Abstract
The dynamic modification of DNA and histones plays a key role in transcriptional regulation through altering the packaging of DNA and modifying the nucleosome surface. These chromatin states, also referred to as the epigenome, are distinctive for different tissues, developmental stages, and disease states and can also be altered by environmental influences. New technologies allow the genome-wide visualization of the information encoded in the epigenome. For example, the chromatin immunoprecipitation (ChIP) assay allows investigators to characterize DNA–protein interactions in vivo. ChIP followed by hybridization to microarrays (ChIP-chip) or by high-throughput sequencing (ChIP-seq) are both powerful tools to identify genome-wide profiles of transcription factors, histone modifications, DNA methylation, and nucleosome positioning. ChIP-seq technology, which can now interrogate the entire human genome at high resolution with only one lane of sequencing, has recently surpassed ChIP-chip technology for epigenomic analyses. Importantly, for the study of primary cells and tissues, epigenetic profiles can be generated using as little as 1 μg of chromatin. In this chapter, we describe in detail the steps involved in performing ChIP assays (with a focus on characterizing histone modifications in primary cells) either manually or using the IP-Star ChIP robot, followed by a detailed protocol to prepare successful libraries for Illumina sequencing. Critical quality control checkpoints are discussed. Although not a focus of this chapter, we also point the reader to several methods by which massive ChIP-seq data sets can be analyzed to extract the tremendous information contained within.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Crews, D. Epigenetics, brain, behavior, and the environment, Hormones (Athens) 9, 41–50.
Grayson, D. R., Chen, Y., Dong, E., Kundakovic, M., and Guidotti, A. (2009) From trans-methylation to cytosine methylation: evolution of the methylation hypothesis of schizophrenia, Epigenetics 4, 144–149.
Iwamoto, K., and Kato, T. (2009) Epigenetic profiling in schizophrenia and major mental disorders, Neuropsychobiology 60, 5–11.
Jiang, Y., Langley, B., Lubin, F. D., Renthal, W., Wood, M. A., Yasui, D. H., Kumar, A., Nestler, E. J., Akbarian, S., and Beckel-Mitchener, A. C. (2008) Epigenetics in the nervous system, J Neurosci 28, 11753–11759.
McCarthy, M. M., Auger, A. P., Bale, T. L., De Vries, G. J., Dunn, G. A., Forger, N. G., Murray, E. K., Nugent, B. M., Schwarz, J. M., and Wilson, M. E. (2009) The epigenetics of sex differences in the brain, J Neurosci 29, 12815–12823.
Jones, P. A., and Baylin, S. B. (2007) The epigenomics of cancer, Cell 128, 683–692.
Ellis, L., Atadja, P. W., and Johnstone, R. W. (2009) Epigenetics in cancer: targeting chromatin modifications, Mol Cancer Ther 8, 1409–1420.
Lund, A. H., and van Lohuizen, M. (2004) Epigenetics and cancer, Genes Dev 18, 2315–2335.
Barski, A., Cuddapah, S., Cui, K., Roh, T. Y., Schones, D. E., Wang, Z., Wei, G., Chepelev, I., and Zhao, K. (2007) High-resolution profiling of histone methylations in the human genome, Cell 129, 823–837.
Kouzarides, T. (2007) Chromatin modifications and their function, Cell 128, 693–705.
Heintzman, N. D., Hon, G. C., Hawkins, R. D., Kheradpour, P., Stark, A., Harp, L. F., Ye, Z., Lee, L. K., Stuart, R. K., Ching, C. W., Ching, K. A., Antosiewicz-Bourget, J. E., Liu, H., Zhang, X., Green, R. D., Lobanenkov, V. V., Stewart, R., Thomson, J. A., Crawford, G. E., Kellis, M., and Ren, B. (2009) Histone modifications at human enhancers reflect global cell-type-specific gene expression, Nature 459, 108–112.
