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. 1997 Feb 1;25(3):466–474. doi: 10.1093/nar/25.3.466

Molecular weight abnormalities of the CTCF transcription factor: CTCF migrates aberrantly in SDS-PAGE and the size of the expressed protein is affected by the UTRs and sequences within the coding region of the CTCF gene.

E M Klenova 1, R H Nicolas 1, S U 1, A F Carne 1, R E Lee 1, V V Lobanenkov 1, G H Goodwin 1
PMCID: PMC146483  PMID: 9016583

Abstract

CTCF belongs to the Zn finger transcription factors family and binds to the promoter region of c-myc. CTCF is highly conserved between species, ubiquitous and localised in nuclei. The endogenous CTCF migrates as a 130 kDa (CTCF-130) protein on SDS-PAGE, however, the open reading frame (ORF) of the CTCF cDNA encodes only a 82 kDa protein (CTCF-82). In the present study we investigate this phenomenon and show with mass-spectra analysis that this occurs due to aberrant mobility of the CTCF protein. Another paradox is that our original cDNA, composed of the ORF and 3'-untranslated region (3'-UTR), produces a protein with the apparent molecular weight of 70 kDa (CTCF-70). This paradox has been found to be an effect of the UTRs and sequences within the coding region of the CTCF gene resulting in C-terminal truncation of CTCF-130. The potential attenuator has been identified and point-mutated. This restored the electrophoretic mobility of the CTCF protein to 130 kDa. CTCF-70, the aberrantly migrating CTCF N-terminus per se, is also detected in some cell types and therefore may have some biological implications. In particular, CTCF-70 interferes with CTCF-130 normal function, enhancing transactivation induced by CTCF-130 in COS6 cells. The mechanism of CTCF-70 action and other possible functions of CTCF-70 are discussed.

