Summary
Chromatin immunoprecipitation has been widely used to determine the status of histone covalent modifications and also to investigate DNA-protein and protein-protein associations to a particular genomic location in vivo. Generally, DNA regulatory elements nucleate the interaction of several transcription factors in conjunction with ubiquitous and/or tissue-specific cofactors in order to regulate gene transcription. Therefore, it has become relevant to determine the cohabitation of several proteins in a particular developmental stage and cell type. Furthermore, multiple post-translational histone modifications can be analyzed on the same genomic location with the aim of deciphering the combinatorial pattern of histone modifications associated to specific transcriptional stages during cell commitment. Here we describe the ChIP–reChIP assay that represents a direct strategy to determine the in vivo colocalization of proteins interacting or in close contact in a chromatinized template on the basis of double and independent rounds of immunoprecipitations with high-quality ChIP grade antibodies.
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References
Hatzis, P. and Talinidis, I. (2002). Dynamics of enhancer-promoter communication during differentiation-induced gene activation. Mol. Cell 10, 1467–1477.
Rincón-Arano, H., Valadez-Graham, V., Guerrero, G., Escamilla-Del-Arenal, M. and Recillas-Targa, F. (2005). YY1 and GATA-1 interaction modulate the chicken 3’-side α-globin enhancer activity. J. Mol. Biol 349, 961–975.
Song, S.H., Hou, C. and Dean, A. (2007). A positive role for NLI/Ldb1 in long-range β-globin locus control region function. Mol. Cell. 28, 810–822.
Surani, M.A., Hayashi, K. and Hajkova, P. (2007). Genetic and epigenetic regulators of pluripotency. Cell 128, 747–762.
Spivakov, M. and Fisher, A.G. (2007) Epigenetic signatures of stem-cell identity. Nat. Rev. Genet 8, 263–271.
Misteli, T. (2007) Beyond the sequence: cellular organization of genome function. Cell 128, 787–800.
Schneider, R. and Grosschedl, R. (2007). Dynamics and interplay of nuclear architecture, genome organization and gene expression. Genes Dev 21, 3027–3043.
Bernstein, B.E., Mikkersen, T.S., Xie, X., Kamal, M., Huebert, D.J., Cuff, J., Fry, B., Meissner, A., Werning, M., Plath, K., Jaenisch, R., Wagschal, A., Feil, R., Schreiber, S.L. and Lander, E.S. (2006). A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125, 315–326.
Mikkersen, T.S., Ku, M., Jaffe, D.B., Issac, B., Lieberman, E., Giannoukos, G., Alvarez, P., Brockman, W., Kim, T.-K., Koche, R.P., Lee, W., Mendenhall, E., O’Donovan, A., Presser, A., Russ, C., Xie, X., Meissner, A., Werning, M., Jaenisch, R., Nusbaum, C., Lander, E.S. and Bernstein, B.E. (2007). Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448, 553–560.
Rincón-Arano, H., Furlan-Magaril, M. and Recillas-Targa, F. (2007). Protection against telomeric-position effects by the chicken cHS4 β-globin insulator. Proc. Natl. Acad. Sci. USA 104, 14044–14049.
Escamilla-Del-Arenal, M. and Recillas-Targa, F. (2008). GATA-1 modulates the chromatin structure and activity of the chicken α-globin enhancer. Mol. Cell. Biol 28, 575–586.
Dorsett, D. (1999). Distant liaisons: long range enhancer-promoter interaction in Drosophila. Curr. Opin. Genet. Dev. 9, 505–514.
Lee, T.I., Johstone, S.E. and Young, R.A. (2006). Chromatin immunoprecipitation and microarray-based analysis of protein location. Nat. Protocol 1, 729–748.
Euskirchen, G.M., Rozowsky, J.S., Wei, C.-L., Lee, W.H., Zhang, Z.D., Hartman, S., Emanuelsson, O., Stola, V., Weissman, S., Gerstein, M.B., Ruan, Y. and Snyder, M. (2007). Mapping of transcription factor binding regions in mammalian cells by ChIP: comparison of array- and sequencing-based technologies. Genome Res 17, 898–909.
Acknowledgments
We would like to thank Georgina Guerrero for her excellent technical assistance. This work was supported by grants from the Dirección General de Asuntos del Personal Académico-UNAM (IN209403 and IN214407), Consejo Nacional de Ciencia y Tecnología, CONACyT (42653-Q and 58767), and the Third World Academy of Sciences (TWAS, Grant 01–055 RG/BIO/LA). MF-M and HR-A were fellowship recipients from CONACyT.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Furlan-Magaril, M., Rincón-Arano, H., Recillas-Targa, F. (2009). Sequential Chromatin Immunoprecipitation Protocol: ChIP-reChIP. In: Leblanc, B., Moss, T. (eds) DNA-Protein Interactions. Methods in Molecular Biology™, vol 543. Humana Press. https://doi.org/10.1007/978-1-60327-015-1_17
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DOI: https://doi.org/10.1007/978-1-60327-015-1_17
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