Abstract
Mammalian cell nuclei contain three RNA polymerases (RNAP I, RNAP II and RNAP III), which transcribe different gene subsets, and whose active forms are contained in supramolecular complexes known as 'transcription factories.' These complexes are difficult to isolate because they are embedded in the 3D structure of the nucleus. Factories exchange components with the soluble nucleoplasmic pool over time as gene expression programs change during development or disease. Analysis of their content can provide information on the nascent transcriptome and its regulators. Here we describe a protocol for the isolation of large factory fragments under isotonic salt concentrations in <72 h. It relies on DNase I–mediated detachment of chromatin from the nuclear substructure of freshly isolated, unfixed cells, followed by caspase treatment to release multi-megadalton factory complexes. These complexes retain transcriptional activity, and isolation of their contents is compatible with downstream analyses by mass spectrometry (MS) or RNA-sequencing (RNA-seq) to catalog the proteins and RNA associated with sites of active transcription.
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Acknowledgements
We thank B. Deng and J. Bartlett for their help, and the Sir William Dunn School of Pathology (SWDSOP) proteomics and BioQuant/DKFZ sequencing facilities for the high-throughput analyses. This work was supported by a Medical Research Council grant (to P.R.C.), by the ERASysBio+/FP7 initiative (to K.R. and P.R.C.), by a DKFZ intramural grant (to M.C.-H.) and by Center for Molecular Medicine core funding (to A.P.).
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S.M. and P.R.C. conceived and developed the factory isolation procedure. S.M., A.P., I.M.C. and P.R.C. implemented and validated the procedure. M.C.-H., L.B., K.R. and A.P. adapted and implemented the protocol for nascent RNA isolation. All authors analyzed data and wrote the manuscript.
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Melnik, S., Caudron-Herger, M., Brant, L. et al. Isolation of the protein and RNA content of active sites of transcription from mammalian cells. Nat Protoc 11, 553–565 (2016). https://doi.org/10.1038/nprot.2016.032
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DOI: https://doi.org/10.1038/nprot.2016.032
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