Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Short Communication
  • Published:

Transcription factor plasmid binding modulates microtubule interactions and intracellular trafficking during gene transfer

Gene Therapy volume 19, pages 338–346 (2012)Cite this article

A Corrigendum to this article was published on 21 July 2011

Abstract

For non-viral gene delivery to be successful, plasmids must move through the cytoplasm to the nucleus in order to be transcribed. While the cytoskeletal meshwork acts as a barrier to plasmid DNA movement in the cytoplasm, the microtubule network is required for directed plasmid trafficking to the nucleus. We have shown previously that plasmid–microtubule interactions require cytoplasmic adapter proteins such as molecular motors, transcription factors (TFs) and importins. However, not all plasmid sequences support these interactions to allow movement to the nucleus. We now demonstrate that microtubule–DNA interactions can show sequence specificity with promoters containing binding sites for cyclic AMP response-element binding protein (CREB), including the cytomegalovirus immediate early promoter (CMViep). Plasmids containing CREB-binding sites showed stringent interactions in an in vitro microtubule-binding assay. Using microinjection and real-time particle tracking, we show that the inclusion of TF binding sites within plasmids permits cytoplasmic trafficking of plasmids during gene transfer. We found that CREB-binding sites are bound by CREB in the cytoplasm during transfection, and allow for enhanced rates of movement and subsequent nuclear accumulation. Moreover, small interfering RNA knockdown of CREB prevented this enhanced trafficking. Therefore, TF binding sites within plasmids are necessary for interactions with microtubules and enhance movement to the nucleus.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  1. Mesika A, Kiss V, Brumfeld V, Ghosh G, Reich Z . Enhanced intracellular mobility and nuclear accumulation of DNA plasmids associated with a karyophilic protein. Hum Gene Ther 2005; 16: 200–208.

    Article  CAS  Google Scholar 

  2. Vaughan EE, Dean DA . Intracellular trafficking of plasmids during transfection is mediated by microtubules. Mol Ther 2006; 13: 422–428.

    Article  CAS  Google Scholar 

  3. Leopold PL, Kreitzer G, Miyazawa N, Rempel S, Pfister KK, Rodriguez-Boulan E et al. Dynein- and microtubule-mediated translocation of adenovirus serotype 5 occurs after endosomal lysis. Hum Gene Ther 2000; 11: 151–165.

    Article  CAS  Google Scholar 

  4. Vaughan EE, DeGiulio JV, Dean DA . Intracellular trafficking of plasmids for gene therapy: mechanisms of cytoplasmic movement and nuclear import. Curr Gene Ther 2006; 6: 671–681.

    Article  CAS  Google Scholar 

  5. Wilson GL, Dean BS, Wang G, Dean DA . Nuclear import of plasmid DNA in digitonin-permeabilized cells requires both cytoplasmic factors and specific DNA sequences. J Biol Chem 1999; 274: 22025–22032.

    Article  CAS  Google Scholar 

  6. Miller AM, Munkonge FM, Alton EW, Dean DA . Identification of protein cofactors necessary for sequence-specific plasmid DNA nuclear import. Mol Ther 2009; 17: 1897–1903.

    Article  CAS  Google Scholar 

  7. Lachish-Zalait A, Lau CK, Fichtman B, Zimmerman E, Harel A, Gaylord MR et al. Transportin mediates nuclear entry of DNA in vertebrate systems. Traffic 2009; 10: 1414–1428.

    Article  CAS  Google Scholar 

  8. Dean DA . Import of plasmid DNA into the nucleus is sequence specific. Exp Cell Res 1997; 230: 293–302.

    Article  CAS  Google Scholar 

  9. Dean DA, Dean BS, Muller S, Smith LC . Sequence requirements for plasmid nuclear import. Exp Cell Res 1999; 253: 713–722.

    Article  CAS  Google Scholar 

  10. Vaughan EE, Geiger RC, Miller AM, Loh-Marley PL, Suzuki T, Miyata N et al. Microtubule acetylation through HDAC6 inhibition results in increased transfection efficiency. Mol Ther 2008; 16: 1841–1847.

    Article  CAS  Google Scholar 

  11. Mercurio F, Karin M . Transcription factors AP-3 and AP-2 interact with the SV40 enhancer in a mutually exclusive manner. EMBO J 1989; 8: 1455–1460.

    Article  CAS  Google Scholar 

  12. Turner WJ, Woodworth ME . DNA replication efficiency depends on transcription factor-binding sites. J Virol 2001; 75: 5638–5645.

