Abstract
Regulated expression of genes in the beta-globin cluster depends upon sequences located between 5 and 20 kb upstream of the epsilon gene, known as the locus control region (LCR). beta-Globin expression in murine erythroleukemia (MEL) cells depends on NF-E2, a transcription factor which binds to enhancer sequences in the LCR. To gain insight into the mechanism of globin gene activation by NF-E2, an NF-E2 null MEL cell line was used to map regions of NF-E2 required for beta-globin expression. Within the transactivation domain, two discrete proline-rich regions were required for rescue of beta-globin expression. The first was located at the N-terminus of NF-E2, while the second was located N-terminal of the cap 'n collar (CNC) domain. Other proline-rich sequences were dispensable, indicating that proline content per se does not determine NF-E2 activity. Mutations within the conserved CNC domain markedly diminished rescue of beta-globin expression. This domain was required, in addition to the basic leucine zipper domain, for DNA binding activity. The requirement for discrete proline-rich sequences within the transactivation domain suggests that globin gene expression in MEL cells depends on specific interactions between NF-E2 and downstream effector molecules.
Full Text
The Full Text of this article is available as a PDF (245.7 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Adzhubei A. A., Sternberg M. J. Left-handed polyproline II helices commonly occur in globular proteins. J Mol Biol. 1993 Jan 20;229(2):472–493. doi: 10.1006/jmbi.1993.1047. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Armstrong J. A., Emerson B. M. NF-E2 disrupts chromatin structure at human beta-globin locus control region hypersensitive site 2 in vitro. Mol Cell Biol. 1996 Oct;16(10):5634–5644. doi: 10.1128/mcb.16.10.5634. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blackwell T. K., Bowerman B., Priess J. R., Weintraub H. Formation of a monomeric DNA binding domain by Skn-1 bZIP and homeodomain elements. Science. 1994 Oct 28;266(5185):621–628. doi: 10.1126/science.7939715. [DOI] [PubMed] [Google Scholar]
- 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]
- Caterina J. J., Ryan T. M., Pawlik K. M., Palmiter R. D., Brinster R. L., Behringer R. R., Townes T. M. Human beta-globin locus control region: analysis of the 5' DNase I hypersensitive site HS 2 in transgenic mice. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1626–1630. doi: 10.1073/pnas.88.5.1626. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Chan J. Y., Han X. L., Kan Y. W. Isolation of cDNA encoding the human NF-E2 protein. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11366–11370. doi: 10.1073/pnas.90.23.11366. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chan K., Lu R., Chang J. C., Kan Y. W. NRF2, a member of the NFE2 family of transcription factors, is not essential for murine erythropoiesis, growth, and development. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13943–13948. doi: 10.1073/pnas.93.24.13943. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cheng X., Reginato M. J., Andrews N. C., Lazar M. A. The transcriptional integrator CREB-binding protein mediates positive cross talk between nuclear hormone receptors and the hematopoietic bZip protein p45/NF-E2. Mol Cell Biol. 1997 Mar;17(3):1407–1416. doi: 10.1128/mcb.17.3.1407. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
- Fiering S., Epner E., Robinson K., Zhuang Y., Telling A., Hu M., Martin D. I., Enver T., Ley T. J., Groudine M. Targeted deletion of 5'HS2 of the murine beta-globin LCR reveals that it is not essential for proper regulation of the beta-globin locus. Genes Dev. 1995 Sep 15;9(18):2203–2213. doi: 10.1101/gad.9.18.2203. [DOI] [PubMed] [Google Scholar]
- Forrester W. C., Takegawa S., Papayannopoulou T., Stamatoyannopoulos G., Groudine M. Evidence for a locus activation region: the formation of developmentally stable hypersensitive sites in globin-expressing hybrids. Nucleic Acids Res. 1987 Dec 23;15(24):10159–10177. doi: 10.1093/nar/15.24.10159. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Garingo A. D., Suhasini M., Andrews N. C., Pilz R. B. cAMP-dependent protein kinase is necessary for increased NF-E2.DNA complex formation during erythroleukemia cell differentiation. J Biol Chem. 1995 Apr 21;270(16):9169–9177. doi: 10.1074/jbc.270.16.9169. [DOI] [PubMed] [Google Scholar]
- 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]
- Hug B. A., Moon A. M., Ley T. J. Structure and function of the murine beta-globin locus control region 5' HS-3. Nucleic Acids Res. 1992 Nov 11;20(21):5771–5778. doi: 10.1093/nar/20.21.5771. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hug B. A., Wesselschmidt R. L., Fiering S., Bender M. A., Epner E., Groudine M., Ley T. J. Analysis of mice containing a targeted deletion of beta-globin locus control region 5' hypersensitive site 3. Mol Cell Biol. 1996 Jun;16(6):2906–2912. doi: 10.1128/mcb.16.6.2906. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Jiménez G., Gale K. B., Enver T. The mouse beta-globin locus control region: hypersensitive sites 3 and 4. Nucleic Acids Res. 1992 Nov 11;20(21):5797–5803. doi: 10.1093/nar/20.21.5797. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kielman M. F., Smits R., Bernini L. F. Localization and characterization of the mouse alpha-globin locus control region. Genomics. 1994 May 15;21(2):431–433. doi: 10.1006/geno.1994.1289. [DOI] [PubMed] [Google Scholar]
- Kotkow K. J., Orkin S. H. Complexity of the erythroid transcription factor NF-E2 as revealed by gene targeting of the mouse p18 NF-E2 locus. Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3514–3518. doi: 10.1073/pnas.93.8.3514. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Li Q. L., Zhou B., Powers P., Enver T., Stamatoyannopoulos G. Beta-globin locus activation regions: conservation of organization, structure, and function. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8207–8211. doi: 10.1073/pnas.87.21.8207. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Mignotte V., Eleouet J. F., Raich N., Romeo P. H. Cis- and trans-acting elements involved in the regulation of the erythroid promoter of the human porphobilinogen deaminase gene. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6548–6552. doi: 10.1073/pnas.86.17.6548. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Mizushima S., Nagata S. pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res. 1990 Sep 11;18(17):5322–5322. doi: 10.1093/nar/18.17.5322. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Moon A. M., Ley T. J. Conservation of the primary structure, organization, and function of the human and mouse beta-globin locus-activating regions. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7693–7697. doi: 10.1073/pnas.87.19.7693. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Ney P. A., Sorrentino B. P., Lowrey C. H., Nienhuis A. W. Inducibility of the HS II enhancer depends on binding of an erythroid specific nuclear protein. Nucleic Acids Res. 1990 Oct 25;18(20):6011–6017. doi: 10.1093/nar/18.20.6011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Philipsen S., Talbot D., Fraser P., Grosveld F. The beta-globin dominant control region: hypersensitive site 2. EMBO J. 1990 Jul;9(7):2159–2167. doi: 10.1002/j.1460-2075.1990.tb07385.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pruzina S., Hanscombe O., Whyatt D., Grosveld F., Philipsen S. Hypersensitive site 4 of the human beta globin locus control region. Nucleic Acids Res. 1991 Apr 11;19(7):1413–1419. doi: 10.1093/nar/19.7.1413. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Shivdasani R. A., Rosenblatt M. F., Zucker-Franklin D., Jackson C. W., Hunt P., Saris C. J., Orkin S. H. Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development. Cell. 1995 Jun 2;81(5):695–704. doi: 10.1016/0092-8674(95)90531-6. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- Yu H., Chen J. K., Feng S., Dalgarno D. C., Brauer A. W., Schreiber S. L. Structural basis for the binding of proline-rich peptides to SH3 domains. Cell. 1994 Mar 11;76(5):933–945. doi: 10.1016/0092-8674(94)90367-0. [DOI] [PubMed] [Google Scholar]
- Zhang Q., Rombel I., Reddy G. N., Gang J. B., Shen C. K. Functional roles of in vivo footprinted DNA motifs within an alpha-globin enhancer. Erythroid lineage and developmental stage specificities. J Biol Chem. 1995 Apr 14;270(15):8501–8505. doi: 10.1074/jbc.270.15.8501. [DOI] [PubMed] [Google Scholar]