Abstract
Gene transcription is a complex process that involves a large number of proteins. These proteins can be brought to their target genes by a variety of different mechanisms: many transcription factors interact with specific DNA sequences in promoters or enhancers, several epigenetic regulators bind histones bearing specific modifications, elongation factors and some RNA processing factors bind to the transcribing RNA polymerase, and other factors interact directly with nascent transcripts or noncoding RNA. Immunostaining of Drosophila polytene chromosomes allows the genome-wide localization of factors involved at different stages of transcriptional regulation. In this chapter, we present protocols that adapt the general technique to probe different recruitment mechanisms employed by these factors, including specific interactions with phosphorylated RNA polymerase II and RNA-mediated chromatin associations.
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Acknowledgments
We are grateful to R. Renkawitz and H. Saumweber and Fred Winston for the gift of antibodies and to R. Renkawitz-Pohl for access to microscopes. M.M. was supported by a fellowship from the International Research Training Group 1384. Work in A.B.’s lab is supported by the Deutsche Forschungsgemeinschaft.
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Murawska, M., Brehm, A. (2012). Immunostaining of Drosophila Polytene Chromosomes to Investigate Recruitment of Chromatin-Binding Proteins. In: Vancura, A. (eds) Transcriptional Regulation. Methods in Molecular Biology, vol 809. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-376-9_18
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DOI: https://doi.org/10.1007/978-1-61779-376-9_18
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