Abstract
Most methods currently available for the analysis of chromatin in vivo rely on a priori knowledge of putative chromatin components or their posttranslational modification state. The isolation of defined native chromosomal regions provides an attractive alternative to obtain a largely unbiased molecular description of chromatin. Here, we describe a strategy combining site-specific recombination at the chromosome with an efficient tandem affinity purification protocol to isolate a single-copy gene locus from the yeast Saccharomyces cerevisiae. The method allows robust enrichment of a targeted chromatin domain, making it amenable to compositional, structural, and biochemical analyses. This technique appears to be suitable to obtain a detailed description of chromatin composition and specific posttranslational histone modification state at virtually any genomic locus in yeast.
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References
Higashinakagawa T, Wahn H, Reeder RH (1977) Isolation of ribosomal gene chromatin. Dev Biol 55:375–386
Zhang XY, Hörz W (1982) Analysis of highly purified satellite DNA containing chromatin from the mouse. Nucleic Acids Res 10: 1481–1494
Workman JL, Langmore JP (1985) Nucleoprotein hybridization: a method for isolating specific genes as high molecular weight chromatin. Biochemistry 24:7486–7497
Boffa LC, Carpaneto EM, Allfrey VG (1995) Isolation of active genes containing CAG repeats by DNA strand invasion by a peptide nucleic acid. Proc Natl Acad Sci U S A 92: 1901–1905
Griesenbeck J, Boeger H, Strattan JS et al (2003) Affinity purification of specific chromatin segments from chromosomal loci in yeast. Mol Cell Biol 23:9275–9282
Simpson RT, Ducker CE, Diller JD et al (2004) Purification of native, defined chromatin segments. Methods Enzymol 375:158–170
Ghirlando R, Felsenfeld G (2008) Hydrodynamic studies on defined heterochromatin fragments support a 30-nm fiber having six nucleosomes per turn. J Mol Biol 376:1417–1425
Déjardin J, Kingston RE (2009) Purification of proteins associated with specific genomic loci. Cell 136:175–186
Unnikrishnan A, Gafken PR, Tsukiyama T (2010) Dynamic changes in histone acetylation regulate origins of DNA replication. Nat Struct Mol Biol 17:430–437
Antão JM, Mason JM, Déjardin J et al (2012) Protein landscape at Drosophila melanogaster telomere-associated sequence repeats. Mol Cell Biol 32:2170–2182
Byrum SD, Raman A, Taverna SD et al (2012) ChAP-MS: a method for identification of proteins and histone posttranslational modifications at a single genomic locus. Cell Rep 2: 198–205
Unnikrishnan A, Akiyoshi B, Biggins S et al (2012) An efficient purification system for native minichromosome from Saccharomyces cerevisiae. Methods Mol Biol 833:115–123
Rigaut G, Shevchenko A, Rutz B et al (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17: 1030–1032
Puig O, Caspary F, Rigaut G et al (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24:218–229
Brown CR, Mao C, Falkovskaia E et al (2013) Linking stochastic fluctuations in chromatin structure and gene expression, PLoS biology 11, e1001621
Hamperl S, Brown C, Perez-Fernandez J et al (in revision) Compositional and structural analysis of selected chromosomal domains from S. cerevisiae
Oeffinger M, Wei KE, Rogers R et al (2007) Comprehensive analysis of diverse ribonucleoprotein complexes. Nat Methods 4:951–956
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354
Mumberg D, Müller R, Funk M (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156:119–122
Flick JS, Johnston M (1990) Two systems of glucose repression of the GAL1 promoter in Saccharomyces cerevisiae. Mol Cell Biol 10:4757–4769
Johnston M (1987) A model fungal gene regulatory mechanism: the GAL genes of Saccharomyces cerevisiae. Microbiol Rev 51: 458–476
Acknowledgements
This work was supported by a grant to H.T, P.M, and J.G. in the context of the SFB960 DFG research center. We thank all the members of the Institute of Biochemistry III for their constant support.
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Hamperl, S. et al. (2014). Purification of Specific Chromatin Domains from Single-Copy Gene Loci in Saccharomyces cerevisiae . In: Stockert, J., Espada, J., Blázquez-Castro, A. (eds) Functional Analysis of DNA and Chromatin. Methods in Molecular Biology, vol 1094. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-706-8_26
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DOI: https://doi.org/10.1007/978-1-62703-706-8_26
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Publisher Name: Humana Press, Totowa, NJ
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