Abstract
The DEAD box RNA helicase p68 (Ddx5) has been demonstrated to act as a transcriptional co-activator for a number of highly regulated transcription factors (e.g. estrogen receptor alpha and the tumour suppressor p53) and to be recruited to promoters of genes that are responsive to activation of these transcription factors, suggesting that it may play a role in transcription initiation. We have investigated the function of p68 as a co-activator of the tumour suppressor p53, with a particular emphasis on the importance of p68 in the induction of p53 transcriptional activity by DNA damage. These studies have involved RNAi-mediated suppression of p68 in cells expressing wild-type p53 and determining its effect on the expression of cellular p53 target genes in response to DNA damage. Additionally a significant amount of our research has focused on the study of the role of p68 in transcriptional initiation; this has included an investigation of the recruitment of p68 to the promoters of p53-responsive genes and of the importance of p68 in influencing recruitment of p53. Here we present detailed methods for RNAi knock-down of p68 expression, determination of its effect on expression of p53-responsive genes by quantitative RT-PCR and Western blotting, and chromatin immunoprecipitation techniques for determining recruitment of p68 and p53 to p53-responsive promoters.
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References
Fuller-Pace F. V. (2006) DExD/H box RNA helicases: multifunctional proteins with important roles in transcriptional regulation. Nucleic Acids Res. 34, 4206–4215.
Endoh H., Maruyama K., Masuhiro Y., Kobayashi Y., Goto M., Tai H., Yanagisawa J., Metzger D., Hashimoto S. and Kato S. (1999) Purification and identification of p68 RNA helicase acting as a transcriptional coactivator specific for the activation function 1 of human estrogen receptor alpha. Mol. Cell Biol. 19, 5363–5372.
Clark E. L., Coulson A., Dalgliesh C., Rajan P., Nicol S. M, Fleming S., Heer R., Gaughan L., Leung H. Y., Elliott D. J. et al. (2008) The RNA helicase p68 is a novel androgen receptor coactivator involved in splicing and is overexpressed in prostate cancer. Cancer Res. 68, 7938–7946.
Caretti G., Schiltz R. L., Dilworth F. J., Di Padova M., Zhao P., Ogryzko V., Fuller-Pace F. V., Hoffman E. P., Tapscott S. J. and Sartorelli V. (2006) The RNA helicases p68/p72 and the noncoding RNA SRA are coregulators of MyoD and skeletal muscle differentiation. Dev. Cell 11, 547–560.
Bates G. J., Nicol S. M, Wilson B. J., Jacobs A. M., Bourdon J. C., Wardrop J., Gregory D. J., Lane D. P, Perkins N. D and Fuller-Pace F. V. (2005) The DEAD box protein p68: a novel transcriptional coactivator of the p53 tumour suppressor. EMBO J. 24, 543–553.
Metivier R., Penot G., Hubner M. R., Reid G., Brand H., Kos M. and Gannon F. (2003) Estrogen receptor-alpha directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell 115, 751–763.
Wilson B. J., Bates G. J., Nicol S. M., Gregory D. J., Perkins N. D. and Fuller-Pace F. V. (2004) The p68 and p72 DEAD box RNA helicases interact with HDAC1 and repress transcription in a promoter-specific manner. BMC Mol. Biol. 5, 11.
Rossow K. L. and Janknecht R. (2003) Synergism between p68 RNA helicase and the transcriptional coactivators CBP and p300. Oncogene 22, 151–156.
Espinosa J. M., Verdun R. E. and Emerson B. M. (2003) p53 functions through stress- and promoter-specific recruitment of transcription initiation components before and after DNA damage. Mol. Cell 12, 1015–1027.
Kaeser M. D. and Iggo R. D. (2002) Chromatin immunoprecipitation analysis fails to support the latency model for regulation of p53 DNA binding activity in vivo. Proc. Natl. Acad. Sci. U.S.A. 99, 95–100.
Donner A. J., Szostek S., Hoover J. M. and Espinosa J. M. (2007) CDK8 is a stimulus-specific positive coregulator of p53 target genes. Mol. Cell 27, 121–133.
Acknowledgements
This work is supported by the Association for International Cancer Research (06-613).
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Nicol, S.M., Fuller-Pace, F.V. (2009). Analysis of the RNA Helicase p68 (Ddx5) as a Transcriptional Regulator. In: Abdelhaleem, M. (eds) Helicases. Methods in Molecular Biology, vol 587. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-355-8_19
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DOI: https://doi.org/10.1007/978-1-60327-355-8_19
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