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Selected References

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  1. Armstrong D. J., Roman A. The anomalous electrophoretic behavior of the human papillomavirus type 16 E7 protein is due to the high content of acidic amino acid residues. Biochem Biophys Res Commun. 1993 May 14;192(3):1380–1387. doi: 10.1006/bbrc.1993.1569. [DOI] [PubMed] [Google Scholar]
  2. Bickmore W. A., Oghene K., Little M. H., Seawright A., van Heyningen V., Hastie N. D. Modulation of DNA binding specificity by alternative splicing of the Wilms tumor wt1 gene transcript. Science. 1992 Jul 10;257(5067):235–237. doi: 10.1126/science.1321494. [DOI] [PubMed] [Google Scholar]
  3. Blank V., Kourilsky P., Israël A. Cytoplasmic retention, DNA binding and processing of the NF-kappa B p50 precursor are controlled by a small region in its C-terminus. EMBO J. 1991 Dec;10(13):4159–4167. doi: 10.1002/j.1460-2075.1991.tb04994.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Casarégola S., Jacq A., Laoudj D., McGurk G., Margarson S., Tempête M., Norris V., Holland I. B. Cloning and analysis of the entire Escherichia coli ams gene. ams is identical to hmp1 and encodes a 114 kDa protein that migrates as a 180 kDa protein. J Mol Biol. 1992 Nov 5;228(1):30–40. doi: 10.1016/0022-2836(92)90489-7. [DOI] [PubMed] [Google Scholar]
  5. Challberg M. D., Desiderio S. V., Kelly T. J., Jr Adenovirus DNA replication in vitro: characterization of a protein covalently linked to nascent DNA strands. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5105–5109. doi: 10.1073/pnas.77.9.5105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cooper A. A., Stevens T. H. Protein splicing: excision of intervening sequences at the protein level. Bioessays. 1993 Oct;15(10):667–674. doi: 10.1002/bies.950151006. [DOI] [PubMed] [Google Scholar]
  7. Eitan S., Schwartz M. A transglutaminase that converts interleukin-2 into a factor cytotoxic to oligodendrocytes. Science. 1993 Jul 2;261(5117):106–108. doi: 10.1126/science.8100369. [DOI] [PubMed] [Google Scholar]
  8. Epstein J. A., Glaser T., Cai J., Jepeal L., Walton D. S., Maas R. L. Two independent and interactive DNA-binding subdomains of the Pax6 paired domain are regulated by alternative splicing. Genes Dev. 1994 Sep 1;8(17):2022–2034. doi: 10.1101/gad.8.17.2022. [DOI] [PubMed] [Google Scholar]
  9. Evan G. I., Lewis G. K., Ramsay G., Bishop J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. doi: 10.1128/mcb.5.12.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Evan G. I., Littlewood T. D. The role of c-myc in cell growth. Curr Opin Genet Dev. 1993 Feb;3(1):44–49. doi: 10.1016/s0959-437x(05)80339-9. [DOI] [PubMed] [Google Scholar]
  11. Filippova G. N., Fagerlie S., Klenova E. M., Myers C., Dehner Y., Goodwin G., Neiman P. E., Collins S. J., Lobanenkov V. V. An exceptionally conserved transcriptional repressor, CTCF, employs different combinations of zinc fingers to bind diverged promoter sequences of avian and mammalian c-myc oncogenes. Mol Cell Biol. 1996 Jun;16(6):2802–2813. doi: 10.1128/mcb.16.6.2802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fontoura B. M., Sorokina E. A., David E., Carroll R. B. p53 is covalently linked to 5.8S rRNA. Mol Cell Biol. 1992 Nov;12(11):5145–5151. doi: 10.1128/mcb.12.11.5145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gopal V., Ma H. W., Kumaran M. K., Chatterji D. A point mutation at the junction of domain 2.3/2.4 of transcription factor sigma 70 abrogates productive transcription and restores its expected mobility on a denaturing gel. J Mol Biol. 1994 Sep 9;242(1):9–22. doi: 10.1006/jmbi.1994.1553. [DOI] [PubMed] [Google Scholar]
  14. Grumont R. J., Gerondakis S. Alternative splicing of RNA transcripts encoded by the murine p105 NF-kappa B gene generates I kappa B gamma isoforms with different inhibitory activities. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4367–4371. doi: 10.1073/pnas.91.10.4367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hershey J. W. Translational control in mammalian cells. Annu Rev Biochem. 1991;60:717–755. doi: 10.1146/annurev.bi.60.070191.003441. [DOI] [PubMed] [Google Scholar]
  16. Jackson R. J., Standart N. Do the poly(A) tail and 3' untranslated region control mRNA translation? Cell. 1990 Jul 13;62(1):15–24. doi: 10.1016/0092-8674(90)90235-7. [DOI] [PubMed] [Google Scholar]
  17. Kato G. J., Dang C. V. Function of the c-Myc oncoprotein. FASEB J. 1992 Sep;6(12):3065–3072. doi: 10.1096/fasebj.6.12.1521738. [DOI] [PubMed] [Google Scholar]
  18. Klenova E. M., Nicolas R. H., Paterson H. F., Carne A. F., Heath C. M., Goodwin G. H., Neiman P. E., Lobanenkov V. V. 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. Mol Cell Biol. 1993 Dec;13(12):7612–7624. doi: 10.1128/mcb.13.12.7612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kozak M. Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. Proc Natl Acad Sci U S A. 1986 May;83(9):2850–2854. doi: 10.1073/pnas.83.9.2850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lee Y. F., Nomoto A., Detjen B. M., Wimmer E. A protein covalently linked to poliovirus genome RNA. Proc Natl Acad Sci U S A. 1977 Jan;74(1):59–63. doi: 10.1073/pnas.74.1.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lobanenkov V. V., Nicolas R. H., Adler V. V., Paterson H., Klenova E. M., Polotskaja A. V., Goodwin G. H. 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] [Google Scholar]
