Skip to main content

Advertisement

SpringerLink
Log in

Characterization of an In Vivo Z-DNA Detection Probe Based on a Cell Nucleus Accumulating Intrabody

  • Original Paper
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Left-handed Z-DNA is a physiologically unstable DNA conformation, and its existence in vivo can be attributed to localized torsional distress. Despite evidence for the existence of Z-DNA in vivo, its precise role in the control of gene expression is not fully understood. Here, an in vivo probe based on an anti-Z-DNA intrabody is proposed for native Z-DNA detection. The probe was used for chromatin immunoprecipitation of potential Z-DNA-forming sequences in the human genome. One of the isolated putative Z-DNA-forming sequences was cloned upstream of a reporter gene expression cassette under control of the CMV promoter. The reporter gene encoded an antibody fragment fused to GFP. Transient co-transfection of this vector along with the Z-probe coding vector improved reporter gene expression. This improvement was demonstrated by measuring reporter gene mRNA and protein levels and the amount of fluorescence in co-transfected CHO-K1 cells. These results suggest that the presence of the anti-Z-DNA intrabody can interfere with a Z-DNA-containing reporter gene expression. Therefore, this in vivo probe for the detection of Z-DNA could be used for global correlation of Z-DNA-forming sequences and gene expression regulation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Wang, A. H., Quigley, G. J., Kolpak, F. J., Crawford, J. L., van Boom, J. H., van der Marel, G., et al. (1979). Molecular structure of a left-handed double helical DNA fragment at atomic resolution. Nature, 282, 680–686.

    Article  CAS  Google Scholar 

  2. Lancillotti, F., Lopez, M. C., Alonso, C., & Stollar, B. D. (1985). Locations of Z-DNA in polytene chromosomes. Journal of Cell Biology, 100, 1759–1766.

    Article  CAS  Google Scholar 

  3. Rich, A., Nordheim, A., & Wang, A. H. (1984). The chemistry and biology of left-handed Z-DNA. Annual Review of Biochemistry, 53, 791–846.

    Article  CAS  Google Scholar 

  4. Pohl, F. M., & Jovin, T. M. (1972). Salt-induced co-operative conformational change of a synthetic DNA: Equilibrium and kinetic studies with poly (dG-dC). Journal of Molecular Biology, 67, 375–396.

    Article  CAS  Google Scholar 

  5. Patel, D. J., Canuel, L. L., & Pohl, F. M. (1979). “Alternating B-DNA” conformation for the oligo (dG-dC) duplex in high-salt solution. Proceedings of the National Academy of Sciences USA, 76, 2508–2511.

    Article  CAS  Google Scholar 

  6. Staiano-Coico, L., Stollar, B. D., Darzynkiewicz, Z., Dutkowski, R., & Weksler, M. E. (1985). Binding of anti-Z-DNA antibodies in quiescent and activated lymphocytes: Relationship to cell cycle progression and chromatin changes. Molecular and Cellular Biology, 5, 3270–3273.

    Article  CAS  Google Scholar 

  7. Wong, B., Chen, S., Kwon, J. A., & Rich, A. (2007). Characterization of Z-DNA as a nucleosome-boundary element in yeast Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences USA, 104, 2229–2234.

    Article  CAS  Google Scholar 

  8. Liu, R., Liu, H., Chen, X., Kirby, M., Brown, P. O., & Zhao, K. (2001). Regulation of CSF1 promoter by the SWI/SNF-like BAF complex. Cell, 106, 309–318.

    Article  CAS  Google Scholar 

  9. Rothenburg, S., Koch-Nolte, F., Rich, A., & Haag, F. (2001). A polymorphic dinucleotide repeat in the rat nucleolin gene forms Z-DNA and inhibits promoter activity. Proceedings of the National Academy of Sciences USA, 98, 8985–8990.

    Article  CAS  Google Scholar 

  10. Oh, D. B., Kim, Y. G., & Rich, A. (2002). Z-DNA-binding proteins can act as potent effectors of gene expression in vivo. Proceedings of the National Academy of Sciences USA, 99, 16666–16671.

    Article  CAS  Google Scholar 

  11. Li, H., Xiao, J., Li, J., Lu, L., Feng, S., & Dröge, P. (2009). Human genomic Z-DNA segments probed by the Z alpha domain of ADAR1. Nucleic Acids Research, 37, 2737–2746.

