Search tips
Search criteria 


Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. 1992 November; 12(11): 4919–4929.
PMCID: PMC360424

Phylogenetic footprinting reveals a nuclear protein which binds to silencer sequences in the human gamma and epsilon globin genes.


Tissue- and developmental stage-specific expression of the human beta-like globin genes is regulated by a combination of ubiquitous and erythroid-restricted trans factors that bind to cis elements near each of the five active genes. Additional interactions of these cis and trans factors with sequences located in the far 5' end of the cluster occur by as yet obscure mechanisms. Because of the complexity of this regulatory puzzle, precise identification of the determinants that control hemoglobin switching has proven difficult. Phylogenetic footprinting is an evolutionary approach to this problem which is based on the supposition that the basic mechanisms of switching are conserved throughout mammalian phylogeny. Alignment of the 5' flanking regions of the gamma genes of several species allows the identification of footprints of 100% conserved sequence. We have now tested oligomers spanning 13 such phylogenetic footprints and find that 12 are bound by nuclear proteins. One conserved element located at -1086 from the gamma genes exhibits repressor activity in transient transfection studies. The protein that binds this element, CSBP-1 (conserved sequence-binding protein 1), also binds at three sites within a silencer element upstream from the epsilon globin gene. Further analysis reveals that the CSBP-1 binding activity is identical to that of a recently cloned zinc finger protein that has been shown to act as a repressor in other systems. The binding of CSPB-1 to silencer sequences in the epsilon and gamma globin genes may be important in the stage-specific silencing of these genes.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (3.0M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Becker DM, Fikes JD, Guarente L. A cDNA encoding a human CCAAT-binding protein cloned by functional complementation in yeast. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1968–1972. [PubMed]
  • Behringer RR, Ryan TM, Palmiter RD, Brinster RL, Townes TM. Human gamma- to beta-globin gene switching in transgenic mice. Genes Dev. 1990 Mar;4(3):380–389. [PubMed]
  • Cao SX, Gutman PD, Dave HP, Schechter AN. 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. [PubMed]
  • Chada K, Magram J, Costantini F. An embryonic pattern of expression of a human fetal globin gene in transgenic mice. Nature. 1986 Feb 20;319(6055):685–689. [PubMed]
  • Choi OR, Engel JD. Developmental regulation of beta-globin gene switching. Cell. 1988 Oct 7;55(1):17–26. [PubMed]
  • Collins FS, Weissman SM. The molecular genetics of human hemoglobin. Prog Nucleic Acid Res Mol Biol. 1984;31:315–462. [PubMed]
  • Curtin PT, Liu DP, Liu W, Chang JC, Kan YW. Human beta-globin gene expression in transgenic mice is enhanced by a distant DNase I hypersensitive site. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7082–7086. [PubMed]
  • de Wet JR, Wood KV, DeLuca M, Helinski DR, Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. [PMC free article] [PubMed]
  • Didier DK, Schiffenbauer J, Woulfe SL, Zacheis M, Schwartz BD. Characterization of the cDNA encoding a protein binding to the major histocompatibility complex class II Y box. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7322–7326. [PubMed]
  • Dillon N, Grosveld F. Human gamma-globin genes silenced independently of other genes in the beta-globin locus. Nature. 1991 Mar 21;350(6315):252–254. [PubMed]
  • Enver T, Raich N, Ebens AJ, 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. [PubMed]
  • Flanagan JR, Becker KG, Ennist DL, Gleason SL, Driggers PH, Levi BZ, Appella E, Ozato K. Cloning of a negative transcription factor that binds to the upstream conserved region of Moloney murine leukemia virus. Mol Cell Biol. 1992 Jan;12(1):38–44. [PMC free article] [PubMed]
  • Forrester WC, Thompson C, Elder JT, Groudine M. A developmentally stable chromatin structure in the human beta-globin gene cluster. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1359–1363. [PubMed]
  • Fraser P, Hurst J, Collis P, Grosveld F. 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):3503–3508. [PMC free article] [PubMed]
  • Friend C, Scher W, Holland JG, Sato T. Hemoglobin synthesis in murine virus-induced leukemic cells in vitro: stimulation of erythroid differentiation by dimethyl sulfoxide. Proc Natl Acad Sci U S A. 1971 Feb;68(2):378–382. [PubMed]
  • Gorman CM, Moffat LF, Howard BH. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. [PMC free article] [PubMed]
  • Gumucio DL, Lockwood WK, Weber JL, Saulino AM, Delgrosso K, Surrey S, Schwartz E, Goodman M, Collins FS. The -175T----C mutation increases promoter strength in erythroid cells: correlation with evolutionary conservation of binding sites for two trans-acting factors. Blood. 1990 Feb 1;75(3):756–761. [PubMed]
  • Gumucio DL, Rood KL, Blanchard-McQuate KL, Gray TA, Saulino A, Collins FS. Interaction of Sp1 with the human gamma globin promoter: binding and transactivation of normal and mutant promoters. Blood. 1991 Oct 1;78(7):1853–1863. [PubMed]