Heintzman, N. D., Stuart, R. K., Hon, G., Fu, Y., Ching, C. W., Hawkins, R. D., Barrera, L. O., Calcar, S. V., Qu, C., Ching, K. A., Wang, W., Weng, Z., Green, R. D., Crawford, G. E., and Ren, B. (2007) Distinct predictive chromatin signatures of transcriptional promoters and enhancers in the human genome, Nature Genetics published online Feb 4, 2007.
O’Geen, H., Squazzo, S. L., Iyengar, S., Blahnik, K., Rinn, J. L., Chang, H. Y., Green, R., and Farnham, P. J. (2007) Genome-Wide Analysis of KAP1 Binding Suggests Autoregulation of KRAB-ZNFs, PLoS Genet 3, e89.
Rinn, J. L., Kertesz, M., Wang, J. K., Squazzo, S. L., Xu, X., Brugmann, S. A., Goodnough, L. H., Helms, J. A., Farnham, P. J., Segal, E., and Chang, H. Y. (2007) Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs, Cell 129, 1311–1323.
Chen, L., and Daley, G. Q. (2008) Molecular basis of pluripotency, Hum Mol Genet 17, R23–27.
Hemberger, M., Dean, W., and Reik, W. (2009) Epigenetic dynamics of stem cells and cell lineage commitment: digging Waddington’s canal, Nat Rev Mol Cell Biol 10, 526–537.
Lanzuolo, C., and Orlando, V. (2007) The function of the epigenome in cell reprogramming, Cell Mol Life Sci 64, 1043–1062.
Mikkelsen, T. S., Ku, M., Jaffe, D. B., Issac, B., Lieberman, E., Giannoukos, G., Alverez, P., Brockman, W., Kim, T.-K., Koche, R. P., Lee, W., Mendenhall, E., O’Donovan, A., Presser, A., Russ, C., Xie, X., Meissner, A., Wernig, 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.
Park, P. J. (2009) ChIP–seq: advantages and challenges of a maturing technology, Nature Rev Genet, 669–680.
Wei, C. L., Wu, Q., Vega, V. B., Chiu, K. P., Ng, P., Zhang, T., Shahab, A., Yong, H. C., Fu, Y., Weng, Z., Liu, J., Zhao, X. D., Chew, J. L., Lee, Y. L., Kuznetsov, V. A., Sung, W. K., Miller, L. D., Lim, B., Liu, E. T., Yu, Q., Ng, H. H., and Ruan, Y. (2006) A global map of p53 transcription-factor binding sites in the human genome, Cell 124, 207–219.
Bhinge, A. A., Kim, J., Euskirchen, G. M., Snyder, M., and Iyer, V. R. (2007) Mapping the chromosomal targets of STAT1 by Sequence Tag Analysis of Genomic Enrichment (STAGE), Genome Res 17, 910–916.
Hawkins, R. D., Hon, G. C., Lee, L. K., Ngo, Q., Lister, R., Pelizzola, M., Edsall, L. E., Kuan, S., Luu, Y., Klugman, S., Antosiewicz-Bourget, J., Ye, Z., Espinoza, C., Agarwahl, S., Shen, L., Ruotti, V., Wang, W., Stewart, R., Thomson, J. A., Ecker, J. R., and Ren, B. (2010) Distinct epigenomic landscapes of pluripotent and lineage-committed human cells, Cell Stem Cell 6, 479–491.
Motallebipour, M., Ameur, A., Reddy Bysani, M. S., Patra, K., Wallerman, O., Mangion, J., Barker, M. A., McKernan, K. J., Komorowski, J., and Wadelius, C. (2009) Differential binding and co-binding pattern of FOXA1 and FOXA3 and their relation to H3K4me3 in HepG2 cells revealed by ChIP-seq, Genome Biol 10, R129.
Koerber, R. T., Rhee, H. S., Jiang, C., and Pugh, B. F. (2009) Interaction of transcriptional regulators with specific nucleosomes across the Saccharomyces genome, Mol Cell 35, 889–902.
Goren, A., Ozsolak, F., Shoresh, N., Ku, M., Adli, M., Hart, C., Gymrek, M., Zuk, O., Regev, A., Milos, P. M., and Bernstein, B. E. (2010) Chromatin profiling by directly sequencing small quantities of immunoprecipitated DNA, Nat Methods 7, 47–49.
Fejes, A. P., Robertson, G., Bilenky, M., Varhol, R., Bainbridge, M., and Jones, S. J. M. (2008) FindPeaks 3.1: a tool for identifying areas of enrichment from massively parallel short-read sequencing technology, Bioinformatics 24, 1729–1730.
Xu, H., Wei, C.-L., Lin, F., and Sung, W.-K. (2008) An HMM approach to genome-wide identification of differential histone modification sites from ChIP-seq data, Bioinformatics 24, 2344–2349.
Zhang, Y., Liu, T., Meyer, C. A., Eeckhoute, J., Johnson, D. S., Bernstein, B. E., Nussbaum, C., Myers, R. M., Brown, M., Li, W., and Liu, X. S. (2008) Model-based analysis of ChIP-Seq (MACS), Genome Biology 9, R137.
Johnson, D. S., Mortazavi, A., Myers, R. M., and Wold, B. (2007) Genome-wide mapping of in vivo protein-DNA interactions., Science 316, 1497–1502.
Robertson, G., Hirst, M., Bainbridge, M., Bilenky, M., Zhao, Y., Zeng, T., Euskirchen, G., Bernier, B., Varhol, R., Delaney, A., Thiessen, N., Griffith, O. L., He, A., Marra, M., Snyder, M., and Jones, S. (2007) Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing, Nat Methods 4, 1–7.
Jothi, R., Cuddapah, S., Barski, A., Cui, K., and Zhao, K. (2008) Genome-wide identification of in vivo protein-DNA binding sites from ChIP-seq data, Nucleic Acids Res 36, 5221–5231.
Rozowsky, J., Euskirchen, G., Auerbach, R. K., Zhang, Z. D., Gibson, T., Bjornson, R., Carriero, N., Snyder, M., and Gerstein, M. B. (2009) PeakSeq enables systematic scoring of ChIP-seq experiments relative to controls, Nat Biotechnol 27, 66–75.
Valouev, A., Johnson, D. S., Sundquist, A., Medina, C., Anton, E., Batzoglou, S., Myers, R. M., and Sidow, A. (2008) Genome-wide analysis of transcription factor binding sites based on ChIP-seq data, Nature Methods 5, 829–834.
Blahnik, K. R., Dou, L., O’Geen, H., McPhillips, T., Xu, X., Cao, A. R., Iyengar, S., Nicolet, C. M., Ludaescher, B., Korf, I., and Farnham, P. J. (2010) Sole-search: An integrated analysis program for peak detection and functional annotation using ChIP-seq data, Nucleic Acids Res 38, e13.
Lefrancois, P., Euskirchen, G. M., Auerbach, R. K., Rozowsky, J., Gibson, T., Yellman, C. M., Gerstein, M., and Snyder, M. (2009) Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing, BMC Genomics 10, 37.
Rozen, S., and Skaletsky, H. J. (2000) Primer3 on the WWW for general users and for biologist programmers, in Bioinformatics Methods and Protocols: Methods in Molecular Biology (Krawetz, S., and Misener, S., Eds.), pp 365–386, Humana Press, Totowa, NJ.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
O’Geen, H., Echipare, L., Farnham, P.J. (2011). Using ChIP-Seq Technology to Generate High-Resolution Profiles of Histone Modifications. In: Tollefsbol, T. (eds) Epigenetics Protocols. Methods in Molecular Biology, vol 791. Humana Press. https://doi.org/10.1007/978-1-61779-316-5_20
Download citation
DOI: https://doi.org/10.1007/978-1-61779-316-5_20
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-315-8
Online ISBN: 978-1-61779-316-5
eBook Packages: Springer Protocols