A General Non‐Radioactive ATPase Assay for Chromatin Remodeling Complexes

Benjamin Z. Stanton1, Courtney Hodges2, Gerald R. Crabtree3, Keji Zhao1

1 Systems Biology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, 2 Department of Pathology, Stanford University School of Medicine, Stanford, California, 3 Howard Hughes Medical Institute, Maryland
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.16
Online Posting Date:  March, 2017
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Abstract

Chromatin remodeling complexes couple the energy released from ATP hydrolysis to facilitate transcription, recombination, and repair mechanisms essential for a wide variety of biologic responses. While recombinant expression of the regulatory subunits of these enzymes is possible, measuring catalytic (ATPase) activity of the intact complexes recovered from normal or mutant cells is critical for understanding their mechanisms. SWI/SNF‐like remodeling complexes can be megadaltons in size and include many regulatory subunits, making reconstitution of purified subunits challenging for recapitulating in vivo function. The protocol in this article defines the first highly quantitative ATPase assay for intact remodeling complexes that does not require radiation or reconstitution of recombinantly expressed subunits. This protocol is specifically useful for defining the catalytic role of active‐site mutations in the context of other regulatory subunits and quantitatively rank‐ordering inactivating catalytic‐site mutations. © 2017 by John Wiley & Sons, Inc.

Keywords: chromatin remodeling; epigenetic regulators; ATPase activity; luciferase; catalysis

     
 
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Table of Contents

  • Basic Protocol 1: Introduction: The Need for a Quantitative Highly Reproducible ATPase Assay for Intact Chromatin Remodeling Complexes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Introduction: The Need for a Quantitative Highly Reproducible ATPase Assay for Intact Chromatin Remodeling Complexes

  Materials
  • 293T cells (ATCC, cat no. CRL‐11268)
  • Complete DMEM medium (see recipe)
  • DNA encoding desired chromatin remodeler (e.g., Brg [Mut] V5; Brg‐WT; BrgK785R; BrgR801H; available from the authors at )
  • Opti‐MEM medium (ThermoFisher, cat. no. 51985091)
  • 1 mg/ml PEI (see recipe)
  • Buffer A (see recipe)
  • Lysis buffer (see recipe)
  • Anti‐Brg (G7) antibody (Santa Cruz Biotechnology, cat. no. sc‐17796)
  • Protein A Dynabeads (Life Technologies, cat. no. 10001D)
  • Wash buffer (see recipe)
  • 1× ATPase assay reaction buffer (see recipe)
  • ADP‐Glo MAX assay kit (Promega, cat. no. V7001; see recipe for reconstituting components)
  • 10‐cm tissue culture dishes
  • Eppendorf Thermomixer (ThermoFisher, cat. no. 05‐400‐201)
  • End‐over‐end rotator
  • Magnetic stand (e.g., Miltenyi Biotec)
  • Opaque‐bottom non‐treated round‐bottom 96‐well plates (Corning. cat. no. CLS3789‐100EA)
  • EnVision Plate Reader (Perkin Elmer, cat. no. 2104‐0010)
  • Additional reagents and equipment for cell culture, including trypsinization and counting cells (Phelan, ) and immunoprecipitation (Bonifacino and Dell'Angelica, )
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Figures

Videos

Literature Cited

Literature Cited
  Bonifacino, J.S., Dell'Angelica, E.C., and Springer, T.A. 2001. Immunoprecipitation. Curr. Protoc. Immunol. 41:8.3.1‐8.3.28.
  Bultman, S.J., Gebuhr, T.C., and Magnuson, T. 2005. A Brg1 mutation that uncouples ATPase activity from chromatin remodeling reveals an essential role for SWI/SNF‐related complexes in beta‐globin expression and erythroid development. Genes Dev. 19:2849‐2861. doi: 10.1101/gad.1364105.
  Cote, J., Quinn, J., Workman, J.L., and Peterson, C.L. 1994. Stimulation of GAL4 derivative binding to nucleosomal DNA by the yeast SWI/SNF complex. Science 265:53‐60. doi: 10.1126/science.8016655.
  Dykhuizen, E.C., Hargreaves, D.C., Miller, E.L., Cui, K., Korshunov, A., Kool, M., Pfister, S., Cho, Y.J., Zhao, K., and Crabtree, G.R. 2013. BAF complexes facilitate decatenation of DNA by topoisomerase IIα. Nature 497:624‐627. doi: 10.1038/nature12146.
  Kadoch, C., Hargreaves, D.C., Hodges, C., Elias, L., Ho, L., Ranish, J., and Crabtree, G.R. 2013. Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy. Nat. Genet. 45:592‐601. doi: 10.1038/ng.2628.
  Kadoch, C., Williams, R.T., Calarco, J.P., Miller, E.L., Weber, C.M., Braun, S.M., Pulice, J.L., Chory, E.J., and Crabtree, G.R. 2016. Dynamics of BAF‐Polycomb complex opposition on heterochromatin in normal and oncogenic states. Nat. Genet. doi: 10.1038/ng.3734.
  Laurent, B.C., Treich, I., and Carlson, M. 1993. The yeast SNF2/SWI2 protein has DNA‐stimulated ATPase activity required for transcriptional activation. Genes Dev. 7:583‐591. doi: 10.1101/gad.7.4.583.
  Phelan, M. C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.18.
  Son, E.Y. and Crabtree, G.R. 2014. The role of BAF (mSWI/SNF) complexes in mammalian neural development. Am. J. Med. Genet. C Semin. Med. Genet. 166C:333‐349. doi: 10.1002/ajmg.c.31416.
  Stanton, B.Z., Hodges, C.H., Calarco, J.P., Braun, S.M.G., Ku, W.L., Kadoch, C., Zhao, K., and Crabtree, G.R. 2016. Smarca4 ATPase mutations disrupt direct eviction of PRC1 from chromatin. Nat. Genet. doi: 10.1038/ng.3735.
  Zhao, K., Wang, W., Rando, O.J., Xue, Y., Swiderek, K., Kuo, A., and Crabtree, G.R. 1998. Rapid and phosphoinositol‐dependent binding of the SWI/SNF‐like BAF complex to chromatin after T lymphocyte receptor signaling. Cell 95:625‐636. doi: 10.1016/S0092‐8674(00)81633‐5.
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