Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Oct;15(10):5552–5562. doi: 10.1128/mcb.15.10.5552

Regulation of the DNA-binding and transcriptional activities of Xenopus laevis NFI-X by a novel C-terminal domain.

E Roulet 1, M T Armentero 1, G Krey 1, B Corthésy 1, C Dreyer 1, N Mermod 1, W Wahli 1
PMCID: PMC230806  PMID: 7565707

Abstract

The nuclear factor I (NFI) family consists of sequence-specific DNA-binding proteins that activate both transcription and adenovirus DNA replication. We have characterized three new members of the NFI family that belong to the Xenopus laevis NFI-X subtype and differ in their C-termini. We show that these polypeptides can activate transcription in HeLa and Drosophila Schneider line 2 cells, using an activation domain that is subdivided into adjacent variable and subtype-specific domains each having independent activation properties in chimeric proteins. Together, these two domains constitute the full NFI-X transactivation potential. In addition, we find that the X. laevis NFI-X proteins are capable of activating adenovirus DNA replication through their conserved N-terminal DNA-binding domains. Surprisingly, their in vitro DNA-binding activities are specifically inhibited by a novel repressor domain contained within the C-terminal part, while the dimerization and replication functions per se are not affected. However, inhibition of DNA-binding activity in vitro is relieved within the cell, as transcriptional activation occurs irrespective of the presence of the repressor domain. Moreover, the region comprising the repressor domain participates in transactivation. Mechanisms that may allow the relief of DNA-binding inhibition in vivo and trigger transcriptional activation are discussed.

