Use of protein biotinylation in vivo for chromatin immunoprecipitation

ANALYTICAL

Analytical Biochemistry 325 (2004) 68–76

BIOCHEMISTRY

www.elsevier.com/locate/yabio

Use of protein biotinylation in vivofor chromatin immunoprecipitation

Antoine Viens, Undine Mechold, Heike Lehrmann, Annick Harel-Bellan,and Vasily Ogryzko*

Laboratoire Oncog�ense, Diff�erenciation et Transduction du Signal, Institut Andr�e Lwoff, 7 rue Guy Moquet, 94800 Villejuif, France

Received 4 June 2003

Abstract

We describe a system designed to express biotinylated proteins in mammalian cells in vivo and its application to the study of

protein–DNA interactions in vivo by chromatin immunoprecipitation (ChIP). The system is based on coexpression 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 allows one to employ more stringent washing conditions in the ChIP protocol, resulting in a

better signal/noise ratio.

� 2003 Elsevier Inc. All rights reserved.

Keywords: Epitope tagging; Biotinylation; Chromatin immunoprecipitation; Protein–DNA interaction

Protein–DNA interactions play major roles in the

regulation of gene activity and other functions of the

genome. A number of methods, such as electropho-

retic gel mobility shift assay (EMSA)1 [1,2] and

others, have been developed to study different aspects

of protein–DNA interactions. However, in most ofthese approaches, the DNA–protein interaction is

studied in vitro, out of the context of chromatin and

the nuclear environment, which certainly limits their

value.

Chromatin immunoprecipitation (ChIP) [3,4] is a

method designed to analyze the protein–DNA interac-

tions in a more physiological context, as it detects them

directly in the living cell. In the past several years it has

* Corresponding author. Fax: +33-1-49-58-34-01.

E-mail address: [email protected] (V. Ogryzko).1 Abbreviations used: EMSA, electrophoretic gel mobility shelt

assay; ChIp chromatin immunoprecipitation; DMEM, Dulbecco�smodified Eagle�s medium; FBS, fetal bovine serum; FCS, fetal calf

serum; NP-40, Nonedet p-40; PBS, phosphate-buffered saline; GEP,

green fluorescent protein; FITC, fluorescein isothiocyanate; BAD,

biotin acceptor domain; ORF, open reading frame; IRES, internal

ribosome entrance sequence.

0003-2697/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.ab.2003.10.015

been widely used to study the in vivo association of a

particular DNA sequence with regulatory proteins and

posttranslationally modified histones. This method has

its own limitations. In particular, it requires antibodies

of a quality that is sufficient for a specific interaction and

at the same time can tolerate stringent binding andwashing conditions.

The interaction between biotin and streptavidin is one

of the strongest known noncovalent interactions [5].

Many commercially available reagents for purification

and detection of biotinylated macromolecules have been

developed. Correspondingly, we wanted to take advan-

tage of this interaction, in the particular application of

ChIP, as it would permit more stringent washing con-ditions than those usually utilized in ChIP experiments.

Here, we describe a system for expression of biotinylated

proteins of choice in mammalian cells in vivo. We

demonstrate that, in our system, the chromatin immu-

noprecipitation experiments can be performed under

conditions more stringent than usual, which significantly

improves the signal to noise ratio. We believe that ChIP

with in vivo biotinylated proteins could have manyapplications relevant for the study of protein–DNA

interactions in vivo.

A. Viens et al. / Analytical Biochemistry 325 (2004) 68–76 69

Materials and methods

Stable cell line generation and transient transfection

All cells were grown in Dulbecco�s modified

Eagle�s medium (DMEM, Gibco) with 10% fetal

bovine serum (FCS, PAN Biotech). For transient

transfection, a standard calcium phosphate precipita-

tion method was used, and the cells were analyzed 1or 2 days after transfection. In some experiments we

also used Polyfect (Qiagen). For retroviral transduc-

tion, the packaging cell line Phenix-E was transfected

with the plasmid DNA of the retroviral vector; 2

days later the retroviral supernatant was harvested,

filter-sterilized, and added to the HeLa or HEK 293

cells. Two days afterward, the transduced cells were

sorted using anti-IL2R antibody coupled to magneticaffinity beads [6].

Western analysis

Total cell extract for the analysis was prepared

using RIPA buffer (50mM Tris–HCl, pH 8.0, 50mM

NaCl, 1% NP-40, 0.5% Na-deoxyholate, 0.1% SDS,

1mM EDTA). First, cells were washed with phos-phate-buffered saline (PBS) and then incubated with

RIPA buffer directly in the six well plates for 5min.

