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Mol Cell Biol. 1993 Jan; 13(1): 44–56.
PMCID: PMC358883

Discrimination among potential activators of the beta-globin CACCC element by correlation of binding and transcriptional properties.


Adult beta-globin-like promoters contain a cis-acting element, CCACACCC, that is conserved across species and is required for wild-type levels of transcription. We have studied the contribution of this element and proteins that interact with it to activate beta-globin transcription. We found that an erythroid-like cell line, MEL, contains several proteins that specifically bind the CACCC element. By comparing the DNA-binding properties of promoters with mutations in the CACCC element with the transcriptional activities of these mutant promoters, we found that two CACCC-binding proteins did not bind to mutant promoters that direct decreased levels of transcription. One of these proteins is the transcriptional activator Sp1, and the other we have designated CACD (CACCC-binding species D). We subjected CACD to a binding site selection procedure and obtained high-affinity CACD binding sites that are identical to that of the beta-globin CACCC element. This result, combined with our finding that CACD binds the CACCC element with a higher affinity than does Sp1, argues that the CACCC element is a target of CACD rather than Sp1. The strategy of correlating the results of a binding site selection experiment with those of in vivo expression and in vitro binding studies may allow evaluation of the relative potential of different proteins to activate transcription through a single cis-acting site.

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  • Antoniou M, Grosveld F. beta-globin dominant control region interacts differently with distal and proximal promoter elements. Genes Dev. 1990 Jun;4(6):1007–1013. [PubMed]
  • Blackwell TK, Weintraub H. Differences and similarities in DNA-binding preferences of MyoD and E2A protein complexes revealed by binding site selection. Science. 1990 Nov 23;250(4984):1104–1110. [PubMed]
  • Catala F, deBoer E, Habets G, Grosveld F. Nuclear protein factors and erythroid transcription of the human A gamma-globin gene. Nucleic Acids Res. 1989 May 25;17(10):3811–3827. [PMC free article] [PubMed]
  • Chao MV, 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. [PubMed]
  • Charnay P, Mellon P, Maniatis T. Linker scanning mutagenesis of the 5'-flanking region of the mouse beta-major-globin gene: sequence requirements for transcription in erythroid and nonerythroid cells. Mol Cell Biol. 1985 Jun;5(6):1498–1511. [PMC free article] [PubMed]
  • Cowie A, Myers RM. DNA sequences involved in transcriptional regulation of the mouse beta-globin promoter in murine erythroleukemia cells. Mol Cell Biol. 1988 Aug;8(8):3122–3128. [PMC free article] [PubMed]
  • Davidson I, Xiao JH, Rosales R, Staub A, Chambon P. The HeLa cell protein TEF-1 binds specifically and cooperatively to two SV40 enhancer motifs of unrelated sequence. Cell. 1988 Sep 23;54(7):931–942. [PubMed]
  • deBoer E, Antoniou M, Mignotte V, Wall L, Grosveld F. The human beta-globin promoter; nuclear protein factors and erythroid specific induction of transcription. EMBO J. 1988 Dec 20;7(13):4203–4212. [PMC free article] [PubMed]
  • Deisseroth A, Hendrick D. Human alpha-globin gene expression following chromosomal dependent gene transfer into mouse erythroleukemia cells. Cell. 1978 Sep;15(1):55–63. [PubMed]
  • Dessain S, Gross CT, Kuziora MA, McGinnis W. Antp-type homeodomains have distinct DNA binding specificities that correlate with their different regulatory functions in embryos. EMBO J. 1992 Mar;11(3):991–1002. [PMC free article] [PubMed]
  • Dierks P, van Ooyen A, Cochran MD, Dobkin C, Reiser J, Weissmann C. Three regions upstream from the cap site are required for efficient and accurate transcription of the rabbit beta-globin gene in mouse 3T6 cells. Cell. 1983 Mar;32(3):695–706. [PubMed]