Full Text

The Full Text of this article is available as a PDF (735.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Apt D., Chong T., Liu Y., Bernard H. U. Nuclear factor I and epithelial cell-specific transcription of human papillomavirus type 16. J Virol. 1993 Aug;67(8):4455–4463. doi: 10.1128/jvi.67.8.4455-4463.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Apt D., Liu Y., Bernard H. U. Cloning and functional analysis of spliced isoforms of human nuclear factor I-X: interference with transcriptional activation by NFI/CTF in a cell-type specific manner. Nucleic Acids Res. 1994 Sep 25;22(19):3825–3833. doi: 10.1093/nar/22.19.3825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Armentero M. T., Horwitz M., Mermod N. Targeting of DNA polymerase to the adenovirus origin of DNA replication by interaction with nuclear factor I. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11537–11541. doi: 10.1073/pnas.91.24.11537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bagni C., Mariottini P., Annesi F., Amaldi F. Structure of Xenopus laevis ribosomal protein L32 and its expression during development. Nucleic Acids Res. 1990 Aug 11;18(15):4423–4426. doi: 10.1093/nar/18.15.4423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bardwell V. J., Treisman R. The POZ domain: a conserved protein-protein interaction motif. Genes Dev. 1994 Jul 15;8(14):1664–1677. doi: 10.1101/gad.8.14.1664. [DOI] [PubMed] [Google Scholar]
  6. Bertholet C., Drillien R., Wittek R. One hundred base pairs of 5' flanking sequence of a vaccinia virus late gene are sufficient to temporally regulate late transcription. Proc Natl Acad Sci U S A. 1985 Apr;82(7):2096–2100. doi: 10.1073/pnas.82.7.2096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bosher J., Dawson A., Hay R. T. Nuclear factor I is specifically targeted to discrete subnuclear sites in adenovirus type 2-infected cells. J Virol. 1992 May;66(5):3140–3150. doi: 10.1128/jvi.66.5.3140-3150.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carey M., Kakidani H., Leatherwood J., Mostashari F., Ptashne M. An amino-terminal fragment of GAL4 binds DNA as a dimer. J Mol Biol. 1989 Oct 5;209(3):423–432. doi: 10.1016/0022-2836(89)90007-7. [DOI] [PubMed] [Google Scholar]
  9. Carey M. Mechanistic advances in eukaryotic gene activation. Curr Opin Cell Biol. 1991 Jun;3(3):452–460. doi: 10.1016/0955-0674(91)90073-8. [DOI] [PubMed] [Google Scholar]
  10. Chen M., Horwitz M. S. Dissection of functional domains of adenovirus DNA polymerase by linker-insertion mutagenesis. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6116–6120. doi: 10.1073/pnas.86.16.6116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chen M., Mermod N., Horwitz M. S. Protein-protein interactions between adenovirus DNA polymerase and nuclear factor I mediate formation of the DNA replication preinitiation complex. J Biol Chem. 1990 Oct 25;265(30):18634–18642. [PubMed] [Google Scholar]
  12. 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. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  13. Clark H. F., Michalski F., Tweedell K. S., Yohn D., Zeigel R. F. An adenovirus, FAV-1, isolated from the kidney of a frog (Rana pipiens). Virology. 1973 Feb;51(2):392–400. doi: 10.1016/0042-6822(73)90438-8. [DOI] [PubMed] [Google Scholar]
  14. Courey A. J., Tjian R. Analysis of Sp1 in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif. Cell. 1988 Dec 2;55(5):887–898. doi: 10.1016/0092-8674(88)90144-4. [DOI] [PubMed] [Google Scholar]
  15. Ellinger-Ziegelbauer H., Dreyer C. A retinoic acid receptor expressed in the early development of Xenopus laevis. Genes Dev. 1991 Jan;5(1):94–104. doi: 10.1101/gad.5.1.94. [DOI] [PubMed] [Google Scholar]
  16. Frankel A. D., Kim P. S. Modular structure of transcription factors: implications for gene regulation. Cell. 1991 May 31;65(5):717–719. doi: 10.1016/0092-8674(91)90378-c. [DOI] [PubMed] [Google Scholar]
  17. Gil G., Smith J. R., Goldstein J. L., Slaughter C. A., Orth K., Brown M. S., Osborne T. F. Multiple genes encode nuclear factor 1-like proteins that bind to the promoter for 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8963–8967. doi: 10.1073/pnas.85.23.8963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gounari F., De Francesco R., Schmitt J., van der Vliet P., Cortese R., Stunnenberg H. Amino-terminal domain of NF1 binds to DNA as a dimer and activates adenovirus DNA replication. EMBO J. 1990 Feb;9(2):559–566. doi: 10.1002/j.1460-2075.1990.tb08143.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Inoue T., Tamura T., Furuichi T., Mikoshiba K. Isolation of complementary DNAs encoding a cerebellum-enriched nuclear factor I family that activates transcription from the mouse myelin basic protein promoter. J Biol Chem. 1990 Nov 5;265(31):19065–19070. [PubMed] [Google Scholar]
  21. Jones K. A., Kadonaga J. T., Rosenfeld P. J., Kelly T. J., Tjian R. A cellular DNA-binding protein that activates eukaryotic transcription and DNA replication. Cell. 1987 Jan 16;48(1):79–89. doi: 10.1016/0092-8674(87)90358-8. [DOI] [PubMed] [Google Scholar]
  22. Jones K. A., Yamamoto K. R., Tjian R. Two distinct transcription factors bind to the HSV thymidine kinase promoter in vitro. Cell. 1985 Sep;42(2):559–572. doi: 10.1016/0092-8674(85)90113-8. [DOI] [PubMed] [Google Scholar]
