Use of In Vivo Biotinylation for Chromatin Immunoprecipitation

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

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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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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Materials

Basic Protocol 1: Expression of Biotinylated Proteins in Mammalian Cells In Vivo and Its Application to Chromatin Immunoprecipitation (ChIP)

Materials

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 CaCl₂ (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

Materials

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

Materials

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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Figures

Figure 17.12.1 General principles of chromatin immunoprecipitation (ChIP) and in vivo biotinylation. (A ) General scheme of ChIP. The proteins are cross‐linked to DNA in vivo, the chromatin is fragmented, and the chromatin fragments with the protein of interest are affinity‐purified. The cross‐links are reversed, and the DNA is usually analyzed by PCR, DNA arrays, or high‐throughput sequencing. (B ) Epitope tagging by in vivo biotinylation. Two recombinant constructs are co‐expressed in the cells: BAP‐X, comprising the protein of interest X fused to a minimal biotin acceptor peptide (BAP, sequence shown), and bacterial biotin ligase BirA, specifically transferring the biotin moiety on the lysine residue of BAP. The reaction requires the presence of ATP and biotin in the cells.

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Figure 17.12.2 Streptavidin gel‐shift. Immunoblot analysis with anti‐GFP antibody of extracts from the HEK 293 cells expressing N‐terminal BAP fusion of GFP. Streptavidin was added before SDS‐PAGE in lanes 3 and 6 (+). Left panel: both BAP‐GFP and BirA were expressed from the same mRNA ( cis ‐biotinylation). Right panel: BAP‐GFP and BirA were expressed from two different plasmids that were cotransfected into HEK 293 cells ( trans ‐biotinylation). The positions of GFP and its streptavidin‐shifted version are indicated by arrows. Abbreviations: GFP, green‐fluorescent protein; NS, nonspecific signal corresponding to endogenously biotinylated proteins; UT, extract from untransfected or untransduced cells.

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Figure 17.12.3 Use of biotinylated proteins is compatible with cross‐linking by formaldehyde. Since biotin possesses two amino groups, it can react with formaldehyde, thus losing its affinity to streptavidin. To test whether formaldehyde treatment impairs the biotin‐streptavidin interaction, biotinylated bovine serum albumin (BSA) was incubated in cell culture medium and treated with 1% formaldehyde for 10 to 60 min. The effect of the treatment on the ability of streptavidin to recognize biotinylated BSA was monitored by immunoblotting using streptavidin‐conjugated peroxidase as a detection reagent. No effect of the formaldehyde treatment on the biotin‐streptavidin interaction was observed, based on the intensity of the chemiluminescent signal, even for long incubations. Thus, the biotin tag is compatible with formaldehyde cross‐linking. Abbreviation: NT, not treated.

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Figure 17.12.4 Use of stringent washing conditions in ChIP experiments significantly improves specificity (signal‐to‐noise ratio). (A ) Silver stain of biotinylated protein expressed in HEK 293 cells, separated by SDS‐PAGE and washed with either standard buffer or 2% SDS. (B ) Interaction of histone H2A.Z with the total genome of NIH3T3 cells. NIH3T3 cells were transiently transfected with biotinylated H2A.Z‐ or eGFP‐expressing plasmids, and biotin ChIP was performed. Quantitative PCR analysis was performed using genomic DNA present in the immunoprecipitates from the cells. Primers B1 5′: gcc ggg tgt ggt ggc gca cac ctt t and B1 3′: gag aca ggg ttt ctc tgt gta gcc ct were used to amplify short, abundant (∼106 copies per genome), randomly distributed, repetitive B1 sequences, allowing for very sensitive detection of mouse genomic DNA in the sample. Shown are results representative of four completely separate experiments. A comparison of the ratio between amounts of DNA pulled down from an H2A.Z‐transfected sample and an eGFP‐transfected sample (nonspecific background) shows that with the more stringent washing conditions possible with the biotin‐ChIP method, the signal‐to‐noise ratio is significantly increased and the specificity of interaction detection is improved.

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Figure 17.12.5 DNA shearing by sonication. Typical results after electrophoresis (1% agarose gel) of DNA sheared by sonication in a chromatin immunoprecipitation experiment. Left: DNA ladder, Right: sonicated DNA.

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Figure 17.12.6 Typical result from a biotin‐chromatin immunoprecipitation (biotin‐ChIP) experiment analyzed by qPCR. (A ) Interaction of transcription factor DP1 with its cognate DHFR promoter. The cells were transiently transfected with pBBHN.DP1 vector. Shown is the ratio between amounts of DNA pulled down from BAP‐DP1 transfected sample and BAP‐eGFP transfected sample, considered as a nonspecific background (mean of three experiments, standard deviations indicated by error bars). The sequences of primers for qPCR analysis are: DHFR 5′: gcg gag cct tag ctg cac aa, DHFR 3′: tac cag cct tca cgc tag ga. (B ) DP1 interaction with irrelevant GAPDH (left) and β‐actin (right) promoters. Immunoprecipitate from the same experiment as in (A) was tested with the GAPDH‐ and β‐actin‐specific primers. The sequences are: β‐actin 5′: acc gag cgt ggc tac agc tt, b‐actin 3′: tgg ccg tca ggc agc tca ta, GAPDH 5′: cca atg tgt ccg tcg tgg atc t, and GAPDH 3′: gtt gaa gtc gca gga gac acc.

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Figure 17.12.7 Amplification of complex mixtures of DNA fragments by isothermal multiple strand displacement amplification (IMDA.) (A ) The principle of concatemer‐mediated multiple displacement amplification. (1) Religation of DNA fragments with T4 DNA ligase leads to two types of products: (2) linear concatemers and (3) circular concatemers. Annealing of random hexamer primers and addition of phi29‐DNA polymerase leads to concatemer‐mediated multiple strand displacement amplification from (4) linear and (5) circular concatemers respectively. (B ) The principle of stuffer DNA. (1) In the case of much‐diluted samples, religation preferentially leads to self‐circularization of fragments, which are then amplified as individual molecules. (2) The addition of stuffer DNA (derived from an evolutionarily distant source; indicated by heavy lines) to the sample DNA favors intermolecular ligation and leads to the formation of long concatemers, both linear and circular. These long concatemers can then be reliably amplified using the IMDA technique. The resulting DNA can be analyzed by PCR and DNA microarrays; however, high‐throughput sequencing will produce mostly irrelevant information due to the excess of foreign DNA in the amplified mixture.

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Videos

Literature Cited

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Use of In Vivo Biotinylation for Chromatin Immunoprecipitation

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited