Use of In Vivo Biotinylation for Chromatin Immunoprecipitation

Arman Kulyyassov1, Muhammad Shoaib2, Vasily Ogryzko2

1 National Center for Biotechnology of the Republic of Kazakhstan, Astana, Republic of Kazakhstan, 2 CNRS, Université Paris, Villejuif, France
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 17.12
DOI:  10.1002/0471143030.cb1712s51
Online Posting Date:  June, 2011
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This unit describes a system for expression of biotinylated proteins in mammalian cells in vivo, and its application to chromatin immunoprecipitation (ChIP). The system is based on co‐expression of the target protein fused to a short biotin acceptor domain, together with the biotinylating enzyme BirA from Escherichia coli. The superior strength of the biotin‐avidin interaction in the modified ChIP protocol presented here allows one to employ more stringent washing conditions, resulting in a better signal/noise ratio. Methods for interpreting the data obtained from ChIP samples analyzed by qPCR, and methods for testing the efficiency of biotinylation using a streptavidin gel‐shift are also presented. In addition, a complementary method, based on isothermal multiple strand displacement amplification (IMDA) of circular concatemers generated from the DNA fragments obtained after ChIP, is described. This method helps to decrease bias in DNA amplification and is useful for the analysis of complex mixtures of DNA fragments typically generated in miniscale ChIP experiments. Curr. Protoc. Cell Biol. 51:17.12.1‐17.12.22. © 2011 by John Wiley & Sons, Inc.

Keywords: chromatin immunoprecipitation; biotinylation in vivo; BirA; IMDA; amplification bias

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Expression of Biotinylated Proteins in Mammalian Cells In Vivo and Its Application to Chromatin Immunoprecipitation (ChIP)
  • Support Protocol 1: Analysis and Data Interpretation by qPCR
  • Support Protocol 2: Testing the Efficiency of Biotinylation using a Streptavidin Gel‐Shift
  • Basic Protocol 2: Unbiased Amplification of ChIP Sample by Isothermal Multiple Strand Displacement Amplification (IMDA)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Expression of Biotinylated Proteins in Mammalian Cells In Vivo and Its Application to Chromatin Immunoprecipitation (ChIP)

  • Human embryonic kidney (HEK) 293 cells (ATCC)
  • Dulbecco's modified Eagle's medium ( appendix 2A)/10% (v/v) fetal bovine serum (DMEM‐10)
  • 2 M CaCl 2 (store up to 6 months At –20°C)
  • Plasmid DNA with expression system for target protein (see ) and controls (plasmid expressing BAP‐GFP; Viens et al., ), checked for biotinylation efficiency ( protocol 3)
  • 2×HeBS ( appendix 2A)
  • Biotin (Sigma cat. no. B4639)
  • 37% (v/v) formaldehyde stock solution (Electron Microscopy Services)
  • 1.25 M glycine stock (store up to 3 months at room temperature)
  • Phosphate‐buffered saline (PBS, appendix 2A), ice‐cold
  • 25× protease inhibitor stock (see recipe)
  • PBS + protease inhibitors, ice cold: dilute 25× protease inhibitor stock (see recipe) 1:1000 with PBS just before use
  • 1% (w/v) agarose gel (see Voytas, )
  • ChIP Buffer (see recipe)
  • 70% (v/v) ethanol
  • 5 M and 300 mM NaCl
  • 10 mg/ml RNase A (e.g., Invitrogen)
  • Streptavidin‐coupled magnetic beads (Dynabeads M‐280 Streptavidin; Invitrogen)
  • Washing buffer 1: 2% (w/v) SDS; store up to 2 months at room temperature
  • Washing buffer 2: 10 mM Tris⋅Cl, pH 8 (see appendix 2A)/1 mM EDTA (see appendix 2A)/0.25 mM LiCl/1% (w/v) Nonidet P‐40/1% (v/v)/sodium deoxycholic acid; store up to several months at 4°C
  • Washing buffer 3: 20 mM Tris⋅Cl, pH 7.6 (see appendix 2A)/50 mM NaCl/1 mM EDTA (see appendix 2A); store up to 2 months at room temperature
  • 20 mg/ml proteinase K (Roche Applied Science) in 10 mM Tris⋅Cl, pH 7.5 (see appendix 2A)/20 mM calcium chloride/5% (v/v) glycerol
  • 5× proteinase K buffer: 50 mM Tris⋅Cl, pH 7.5 (see appendix 2A)/25 mM EDTA (see appendix 2A)/1.25% (w/v) SDS
  • 3 M sodium acetate (NaOAc), pH 5 to 5.2
  • DNA purification kit for PCR (e.g., Qiagen): includes MiniElute columns, PBI buffer, PE buffer, and EB buffer
  • 1.5‐ml microcentrifuge tubes
  • 14‐ml round‐bottom tube (Falcon 2059, or equivalent).
  • 15‐ml conical polystyrene tubes (Falcon)
  • Sonicator, with appropriate tube holder and accessories (e.g., Diagenode)
  • Magnetic separator for 1.5‐ml tubes (e.g., Dynal MPC, Invitrogen)
  • Test‐tube rotators, at room temperature and in a cold room
  • 1.5‐ml screw‐cap tubes (VWR)
  • 67°C heating block or thermal cycler
  • Additional reagents and equipment for culturing mammalian cells (unit 1.1) performing agarose gel electrophoresis (Voytas, )

Support Protocol 1: Analysis and Data Interpretation by qPCR

  • 5 × 106 HEK 293 cells expressing the biotinylated protein of interest (see protocol 1, steps 1 to 5) grown in one well of a 6‐well tissue culture plate
  • Phosphate‐buffered saline (PBS, appendix 2A)
  • CSK buffer (see recipe)
  • 1× NuPAGE LDS sample buffer (Invitrogen)
  • 5 mg/ml streptavidin (Sigma)
  • 4 to 12% gradient Novex Tris⋅glycine precast gels (Invitrogen)
  • Antibody for detection of the protein of interest (e.g., anti‐GFP)
  • 1.5‐ml microcentrifuge tube
  • Sonicator (e.g., Diagenode), with appropriate tube holder and accessories
  • Heating block set at 96°C
  • Additional reagents and equipment for performing immunoblot analysis (unit 6.2)

Support Protocol 2: Testing the Efficiency of Biotinylation using a Streptavidin Gel‐Shift

  • ChIP DNA sample ( protocol 1)
  • ∼500‐bp fragments of sonicated E. coli DNA, optional (see protocol 1, step 18)
  • Takara Mighty Cloning Kit, blunt end (Takara), including
    • 10× Blunting Kination Buffer
    • Blunting Kination Enzyme Mix
    • Ligation Mighty Mix
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 3 M sodium acetate
  • 20 mg/ml glycogen
  • 70% (v/v) and 100% ethanol, cold
  • TE (Tris/EDTA) buffer ( appendix 2A)
  • 5 U/µl Ø29‐DNA polymerase: dilute 1000 U/µl Ø29‐DNA polymerase (Epicentre Biotechnologies) 1:200 with the supplied buffer and store up to 3 months at −20°C
  • 2× IMDA reaction buffer (see recipe)
  • 200 µM random hexamer primers (NEB)
  • Mineral oil, biotechnology grade
  • NanoDrop spectrophotometer (NanoDrop)
  • 1.5‐ml microcentrifuge tubes
  • 16°C, 30°C, 37°C, 65°C, and 70°C cooling/heating blocks or thermal cycler
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Literature Cited

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