  23. Kenny M. K., Hurwitz J. Initiation of adenovirus DNA replication. II. Structural requirements using synthetic oligonucleotide adenovirus templates. J Biol Chem. 1988 Jul 15;263(20):9809–9817. [PubMed] [Google Scholar]
  24. Kruse U., Qian F., Sippel A. E. Identification of a fourth nuclear factor I gene in chicken by cDNA cloning: NFI-X. Nucleic Acids Res. 1991 Dec 11;19(23):6641–6641. doi: 10.1093/nar/19.23.6641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kumar V., Chambon P. The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer. Cell. 1988 Oct 7;55(1):145–156. doi: 10.1016/0092-8674(88)90017-7. [DOI] [PubMed] [Google Scholar]
  26. Lillie J. W., Green M. R. Transcription activation by the adenovirus E1a protein. Nature. 1989 Mar 2;338(6210):39–44. doi: 10.1038/338039a0. [DOI] [PubMed] [Google Scholar]
  27. Lim F., Kraut N., Framptom J., Graf T. DNA binding by c-Ets-1, but not v-Ets, is repressed by an intramolecular mechanism. EMBO J. 1992 Feb;11(2):643–652. doi: 10.1002/j.1460-2075.1992.tb05096.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Madden S. L., Cook D. M., Rauscher F. J., 3rd A structure-function analysis of transcriptional repression mediated by the WT1, Wilms' tumor suppressor protein. Oncogene. 1993 Jul;8(7):1713–1720. [PubMed] [Google Scholar]
  29. Marmorstein R., Carey M., Ptashne M., Harrison S. C. DNA recognition by GAL4: structure of a protein-DNA complex. Nature. 1992 Apr 2;356(6368):408–414. doi: 10.1038/356408a0. [DOI] [PubMed] [Google Scholar]
  30. Martinez E., Dusserre Y., Wahli W., Mermod N. Synergistic transcriptional activation by CTF/NF-I and the estrogen receptor involves stabilized interactions with a limiting target factor. Mol Cell Biol. 1991 Jun;11(6):2937–2945. doi: 10.1128/mcb.11.6.2937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mermod N., O'Neill E. A., Kelly T. J., Tjian R. The proline-rich transcriptional activator of CTF/NF-I is distinct from the replication and DNA binding domain. Cell. 1989 Aug 25;58(4):741–753. doi: 10.1016/0092-8674(89)90108-6. [DOI] [PubMed] [Google Scholar]
  32. Nagata K., Guggenheimer R. A., Enomoto T., Lichy J. H., Hurwitz J. Adenovirus DNA replication in vitro: identification of a host factor that stimulates synthesis of the preterminal protein-dCMP complex. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6438–6442. doi: 10.1073/pnas.79.21.6438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nebl G., Mermod N., Cato A. C. Post-transcriptional down-regulation of expression of transcription factor NF1 by Ha-ras oncogene. J Biol Chem. 1994 Mar 11;269(10):7371–7378. [PubMed] [Google Scholar]
  34. Norman C., Runswick M., Pollock R., Treisman R. Isolation and properties of cDNA clones encoding SRF, a transcription factor that binds to the c-fos serum response element. Cell. 1988 Dec 23;55(6):989–1003. doi: 10.1016/0092-8674(88)90244-9. [DOI] [PubMed] [Google Scholar]
  35. Paonessa G., Gounari F., Frank R., Cortese R. Purification of a NF1-like DNA-binding protein from rat liver and cloning of the corresponding cDNA. EMBO J. 1988 Oct;7(10):3115–3123. doi: 10.1002/j.1460-2075.1988.tb03178.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rossi P., Karsenty G., Roberts A. B., Roche N. S., Sporn M. B., de Crombrugghe B. A nuclear factor 1 binding site mediates the transcriptional activation of a type I collagen promoter by transforming growth factor-beta. Cell. 1988 Feb 12;52(3):405–414. doi: 10.1016/s0092-8674(88)80033-3. [DOI] [PubMed] [Google Scholar]
  37. Rupp R. A., Kruse U., Multhaup G., Göbel U., Beyreuther K., Sippel A. E. 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. doi: 10.1093/nar/18.9.2607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sadowski I., Ptashne M. A vector for expressing GAL4(1-147) fusions in mammalian cells. Nucleic Acids Res. 1989 Sep 25;17(18):7539–7539. doi: 10.1093/nar/17.18.7539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Santoro C., Mermod N., Andrews P. C., Tjian R. A family of human CCAAT-box-binding proteins active in transcription and DNA replication: cloning and expression of multiple cDNAs. Nature. 1988 Jul 21;334(6179):218–224. doi: 10.1038/334218a0. [DOI] [PubMed] [Google Scholar]
  40. Sperisen P., Wang S. M., Reichenbach P., Nabholz M. A PCR-based assay for reporter gene expression. PCR Methods Appl. 1992 Feb;1(3):164–170. doi: 10.1101/gr.1.3.164. [DOI] [PubMed] [Google Scholar]
  41. Temperley S. M., Hay R. T. Recognition of the adenovirus type 2 origin of DNA replication by the virally encoded DNA polymerase and preterminal proteins. EMBO J. 1992 Feb;11(2):761–768. doi: 10.1002/j.1460-2075.1992.tb05109.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tjian R., Maniatis T. Transcriptional activation: a complex puzzle with few easy pieces. Cell. 1994 Apr 8;77(1):5–8. doi: 10.1016/0092-8674(94)90227-5. [DOI] [PubMed] [Google Scholar]
  43. White J., Brou C., Wu J., Lutz Y., Moncollin V., Chambon P. The acidic transcriptional activator GAL-VP16 acts on preformed template-committed complexes. EMBO J. 1992 Jun;11(6):2229–2240. doi: 10.1002/j.1460-2075.1992.tb05282.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yang B. S., Gilbert J. D., Freytag S. O. Overexpression of Myc suppresses CCAAT transcription factor/nuclear factor 1-dependent promoters in vivo. Mol Cell Biol. 1993 May;13(5):3093–3102. doi: 10.1128/mcb.13.5.3093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES