Using Chromatin Immunoprecipitation in Toxicology: A Step‐by‐Step Guide to Increasing Efficiency, Reducing Variability, and Expanding Applications

Shaun D. McCullough1, Doan M. On2, Emma C. Bowers3

1 National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, North Carolina, 2 Department of Physiology and Biophysics, Medical College of Virginia, Richmond, 3 Curriculum in Toxicology, University of North Carolina–Chapel Hill, Chapel Hill
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 3.14
DOI:  10.1002/cptx.22
Online Posting Date:  May, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Histone modifications work in concert with DNA methylation to regulate cellular structure, function, and response to environmental stimuli. More than 130 unique histone modifications have been described to date, and chromatin immunoprecipitation (ChIP) allows for the exploration of their associations with the regulatory regions of target genes and other DNA/chromatin‚Äźassociated proteins across the genome. Many variations of ChIP have been developed in the 30 years since its earliest version came into use, which makes it challenging for users to integrate the procedure into their research programs. Furthermore, the differences in ChIP protocols can confound efforts to increase reproducibility across studies. The streamlined ChIP procedure presented here can be readily applied to samples from a wide range of in vitro studies (cell lines and primary cells) and clinical samples (peripheral leukocytes) in toxicology. We also provide detailed guidance on the optimization of critical protocol parameters, such as chromatin fixation, fragmentation, and immunoprecipitation, to increase efficiency and improve reproducibility. Expanding toxicoepigenetic studies to more readily include histone modifications will facilitate a more comprehensive understanding of the role of the epigenome in environmental exposure effects and the integration of epigenetic data in mechanistic toxicology, adverse outcome pathways, and risk assessment. ¬© 2017 by John Wiley & Sons, Inc.

Keywords: chromatin; epigenome; histone; immunoprecipitation; leukocytes; transcription factors

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Quantifying the Abundance of Epigenetic Modifications Within the Regulatory Regions of Target Genes
  • Support Protocol 1: Determine Optimal Sonication Conditions for ChIP
  • Support Protocol 2: Isolation of Leukocytes From Peripheral Whole Blood
  • Support Protocol 3: TaqMan Quantitative PCR of ChIP Samples
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Quantifying the Abundance of Epigenetic Modifications Within the Regulatory Regions of Target Genes

  Materials
  • Cells in culture
  • Dulbecco's phosphate buffered saline (DPBS; Life Technologies, cat. no. 14190‐144), room temperature
  • 0.25% trypsin (Life Technologies, cat. no. 25200‐056; appendix 3B; Phelan & May, )
  • DPBS‐TI: Dulbecco's phosphate‐buffered saline (DPBS; Life Technologies, cat. no. 14190‐144) containing 30 mg/ml soybean trypsin inhibitor (see recipe for 30× soybean trypsin inhibitor)
  • 16% paraformaldehyde (in single‐use ampules; Electron Microscopy Sciences, cat. no. 15710)
  • 2.5 M glycine (see recipe)
  • Protease inhibitor solution (PIS; see recipe), ice cold
  • ChIP lysis buffer (see recipe)
  • ChIP dilution buffer (see recipe)
  • ChIP‐grade antibodies (see Table 3.14.1 for antibodies that we have used successfully with this protocol)
  • Protein A/G polyacrylamide beads (Thermo, cat. no. 53133)
  • Low salt wash buffer (see recipe)
  • High salt wash buffer (see recipe)
  • LiCl wash buffer (see recipe)
  • TE buffer (see recipe)
  • SDS elution buffer (see recipe)
  • Elution addition buffer (see recipe)
  • 10 mg/ml proteinase K (see recipe)
  • 25:24:1 (v/v/v) phenol:chloroform:isoamyl alcohol (Life Technologies, cat. no. 15593‐031)
  • 1:1 (v/v) chloroform:isoamyl alcohol
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • 10 mg/ml glycogen (see recipe)
  • 100% and 70% ethanol
  • Liquid nitrogen
  • Refrigerated centrifuge
  • 70‐μm cell strainer (Corning Falcon, cat. no. 352350)
  • Hemacytometer (see appendix 3B; Phelan & May, )
  • Sonicator with microprobe tip (Fisher Scientific Sonic Dismembranator Model 500)
  • Microcentrifuge tubes with snap‐lock caps
  • 15‐ml conical polypropylene tubes (e.g., Corning Falcon)
  • Vacuum aspirator attached to 25‐G needle
  • End‐over‐end rotator
  • Digital temperature controlled dry bath
  • Additional reagents and equipment for basic cell culture techniques including trypsinization and counting cells ( appendix 3B; Phelan & May, )
Table 3.4.1   MaterialsAntibodies Successfully Used in ChIP as Described in the protocol 1Basic Protocol

ChIP target Mono/polyclonal Antibody manufacturer Product no.
H2BK5ac Polyclonal Active Motif 39123
H2BK120ub Monoclonal Active Motif 39623
H2BK120ub Monoclonal Millipore 17‐650
Total H3 Polyclonal Active Motif 39163
Total H3 Monoclonal Active Motif 61475
H3K4me1 Polyclonal Active Motif 61633
H3K4me3 Polyclonal Active Motif 39915
H3K4me3 Monoclonal Active Motif 61379
H3K9ac Monoclonal Active Motif 61251
H3K27me3 Monoclonal Active Motif 61017
H3K27me2/3 Polyclonal Active Motif 39535
H3K27ac Polyclonal Active Motif 39133
H3K27ac Monoclonal Active Motif 39685
H3K79me1 Polyclonal Active Motif 39921
H4ac Polyclonal Active Motif 39925
H4K20me1 Monoclonal Active Motif 39727
5‐hmC Monoclonal Active Motif 39999
RNA PolII Monoclonal Active Motif 39097
RNA PolII Monoclonal Millipore 17‐620
RNA PolII phosphor‐Ser5 Polyclonal Active Motif 39749

Support Protocol 1: Determine Optimal Sonication Conditions for ChIP

  Materials
  • Fixed chromatin ( protocol 1Basic Protocol)
  • SDS elution buffer (see recipe)
  • Elution addition buffer (see recipe)
  • 10 mg/ml proteinase K (see recipe)
  • 25:24:1 (v:v:v) phenol:chloroform:isoamyl alcohol (Life Technologies, cat. no. 15593‐031)
  • 1:1 (v:v) chloroform:isoamyl alcohol
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • 20 mg/ml glycogen (see recipe)
  • 100% ethanol
  • TE buffer (see recipe)
  • 20 mg/ml RNase A (Life Technologies, cat. no. 12091‐021)
  • 3% agarose/TBE gel (see recipe; also see Voytas, )
  • 10 mg/ml ethidium bromide (see recipe)
  • 6× purple gel loading dye (NEB, cat. no. B7025S; also see Voytas, )
  • 100 bp DNA ladder (NEB, cat. no. N3231L)
  • TBE buffer (see recipe)
  • Sonicator with microprobe tip (Fisher Scientific Sonic Dismembranator Model 500)
  • Digital temperature controlled dry bath
  • Vacuum aspirator attached to 25‐G needle
  • Ultraviolet light box
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, )

Support Protocol 2: Isolation of Leukocytes From Peripheral Whole Blood

  Materials
  • Whole blood sample in EDTA or sodium citrate as anticoagulant
  • ACK lysis buffer (see recipe)
  • Cell resuspension solution (see recipe)
  • 15‐ml conical centrifuge tubes (e.g., Corning Falcon)
  • Hemacytometer (see appendix 3B; Phelan & May, )
  • Refrigerated centrifuge
  • Additional reagents and equipment for counting cells ( appendix 3B; Phelan & May, )