  22. Marcu K. B., Bossone S. A., Patel A. J. myc function and regulation. Annu Rev Biochem. 1992;61:809–860. doi: 10.1146/annurev.bi.61.070192.004113. [DOI] [PubMed] [Google Scholar]
  23. Milland J., Christiansen D., Thorley B. R., McKenzie I. F., Loveland B. E. Translation is enhanced after silent nucleotide substitutions in A+T- rich sequences of the coding region of CD46 cDNA. Eur J Biochem. 1996 May 15;238(1):221–230. doi: 10.1111/j.1432-1033.1996.0221q.x. [DOI] [PubMed] [Google Scholar]
  24. Molnár A., Georgopoulos K. The Ikaros gene encodes a family of functionally diverse zinc finger DNA-binding proteins. Mol Cell Biol. 1994 Dec;14(12):8292–8303. doi: 10.1128/mcb.14.12.8292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nakamura Y., Ito K., Isaksson L. A. Emerging understanding of translation termination. Cell. 1996 Oct 18;87(2):147–150. doi: 10.1016/s0092-8674(00)81331-8. [DOI] [PubMed] [Google Scholar]
  26. Nordeen S. K. Luciferase reporter gene vectors for analysis of promoters and enhancers. Biotechniques. 1988 May;6(5):454–458. [PubMed] [Google Scholar]
  27. Pham D. Q., Sivasubramanian N. Sequence and in vitro translational analysis of a 1629-nucleotide ORF in Autographa californica nuclear polyhedrosis virus strain E2. Gene. 1992 Dec 15;122(2):345–348. doi: 10.1016/0378-1119(92)90224-d. [DOI] [PubMed] [Google Scholar]
  28. Pulverer B. J., Kyriakis J. M., Avruch J., Nikolakaki E., Woodgett J. R. Phosphorylation of c-jun mediated by MAP kinases. Nature. 1991 Oct 17;353(6345):670–674. doi: 10.1038/353670a0. [DOI] [PubMed] [Google Scholar]
  29. Query C. C., Bentley R. C., Keene J. D. A common RNA recognition motif identified within a defined U1 RNA binding domain of the 70K U1 snRNP protein. Cell. 1989 Apr 7;57(1):89–101. doi: 10.1016/0092-8674(89)90175-x. [DOI] [PubMed] [Google Scholar]
  30. Reason A. J., Morris H. R., Panico M., Marais R., Treisman R. H., Haltiwanger R. S., Hart G. W., Kelly W. G., Dell A. Localization of O-GlcNAc modification on the serum response transcription factor. J Biol Chem. 1992 Aug 25;267(24):16911–16921. [PubMed] [Google Scholar]
  31. Resar L. M., Dolde C., Barrett J. F., Dang C. V. B-myc inhibits neoplastic transformation and transcriptional activation by c-myc. Mol Cell Biol. 1993 Feb;13(2):1130–1136. doi: 10.1128/mcb.13.2.1130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Roth M. B., Zahler A. M., Stolk J. A. A conserved family of nuclear phosphoproteins localized to sites of polymerase II transcription. J Cell Biol. 1991 Nov;115(3):587–596. doi: 10.1083/jcb.115.3.587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Roth M. J., Brown D. R., Hurwitz J. Analysis of bacteriophage phi X174 gene A protein-mediated termination and reinitiation of phi X DNA synthesis. II. Structural characterization of the covalent phi X A protein-DNA complex. J Biol Chem. 1984 Aug 25;259(16):10556–10568. [PubMed] [Google Scholar]
  34. Ruiz-Linares A., Bouloy M., Girard M., Cahour A. Modulations of the in vitro translational efficiencies of Yellow Fever virus mRNAs: interactions between coding and noncoding regions. Nucleic Acids Res. 1989 Apr 11;17(7):2463–2476. doi: 10.1093/nar/17.7.2463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schiavi S. C., Wellington C. L., Shyu A. B., Chen C. Y., Greenberg M. E., Belasco J. G. Multiple elements in the c-fos protein-coding region facilitate mRNA deadenylation and decay by a mechanism coupled to translation. J Biol Chem. 1994 Feb 4;269(5):3441–3448. [PubMed] [Google Scholar]
  36. Shaulian E., Haviv I., Shaul Y., Oren M. Transcriptional repression by the C-terminal domain of p53. Oncogene. 1995 Feb 16;10(4):671–680. [PubMed] [Google Scholar]
  37. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  38. Sonenberg N. mRNA translation: influence of the 5' and 3' untranslated regions. Curr Opin Genet Dev. 1994 Apr;4(2):310–315. doi: 10.1016/s0959-437x(05)80059-0. [DOI] [PubMed] [Google Scholar]
  39. Spencer C. A., Groudine M. Control of c-myc regulation in normal and neoplastic cells. Adv Cancer Res. 1991;56:1–48. doi: 10.1016/s0065-230x(08)60476-5. [DOI] [PubMed] [Google Scholar]
  40. Spencer C. A., Groudine M. Transcription elongation and eukaryotic gene regulation. Oncogene. 1990 Jun;5(6):777–785. [PubMed] [Google Scholar]
  41. Standart N., Jackson R. J. Regulation of translation by specific protein/mRNA interactions. Biochimie. 1994;76(9):867–879. doi: 10.1016/0300-9084(94)90189-9. [DOI] [PubMed] [Google Scholar]
  42. Stansfield I., Jones K. M., Tuite M. F. The end in sight: terminating translation in eukaryotes. Trends Biochem Sci. 1995 Dec;20(12):489–491. doi: 10.1016/s0968-0004(00)89113-6. [DOI] [PubMed] [Google Scholar]
  43. Tuite M. F., Stansfield I. Termination of protein synthesis. Mol Biol Rep. 1994 May;19(3):171–181. doi: 10.1007/BF00986959. [DOI] [PubMed] [Google Scholar]
  44. Whiteside S. T., King P., Goodbourn S. A truncated form of the IRF-2 transcription factor has the properties of a postinduction repressor of interferon-beta gene expression. J Biol Chem. 1994 Oct 28;269(43):27059–27065. [PubMed] [Google Scholar]
  45. Yanofsky C. Transcription attenuation. J Biol Chem. 1988 Jan 15;263(2):609–612. [PubMed] [Google Scholar]

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