• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. Dec 1993; 13(12): 7612–7624.
PMCID: PMC364833

CTCF, a conserved nuclear factor required for optimal transcriptional activity of the chicken c-myc gene, is an 11-Zn-finger protein differentially expressed in multiple forms.

Abstract

A novel sequence-specific DNA-binding protein, CTCF, which interacts with the chicken c-myc gene promoter, has been identified and partially characterized (V. V. Lobanenkov, R. H. Nicolas, V. V. Adler, H. Paterson, E. M. Klenova, A. V. Polotskaja, and G. H. Goodwin, Oncogene 5:1743-1753, 1990). In order to test directly whether binding of CTCF to one specific DNA region of the c-myc promoter is important for chicken c-myc transcription, we have determined which nucleotides within this GC-rich region are responsible for recognition of overlapping sites by CTCF and Sp1-like proteins. Using missing-contact analysis of all four nucleotides in both DNA strands and homogeneous CTCF protein purified by sequence-specific chromatography, we have identified three sets of nucleotides which contact either CTCF or two Sp1-like proteins binding within the same DNA region. Specific mutations of 3 of 15 purines required for CTCF binding were designed to eliminate binding of CTCF without altering the binding of other proteins. Electrophoretic mobility shift assay of nuclear extracts showed that the mutant DNA sequence did not bind CTCF but did bind two Sp1-like proteins. When introduced into a 3.3-kbp-long 5'-flanking noncoding c-myc sequence fused to a reporter CAT gene, the same mutation of the CTCF binding site resulted in 10- and 3-fold reductions, respectively, of transcription in two different (erythroid and myeloid) stably transfected chicken cell lines. Isolation and analysis of the CTCF cDNA encoding an 82-kDa form of CTCF protein shows that DNA-binding domain of CTCF is composed of 11 Zn fingers: 10 are of C2H2 class, and 1 is of C2HC class. CTCF was found to be abundant and conserved in cells of vertebrate species. We detected six major nuclear forms of CTCF protein differentially expressed in different chicken cell lines and tissues. We conclude that isoforms of 11-Zn-finger factor CTCF which are present in chicken hematopoietic HD3 and BM2 cells can act as a positive regulator of the chicken c-myc gene transcription. Possible functions of other CTCF forms are discussed.