    Article  CAS  Google Scholar 

  13. Clark L, Pollock RM, Hay RT . Identification and purification of EBP1: a HeLa cell protein that binds to a region overlapping the ‘core’ of the SV40 enhancer. Genes Dev 1988; 2: 991–1002.

    Article  CAS  Google Scholar 

  14. Kalderon D, Richardson WD, Markham AF, Smith AE . Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature 1984; 311: 33–38.

    Article  CAS  Google Scholar 

  15. Suh J, Wirtz D, Hanes J . Efficient active transport of gene nanocarriers to the cell nucleus. Proc Natl Acad Sci USA 2003; 100: 3878–3882.

    Article  CAS  Google Scholar 

  16. Whiteside ST, Goodbourn S . Signal transduction and nuclear targeting: regulation of transcription factor activity by subcellular localisation. J Cell Sci 1993; 104 (Part 4): 949–955.

    CAS  PubMed  Google Scholar 

  17. Park EA, Roesler WJ, Liu J, Klemm DJ, Gurney AL, Thatcher JD et al. The role of the CCAAT/enhancer-binding protein in the transcriptional regulation of the gene for phosphoenolpyruvate carboxykinase (GTP). Mol Cell Biol 1990; 10: 6264–6272.

    Article  CAS  Google Scholar 

  18. Muchardt C, Li C, Kornuc M, Gaynor R . CREB regulation of cellular cyclic AMP-responsive and adenovirus early promoters. J Virol 1990; 64: 4296–4305.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Desai A, Mitchison TJ . Microtubule polymerization dynamics. Annu Rev Cell Dev Biol 1997; 13: 83–117.

    Article  CAS  Google Scholar 

  20. Miller AM, Dean DA . Cell-specific nuclear import of plasmid DNA in smooth muscle requires tissue-specific transcription factors and DNA sequences. Gene Therapy 2008; 15: 1107–1115.

    Article  CAS  Google Scholar 

  21. Mesika A, Grigoreva I, Zohar M, Reich Z . A regulated, NFkappaB-assisted import of plasmid DNA into mammalian cell nuclei. Mol Ther 2001; 3: 653–657.

    Article  CAS  Google Scholar 

  22. Munkonge FM, Amin V, Hyde SC, Green AM, Pringle IA, Gill DR et al. Identification and functional characterization of cytoplasmic determinants of plasmid DNA nuclear import. J Biol Chem 2009; 284: 26978–26987.

    Article  CAS  Google Scholar 

  23. Breuzard G, Tertil M, Goncalves C, Cheradame H, Geguan P, Pichon C et al. Nuclear delivery of NFkappaB-assisted DNA/polymer complexes: plasmid DNA quantitation by confocal laser scanning microscopy and evidence of nuclear polyplexes by FRET imaging. Nucleic Acids Res 2008; 36: e71.

    Article  Google Scholar 

  24. Hager GL, Elbi C, Becker M . Protein dynamics in the nuclear compartment. Curr Opin Genet Dev 2002; 12: 137–141.

    Article  CAS  Google Scholar 

  25. Misteli T . Protein dynamics: implications for nuclear architecture and gene expression. Science 2001; 291: 843–847.

    Article  CAS  Google Scholar 

  26. Barry ME, Pinto-Gonzalez D, Orson FM, McKenzie GJ, Petry GR, Barry MA . Role of endogenous endonucleases and tissue site in transfection and CpG-mediated immune activation after naked DNA injection. Hum Gene Ther 1999; 10: 2461–2480.

    Article  CAS  Google Scholar 

  27. Lechardeur D, Sohn KJ, Haardt M, Joshi PB, Monck M, Graham RW et al. Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer. Gene Therapy 1999; 6: 482–497.

    Article  CAS  Google Scholar 

  28. Gazzola M, Burckhardt CJ, Bayati B, Engelke M, Greber UF, Koumoutsakos P . A stochastic model for microtubule motors describes the in vivo cytoplasmic transport of human adenovirus. PLoS Comput Biol 2009; 5: e1000623.

    Article  Google Scholar 

  29. King SJ, Schroer TA . Dynactin increases the processivity of the cytoplasmic dynein motor. Nat Cell Biol 2000; 2: 20–24.

    Article  CAS  Google Scholar 

  30. Paschal BM, Shpetner HS, Vallee RB . MAP 1C is a microtubule-activated ATPase which translocates microtubules in vitro and has dynein-like properties. J Cell Biol 1987; 105: 1273–1282.

    Article  CAS  Google Scholar 

  31. Kural C, Kim H, Syed S, Goshima G, Gelfand VI, Selvin PR . Kinesin and dynein move a peroxisome in vivo: a tug-of-war or coordinated movement? Science 2005; 308: 1469–1472.