  • Gumucio DL, Rood KL, Gray TA, Riordan MF, Sartor CI, Collins FS. Nuclear proteins that bind the human gamma-globin gene promoter: alterations in binding produced by point mutations associated with hereditary persistence of fetal hemoglobin. Mol Cell Biol. 1988 Dec;8(12):5310–5322. [PMC free article] [PubMed]
  • Hariharan N, Kelley DE, Perry RP. Delta, a transcription factor that binds to downstream elements in several polymerase II promoters, is a functionally versatile zinc finger protein. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9799–9803. [PubMed]
  • Hooft van Huijsduijnen R, Li XY, Black D, Matthes H, Benoist C, Mathis D. Co-evolution from yeast to mouse: cDNA cloning of the two NF-Y (CP-1/CBF) subunits. EMBO J. 1990 Oct;9(10):3119–3127. [PubMed]
  • Janson L, Pettersson U. Cooperative interactions between transcription factors Sp1 and OTF-1. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4732–4736. [PubMed]
  • Knowles BB, Howe CC, Aden DP. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science. 1980 Jul 25;209(4455):497–499. [PubMed]
  • Kollias G, Wrighton N, Hurst J, Grosveld F. 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. [PubMed]
  • Lloyd JA, Lee RF, Lingrel JB. Mutations in two regions upstream of the A gamma globin gene canonical promoter affect gene expression. Nucleic Acids Res. 1989 Jun 12;17(11):4339–4352. [PMC free article] [PubMed]
  • Lozzio CB, Lozzio BB. Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood. 1975 Mar;45(3):321–334. [PubMed]
  • Mantovani R, Malgaretti N, Nicolis S, Ronchi A, Giglioni B, Ottolenghi S. The effects of HPFH mutations in the human gamma-globin promoter on binding of ubiquitous and erythroid specific nuclear factors. Nucleic Acids Res. 1988 Aug 25;16(16):7783–7797. [PMC free article] [PubMed]
  • Martin DI, Tsai SF, Orkin SH. Increased gamma-globin expression in a nondeletion HPFH mediated by an erythroid-specific DNA-binding factor. Nature. 1989 Mar 30;338(6214):435–438. [PubMed]
  • Martin P, Papayannopoulou T. HEL cells: a new human erythroleukemia cell line with spontaneous and induced globin expression. Science. 1982 Jun 11;216(4551):1233–1235. [PubMed]
  • McDonagh KT, Lin HJ, Lowrey CH, Bodine DM, Nienhuis AW. The upstream region of the human gamma-globin gene promoter. Identification and functional analysis of nuclear protein binding sites. J Biol Chem. 1991 Jun 25;266(18):11965–11974. [PubMed]
  • Morgan DA, Gumucio DL, Brodsky I. Granulocyte-macrophage colony-stimulating factor-dependent growth and erythropoietin-induced differentiation of a human cell line MB-02. Blood. 1991 Dec 1;78(11):2860–2871. [PubMed]
  • Ney PA, Sorrentino BP, McDonagh KT, Nienhuis AW. 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. [PubMed]
  • O'Hare P, Goding CR. Herpes simplex virus regulatory elements and the immunoglobulin octamer domain bind a common factor and are both targets for virion transactivation. Cell. 1988 Feb 12;52(3):435–445. [PubMed]
  • Park K, Atchison ML. Isolation of a candidate repressor/activator, NF-E1 (YY-1, delta), that binds to the immunoglobulin kappa 3' enhancer and the immunoglobulin heavy-chain mu E1 site. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9804–9808. [PubMed]
  • Pious D, Krangel MS, Dixon LL, Parham P, Strominger JL. HLA antigen structural gene mutants selected with an allospecific monoclonal antibody. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7832–7836. [PubMed]
  • 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]
  • Reitman M, Lee E, Westphal H, Felsenfeld G. Site-independent expression of the chicken beta A-globin gene in transgenic mice. Nature. 1990 Dec 20;348(6303):749–752. [PubMed]
  • Ryan TM, Behringer RR, Martin NC, Townes TM, Palmiter RD, Brinster RL. 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. [PubMed]
  • Seed B, Sheen JY. A simple phase-extraction assay for chloramphenicol acyltransferase activity. Gene. 1988 Jul 30;67(2):271–277. [PubMed]
  • Shi Y, Seto E, Chang LS, Shenk T. Transcriptional repression by YY1, a human GLI-Krüppel-related protein, and relief of repression by adenovirus E1A protein. Cell. 1991 Oct 18;67(2):377–388. [PubMed]
  • Tagle DA, Koop BF, Goodman M, Slightom JL, Hess DL, Jones RT. Embryonic epsilon and gamma globin genes of a prosimian primate (Galago crassicaudatus). Nucleotide and amino acid sequences, developmental regulation and phylogenetic footprints. J Mol Biol. 1988 Sep 20;203(2):439–455. [PubMed]
  • Tagle DA, Stanhope MJ, Siemieniak DR, Benson P, Goodman M, Slightom JL. The beta globin gene cluster of the prosimian primate Galago crassicaudatus: nucleotide sequence determination of the 41-kb cluster and comparative sequence analyses. Genomics. 1992 Jul;13(3):741–760. [PubMed]
  • Townes TM, Lingrel JB, Chen HY, Brinster RL, Palmiter RD. Erythroid-specific expression of human beta-globin genes in transgenic mice. EMBO J. 1985 Jul;4(7):1715–1723. [PubMed]
  • Tuan D, Solomon W, Li Q, London IM. The "beta-like-globin" gene domain in human erythroid cells. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6384–6388. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)