Abstract
Cellular DNA is packaged into chromatin, which is the substrate of all DNA-dependent processes in eukaryotes. The regulation of chromatin requires specialized enzyme activities to allow the access of sequence-specific binding proteins and RNA polymerases. In order to dissect chromatin-dependent features of transcription regulation in detail, in vitro systems to generate defined chromatin templates for transcription are required. I present a protocol that allows the assembly of nucleosomes on ribosomal RNA (rRNA) minigenes by salt gradient dialysis and subsequent sucrose gradient centrifugation. This procedure yields high nucleosome occupancy and high dynamic response in subsequent transcriptional analysis. It provides an invaluable tool to study rRNA gene transcription, as transcription on free DNA is clearly different from the more in vivo-like transcription on reconstituted chromatin templates.
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References
Knezetic JA, Luse DS (1986) The presence of nucleosomes on a DNA template prevents initiation by RNA polymerase II in vitro. Cell 45:95–104
Lorch Y, LaPointe JW, Kornberg RD (1987) Nucleosomes inhibit the initiation of transcription but allow chain elongation with the displacement of histones. Cell 49:203–210
Almouzni G, Méchali M, Wolffe AP (1990) Competition between transcription complex assembly and chromatin assembly on replicating DNA. EMBO J 9:573–582
Längst G, Blank TA, Becker PB, Grummt I (1997) RNA polymerase I transcription on nucleosomal templates: the transcription termination factor TTF-I induces chromatin remodeling and relieves transcriptional repression. EMBO J 16:760–768. doi:10.1093/emboj/16.4.760
Längst G, Becker PB, Grummt I (1998) TTF-I determines the chromatin architecture of the active rDNA promoter. EMBO J 17:3135–3145. doi:10.1093/emboj/17.11.3135
Diermeier S, Grummt I, Németh A et al (2013) Chromatin-specific regulation of mammalian rDNA transcription by clustered TTF-I binding sites. PLoS Genet 9:e1003786. doi:10.1371/journal.pgen.1003786
Grummt I, Längst G (2013) Epigenetic control of RNA polymerase I transcription in mammalian cells. Biochim Biophys Acta 1829:393–404. doi:10.1016/j.bbagrm.2012.10.004
Oudet P, Gross-Bellard M, Chambon P (1975) Electron microscopic and biochemical evidence that chromatin structure is a repeating unit. Cell 4:281–300
Axel R, Melchior W, Sollner-Webb B, Felsenfeld G (1974) Specific sites of interaction between histones and DNA in chromatin. Proc Natl Acad Sci U S A 71:4101–4105
Patterton HG, von Holt C (1993) Negative supercoiling and nucleosome cores. I. The effect of negative supercoiling on the efficiency of nucleosome core formation in vitro. J Mol Biol 229:623–636. doi:10.1006/jmbi.1993.1068
Kaplan N, Moore IK, Moore IK et al (2009) The DNA-encoded nucleosome organization of a eukaryotic genome. Nature 458:362–366. doi:10.1038/nature07667
Simpson RT, Stafford DW (1983) Structural features of a phased nucleosome core particle. Proc Natl Acad Sci U S A 80:51–55
Neubauer B, Linxweiler W, Hörz W (1986) DNA engineering shows that nucleosome phasing on the African green monkey alpha-satellite is the result of multiple additive histone-DNA interactions. J Mol Biol 190:639–645
Felle M, Exler J, Merkl R et al (2010) DNA sequence encoded repression of rRNA gene transcription in chromatin. Nucleic Acids Res 38:5304–5314. doi:10.1093/nar/gkq263
Schroth GP, Siino JS, Cooney CA et al (1992) Intrinsically bent DNA flanks both sides of an RNA polymerase I transcription start site. Both regions display novel electrophoretic mobility. J Biol Chem 267:9958–9964
Marilley M, Pasero P (1996) Common DNA structural features exhibited by eukaryotic ribosomal gene promoters. Nucleic Acids Res 24:2204–2211
Becker PB, Wu C (1992) Cell-free system for assembly of transcriptionally repressed chromatin from Drosophila embryos. Mol Cell Biol 12:2241–2249
Lusser A, Urwin DL, Kadonaga JT (2005) Distinct activities of CHD1 and ACF in ATP-dependent chromatin assembly. Nat Struct Mol Biol 12:160–166. doi:10.1038/nsmb884
Pazin MJ, Bhargava P, Geiduschek EP, Kadonaga JT (1997) Nucleosome mobility and the maintenance of nucleosome positioning. Science 276:809–812
Strohner R, Németh A, Nightingale KP et al (2004) Recruitment of the nucleolar remodeling complex NoRC establishes ribosomal DNA silencing in chromatin. Mol Cell Biol 24:1791–1798. doi:10.1128/MCB.24.4.1791-1798.2004
Simon RH, Felsenfeld G (1979) A new procedure for purifying histone pairs H2A + H2B and H3 + H4 from chromatin using hydroxylapatite. Nucleic Acids Res 6:689–696
Luger K, Rechsteiner TJ, Richmond TJ (1999) Expression and purification of recombinant histones and nucleosome reconstitution. Methods Mol Biol 119:1–16. doi:10.1385/1-59259-681-9:1
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Work in our laboratory is funded by the German Research Community (DFG, grants within the SFB960)
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Längst, G. (2016). Preparation of Chromatin Templates to Study RNA Polymerase I Transcription In Vitro. In: Németh, A. (eds) The Nucleolus. Methods in Molecular Biology, vol 1455. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3792-9_9
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DOI: https://doi.org/10.1007/978-1-4939-3792-9_9
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