Analysis of the RNA helicase p68 (Ddx5) as a transcriptional regulator

Samantha M. Nicol, Frances V. Fuller-Pace

    Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)

    12 Citations (Scopus)

    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.
    Original languageEnglish
    Title of host publicationHelicases
    Subtitle of host publicationmethods and protocols
    EditorsMohamed M. Abdelhaleem
    PublisherHumana Press
    Pages265-279
    Number of pages15
    ISBN (Electronic)9781603273558
    ISBN (Print)9781603273541
    DOIs
    Publication statusPublished - 2010

    Publication series

    NameMethods in Molecular Biology
    PublisherHumana Press
    Volume587
    ISSN (Print)1064-3745

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