Support Protocol 3: TaqMan Quantitative PCR of ChIP Samples

  Materials
  • One aliquot of undiluted genomic DNA (gDNA) to use for the preparation of a standard curve; can be input material from another ChIP or generated from protocol 2
  • Nuclease‐free water
  • iTaq Universal Probes 2× Master Mix (BioRad, cat. no. 172‐5134)
  • Primers and probes resuspended at 100 μM in 10 mM Tris⋅Cl, pH 8.0/0.1 mM EDTA and used to prepare working stocks at 6.0 μM (primers) and 6.8 μM (probes)
  • Samples
  • 96‐well PCR plate and optical film compatible with available real‐time qPCR apparatus
  • Tabletop centrifuge with plate adapter
  • Quantitative PCR machine
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Baccarelli, A., & Bollati, V. (2009). Epigenetics and environmental chemicals. Current Opinion in Pediatrics, 21, 243–251. doi: 10.1097/MOP.0b013e32832925cc.
  Brutlag, D., Schlehuber, C., & Bonner, J. (1969). Properties of formaldehyde‐treated nucleohistone. Biochemistry, 8, 3214–3218. doi: 10.1021/bi00836a013.
  Burris, H. H., & Baccarelli, A. (2014). Environmental epigenetics: From novelty to scientific discipline. Journal of Applied Toxicology, 34, 113–116. doi: 10.1002/jat.2904.
  Carey, M. F., Peterson, C. L., & Smale, S. T. (2009). Chromatin immunoprecipitation (ChIP). Cold Spring Harbor Protocols, 2009 Sep;2009(9):pdb.prot5279. doi: 10.1101/pdb.prot5279.
  Cedar, H., & Bergman, Y. (2009). Linking DNA methylation and histone modification: Patterns and paradigms. Nature Reviews. Genetics, 10, 295–304. doi: 10.1038/nrg2540.
  Dahl, J. A., & Collas, P. (2008). A rapid micro chromatin immunoprecipitation assay (μChIP). Nature Protocols, 3, 1032–1045. doi: 10.1038/nprot.2008.68.
  Gilmour, D. S., & Lis, J. T. (1984). Detecting protein‐DNA interactions in vivo: Distribution of RNA polymerase in specific bacterial genes. Proceedings of the National Academy of Sciences of the United States of America, 81, 4275–4279. doi: 10.1073/pnas.81.14.4275.
  Gilmour, D. S., & Lis, J. T. (1985). In vivo interactions of RNA polymerase II with genes of Drosophila melanogaster. Molecular and Cell Biology of Human Diseases Series, 5, 2009–2018. doi: 10.1128/MCB.5.8.2009.
  Gilmour, D. S., & Lis, J. T. (1986). RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells. Molecular and Cell Biology of Human Diseases Series, 6, 3984–3989. doi: 10.1128/MCB.6.11.3984.
  Hecht, A., Strahl‐Bolsinger, S., & Grunstein, M. (1996). Spreading of transcriptional repressor SIR3 from telomeric heterochromatin. Nature, 383, 92–96. doi: 10.1038/383092a0.
  Hoffman, E. A., Frey, B. L., Smith, L. M., & Auble, D. T. (2015). Formaldehyde crosslinking: A tool for the study of chromatin complexes. The Journal of Biological Chemistry, 290, 26404–26411. doi: 10.1074/jbc.R115.651679.
  Ilyin, Y. V., & Georgiev, G. P. (1969). Heterogeneity of deoxynucleoprotein particles as evidenced by ultracentrifugation in cesium chloride density gradient. Journal of Molecular Biology, 41, 299–303. doi: 10.1016/0022‐2836(69)90395‐7.
  Jackson, V. (1978). Studies on histone organization in the nucleosome using formaldehyde as a reversible cross‐linking agent. Cell, 15, 945–954. doi: 10.1016/0092‐8674(78)90278‐7.
  Jackson, V., & Chalkley, R. (1981). A new method for the isolation of replicative chromatin: Selective deposition of histone on both new and old DNA. Cell, 23, 121–134. doi: 10.1016/0092‐8674(81)90277‐4.
  Jenuwein, T., & Allis, C. D. (2001). Translating the histone code. Science, 293, 1074–1080. doi: 10.1126/science.1063127.
  Kouzarides, T. (2007). Chromatin modifications and their function. Cell, 128, 693–705. doi: 10.1016/j.cell.2007.02.005.
  Phelan, K. and May, K. M. (2016). Basic techniques in mammalian cell tissue culture. Current Protocols in Toxicology, 70, A.3B.1‐A.3B.22. doi: 10.1002/cptx.13.
  Roadmap Epigenomics Consortium, Kundaje, A., Meuleman, W., Ernst, J., Bilenky, M., Yen, A., Heravi Moussavi, A., … Kellis, M. (2015). Integrative analysis of 111 reference human epigenomes. Nature, 518, 317–330. doi: 10.1038/nature14248.
  McCullough, S. D., Bowers, E. C., On, D. M., Morgan, D. S., Dailey, L. A., Hines, R. N., … Diaz‐Sanchez, D. (2016). Baseline chromatin modification levels may predict interindividual variability in ozone‐induced gene expression. The Journal of Toxicological Sciences, 150, 216–224. doi: 10.1093/toxsci/kfv324.
  Milne, T. A., Zhao, K., & Hess, J. L. (2009). Chromatin immunoprecipitation (ChIP) for analysis of histone modifications and chromatin‐associated proteins. Methods in Molecular Biology, 538, 409–423. doi: 10.1007/978‐1‐59745‐418‐6_21.
  O'Neill, L. P., & Turner, B. M. (2003). Immunoprecipitation of native chromatin: NChIP. Methods, 31, 76–82. doi: 10.1016/S1046‐2023(03)00090‐2.
  Solomon, M. J., Larsen, P. L., & Varshavsky, A. (1988). Mapping protein‐DNA interactions in vivo with formaldehyde: Evidence that histone H4 is retained on a highly transcribed gene. Cell, 53, 937–947. doi: 10.1016/S0092‐8674(88)90469‐2.
  Solomon, M. J., & Varshavsky, A. (1985). Formaldehyde‐mediated DNA‐protein crosslinking: A probe for in vivo chromatin structures. Proceedings of the National Academy of Sciences of the United States of America, 82, 6470–6474. doi: 10.1073/pnas.82.19.6470.
  Strahl, B. D., & Allis, C. D. (2000). The language of covalent histone modifications. Nature, 403, 41–45. doi: 10.1038/47412.
  Tan, M., Luo, H., Lee, S., Jin, F., Yang, J. S., Montellier, E., … Zhao, Y. (2011). Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification. Cell, 146, 1016–1028. doi: 10.1016/j.cell.2011.08.008.
  Van Lente, F., Jackson, J. F., & Weintraub, H. (1975). Identification of specific crosslinked histones after treatment of chromatin with formaldehyde, Cell, 5, 45–50. doi: 10.1016/0092‐8674(75)90090‐2.
  Voytas, D., (2000). Agarose gel electrophoresis. Current Protocols in Molecular Biology, 51, 2.5A.1‐2.5A.9. doi: 10.1002/0471142727.mb0205as51.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library