In Vitro Histone Acetylation Assay

James A.L. Brown1

1 Discipline of Surgery, Lambe Institute for Translational Research, School of Medicine, National University of Ireland, Galway
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 3.14
DOI:  10.1002/cpph.31
Online Posting Date:  December, 2017
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Abstract

Acetylation is a core cellular process involved in maintaining genomic integrity, gene regulation, and metabolism. Histone acetyltransferases (HATs) are an enzyme family that regulates these processes by catalyzing the transfer of an acetyl moiety onto target proteins. Perturbations of cellular acetylation profiles have been associated with a variety of disease states, including cancer. Changes in acetylation profiles can be achieved by mechanisms associated with acetyltransferases, such as gene down‐regulation or alterations in the activity of key acetyltransferase enzymes. An important set of tools for quantifying enzyme activity are in vitro histone acetylation assays, using either endogenous or tagged overexpressed proteins. Detailed in this unit is an in vitro acetylation assay used to quantify HAT activity. © 2017 by John Wiley & Sons, Inc.

Keywords: acetylation; acetyltransferase; analysis; assay; histone; in vitro; quantitative; HAT

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Cell Lysis
  • Basic Protocol 2: Immunoprecipitation of Target Acetyltransferase
  • Alternate Protocol 1: Immunoprecipitation of Endogenous or Untagged Proteins
  • Basic Protocol 3: In Vitro Histone Acetylation Assay
  • Basic Protocol 4: Polyacrylamide Gel Separation of Proteins and Transfer to Nitrocellulose
  • Basic Protocol 5: Immunoblotting
  • Basic Protocol 6: Densitometry: Quantification of Immunoblot
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Cell Lysis

  Materials
  • Cultured cells grown in cell culture flask
  • Phosphate‐buffered saline (PBS; see Moore, )
  • 0.25% trypsin‐EDTA
  • Cell culture medium appropriate for cells of interest
  • Lysis buffer (see recipe)
  • 15‐ml conical tubes
  • Centrifuge, suitable for handling 15‐ml tubes
  • 1.5‐ml microcentrifuge tubes
  • Refrigerated microcentrifuge
  • Ice bucket and ice
  • Microtube rotator
  • Additional reagents and equipment for determination of protein concentration (see appendix 3A; Olson, )

Basic Protocol 2: Immunoprecipitation of Target Acetyltransferase

  Materials
  • Soluble protein extract (see protocol 1)
  • GFP‐agarose/G4 sepharose mix (see recipe)
  • Lysis buffer (see recipe)
  • Vortex
  • Microtube rotator
  • Refrigerated microcentrifuge
  • 1.5‐ml microcentrifuge tubes

Alternate Protocol 1: Immunoprecipitation of Endogenous or Untagged Proteins

  Materials
  • Soluble protein extract (see protocol 1)
  • Protein G beads/G4 sepharose mix (see recipe)
  • Antibody for protein of interest
  • Control antibody
  • Lysis buffer (see recipe)
  • Vortex
  • Microtube rotator
  • Refrigerated microcentrifuge
  • 1.5‐ml microcentrifuge tubes

Basic Protocol 3: In Vitro Histone Acetylation Assay

  Materials
  • Beads with bound target HAT protein (see protocol 2)
  • HAT assay buffer 1 (see recipe)
  • HAT assay buffer 2 (see recipe)
  • 2× SDS loading buffer (see recipe)
  • Heating block
  • Refrigerated microcentrifuge
  • Ice bucket and ice

Basic Protocol 4: Polyacrylamide Gel Separation of Proteins and Transfer to Nitrocellulose

  Materials
  • Protein samples containing SDS loading buffer (see protocol 4)
  • Polyacrylamide gels (pre‐cast or freshly prepared)
  • Tris‐glycine SDS‐PAGE running buffer (see recipe)
  • 1× transfer buffer (pre‐cooled to 4°C; see recipe)
  • Wet protein blotting apparatus (e.g., BioRad Mini Trans‐Blot system, cat. no. 170‐3930) containing:
    • Running apparatus
    • Running tank
    • Transfer cassette
  • 3‐mm‐thick blotting paper
  • Nitrocellulose membrane (e.g., Whatman Protran nitrocellulose membrane; Sigma‐Aldrich, cat no. Z613630)
  • Sponges
  • Scalpel
  • 10‐ml transfer pipette
  • Ice pack

Basic Protocol 5: Immunoblotting

  Materials
  • Nitrocellulose membrane with transferred proteins (see protocol 5)
  • Tris‐buffered saline containing Tween 20 (TBST; see recipe)
  • TBST containing bovine serum albumin (TBST/BSA; see recipe)
  • Antibodies:
    • Target‐specific primary antibody: Anti‐Tip60 (K17) (e.g., Santa Cruz Biotechnology) or anti‐GFP (e.g., Roche, cat. no. 11814460001)
    • HRP‐conjugated species‐specific secondary antibody (e.g., Jackson ImmunoResearch)
    • Nonspecific anti‐acetylation antibody or residue‐specific anti‐acetylation antibody (e.g., anti‐acetyl lysine, Millipore, cat. no. AB3879; H4K8, Millipore, cat. no 07‐328; H4K16, Millipore, cat. no. 07‐329)
    • Substrate‐specific primary antibody (e.g., H3, Abcam, cat. no. AB1791)
  • Blotto (see recipe)
  • Enhanced Chemiluminescence (ECL) kit (e.g., SuperSignal West Pico Chemiluminescent Substrate, Thermo Fisher Scientific)
  • Square petri dish (optimally: 120 mm l × 120 mm w × 17 mm h)
  • Blotting paper
  • Laboratory rocker or platform shaker (e.g., Stuart See‐saw rocker SSM4)
  • Polypropylene sheet protector
  • X‐ray film cassette
  • Tissue paper
  • X‐ray film (e.g., Kodak Scientific Imaging film)
  • Dark room
  • Developing solutions and trays

Basic Protocol 6: Densitometry: Quantification of Immunoblot

  Materials
  • X‐ray film–developed immunoblots
  • High‐resolution flatbed scanner
  • Immunoblot quantification software (e.g., ImageJ)
  • Statistical software package (e.g., GraphPad Prism)
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Figures

Videos

Literature Cited

 
  Brown, J. A. L., Bourke, E., Eriksson, L. A., & Kerin, M. J. (2016). Targeting cancer using KAT inhibitors to mimic lethal knockouts. Biochemical Society Transactions, 44, 979–986. doi: 10.1042/BST20160081.
  Coffey, K., Blackburn, T. J., Cook, S., Golding, B. T., Griffin, R. J., Hardcastle, I. R., … Robson, C. N. (2012). Characterisation of a Tip60 specific inhibitor, NU9056, in prostate cancer. PloS One, 7, e45539. doi: 10.1371/journal.pone.0045539.t001.
  Cole, P. A. (2008). Chemical probes for histone‐modifying enzymes. Nature Chemical Biology, 4, 590–597. doi: 10.1038/nchembio.111.
  Farria, A., Li, W., & Dent, S. Y. R. (2015). KATs in cancer: Functions and therapies. Oncogene, 34, 4901–4913. doi: 10.1038/onc.2014.453.
  Gao, C., Bourke, E., Scobie, M., Famme, M. A., Koolmeister, T., Helleday, T., … Brown, J. A. (2014). Rational design and validation of a Tip60 histone acetyltransferase inhibitor. Scientific Reports, 4, 1–10. doi: 10.1038/srep05372.
  Ghizzoni, M., Wu, J., Gao, T., Haisma, H. J., Dekker, F. J., & Zheng, Y. G. (2012). 6‐alkylsalicylates are selective Tip60 inhibitors and target the acetyl‐CoA binding site. European Journal of Medicinal Chemistry, 47, 337–344. doi: 10.1016/j.ejmech.2011.11.001.
  Halkidou, K., Gnanapragasam, V. J., Mehta, P. B., Logan, I. R., Brady, M. E., Cook, S., … Robson, C. N. (2003). Expression of Tip60, an androgen receptor coactivator, and its role in prostate cancer development. Oncogene, 22, 2466–2477. doi: 10.1038/sj.onc.1206342.
  Kaidi, A., & Jackson, S. P. (2013). KAT5 tyrosine phosphorylation couples chromatin sensing to ATM signalling. Nature, 498, 70–74. doi: 10.1038/nature12201.
  Kobayashi, J., Kato, A., Ota, Y., Ohba, R., & Komatsu, K. (2010). Bisbenzamidine derivative, pentamidine represses DNA damage response through inhibition of histone H2A acetylation. Molecular Cancer, 9, 34. doi: 10.1186/1476‐4598‐9‐34.
  Lau, O. D., Kundu, T. K., Soccio, R. E., Ait‐Si‐Ali, S., Khalil, E. M., Vassilev, A., … Cole, P. A. (2000). HATs off: Selective synthetic inhibitors of the histone acetyltransferases p300 and PCAF. Molecular Cell, 5, 589–595. doi: 10.1016/S1097‐2765(00)80452‐9.
  Marmorstein, R., & Trievel, R. C. (2009). Histone modifying enzymes: Structures, mechanisms, and specificities. Biochimica Et Biophysica Acta, 1789, 58–68. doi: 10.1016/j.bbagrm.2008.07.009.
  Moore, D.D. (2001). Commonly used reagents and equipment. Current Protocols in Molecular Biology, 35, A.2.1–A.2.9. doi: 10.1002/0471142727.mba02s35.
  Olson, B.J.S.C. (2016). Assays for determination of protein concentration. Current Protocols in Pharmacology, 73, A.3A.1–A.3A.32. doi: 10.1002/cpph.3.
  Roth, S. Y., Denu, J. M., & Allis, C. D. (2001). Histone acetyltransferases. Annual Review of Biochemistry, 70, 81–120. doi: 10.1146/annurev.biochem.70.1.81.
  Sapountzi, V., & Cote, J. (2011). MYST‐family histone acetyltransferases: Beyond chromatin. Cellular and Molecular Life Sciences, 68, 1147–1156. doi: 10.1007/s00018‐010‐0599‐9.
  Selvi, B. R., Chatterjee, S., Modak, R., Eswaramoorthy, M., & Kundu, T. K. (2013). Histone acetylation as a therapeutic target. Sub‐Cellular Biochemistry, 61, 567–596. doi: 10.1007/978‐94‐007‐4525‐4_25.
  Simon, R. P., Robaa, D., Alhalabi, Z., Sippl, W., & Jung, M. (2016). KATching‐up on small molecule modulators of lysine acetyltransferases. Journal of Medicinal Chemistry, 59, 1249–1270. doi: 10.1021/acs.jmedchem.5b01502.
  Sun, Y., Jiang, X., Chen, S., & Price, B. D. (2006). Inhibition of histone acetyltransferase activity by anacardic acid sensitizes tumor cells to ionizing radiation. FEBS Letters, 580, 4353–4356. doi: 10.1016/j.febslet.2006.06.092.
  Wu, J., Wang, J., Li, M., Yang, Y., Wang, B., & Zheng, Y. G. (2011). Small molecule inhibitors of histone acetyltransferase Tip60. Bioorganic Chemistry, 39, 53–58. doi: 10.1016/j.bioorg.2010.11.003.
  Yang, X.‐J. (2004). The diverse superfamily of lysine acetyltransferases and their roles in leukemia and other diseases. Nucleic Acids Research, 32, 959–976. doi: 10.1093/nar/gkh252.
Key Reference
  Gao et al. (2014). See above.
  The protocols in this unit are based on the HAT assays described in this article.
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