Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Oct;16(10):5634–5644. doi: 10.1128/mcb.16.10.5634

NF-E2 disrupts chromatin structure at human beta-globin locus control region hypersensitive site 2 in vitro.

J A Armstrong 1, B M Emerson 1
PMCID: PMC231563  PMID: 8816476

Abstract

The human beta-globin locus control region (LCR) is responsible for forming an active chromatin structure extending over the 100-kb locus, allowing expression of the beta-globin gene family. The LCR consists of four erythroid-cell-specific DNase I hypersensitive sites (HS1 to -4). DNase I hypersensitive sites are thought to represent nucleosome-free regions of DNA which are bound by trans-acting factors. Of the four hypersensitive sites only HS2 acts as a transcriptional enhancer. In this study, we examine the binding of an erythroid protein to its site within HS2 in chromatin in vitro. NF-E2 is a transcriptional activator consisting of two subunits, the hematopoietic cell-specific p45 and the ubiquitous DNA-binding subunit, p18. NF-E2 binds two tandem AP1-like sites in HS2 which form the core of its enhancer activity. In this study, we show that when bound to in vitro-reconstituted chromatin, NF-E2 forms a DNase I hypersensitive site at HS2 similar to the site observed in vivo. Moreover, NF-E2 binding in vitro results in a disruption of nucleosome structure which can be detected 200 bp away. Although NF-E2 can disrupt nucleosomes when added to preformed chromatin, the disruption is more pronounced when NF-E2 is added to DNA prior to chromatin assembly. Interestingly, the hematopoietic cell-specific subunit, p45, is necessary for binding to chromatin but not to naked DNA. Interaction of NF-E2 with its site in chromatin-reconstituted HS2 allows a second erythroid factor, GATA-1, to bind its nearby sites. Lastly, nucleosome disruption by NF-E2 is an ATP-dependent process, suggesting the involvement of energy-dependent nucleosome remodeling factors.

Full Text

The Full Text of this article is available as a PDF (765.7 KB).