    Article  CAS  Google Scholar 

  12. Maruyama, A., Mimura, J., Harada, N., & Itoh, K. (2013). Nrf2 activation is associated with Z-DNA formation in the human HO-1 promoter. Nucleic Acids Research, 41, 5223–5234.

    Article  CAS  Google Scholar 

  13. Lafer, E. M., Möller, A., Valle, R. P., Nordheim, A., Rich, A., & Stollar, B. D. (1983). Antibody recognition of Z-DNA. Cold Spring Harbor Symposia on Quantitative Biology, 47, 155–162.

    Article  Google Scholar 

  14. Sanford, D. G., & Stollar, B. D. (1990). Characterization of anti-Z-DNA antibody binding sites on Z-DNA by nuclear magnetic resonance spectroscopy. Journal of Biological Chemistry, 265, 18608–18614.

    CAS  Google Scholar 

  15. Brigido, M. M., Polymenis, M., & Stollar, B. D. (1993). Role of mouse VH10 and VL gene segments in the specific binding of antibody to Z-DNA, analyzed with recombinant single chain Fv molecules. The Journal of Immunology, 150, 469–479.

    CAS  Google Scholar 

  16. Polymenis, M., & Stollar, B. D. (1994). Critical binding site amino acids of anti-Z-DNA single chain Fv molecules. Role of heavy and light chain CDR3 and relationship to autoantibody activity. The Journal of Immunology, 152, 5318–5329.

    CAS  Google Scholar 

  17. Chen, Y., & Stollar, B. D. (1999). DNA binding by the VH domain of anti-Z-DNA antibody and its modulation by association of the VL domain. The Journal of Immunology, 162, 4663–4670.

    CAS  Google Scholar 

  18. Andrade, E. V., Freitas, S. M., Ventura, M. M., Maranhão, A. Q., & Brigido, M. M. (2005). Thermodynamic basis for antibody binding to Z-DNA: comparison of a monoclonal antibody and its recombinant derivatives. Biochimica et Biophysica Acta, 1726, 293–301.

    Article  Google Scholar 

  19. Campos-da-Paz, M., Costa, C. S., Quilici, L. S., de CarmoSimões, I., Kyaw, C. M., Maranhão, A. Q., et al. (2008). Production of recombinant human factor VIII in different cell lines and the effect of human XBP1 co-expression. Molecular Biotechnology, 39, 155–158.

    Article  CAS  Google Scholar 

  20. Stallings, C. L., & Silverstein, S. (2005). Dissection of a novel nuclear localization signal in open reading frame 29 of varicella-zoster virus. Journal of Virology, 79, 13070–13081.

    Article  CAS  Google Scholar 

  21. Silva, H. M., Vieira, P. M., Costa, P. L., Pimentel, B. M., Moro, A. M., Kalil, J., et al. (2009). Novel humanized anti-CD3 antibodies induce a predominantly immunoregulatory profile in human peripheral blood mononuclear cells. Immunology Letters, 125, 129–136.

    Article  CAS  Google Scholar 

  22. Quilici, L. S., Silva-Pereira, I., Andrade, A. C., Albuquerque, F. C., Brigido, M. M., & Maranhão, A. Q. (2013). A minimal cytomegalovirus intron A variant can improve transgene expression in different mammalian cell lines. Biotechnology Letters, 35, 21–27.

    Article  CAS  Google Scholar 

  23. Rothenburg, S., Koch-Nolte, F., Rich, A., & Haag, F. (2001). A polymorphic dinucleotide repeat in the rat nucleolin gene forms Z-DNA and inhibits promoter activity. PNAS, 98(16), 8985–8990.

    Article  CAS  Google Scholar 

  24. Hamada, H., Seidman, M., Howard, B. H., & Gorman, C. M. (1984). Enhanced gene expression by the Poly(dT-dG)-Poly(dC-dA) sequence. Molecular and Cell Biology, 4(12), 2622–2630.

    Article  CAS  Google Scholar 

  25. Champ, P. C., Maurice, S., Vargason, J. M., Camp, T., & Ho, P. S. (2004). Distributions of Z-DNA and nuclear factor I in human chromosome 22: a model for coupled transcriptional regulation. Nucleic Acids Research, 32, 6501–6510.

    Article  CAS  Google Scholar 

  26. Gulis, G., Simi, K. C., de Toledo, R. R., Maranhao, A. Q., & Brigido, M. M. (2014). Optimization of heterologous protein production in Chinese hamster ovary cells under overexpression of spliced form of human X-box binding protein. BMC Biotechnology, 14, 26.

