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

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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

Figure 21.20.1 System for expression of biotinylated transcription factors. (A ) Two‐vector system for titratable expression of transcription factors with C‐terminal FLAG and bio peptides. BirA recognizes and biotinylates the bio sequence on the central lysine residue (asterisk). (B ) Titration of tagged transcription factor expression by adjusting the concentration of Dox. Tagged transcription factors are slightly larger than the corresponding endogenous protein due to the epitope tags.

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Figure 21.20.2 Optimization of sonication conditions. A 2‐ml quantity of nuclear extract was sonicated for 5 or 8 min using a Misonix 4000 with the amplitude of 70, cycles 15 sec on and 1 min off. The plot on the right shows size versus intensity profile of sonicated chromatin. A sonication time of 8 min led to the desired size peak of ∼200 bp.

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Figure 21.20.3 Gel size selection of fragmented chromatin and ChIP‐seq library.

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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.