Selected References

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

  1. Adams C. C., Workman J. L. Binding of disparate transcriptional activators to nucleosomal DNA is inherently cooperative. Mol Cell Biol. 1995 Mar;15(3):1405–1421. doi: 10.1128/mcb.15.3.1405. [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. Andrews N. C., Kotkow K. J., Ney P. A., Erdjument-Bromage H., Tempst P., Orkin S. H. The ubiquitous subunit of erythroid transcription factor NF-E2 is a small basic-leucine zipper protein related to the v-maf oncogene. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11488–11492. doi: 10.1073/pnas.90.24.11488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Becker P. B. The establishment of active promoters in chromatin. Bioessays. 1994 Aug;16(8):541–547. doi: 10.1002/bies.950160807. [DOI] [PubMed] [Google Scholar]
  7. Bresnick E. H., Bustin M., Marsaud V., Richard-Foy H., Hager G. L. The transcriptionally-active MMTV promoter is depleted of histone H1. Nucleic Acids Res. 1992 Jan 25;20(2):273–278. doi: 10.1093/nar/20.2.273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Caterina J. J., Donze D., Sun C. W., Ciavatta D. J., Townes T. M. Cloning and functional characterization of LCR-F1: a bZIP transcription factor that activates erythroid-specific, human globin gene expression. Nucleic Acids Res. 1994 Jun 25;22(12):2383–2391. doi: 10.1093/nar/22.12.2383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chan J. Y., Han X. L., Kan Y. W. Cloning of Nrf1, an NF-E2-related transcription factor, by genetic selection in yeast. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11371–11375. doi: 10.1073/pnas.90.23.11371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chung J. H., Whiteley M., Felsenfeld G. A 5' element of the chicken beta-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila. Cell. 1993 Aug 13;74(3):505–514. doi: 10.1016/0092-8674(93)80052-g. [DOI] [PubMed] [Google Scholar]
  11. Croston G. E., Lira L. M., Kadonaga J. T. A general method for purification of H1 histones that are active for repression of basal RNA polymerase II transcription. Protein Expr Purif. 1991 Apr-Jun;2(2-3):162–169. doi: 10.1016/1046-5928(91)90066-r. [DOI] [PubMed] [Google Scholar]
  12. Côté J., Quinn J., Workman J. L., Peterson C. L. Stimulation of GAL4 derivative binding to nucleosomal DNA by the yeast SWI/SNF complex. Science. 1994 Jul 1;265(5168):53–60. doi: 10.1126/science.8016655. [DOI] [PubMed] [Google Scholar]
  13. Enver T., Raich N., Ebens A. J., Papayannopoulou T., Costantini F., Stamatoyannopoulos G. Developmental regulation of human fetal-to-adult globin gene switching in transgenic mice. Nature. 1990 Mar 22;344(6264):309–313. doi: 10.1038/344309a0. [DOI] [PubMed] [Google Scholar]
  14. Forrester W. C., Epner E., Driscoll M. C., Enver T., Brice M., Papayannopoulou T., Groudine M. A deletion of the human beta-globin locus activation region causes a major alteration in chromatin structure and replication across the entire beta-globin locus. Genes Dev. 1990 Oct;4(10):1637–1649. doi: 10.1101/gad.4.10.1637. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Goldman M. A., Holmquist G. P., Gray M. C., Caston L. A., Nag A. Replication timing of genes and middle repetitive sequences. Science. 1984 May 18;224(4650):686–692. doi: 10.1126/science.6719109. [DOI] [PubMed] [Google Scholar]
  17. Gross D. S., Garrard W. T. Nuclease hypersensitive sites in chromatin. Annu Rev Biochem. 1988;57:159–197. doi: 10.1146/annurev.bi.57.070188.001111. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Igarashi K., Kataoka K., Itoh K., Hayashi N., Nishizawa M., Yamamoto M. Regulation of transcription by dimerization of erythroid factor NF-E2 p45 with small Maf proteins. Nature. 1994 Feb 10;367(6463):568–572. doi: 10.1038/367568a0. [DOI] [PubMed] [Google Scholar]
  20. Jane S. M., Ney P. A., Vanin E. F., Gumucio D. L., Nienhuis A. W. Identification of a stage selector element in the human gamma-globin gene promoter that fosters preferential interaction with the 5' HS2 enhancer when in competition with the beta-promoter. EMBO J. 1992 Aug;11(8):2961–2969. doi: 10.1002/j.1460-2075.1992.tb05366.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jiménez G., Griffiths S. D., Ford A. M., Greaves M. F., Enver T. Activation of the beta-globin locus control region precedes commitment to the erythroid lineage. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10618–10622. doi: 10.1073/pnas.89.22.10618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kamakaka R. T., Bulger M., Kadonaga J. T. Potentiation of RNA polymerase II transcription by Gal4-VP16 during but not after DNA replication and chromatin assembly. Genes Dev. 1993 Sep;7(9):1779–1795. doi: 10.1101/gad.7.9.1779. [DOI] [PubMed] [Google Scholar]