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 (3.5M), 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.
  • Adamson P, Paterson HF, Hall A. Intracellular localization of the P21rho proteins. J Cell Biol. 1992 Nov;119(3):617–627. [PMC free article] [PubMed]
  • Askew DS, Ashmun RA, Simmons BC, Cleveland JL. Constitutive c-myc expression in an IL-3-dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis. Oncogene. 1991 Oct;6(10):1915–1922. [PubMed]
  • Basu A, Park K, Atchison ML, Carter RS, Avadhani NG. Identification of a transcriptional initiator element in the cytochrome c oxidase subunit Vb promoter which binds to transcription factors NF-E1 (YY-1, delta) and Sp1. J Biol Chem. 1993 Feb 25;268(6):4188–4196. [PubMed]
  • Berg JM. Zinc fingers and other metal-binding domains. Elements for interactions between macromolecules. J Biol Chem. 1990 Apr 25;265(12):6513–6516. [PubMed]
  • Beug H, Doederlein G, Freudenstein C, Graf T. Erythroblast cell lines transformed by a temperature-sensitive mutant of avian erythroblastosis virus: a model system to study erythroid differentiation in vitro. J Cell Physiol Suppl. 1982;1:195–207. [PubMed]
  • Bickmore WA, Oghene K, Little MH, Seawright A, van Heyningen V, Hastie ND. Modulation of DNA binding specificity by alternative splicing of the Wilms tumor wt1 gene transcript. Science. 1992 Jul 10;257(5067):235–237. [PubMed]
  • Bird AP. CpG-rich islands and the function of DNA methylation. Nature. 1986 May 15;321(6067):209–213. [PubMed]
  • Blackwood EM, Eisenman RN. Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science. 1991 Mar 8;251(4998):1211–1217. [PubMed]
  • Bordereaux D, Fichelson S, Tambourin P, Gisselbrecht S. Alternative splicing of the Evi-1 zinc finger gene generates mRNAs which differ by the number of zinc finger motifs. Oncogene. 1990 Jun;5(6):925–927. [PubMed]
  • Bossone SA, Asselin C, Patel AJ, Marcu KB. MAZ, a zinc finger protein, binds to c-MYC and C2 gene sequences regulating transcriptional initiation and termination. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7452–7456. [PMC free article] [PubMed]
  • Brunelle A, Schleif RF. Missing contact probing of DNA-protein interactions. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6673–6676. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Cole MD. The myc oncogene: its role in transformation and differentiation. Annu Rev Genet. 1986;20:361–384. [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]
  • Dingwall C, Laskey RA. Nuclear targeting sequences--a consensus? Trends Biochem Sci. 1991 Dec;16(12):478–481. [PubMed]
  • el-Baradi T, Pieler T. Zinc finger proteins: what we know and what we would like to know. Mech Dev. 1991 Nov;35(3):155–169. [PubMed]
  • Evan GI, Wyllie AH, Gilbert CS, Littlewood TD, Land H, Brooks M, Waters CM, Penn LZ, Hancock DC. Induction of apoptosis in fibroblasts by c-myc protein. Cell. 1992 Apr 3;69(1):119–128. [PubMed]
  • Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7413–7417. [PMC free article] [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]
  • Giniger E, Ptashne M. Transcription in yeast activated by a putative amphipathic alpha helix linked to a DNA binding unit. Nature. 1987 Dec 17;330(6149):670–672. [PubMed]
  • Helin K, Lees JA, Vidal M, Dyson N, Harlow E, Fattaey A. A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F. Cell. 1992 Jul 24;70(2):337–350. [PubMed]
  • Hirt RP, Poulain-Godefroy O, Billotte J, Kraehenbuhl JP, Fasel N. Highly inducible synthesis of heterologous proteins in epithelial cells carrying a glucocorticoid-responsive vector. Gene. 1992 Feb 15;111(2):199–206. [PubMed]
  • Hsu T, Gogos JA, Kirsh SA, Kafatos FC. Multiple zinc finger forms resulting from developmentally regulated alternative splicing of a transcription factor gene. Science. 1992 Sep 25;257(5078):1946–1950. [PubMed]
  • Hunter T, Karin M. The regulation of transcription by phosphorylation. Cell. 1992 Aug 7;70(3):375–387. [PubMed]
  • Imataka H, Sogawa K, Yasumoto K, Kikuchi Y, Sasano K, Kobayashi A, Hayami M, Fujii-Kuriyama Y. 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):3663–3671. [PMC free article] [PubMed]
  • Kadonaga JT. Purification of sequence-specific binding proteins by DNA affinity chromatography. Methods Enzymol. 1991;208:10–23. [PubMed]
  • Kadonaga JT, Courey AJ, Ladika J, Tjian R. Distinct regions of Sp1 modulate DNA binding and transcriptional activation. Science. 1988 Dec 16;242(4885):1566–1570. [PubMed]
  • Kim S, Humphries EH, Tjoelker L, Carlson L, Thompson CB. Ongoing diversification of the rearranged immunoglobulin light-chain gene in a bursal lymphoma cell line. Mol Cell Biol. 1990 Jun;10(6):3224–3231. [PMC free article] [PubMed]
  • Kingsley C, Winoto A. Cloning of GT box-binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression. Mol Cell Biol. 1992 Oct;12(10):4251–4261. [PMC free article] [PubMed]
  • Kolluri R, Kinniburgh AJ. Full length cDNA sequence encoding a nuclease-sensitive element DNA binding protein. Nucleic Acids Res. 1991 Sep 11;19(17):4771–4771. [PMC free article] [PubMed]
  • Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. [PMC free article] [PubMed]
  • Kretzner L, Blackwood EM, Eisenman RN. Myc and Max proteins possess distinct transcriptional activities. Nature. 1992 Oct 1;359(6394):426–429. [PubMed]
  • Lachmann PJ, Strangeways L, Vyakarnam A, Evan G. Raising antibodies by coupling peptides to PPD and immunizing BCG-sensitized animals. Ciba Found Symp. 1986;119:25–57. [PubMed]