    Article  Google Scholar 

  27. Jackson, D. A., Yuan, J., & Cook, P. R. (1988). A gentle method for preparing cyto- and nucleo-skeletons and associated chromatin. Journal of Cell Science, 90, 365–378.

    CAS  Google Scholar 

  28. Peck, L. J., Wang, J. C., Nordheim, A., & Rich, A. (1986). Rate of B to Z structural transition of supercoiled DNA. Journal of Molecular Biology, 190, 125–127.

    Article  CAS  Google Scholar 

  29. Schwartz, T., Behlke, J., Lowenhaupt, K., Heinemann, U., & Rich, A. (2001). Structure of the DLM-1-Z-DNA complex reveals a conserved family of Z-DNA-binding proteins. Natural Structural Biology, 8, 761–765.

    Article  CAS  Google Scholar 

  30. Takaoka, A., Wang, Z., Choi, M. K., Yanai, H., Negishi, H., Ban, T., et al. (2007). DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. Nature, 448, 501–505.

    Article  CAS  Google Scholar 

  31. Kang, H. J., Le, T. V., Kim, K., Hur, J., Kim, K. K., & Park, H. J. (2014). Novel interaction of the Z-DNA binding domain of human ADAR1 with the oncogenic c-Myc promoter G-quadruplex. Journal of Molecular Biology, 426, 2594–2604.

    Article  CAS  Google Scholar 

  32. Möller, A., Gabriels, J. E., Lafer, E. M., Nordheim, A., Rich, A., & Stollar, B. D. (1982). Monoclonal antibodies recognize different parts of Z-DNA. Journal of Biological Chemistry, 257, 12081–12085.

    Google Scholar 

  33. Wölfl, S., Wittig, B., Dorbic, T., & Rich, A. (1997). Identification of processes that influence negative supercoiling in the human c-myc gene. Biochimica et Biophysica Acta, 1352, 213–221.

    Article  Google Scholar 

  34. Vilela, H., Raiol, T., Maranhão, A. Q., Walter, M. E., & Brígido, M. M. (2012). A bioconductor based workflow for Z-DNA region detection and biological inference. In S. Istrail, P. Pevzner, M. Waterman, M. C. P. Souto, & M. G. Kann (Eds.), Advances in bioinformatics and computational biology (pp. 73–83). Berlin, Heidelberg: Springer.

    Chapter  Google Scholar 

Download references

Acknowledgments

In honor of Alexander Rich, who brought Z-DNA into reality, GG is a PNPD/CAPES fellow. ICRS, HRS, IGS and LSQ were CAPES fellows. MAGB is a fellow of FAP-DF.

Authors’ Contributions

GG, ICRS, AQM and MMB designed the study, interpreted the results and wrote the manuscript. GG, ICRS, HRS, IGS, MAGB and LSQ performed the experiments and interpreted the results. All authors read and approved the final manuscript.

Author information

Authors and Affiliations

  1. Department of Cell Biology, Institute of Biological Sciences, University of Brasilia, Brasilia, Brazil

    Galina Gulis, Andrea Queiroz Maranhao & Marcelo Macedo Brigido

  2. Molecular Pathology Graduation Program, Medicine Faculty, University of Brasilia, Brasilia, Brazil

    Izabel Cristina Rodrigues Silva, Herdson Renney Sousa & Isabel Garcia Sousa

  3. Faculty of Ceilandia, University of Brasilia, Brasilia, Brazil

    Izabel Cristina Rodrigues Silva

  4. Molecular Biology Graduation Program, Institute of Biological Sciences, University of Brasilia, Brasilia, Brazil

    Maryani Andressa Gomes Bezerra & Luana Salgado Quilici

Authors
  1. Galina Gulis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Izabel Cristina Rodrigues Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Herdson Renney Sousa
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Isabel Garcia Sousa
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Maryani Andressa Gomes Bezerra
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Luana Salgado Quilici
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Andrea Queiroz Maranhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Marcelo Macedo Brigido
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Marcelo Macedo Brigido.

Ethics declarations

Competing interests

The authors declare that they have no competing interests.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gulis, G., Silva, I.C.R., Sousa, H.R. et al. Characterization of an In Vivo Z-DNA Detection Probe Based on a Cell Nucleus Accumulating Intrabody. Mol Biotechnol 58, 585–594 (2016). https://doi.org/10.1007/s12033-016-9958-6

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-016-9958-6

Keywords

Search

Navigation