Abstract
Chromatin immunoprecipitation (ChIP) is a powerful technique for examining transcription factor recruitment to chromatin, or histone modifications, at the level of specific genomic sequences. As such, it provides an invaluable tool for elucidating gene regulation at the molecular level. Combined with high-throughput methods such as second generation sequencing (ChIP-Seq), this technique is now commonly used for studying DNA–protein interactions at a genome-wide scale. The ChIP technique is based on covalent cross-linking of DNA and proteins with formaldehyde, followed by chromatin fragmentation, either enzymatic or by sonication, and immunoprecipitation of protein–DNA complexes using antibodies specific for the protein of interest. The immunoprecipitated DNA is then purified and the DNA sequences associated with the immunoprecipitated protein are identified by PCR (ChIP-PCR) or, alternatively, by direct sequencing (ChIP-Seq). Initially, the vast majority of ChIP experiments were performed on cultured cell lines. More recently, this technique has been adapted to a variety of tissues in different model organisms. We describe here a ChIP protocol on freshly isolated mouse embryonic kidneys for in vivo analysis of transcription factor recruitment on chromatin. This protocol has been easily adapted to other mouse embryonic tissues and has also been successfully scaled up to perform ChIP-Seq.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Cao Y, Yao Z, Sarkar D et al (2010) Genome-wide MyoD binding in skeletal muscle cells: a potential for broad cellular reprogramming. Dev Cell 18:662–674
Xu J, Watts JA, Pope SD et al (2009) Transcriptional competence and the active marking of tissue-specific enhancers by defined transcription factors in embryonic and induced pluripotent stem cells. Genes Dev 23:2824–2838
Collas P, Dahl JA (2008) Chop it, ChIP it, check it: the current status of chromatin immunoprecipitation. Front Biosci 13:929–943
Boyd KE, Farnham PJ (1999) Coexamination of site-specific transcription factor binding and promoter activity in living cells. Mol Cell Biol 19:8393–8399
Orlando V, Strutt H, Paro R (1997) Analysis of chromatin structure by in vivo formaldehyde cross-linking. Methods (San Diego, CA) 11:205–214
Lagha M, Kormish JD, Rocancourt D et al (2008) Pax3 regulation of FGF signaling affects the progression of embryonic progenitor cells into the myogenic program. Genes Dev 22:1828–1837
Parrizas M, Maestro MA, Boj SF et al (2001) Hepatic nuclear factor 1-alpha directs nucleosomal hyperacetylation to its tissue-specific transcriptional targets. Mol Cell Biol 21:3234–3243
Havis E, Anselme I, Schneider-Maunoury S (2006) Whole embryo chromatin immunoprecipitation protocol for the in vivo study of zebrafish development. BioTechniques 40:34, 36, 38 passim
Lokmane L, Heliot C, Garcia-Villalba P, Fabre M, Cereghini S (2010) vHNF1 functions in distinct regulatory circuits to control ureteric bud branching and early nephrogenesis. Development (Cambridge, UK) 137:347–357
Stock JK, Giadrossi S, Casanova M et al (2007) Ring1-mediated ubiquitination of H2A restrains poised RNA polymerase II at bivalent genes in mouse ES cells. Nat Cell Biol 9:1428–1435
Haring M, Offermann S, Danker T et al (2007) Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods 3:11
Verdeguer F, Le Corre S, Fischer E et al (2010) A mitotic transcriptional switch in polycystic kidney disease. Nat Med 16:106–110
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C(T)) method. Methods (San Diego, CA) 25:402–408
Acknowledgments
We thank I. Talianidis (Biomedical Sciences Research Center Al. Fleming, Greece.), K. Zaret (University of Pennsylvania School of Medicine, Philadelphia, USA), L. Sachs (UPMC CNRS UMR7221, Paris France), and E. Havis (CNRS UPMC UMR 7622, Paris, France) for their advice during optimization of the ChIP experiment. We thank S. Schneider-Maunoury and C. Haumaitre (UPMC, CNRS UMR 7622 and INSERM U969, Paris, France) for critical reading of the manuscript. This work was supported by Inserm, CNRS, Universite Pierre et Marie Curie and ARC (N° 3911) grants. C. H. is a recipient of a PhD student fellowship from Ministere de la Recherche et de la Technologie and from ARC.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Heliot, C., Cereghini, S. (2012). Analysis of In Vivo Transcription Factor Recruitment by Chromatin Immunoprecipitation of Mouse Embryonic Kidney. In: Michos, O. (eds) Kidney Development. Methods in Molecular Biology™, vol 886. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-851-1_25
Download citation
DOI: https://doi.org/10.1007/978-1-61779-851-1_25
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-850-4
Online ISBN: 978-1-61779-851-1
eBook Packages: Springer Protocols