C., Baron M. H. Upstream regulatory region of the human embryonic beta-like globin gene, epsilon. DNA Seq. 1994;4(6):409–412. doi: 10.3109/10425179409010191. [DOI] [PubMed] [Google Scholar]
  58. Tsai S. F., Martin D. I., Zon L. I., D'Andrea A. D., Wong G. G., Orkin S. H. Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells. Nature. 1989 Jun 8;339(6224):446–451. doi: 10.1038/339446a0. [DOI] [PubMed] [Google Scholar]
  59. Verrijzer C. P., van Oosterhout J. A., van Weperen W. W., van der Vliet P. C. POU proteins bend DNA via the POU-specific domain. EMBO J. 1991 Oct;10(10):3007–3014. doi: 10.1002/j.1460-2075.1991.tb07851.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Vinson C. R., LaMarco K. L., Johnson P. F., Landschulz W. H., McKnight S. L. In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage. Genes Dev. 1988 Jul;2(7):801–806. doi: 10.1101/gad.2.7.801. [DOI] [PubMed] [Google Scholar]
  61. Wada-Kiyama Y., Peters B., Noguchi C. T. The epsilon-globin gene silencer. Characterization by in vitro transcription. J Biol Chem. 1992 Jun 5;267(16):11532–11538. [PubMed] [Google Scholar]
  62. Warren A. J., Colledge W. H., Carlton M. B., Evans M. J., Smith A. J., Rabbitts T. H. The oncogenic cysteine-rich LIM domain protein rbtn2 is essential for erythroid development. Cell. 1994 Jul 15;78(1):45–57. doi: 10.1016/0092-8674(94)90571-1. [DOI] [PubMed] [Google Scholar]
  63. Wolffe A. P. Architectural transcription factors. Science. 1994 May 20;264(5162):1100–1101. doi: 10.1126/science.8178167. [DOI] [PubMed] [Google Scholar]
  64. Wu H. M., Crothers D. M. The locus of sequence-directed and protein-induced DNA bending. Nature. 1984 Apr 5;308(5959):509–513. doi: 10.1038/308509a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES