Home LiteratureArticle Details
PMID: 9744863 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Altered DNA-binding specificity mutants of EKLF and Sp1 show that EKLF is an activator of the beta-globin locus control region in vivo.

Genes & development ·Vol. 12 ·No. 18 ·1998-09-15 ·Pages 2863-73

Gillemans N, Tewari R, Lindeboom F, Rottier R, de Wit T, Wijgerde M, Grosveld F, Philipsen S

Abstract

The locus control region of the beta-globin cluster contains five DNase I hypersensitive sites (5'HS1-5) required for locus activation. 5'HS3 contains six G-rich motifs that are essential for its activity. Members of a protein family, characterized by three zinc fingers highly homologous to those found in transcription factor Sp1, interact with these motifs. Because point mutagenesis cannot distinguish between family members, it is not known which protein activates 5'HS3. We show that the function of such closely related proteins can be distinguished in vivo by matching point mutations in 5'HS3 with amino acid changes in the zinc fingers of Sp1 and EKLF. Testing their activity in transgenic mice shows that EKLF is a direct activator of 5'HS3.

MeSH Terms
Amino Acid Sequence Animals Base Sequence Binding Sites/genetics CHO Cells Cell Line Cricetinae DNA/genetics,metabolism DNA-Binding Proteins/genetics,metabolism Deoxyribonuclease I Erythrocytes/metabolism Gene Expression Genes, Reporter Globins/genetics Humans In Situ Hybridization Kruppel-Like Transcription Factors Locus Control Region Mice Mice, Transgenic Molecular Sequence Data Mutation Sp1 Transcription Factor/genetics,metabolism Trans-Activators/genetics,metabolism Transcription Factors/genetics,metabolism Transfection Zinc Fingers/genetics
Chemicals
DNA-Binding Proteins Kruppel-Like Transcription Factors Sp1 Transcription Factor Trans-Activators Transcription Factors erythroid Kruppel-like factor Globins DNA Deoxyribonuclease I
Authors & Affiliations
8 authors, click to expand affiliations / ORCID
Gillemans N
Erasmus University Rotterdam, Medical Genetics Center-Department of Cell Biology, 3000 DR Rotterdam, The Netherlands.
Tewari R
Lindeboom F
Rottier R
de Wit T
Wijgerde M
Grosveld F
Philipsen S
References (60)
60 references, click to expand
  1. A dominant control region from the human beta-globin locus conferring integration site-independent gene expression.
    Nature. 1989 Mar 23;338(6213):352-5 PMID: 2922063
  2. DNaseI hypersensitive sites 1, 2 and 3 of the human beta-globin dominant control region direct position-independent expression.
    Nucleic Acids Res. 1990 Jun 25;18(12):3503-8 PMID: 2362805
  3. Direct interaction between Sp1 and the BPV enhancer E2 protein mediates synergistic activation of transcription.
    Cell. 1991 May 3;65(3):493-505 PMID: 1850324
  4. Identification and characterization of a gene encoding a gut-enriched Krüppel-like factor expressed during growth arrest.
    J Biol Chem. 1996 Aug 16;271(33):20009-17 PMID: 8702718
  5. Heterochromatin effects on the frequency and duration of LCR-mediated gene transcription.
    Cell. 1996 Oct 4;87(1):105-14 PMID: 8858153
  6. A dominant chromatin-opening activity in 5' hypersensitive site 3 of the human beta-globin locus control region.
    EMBO J. 1996 Feb 1;15(3):562-8 PMID: 8599939
  7. Ligand-inducible and liver-specific target gene expression in transgenic mice.
    Nat Biotechnol. 1997 Mar;15(3):239-43 PMID: 9062922
  8. Cloning by recognition site screening of two novel GT box binding proteins: a family of Sp1 related genes.
    Nucleic Acids Res. 1992 Nov 11;20(21):5519-25 PMID: 1454515
  9. Ecdysone-inducible gene expression in mammalian cells and transgenic mice.
    Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3346-51 PMID: 8622939
  10. The regulation of human globin gene expression.
    Baillieres Clin Haematol. 1993 Mar;6(1):31-55 PMID: 8353317
  11. Use of a zinc-finger consensus sequence framework and specificity rules to design specific DNA binding proteins.
    Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2256-60 PMID: 8460130
  12. The human beta-globin gene 3' enhancer contains multiple binding sites for an erythroid-specific protein.
    Genes Dev. 1988 Sep;2(9):1089-100 PMID: 2461328
  13. beta-Thalassaemia.
    Baillieres Clin Haematol. 1993 Mar;6(1):151-75 PMID: 8353313
  14. Analysis of mice containing a targeted deletion of beta-globin locus control region 5' hypersensitive site 3.
    Mol Cell Biol. 1996 Jun;16(6):2906-12 PMID: 8649401
  15. A general strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites.
    Science. 1997 Jan 31;275(5300):657-61 PMID: 9005850
  16. Erythroid Krüppel-like factor (EKLF) is active in primitive and definitive erythroid cells and is required for the function of 5'HS3 of the beta-globin locus control region.
    EMBO J. 1998 Apr 15;17(8):2334-41 PMID: 9545245
  17. 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):2776-86 PMID: 7682653
  18. Analyses of beta-thalassemia mutant DNA interactions with erythroid Krüppel-like factor (EKLF), an erythroid cell-specific transcription factor.
    J Biol Chem. 1994 Jan 14;269(2):1493-500 PMID: 8288615
  19. Induction of Erythroid-Specific Expression in Murine Erythroleukemia (MEL) Cell Lines.
    Methods Mol Biol. 1991;7:421-34 PMID: 21416373
  20. FOG, a multitype zinc finger protein, acts as a cofactor for transcription factor GATA-1 in erythroid and megakaryocytic differentiation.
    Cell. 1997 Jul 11;90(1):109-19 PMID: 9230307
  21. Synergy between adjacent zinc fingers in sequence-specific DNA recognition.
    Proc Natl Acad Sci U S A. 1997 May 27;94(11):5617-21 PMID: 9159121
  22. Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain.
    Cell. 1987 Dec 24;51(6):1079-90 PMID: 3319186
  23. Transcriptional activation by hypersensitive site three of the human beta-globin locus control region in murine erythroleukemia cells.
    Biochim Biophys Acta. 1994 Oct 18;1219(2):351-60 PMID: 7918632
  24. In vivo repression by a site-specific DNA-binding protein designed against an oncogenic sequence.
    Nature. 1994 Dec 15;372(6507):642-5 PMID: 7990954
  25. Transcription complex stability and chromatin dynamics in vivo.
    Nature. 1995 Sep 21;377(6546):209-13 PMID: 7675106
  26. The role of EKLF in human beta-globin gene competition.
    Genes Dev. 1996 Nov 15;10(22):2894-902 PMID: 8918890
  27. The effect of distance on long-range chromatin interactions.
    Mol Cell. 1997 Dec;1(1):131-9 PMID: 9659910
  28. Transcription factor Sp1 is essential for early embryonic development but dispensable for cell growth and differentiation.
    Cell. 1997 May 16;89(4):619-28 PMID: 9160753
  29. 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):2437-47 PMID: 7739528
  30. Lethal beta-thalassaemia in mice lacking the erythroid CACCC-transcription factor EKLF.
    Nature. 1995 May 25;375(6529):318-22 PMID: 7753195
  31. Silencing of human fetal globin expression is impaired in the absence of the adult beta-globin gene activator protein EKLF.
    Proc Natl Acad Sci U S A. 1996 Oct 29;93(22):12267-71 PMID: 8901569
  32. An interaction between the DNA-binding domains of RelA(p65) and Sp1 mediates human immunodeficiency virus gene activation.
    Mol Cell Biol. 1994 Oct;14(10):6570-83 PMID: 7935378
  33. The human beta-globin locus control region confers an early embryonic erythroid-specific expression pattern to a basic promoter driving the bacterial lacZ gene.
    Development. 1996 Dec;122(12):3991-9 PMID: 9012519
  34. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A.
    Science. 1991 May 10;252(5007):809-17 PMID: 2028256
  35. OVEC, a versatile system to study transcription in mammalian cells and cell-free extracts.
    Nucleic Acids Res. 1987 Sep 11;15(17):6787-98 PMID: 3658668
  36. Transcriptional activation by tetracyclines in mammalian cells.
    Science. 1995 Jun 23;268(5218):1766-9 PMID: 7792603
  37. Isolation and characterization of the cDNA encoding BKLF/TEF-2, a major CACCC-box-binding protein in erythroid cells and selected other cells.
    Mol Cell Biol. 1996 Apr;16(4):1695-705 PMID: 8657145
  38. Sp1-mediated transcriptional activation is repressed by Sp3.
    EMBO J. 1994 Aug 15;13(16):3843-51 PMID: 8070411
  39. The beta-globin dominant control region: hypersensitive site 2.
    EMBO J. 1990 Jul;9(7):2159-67 PMID: 2357964
  40. Cloning of GT box-binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression.
    Mol Cell Biol. 1992 Oct;12(10):4251-61 PMID: 1341900
  41. Transcriptional activation by recruitment.
    Nature. 1997 Apr 10;386(6625):569-77 PMID: 9121580
  42. Direct homeodomain-DNA interaction in the autoregulation of the fushi tarazu gene.
    Nature. 1992 Apr 30;356(6372):804-7 PMID: 1574120
  43. Determination of the base recognition positions of zinc fingers from sequence analysis.
    EMBO J. 1992 Dec;11(12):4507-17 PMID: 1425585
  44. Defective haematopoiesis in fetal liver resulting from inactivation of the EKLF gene.
    Nature. 1995 May 25;375(6529):316-8 PMID: 7753194
  45. Isolation of a gene encoding a functional zinc finger protein homologous to erythroid Krüppel-like factor: identification of a new multigene family.
    Mol Cell Biol. 1995 Nov;15(11):5957-65 PMID: 7565748
  46. In vivo protein-DNA interactions at hypersensitive site 3 of the human beta-globin locus control region.
    Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5809-13 PMID: 1631062
  47. Structures of zinc finger domains from transcription factor Sp1. Insights into sequence-specific protein-DNA recognition.
    J Biol Chem. 1997 Mar 21;272(12):7801-9 PMID: 9065444
  48. Position effects and genetic disease.
    Trends Genet. 1996 Apr;12(4):123-6 PMID: 8901413
  49. Binary system for regulating transgene expression in mice: targeting int-2 gene expression with yeast GAL4/UAS control elements.
    Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):698-702 PMID: 1846961
  50. Ultra-sensitive FISH using peroxidase-mediated deposition of biotin- or fluorochrome tyramides.
    Hum Mol Genet. 1995 Apr;4(4):529-34 PMID: 7633400
  51. LCR/MEL: a versatile system for high-level expression of heterologous proteins in erythroid cells.
    Nucleic Acids Res. 1992 Mar 11;20(5):997-1003 PMID: 1549512
  52. Regulated expression of human A gamma-, beta-, and hybrid gamma beta-globin genes in transgenic mice: manipulation of the developmental expression patterns.
    Cell. 1986 Jul 4;46(1):89-94 PMID: 3719696
  53. Structural and functional analyses of DNA bending induced by Sp1 family transcription factors.
    J Mol Biol. 1997 Apr 4;267(3):490-504 PMID: 9126833
  54. Developmental regulation of a complete 70-kb human beta-globin locus in transgenic mice.
    Genes Dev. 1992 Oct;6(10):1857-64 PMID: 1383089
  55. Measurement of the binding of transcription factor Sp1 to a single GC box recognition sequence.
    Nucleic Acids Res. 1989 Apr 11;17(7):2639-53 PMID: 2717405
  56. A role in DNA binding for the linker sequences of the first three zinc fingers of TFIIIA.
    Nucleic Acids Res. 1993 Jul 25;21(15):3341-6 PMID: 8346014
  57. Octamer binding proteins confer transcriptional activity in early mouse embryogenesis.
    EMBO J. 1989 Sep;8(9):2551-7 PMID: 2573524
  58. A new technique for the assay of infectivity of human adenovirus 5 DNA.
    Virology. 1973 Apr;52(2):456-67 PMID: 4705382
  59. The minimal requirements for activity in transgenic mice of hypersensitive site 3 of the beta globin locus control region.
    EMBO J. 1993 Mar;12(3):1077-85 PMID: 8458325
  60. Two regulatory proteins that bind to the basic transcription element (BTE), a GC box sequence in the promoter region of the rat P-4501A1 gene.
    EMBO J. 1992 Oct;11(10):3663-71 PMID: 1356762
Article Info
Journal
Genes & development
Abbr.
Genes Dev
ISSN
0890-9369
DOI
10.1101/gad.12.18.2863
Published
1998-09-15
Pages
2863-73
Language
English
Region
United States
NLM ID
8711660
PMCID
PMC317172
Subset
IM
External Links
PubMed source DOI: 10.1101/gad.12.18.2863 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2