  • Letovsky J, Dynan WS. Measurement of the binding of transcription factor Sp1 to a single GC box recognition sequence. Nucleic Acids Res. 1989 Apr 11;17(7):2639–2653. [PMC free article] [PubMed]
  • Lobanenkov VV, Nicolas RH, Adler VV, Paterson H, Klenova EM, Polotskaja AV, Goodwin GH. A novel sequence-specific DNA binding protein which interacts with three regularly spaced direct repeats of the CCCTC-motif in the 5'-flanking sequence of the chicken c-myc gene. Oncogene. 1990 Dec;5(12):1743–1753. [PubMed]
  • Lobanenkov VV, Nicolas RH, Plumb MA, Wright CA, Goodwin GH. Sequence-specific DNA-binding proteins which interact with (G + C)-rich sequences flanking the chicken c-myc gene. Eur J Biochem. 1986 Aug 15;159(1):181–188. [PubMed]
  • Marcu KB, Bossone SA, Patel AJ. myc function and regulation. Annu Rev Biochem. 1992;61:809–860. [PubMed]
  • Maxam AM, Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. [PubMed]
  • Moll T, Tebb G, Surana U, Robitsch H, Nasmyth K. The role of phosphorylation and the CDC28 protein kinase in cell cycle-regulated nuclear import of the S. cerevisiae transcription factor SWI5. Cell. 1991 Aug 23;66(4):743–758. [PubMed]
  • Neiman PE, Thomas SJ, Loring G. Induction of apoptosis during normal and neoplastic B-cell development in the bursa of Fabricius. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5857–5861. [PMC free article] [PubMed]
  • Pearson RB, Kemp BE. Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations. Methods Enzymol. 1991;200:62–81. [PubMed]
  • Plumb MA, Lobanenkov VV, Nicolas RH, Wright CA, Zavou S, Goodwin GH. Characterisation of chicken erythroid nuclear proteins which bind to the nuclease hypersensitive regions upstream of the beta A- and beta H-globin genes. Nucleic Acids Res. 1986 Oct 10;14(19):7675–7693. [PMC free article] [PubMed]
  • Pyrc JJ, Moberg KH, Hall DJ. Isolation of a novel cDNA encoding a zinc-finger protein that binds to two sites within the c-myc promoter. Biochemistry. 1992 Apr 28;31(16):4102–4110. [PubMed]
  • Ray R, Miller DM. Cloning and characterization of a human c-myc promoter-binding protein. Mol Cell Biol. 1991 Apr;11(4):2154–2161. [PMC free article] [PubMed]
  • Read D, Manley JL. Alternatively spliced transcripts of the Drosophila tramtrack gene encode zinc finger proteins with distinct DNA binding specificities. EMBO J. 1992 Mar;11(3):1035–1044. [PMC free article] [PubMed]
  • Roman DG, Toledano MB, Leonard WJ. Sp1 represses IL-2 receptor alpha chain gene expression. New Biol. 1990 Jul;2(7):642–647. [PubMed]
  • Roy AL, Meisterernst M, Pognonec P, Roeder RG. Cooperative interaction of an initiator-binding transcription initiation factor and the helix-loop-helix activator USF. Nature. 1991 Nov 21;354(6350):245–248. [PubMed]
  • Rupp RA, Kruse U, Multhaup G, Göbel U, Beyreuther K, Sippel AE. Chicken NFI/TGGCA proteins are encoded by at least three independent genes: NFI-A, NFI-B and NFI-C with homologues in mammalian genomes. Nucleic Acids Res. 1990 May 11;18(9):2607–2616. [PMC free article] [PubMed]
  • Ruppert JM, Kinzler KW, Wong AJ, Bigner SH, Kao FT, Law ML, Seuanez HN, O'Brien SJ, Vogelstein B. The GLI-Kruppel family of human genes. Mol Cell Biol. 1988 Aug;8(8):3104–3113. [PMC free article] [PubMed]
  • Saraste M, Sibbald PR, Wittinghofer A. The P-loop--a common motif in ATP- and GTP-binding proteins. Trends Biochem Sci. 1990 Nov;15(11):430–434. [PubMed]
  • Seto E, Shi Y, Shenk T. YY1 is an initiator sequence-binding protein that directs and activates transcription in vitro. Nature. 1991 Nov 21;354(6350):241–245. [PubMed]
  • Spencer CA, Groudine M. Transcription elongation and eukaryotic gene regulation. Oncogene. 1990 Jun;5(6):777–785. [PubMed]
  • Spencer CA, Groudine M. Control of c-myc regulation in normal and neoplastic cells. Adv Cancer Res. 1991;56:1–48. [PubMed]
  • St-Arnaud R, Moir JM. Wnt-1-inducing factor-1: a novel G/C box-binding transcription factor regulating the expression of Wnt-1 during neuroectodermal differentiation. Mol Cell Biol. 1993 Mar;13(3):1590–1598. [PMC free article] [PubMed]
  • Stappert J, Wirsching J, Kemler R. A PCR method for introducing mutations into cloned DNA by joining an internal primer to a tagged flanking primer. Nucleic Acids Res. 1992 Feb 11;20(3):624–624. [PMC free article] [PubMed]
  • Symonds G, Klempnauer KH, Evan GI, Bishop JM. Induced differentiation of avian myeloblastosis virus-transformed myeloblasts: phenotypic alteration without altered expression of the viral oncogene. Mol Cell Biol. 1984 Dec;4(12):2587–2593. [PMC free article] [PubMed]
  • Tevosian SG, Gromova ES, Adler VV, Lobanenkov VV. Reguliatornyi belkovyi faktor CTCF vzaimodeistvuet s uchastkom promotora onkogena c-myc kur, sposobnym k perekhody v nekanonicheskuiu konformatsiiu. Mol Biol (Mosk) 1991 Jul-Aug;25(4):1013–1023. [PubMed]
  • Thalmeier K, Synovzik H, Mertz R, Winnacker EL, Lipp M. Nuclear factor E2F mediates basic transcription and trans-activation by E1a of the human MYC promoter. Genes Dev. 1989 Apr;3(4):527–536. [PubMed]
  • Tsang SS, Yin X, Guzzo-Arkuran C, Jones VS, Davison AJ. Loss of resolution in gel electrophoresis of RNA: a problem associated with the presence of formaldehyde gradients. Biotechniques. 1993 Mar;14(3):380–381. [PubMed]
  • Walker JE, Saraste M, Runswick MJ, Gay NJ. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. [PMC free article] [PubMed]
  • Weis L, Reinberg D. Transcription by RNA polymerase II: initiator-directed formation of transcription-competent complexes. FASEB J. 1992 Nov;6(14):3300–3309. [PubMed]

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

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links