  • Dignam JD, Lebovitz RM, Roeder RG. 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. [PMC free article] [PubMed]
  • Efstratiadis A, Posakony JW, Maniatis T, Lawn RM, O'Connell C, Spritz RA, DeRiel JK, Forget BG, Weissman SM, Slightom JL, et al. The structure and evolution of the human beta-globin gene family. Cell. 1980 Oct;21(3):653–668. [PubMed]
  • Favaloro J, Treisman R, Kamen R. Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol. 1980;65(1):718–749. [PubMed]
  • Frampton J, Walker M, Plumb M, Harrison PR. Synergy between the NF-E1 erythroid-specific transcription factor and the CACCC factor in the erythroid-specific promoter of the human porphobilinogen deaminase gene. Mol Cell Biol. 1990 Jul;10(7):3838–3842. [PMC free article] [PubMed]
  • Giglioni B, Comi P, Ronchi A, Mantovani R, Ottolenghi S. The same nuclear proteins bind the proximal CACCC box of the human beta-globin promoter and a similar sequence in the enhancer. Biochem Biophys Res Commun. 1989 Oct 16;164(1):149–155. [PubMed]
  • Gonzalez-Redondo JM, Stoming TA, Kutlar A, Kutlar F, Lanclos KD, Howard EF, Fei YJ, Aksoy M, Altay C, Gurgey A, et al. A C----T substitution at nt--101 in a conserved DNA sequence of the promotor region of the beta-globin gene is associated with "silent" beta-thalassemia. Blood. 1989 May 1;73(6):1705–1711. [PubMed]
  • Goutte C, Johnson AD. a1 protein alters the DNA binding specificity of alpha 2 repressor. Cell. 1988 Mar 25;52(6):875–882. [PubMed]
  • Grosveld GC, de Boer E, Shewmaker CK, Flavell RA. DNA sequences necessary for transcription of the rabbit beta-globin gene in vivo. Nature. 1982 Jan 14;295(5845):120–126. [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]
  • Hayashi S, Scott MP. What determines the specificity of action of Drosophila homeodomain proteins? Cell. 1990 Nov 30;63(5):883–894. [PubMed]
  • Jackson SP, Tjian R. Purification and analysis of RNA polymerase II transcription factors by using wheat germ agglutinin affinity chromatography. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1781–1785. [PMC free article] [PubMed]
  • Johnson PF, McKnight SL. Eukaryotic transcriptional regulatory proteins. Annu Rev Biochem. 1989;58:799–839. [PubMed]
  • Jones KA, Kadonaga JT, Rosenfeld PJ, Kelly TJ, Tjian R. A cellular DNA-binding protein that activates eukaryotic transcription and DNA replication. Cell. 1987 Jan 16;48(1):79–89. [PubMed]
  • Kadonaga JT, Carner KR, Masiarz FR, Tjian R. Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell. 1987 Dec 24;51(6):1079–1090. [PubMed]
  • Keleher CA, Goutte C, Johnson AD. The yeast cell-type-specific repressor alpha 2 acts cooperatively with a non-cell-type-specific protein. Cell. 1988 Jun 17;53(6):927–936. [PubMed]
  • Krieg PA, Melton DA. In vitro RNA synthesis with SP6 RNA polymerase. Methods Enzymol. 1987;155:397–415. [PubMed]
  • Kulozik AE, Bellan-Koch A, Bail S, Kohne E, Kleihauer E. Thalassemia intermedia: moderate reduction of beta globin gene transcriptional activity by a novel mutation of the proximal CACCC promoter element. Blood. 1991 May 1;77(9):2054–2058. [PubMed]
  • Kunkel TA, Roberts JD, Zakour RA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. [PubMed]
  • Lewis CD, Clark SP, Felsenfeld G, Gould H. An erythrocyte-specific protein that binds to the poly(dG) region of the chicken beta-globin gene promoter. Genes Dev. 1988 Jul;2(7):863–873. [PubMed]
  • Mantovani R, Malgaretti N, Nicolis S, Giglioni B, Comi P, Cappellini N, Bertero MT, Caligaris-Cappio F, Ottolenghi S. An erythroid specific nuclear factor binding to the proximal CACCC box of the beta-globin gene promoter. Nucleic Acids Res. 1988 May 25;16(10):4299–4313. [PMC free article] [PubMed]