  23. Kotkow K. J., Orkin S. H. Dependence of globin gene expression in mouse erythroleukemia cells on the NF-E2 heterodimer. Mol Cell Biol. 1995 Aug;15(8):4640–4647. doi: 10.1128/mcb.15.8.4640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kwon H., Imbalzano A. N., Khavari P. A., Kingston R. E., Green M. R. Nucleosome disruption and enhancement of activator binding by a human SW1/SNF complex. Nature. 1994 Aug 11;370(6489):477–481. doi: 10.1038/370477a0. [DOI] [PubMed] [Google Scholar]
  25. Labbaye C., Valtieri M., Barberi T., Meccia E., Masella B., Pelosi E., Condorelli G. L., Testa U., Peschle C. Differential expression and functional role of GATA-2, NF-E2, and GATA-1 in normal adult hematopoiesis. J Clin Invest. 1995 May;95(5):2346–2358. doi: 10.1172/JCI117927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Laybourn P. J., Kadonaga J. T. Role of nucleosomal cores and histone H1 in regulation of transcription by RNA polymerase II. Science. 1991 Oct 11;254(5029):238–245. doi: 10.1126/science.254.5029.238. [DOI] [PubMed] [Google Scholar]
  27. Li Q., Stamatoyannopoulos G. Hypersensitive site 5 of the human beta locus control region functions as a chromatin insulator. Blood. 1994 Sep 1;84(5):1399–1401. [PubMed] [Google Scholar]
  28. Liu D., Chang J. C., Moi P., Liu W., Kan Y. W., Curtin P. T. Dissection of the enhancer activity of beta-globin 5' DNase I-hypersensitive site 2 in transgenic mice. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3899–3903. doi: 10.1073/pnas.89.9.3899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lozzio C. B., Lozzio B. B. Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood. 1975 Mar;45(3):321–334. [PubMed] [Google Scholar]
  30. Lu S. J., Rowan S., Bani M. R., Ben-David Y. Retroviral integration within the Fli-2 locus results in inactivation of the erythroid transcription factor NF-E2 in Friend erythroleukemias: evidence that NF-E2 is essential for globin expression. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8398–8402. doi: 10.1073/pnas.91.18.8398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Martin D. I., Orkin S. H. Transcriptional activation and DNA binding by the erythroid factor GF-1/NF-E1/Eryf 1. Genes Dev. 1990 Nov;4(11):1886–1898. doi: 10.1101/gad.4.11.1886. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Moi P., Chan K., Asunis I., Cao A., Kan Y. W. Isolation of NF-E2-related factor 2 (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the beta-globin locus control region. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9926–9930. doi: 10.1073/pnas.91.21.9926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ney P. A., Andrews N. C., Jane S. M., Safer B., Purucker M. E., Weremowicz S., Morton C. C., Goff S. C., Orkin S. H., Nienhuis A. W. Purification of the human NF-E2 complex: cDNA cloning of the hematopoietic cell-specific subunit and evidence for an associated partner. Mol Cell Biol. 1993 Sep;13(9):5604–5612. doi: 10.1128/mcb.13.9.5604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ney P. A., Sorrentino B. P., McDonagh K. T., Nienhuis A. W. Tandem AP-1-binding sites within the human beta-globin dominant control region function as an inducible enhancer in erythroid cells. Genes Dev. 1990 Jun;4(6):993–1006. doi: 10.1101/gad.4.6.993. [DOI] [PubMed] [Google Scholar]
  36. Orkin S. H. Globin gene regulation and switching: circa 1990. Cell. 1990 Nov 16;63(4):665–672. doi: 10.1016/0092-8674(90)90133-y. [DOI] [PubMed] [Google Scholar]
  37. Orkin S. H. Regulation of globin gene expression in erythroid cells. Eur J Biochem. 1995 Jul 15;231(2):271–281. doi: 10.1111/j.1432-1033.1995.tb20697.x. [DOI] [PubMed] [Google Scholar]
  38. Orlic D., Anderson S., Biesecker L. G., Sorrentino B. P., Bodine D. M. Pluripotent hematopoietic stem cells contain high levels of mRNA for c-kit, GATA-2, p45 NF-E2, and c-myb and low levels or no mRNA for c-fms and the receptors for granulocyte colony-stimulating factor and interleukins 5 and 7. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4601–4605. doi: 10.1073/pnas.92.10.4601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pazin M. J., Kamakaka R. T., Kadonaga J. T. ATP-dependent nucleosome reconfiguration and transcriptional activation from preassembled chromatin templates. Science. 1994 Dec 23;266(5193):2007–2011. doi: 10.1126/science.7801129. [DOI] [PubMed] [Google Scholar]
