Genome‐Wide Location Analysis by Pull Down of In Vivo Biotinylated Transcription Factors

Aibin He1, William T. Pu1

1 Harvard Stem Cell Institute, Harvard University, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.20
DOI:  10.1002/0471142727.mb2120s92
Online Posting Date:  October, 2010
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Abstract

Recent development of methods for genome‐wide identification of transcription factor binding sites by chromatin immunoprecipitation (ChIP) has led to novel insights into transcriptional regulation and greater understanding of the function of individual transcription factors. ChIP requires highly specific antibody against the transcriptional regulator of interest, and availability of suitable antibodies is a significant impediment to broader application of this approach. This limitation can be circumvented by tagging the transcriptional regulator of interest with a short bio epitope which is specifically biotinylated by the E. coli enzyme BirA. The biotinylated transcription factor can then be selectively pulled down on streptavidin beads under stringent conditions. This unit provides a detailed protocol for genome‐wide location analysis of in vivo biotinylated transcription factors by streptavidin pull‐down followed by high‐throughput sequencing (bioChIP‐seq). Curr. Protoc. Mol. Biol. 92:21.20.1‐21.20.15. © 2010 by John Wiley & Sons, Inc.

Keywords: ChIP‐seq; transcription factor; biotinylation; genome‐wide location analysis

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Genome‐Wide Location Analysis of In Vivo Biotinylated Transcription Factors by Streptavidin Pull‐Down Followed by High‐Throughput Sequencing (bioChIP‐seq)
  • Support Protocol 1: Optimization of Sonication Conditions for bioChIP‐seq
  • Support Protocol 2: Conversion of bioChIP‐seq DNA to a Library for Sequencing on the Illumina Genome Analyzer 2
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Genome‐Wide Location Analysis of In Vivo Biotinylated Transcription Factors by Streptavidin Pull‐Down Followed by High‐Throughput Sequencing (bioChIP‐seq)

  Materials
  • Cell type of interest, grown under appropriate cell culture conditions in appropriate medium, and expressing BirA and the bio tagged transcription factor (see Strategic Planning)
  • Negative control: cell type of interest expressing BirA only
  • 37% formaldehyde (Fisher Scientific, cat. no. F79‐500)
  • 2.5 M glycine (American Bioanalytic, cat. no. AB00730)
  • Phosphate‐buffered saline (PBS; appendix 22), cold
  • Hypotonic buffer (see recipe)
  • ChIP dilution buffer (see recipe)
  • TE Buffer (see recipe)
  • PBS ( appendix 22) containing 1% (w/v) BSA
  • Protein A magnetic beads (Invitrogen)
  • M‐280 streptavidin magnetic beads (Invitrogen)
  • SDS wash buffer (see recipe)
  • High‐salt buffer (see recipe)
  • LiCl buffer (see recipe)
  • SDS ChIP elution buffer (see recipe)
  • 20 mg/ml proteinase K, DNase‐free
  • 10 mg/ml RNase A, DNase‐free
  • QIAquick PCR purification kit (Qiagen)
  • Quant‐It PicoGreen dsDNA DNA reagent (Invitrogen), for qPCR (see unit 15.8)
  • 15‐cm tissue culture dishes
  • Cell culture incubator
  • Cell lifter
  • 15‐ and 50‐ml conical centrifuge tubes
  • Tissue culture centrifuge
  • Glass Dounce homogenizer (2‐ml size, Fisher, cat. no. K885300)
  • 1.7‐ml microcentrifuge tubes, prechilled
  • Refrigerated microcentrifuge
  • Misonix Sonicator 4000 with microtip (part no. 418, Qsonica, http://nano.sonicator.com/)
  • Nanodrop spectrophotometer
  • Siliconized nonstick microcentrifuge tubes, precooled
  • Magnetic stand (Ambion, cat. no. am10055)
  • 37°C, 55°C, and 70°C water baths
  • Applied Biosystems 7500 Real‐Time PCR system or equivalent (also see unit 15.8)
  • Additional reagents and equipment for optimizing sonication conditions for bioChIP‐seq ( protocol 2) and qPCR (unit 15.8)

