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Analysis of epigenetic modifications of chromatin at specific gene loci by native chromatin immunoprecipitation of nucleosomes isolated using hydroxyapatite chromatography

Nature Protocols volume 3, pages 398–409 (2008)Cite this article

Abstract

Chromatin immunoprecipitation (ChIP) is routinely used to examine epigenetic modification of histones at specific genomic locations. However, covalent modifications of histone tails can serve as docking sites for chromatin regulatory factors. As such, association of these regulatory factors with chromatin could cause steric hindrance for antibody recognition, resulting in an underestimation of the relative enrichment of a given histone modification at specific loci. To overcome this problem, we have developed a native ChIP protocol to study covalent modification of histones that takes advantage of hydroxyapatite (HAP) chromatography to wash away chromatin-associated proteins before the immunoprecipitation of nucleosomes. This fast and simple procedure consists of five steps: nuclei isolation from cultured cells; fragmentation of chromatin using MNase; purification of nucleosomes using HAP; immunoprecipitation of modified nucleosomes; and qPCR analysis of DNA associated with modified histones. Nucleosomes prepared in this manner are free of contaminating proteins and permit an accurate evaluation of relative abundance of different covalent histone modifications at specific genomic loci. Completion of this protocol requires ∼1.5 d.

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Figure 1
Figure 2: Analysis of loci representation in chromatin eluted from HAP column.
Figure 3: Analysis of nucleosomes isolated by hydroxyapatite chromatography.
Figure 4: Distribution of H3K4me2 and H3K4me3 across the Myog locus.

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Acknowledgements

This work was supported by grants from the Canadian Institutes of Health Research, Muscular Dystrophy Association and the Stem Cell Network (to F.J.D.); and Terry Fox Foundation/National Cancer Institute of Canada, and Human Frontiers Science Program (to M.B.). F.J.D. holds a Canadian Research Chair in Epigenetic Regulation of Transcription. M.B. holds a Canadian Research Chair in the Regulation of Gene Expression.

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  1. Sprott Center for Stem Cell Research, Regenerative Medicine Program, Ottawa Health Research Institute, 501 Smyth Rd, Mailbox 511, Ottawa, K1H 8L6, Ontario, Canada

    Marjorie Brand, Shravanti Rampalli, Chandra-Prakash Chaturvedi & F Jeffrey Dilworth

  2. Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, K1H 8L6, Ontario, Canada

    Marjorie Brand & F Jeffrey Dilworth

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  1. Marjorie Brand
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Correspondence to Marjorie Brand or F Jeffrey Dilworth.

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Brand, M., Rampalli, S., Chaturvedi, CP. et al. Analysis of epigenetic modifications of chromatin at specific gene loci by native chromatin immunoprecipitation of nucleosomes isolated using hydroxyapatite chromatography. Nat Protoc 3, 398–409 (2008). https://doi.org/10.1038/nprot.2008.8

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