    Article  CAS  Google Scholar 

  32. Rapp S, Saffrich R, Jakle U, Ansorge W, Gorgas K, Just WW . Microtubule-mediated peroxisomal saltations. Ann N Y Acad Sci 1996; 804: 666–668.

    Article  CAS  Google Scholar 

  33. Suomalainen M, Nakano MY, Boucke K, Keller S, Greber UF . Adenovirus-activated PKA and p38/MAPK pathways boost microtubule-mediated nuclear targeting of virus. EMBO J 2001; 20: 1310–1319.

    Article  CAS  Google Scholar 

  34. Lee GE, Murray JW, Wolkoff AW, Wilson DW . Reconstitution of herpes simplex virus microtubule-dependent trafficking in vitro. J Virol 2006; 80: 4264–4275.

    Article  CAS  Google Scholar 

  35. Suomalainen M, Nakano MY, Keller S, Boucke K, Stidwill RP, Greber UF . Microtubule-dependent plus- and minus end-directed motilities are competing processes for nuclear targeting of adenovirus. J Cell Biol 1999; 144: 657–672.

    Article  CAS  Google Scholar 

  36. Lochner JE, Kingma M, Kuhn S, Meliza CD, Cutler B, Scalettar BA . Real-time imaging of the axonal transport of granules containing a tissue plasminogen activator/green fluorescent protein hybrid. Mol Biol Cell 1998; 9: 2463–2476.

    Article  CAS  Google Scholar 

  37. Ondrej V, Lukasova E, Falk M, Kozubek S . The role of actin and microtubule networks in plasmid DNA intracellular trafficking. Acta Biochim Pol 2007; 54: 657–663.

    CAS  PubMed  Google Scholar 

  38. Dean DA . Nucleocytoplasmic trafficking, in pharmaceutical perspectives of nucleic acid-based therapeutics. In: Mahato RI (ed). Harwood Academic Publishers: London, 2002, pp 229–260.

  39. Guilley H, Dudley RK, Jonard G, Balazs E, Richards KE . Transcription of Cauliflower mosaic virus DNA: detection of promoter sequences, and characterization of transcripts. Cell 1982; 30: 763–773.

    Article  CAS  Google Scholar 

  40. Obata H, Hayashi K, Nishida W, Momiyama T, Uchida A, Ochi T et al. Smooth muscle cell phenotype-dependent transcriptional regulation of the alpha1 integrin gene. J Biol Chem 1997; 272: 26643–26651.

    Article  CAS  Google Scholar 

  41. Gasiorowski JZ, Dean DA . Intranuclear trafficking of episomal DNA is transcription-dependent. Mol Ther 2007; 15: 2132–2139.

    Article  CAS  Google Scholar 

  42. Gasiorowski JZ, Dean DA . Postmitotic nuclear retention of episomal plasmids is altered by DNA labeling and detection methods. Mol Ther 2005; 12: 460–467.

    Article  CAS  Google Scholar 

  43. Rogers SS, Waigh TA, Zhao X, Lu JR . Precise particle tracking against a complicated background: polynomial fitting with Gaussian weight. Phys Biol 2007; 4: 220–227.

    Article  CAS  Google Scholar 

  44. Greenberg ME, Beader TP . Identification of newly transcribed RNA. In: Ausubel FM, Brents R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds). Current Protocols in Molecular Biology, Vol 1. John Wiley & Sons, Inc.: New York, 1994, pp 4.10.1-4.10.11.

    Google Scholar 

Download references

Acknowledgements

We thank doctors Aaron Miller, Rui Zhou and Josh Gasiorowski, along with Mootaz Eldib for insightful discussions and technical advice. This work was supported in part by predoctoral fellowships from the Founders (MAB) and the Midwest (EEV) Affiliates of the American Heart Association, and Grants EB9903, HL71643, ES07026 and center Grant ES01247 from the National Institutes of Health.

Author information

Authors and Affiliations

  1. Division of Neonatology, Department of Pediatrics, School of Medicine and Dentistry, University of Rochester, Rochester, NY, USA

    M A Badding & D A Dean

  2. Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, IL, USA

    E E Vaughan & D A Dean

Authors
  1. M A Badding
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. E E Vaughan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. D A Dean
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to D A Dean.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Badding, M., Vaughan, E. & Dean, D. Transcription factor plasmid binding modulates microtubule interactions and intracellular trafficking during gene transfer. Gene Ther 19, 338–346 (2012). https://doi.org/10.1038/gt.2011.96

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2011.96

Keywords

This article is cited by

Search

Advanced search

Quick links