  • Marks PA, Rifkind RA. Erythroleukemic differentiation. Annu Rev Biochem. 1978;47:419–448. [PubMed]
  • Maxam AM, Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. [PubMed]
  • Mitchell PJ, Wang C, Tjian R. Positive and negative regulation of transcription in vitro: enhancer-binding protein AP-2 is inhibited by SV40 T antigen. Cell. 1987 Sep 11;50(6):847–861. [PubMed]
  • Myers RM, Tilly K, Maniatis T. Fine structure genetic analysis of a beta-globin promoter. Science. 1986 May 2;232(4750):613–618. [PubMed]
  • Orkin SH, Antonarakis SE, Kazazian HH., Jr Base substitution at position -88 in a beta-thalassemic globin gene. Further evidence for the role of distal promoter element ACACCC. J Biol Chem. 1984 Jul 25;259(14):8679–8681. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Ristaldi MS, Murru S, Loudianos G, Casula L, Porcu S, Pigheddu D, Fanni B, Sciarratta GV, Agosti S, Parodi MI, et al. The C-T substitution in the distal CACCC box of the beta-globin gene promoter is a common cause of silent beta thalassaemia in the Italian population. Br J Haematol. 1990 Apr;74(4):480–486. [PubMed]
  • Saffer JD, Thurston SJ, Annarella MB, Compton JG. Localization of the gene for the trans-acting transcription factor Sp1 to the distal end of mouse chromosome 15. Genomics. 1990 Nov;8(3):571–574. [PubMed]
  • Schreiber E, Matthias P, Müller MM, Schaffner W. Identification of a novel lymphoid specific octamer binding protein (OTF-2B) by proteolytic clipping bandshift assay (PCBA). EMBO J. 1988 Dec 20;7(13):4221–4229. [PMC free article] [PubMed]
  • Schüle R, Muller M, Otsuka-Murakami H, Renkawitz R. Cooperativity of the glucocorticoid receptor and the CACCC-box binding factor. Nature. 1988 Mar 3;332(6159):87–90. [PubMed]
  • Spanopoulou E, Giguere V, Grosveld F. The functional domains of the murine Thy-1 gene promoter. Mol Cell Biol. 1991 Apr;11(4):2216–2228. [PMC free article] [PubMed]
  • Strauss EC, Andrews NC, Higgs DR, Orkin SH. In vivo footprinting of the human alpha-globin locus upstream regulatory element by guanine and adenine ligation-mediated polymerase chain reaction. Mol Cell Biol. 1992 May;12(5):2135–2142. [PMC free article] [PubMed]
  • Stuve LL, Myers RM. A directly repeated sequence in the beta-globin promoter regulates transcription in murine erythroleukemia cells. Mol Cell Biol. 1990 Mar;10(3):972–981. [PMC free article] [PubMed]
  • Thiesen HJ, Bach C. Target Detection Assay (TDA): a versatile procedure to determine DNA binding sites as demonstrated on SP1 protein. Nucleic Acids Res. 1990 Jun 11;18(11):3203–3209. [PMC free article] [PubMed]
  • Treisman R, Orkin SH, Maniatis T. Specific transcription and RNA splicing defects in five cloned beta-thalassaemia genes. Nature. 1983 Apr 14;302(5909):591–596. [PubMed]
  • Tsai SF, Strauss E, Orkin SH. Functional analysis and in vivo footprinting implicate the erythroid transcription factor GATA-1 as a positive regulator of its own promoter. Genes Dev. 1991 Jun;5(6):919–931. [PubMed]
  • Wang WD, Gralla JD. Differential ability of proximal and remote element pairs to cooperate in activating RNA polymerase II transcription. Mol Cell Biol. 1991 Sep;11(9):4561–4571. [PMC free article] [PubMed]
  • Wright S, Rosenthal A, Flavell R, Grosveld F. DNA sequences required for regulated expression of beta-globin genes in murine erythroleukemia cells. Cell. 1984 Aug;38(1):265–273. [PubMed]
  • Xiao JH, Davidson I, Macchi M, Rosales R, Vigneron M, Staub A, Chambon P. In vitro binding of several cell-specific and ubiquitous nuclear proteins to the GT-I motif of the SV40 enhancer. Genes Dev. 1987 Oct;1(8):794–807. [PubMed]
  • Yu CY, Motamed K, Chen J, Bailey AD, Shen CK. The CACC box upstream of human embryonic epsilon globin gene binds Sp1 and is a functional promoter element in vitro and in vivo. J Biol Chem. 1991 May 15;266(14):8907–8915. [PubMed]

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