Support Protocol 1: Optimization of Sonication Conditions for bioChIP‐seq

  Materials
  • bioChIP DNA samples (from protocol 1)
  • 10× T4 DNA ligase buffer (from Illumina kit) containing 10 mM ATP
  • QIAquick PCR Purification Kit (Qiagen)
  • ChIP‐seq library preparation kit (Illumina) including:
    • Adapter oligo mix
    • T4 DNA ligase
    • Klenow DNA polymerase
    • T4 polynucleotide kinase (PNK)
    • 1 mM dATP
    • T4 DNA ligase buffer
    • 5× Phusion buffer
    • 10 mM dNTP mix
    • Klenow buffer
    • Klenow exo (3′ to 5′ exo minus )
    • Illumina PCR primers 1.1
    • Illumina PCR primers 2.1
    • Phusion DNA polymerase
  • Agarose
  • 1× TAE buffer ( appendix 22)
  • Ethidium bromide (EtBr)
  • 10× DNA loading buffer (unit 2.5)
  • 80% (v/v) glycerol
  • 100‐bp DNA ladder (New England Biolabs)
  • QIAquick Gel Extraction Kit (Qiagen)
  • MinElute PCR purification kit (Qiagen)
  • Ethidium bromide
  • Siliconized microcentrifuge tubes
  • Thermal cycler
  • Long‐wavelength UV transilluminator or Dark Reader (Clare Chemical)
  • Razor blades
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5)
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Figures

Videos

Literature Cited

   Beckett, D., Kovaleva, E., and Schatz, P.J. 1999. A minimal peptide substrate in biotin holoenzyme synthetase‐catalyzed biotinylation. Protein Sci. 8:921‐929.
   Blahnik, K.R., Dou, L., O'Geen, H., McPhillips, T., Xu, X., Cao, A.R., Iyengar, S., Nicolet, C.M., Ludascher, 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.
   de Boer, E., Rodriguez, P., Bonte, E., Krijgsveld, J., Katsantoni, E., Heck, A., Grosveld, F., and Strouboulis, J. 2003. Efficient biotinylation and single‐step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 100:7480‐7485.
   Farnham, P.J. 2009. Insights from genomic profiling of transcription factors. Nat. Rev. Genet. 10:605‐616.
   Ji, H., Jiang, H., Ma, W., Johnson, D.S., Myers, R.M., and Wong, W.H. 2008. An integrated software system for analyzing ChIP‐chip and ChIP‐seq data. Nat. Biotechnol. 26:1293‐1300.
   Kim, J., Chu, J., Shen, X., Wang, J., and Orkin, S.H. 2008. An extended transcriptional network for pluripotency of embryonic stem cells. Cell 132:1049‐1061.
   Park, P.J. 2009. ChIP‐seq: Advantages and challenges of a maturing technology. Nat. Rev. Genet. 10:669‐680.
   Pepke, S., Wold, B., and Mortazavi, A. 2009. Computation for ChIP‐seq and RNA‐seq studies. Nat. Methods 6:S22‐S32.
Key References
   de Boer et al., 2003. See above.
  Application of the bio epitope tag for one step purification of mammalian proteins.
   Blahnik et al., 2010. See above.
  Describes a bioinformatic tool for read alignment, peak calling, and peak annotation.
   Kim et al., 2008. See above.
  Application of in vivo transcription factor biotinylation to genome‐wide location analysis by bioChIP‐chip.
Internet Resources
  http://chipseq.genomecenter.ucdavis.edu/cgi‐bin/chipseq.cgi
  Sole‐Search: Web‐based tool for read alignment, peak calling, and peak annotation.
  http://frodo.wi.mit.edu/primer3/
  Primer3: Web‐based tool for designing PCR primers.
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