  40. Ryan T. M., Behringer R. R., Martin N. C., Townes T. M., Palmiter R. D., Brinster R. L. A single erythroid-specific DNase I super-hypersensitive site activates high levels of human beta-globin gene expression in transgenic mice. Genes Dev. 1989 Mar;3(3):314–323. doi: 10.1101/gad.3.3.314. [DOI] [PubMed] [Google Scholar]
  41. Shivdasani R. A., Orkin S. H. Erythropoiesis and globin gene expression in mice lacking the transcription factor NF-E2. Proc Natl Acad Sci U S A. 1995 Sep 12;92(19):8690–8694. doi: 10.1073/pnas.92.19.8690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stamatoyannopoulos J. A., Goodwin A., Joyce T., Lowrey C. H. NF-E2 and GATA binding motifs are required for the formation of DNase I hypersensitive site 4 of the human beta-globin locus control region. EMBO J. 1995 Jan 3;14(1):106–116. doi: 10.1002/j.1460-2075.1995.tb06980.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Talbot D., Collis P., Antoniou M., Vidal M., Grosveld F., Greaves D. R. A dominant control region from the human beta-globin locus conferring integration site-independent gene expression. Nature. 1989 Mar 23;338(6213):352–355. doi: 10.1038/338352a0. [DOI] [PubMed] [Google Scholar]
  44. Talbot D., Grosveld F. The 5'HS2 of the globin locus control region enhances transcription through the interaction of a multimeric complex binding at two functionally distinct NF-E2 binding sites. EMBO J. 1991 Jun;10(6):1391–1398. doi: 10.1002/j.1460-2075.1991.tb07659.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Talbot D., Philipsen S., Fraser P., Grosveld F. Detailed analysis of the site 3 region of the human beta-globin dominant control region. EMBO J. 1990 Jul;9(7):2169–2177. doi: 10.1002/j.1460-2075.1990.tb07386.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Trainor C. D., Evans T., Felsenfeld G., Boguski M. S. Structure and evolution of a human erythroid transcription factor. Nature. 1990 Jan 4;343(6253):92–96. doi: 10.1038/343092a0. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Tsukiyama T., Becker P. B., Wu C. ATP-dependent nucleosome disruption at a heat-shock promoter mediated by binding of GAGA transcription factor. Nature. 1994 Feb 10;367(6463):525–532. doi: 10.1038/367525a0. [DOI] [PubMed] [Google Scholar]
  49. Tsukiyama T., Wu C. Purification and properties of an ATP-dependent nucleosome remodeling factor. Cell. 1995 Dec 15;83(6):1011–1020. doi: 10.1016/0092-8674(95)90216-3. [DOI] [PubMed] [Google Scholar]
  50. Tuan D. Y., Solomon W. B., London I. M., Lee D. P. An erythroid-specific, developmental-stage-independent enhancer far upstream of the human "beta-like globin" genes. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2554–2558. doi: 10.1073/pnas.86.8.2554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tuan D., Solomon W., Li Q., London I. M. The "beta-like-globin" gene domain in human erythroid cells. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6384–6388. doi: 10.1073/pnas.82.19.6384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Varga-Weisz P. D., Blank T. A., Becker P. B. Energy-dependent chromatin accessibility and nucleosome mobility in a cell-free system. EMBO J. 1995 May 15;14(10):2209–2216. doi: 10.1002/j.1460-2075.1995.tb07215.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wall G., Varga-Weisz P. D., Sandaltzopoulos R., Becker P. B. Chromatin remodeling by GAGA factor and heat shock factor at the hypersensitive Drosophila hsp26 promoter in vitro. EMBO J. 1995 Apr 18;14(8):1727–1736. doi: 10.1002/j.1460-2075.1995.tb07162.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Weintraub H., Groudine M. Chromosomal subunits in active genes have an altered conformation. Science. 1976 Sep 3;193(4256):848–856. doi: 10.1126/science.948749. [DOI] [PubMed] [Google Scholar]
  55. Wolffe A. P. Transcription: in tune with the histones. Cell. 1994 Apr 8;77(1):13–16. doi: 10.1016/0092-8674(94)90229-1. [DOI] [PubMed] [Google Scholar]
  56. Workman J. L., Buchman A. R. Multiple functions of nucleosomes and regulatory factors in transcription. Trends Biochem Sci. 1993 Mar;18(3):90–95. doi: 10.1016/0968-0004(93)90160-o. [DOI] [PubMed] [Google Scholar]

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

RESOURCES