The extract was harvested and spun at high speed on

a microcentrifuge. SDP–PAGE separation, transfer to

a nitrocellulose membrane, blocking, incubation with

antibody, and electrochemiluminescence detection

were performed according to a standard protocol,

except that for the detection of biotinylated proteins,500mM NaCl was added to the washing buffer

(PBS+0.1% Tween). The anti-GFP antibodies were

purchased from Clontech, the HP1 antibodies were

from Euromedex, and the streptavidin–HPO conju-

gate was from Sigma.

Immunofluorescence

NIH3T3 cells were grown and transfected with

Polyfect on coverslips. The day before fixation, 0.2%

biotin was added to the medium. Fixation was ac-

complished with 4% formaldehyde in PBS for 15min at4 �C. Fixed cells were permeabilized with 0.5% Triton

for 15min, blocked with 1% BSA and 1% FBS, and

incubated with anti-HP1c antibodies. They were then

washed with PBS and incubated with secondary anti-

body conjugated to rhodamine. For detection of bio-

tinylated proteins, the cells were incubated with

streptavidin–FITC for 1 h at 37 �C, and then washed

with PBS. The cells were observed using a fluorescencemicroscope, and images were acquired using a charge-

coupled device camera.

Chromatin immunoprecipitation

NIH 3T3 cells from a six-well plate well or a 10-cm

dish at 50–70% confluency were transfected with Poly-

fect (Qiagen). The next day the cells were split (to keep

them asynchronous) and put into DMEM with 10%

FBS and 0.2mg/L biotin. The next day this medium was

removed and replaced by fresh DMEM, 10% FBS

without biotin. Formaldehyde (Sigma, Cat. No. F-8775)was added to a final concentration of 1%. Cross-linking

was performed at 37 �C for 10min and stopped by ad-

dition of glycine to a final concentration of 0.125M,

followed by 5-min incubation at room temperature.

Fixed cells were washed twice with cold PBS and har-

vested in cold PBS containing protease inhibitors. Cells

were pelleted by centrifugation, suspended in 1mL of

SDS buffer (50mM Tris at pH 8.1, 0.5% SDS, 100mMNaCl, 5mM EDTA and protease inhibitors), and in-

cubated for 10min on ice. Cells were then pelleted by

centrifugation, resuspended in 400 lL of IP buffer (0.3%

SDS, 1.1% Triton X-100, 1.2mM EDTA, 16.7mM Tris

at pH 8.1, 167mM NaCl, and protease inhibitors), and

disrupted by sonication, yielding genomic DNA frag-

ments of a size of 100–500 bp. For each immunopre-

cipitation, 50 lg of chromatin was diluted to a finalvolume of 300 lL in IP buffer. Chromatin was precle-

ared for 1 h by addition of 40 lL of blocked protein A

beads (Pierce, Cat. No. 20334) (50% slurry proteinA–

agarose, 3mg/mL BSA, 0.1mg/mL salmon sperm DNA,

in IP buffer). Samples were next incubated for 3 h with

40 lL of streptavidin-coated magnetic particles (Pro-

mega, Cat. No. Z5481). Beads were then washed twice

with 2% SDS (or with 100mM NaOH, or 3M guanidin–Cl, or 8M urea, or 0.5% SDS+100mM NaOH, or 5M

NaCl) followed by three washes with LiCl wash buffer

(100mM Tris at pH 8.0, 500mM LiCl, 1% NP-40, 1%

deoxycholic acid), or five washes with the LiCl wash

buffer (normal wash [7]). Samples were then decross-

linked by an overnight incubation at 67 �C in 60 lL of

300mM NaCl solution. Proteinase K and 5X Proteinase

K buffer (50mM Tris at pH 7.5, 25mM EDTA, 1.25%SDS) were then added to the samples. After a 2-h in-

cubation at 45 �C, DNA from the samples was purified

using Qiagen miniprep columns (Cat. No. 27106). Pu-

rified DNA was recovered in 50 lL of water. In addition

to the precipitated samples, 5% of the input chromatin

for each sample was decross-linked, processed, and an-

alyzed in the same way. For the immunoprecipitation

with anti-HA antibody (Roche, Cat. No.1867423), thesame procedure was used, except that biotin was omit-

ted from the medium, and chromatin was incubated

overnight with 1 lL of anti-HA antibody and then in-

cubated with 80 lL of blocked protein A beads. After

washing, the immunoprecipitate was eluted in 200 lLof elution buffer (50mM NaHCO3, 1% SDS) before

decross-linking.

70 A. Viens et al. / Analytical Biochemistry 325 (2004) 68–76

Quantitative PCR

One microliter of DNA was used in 20 ll of quanti-tative PCR (using a Quantitect SYBR Green PCR kit

from Qiagen) on the LightCycler (Roche). Each reaction

was performed in duplicate. The PCR primer sequences

were B1 50: gcc ggg tgt ggt ggc gca cac ctt t, B1 30: gagaca ggg ttt ctc tgt gta gcc ct, DHFR 50: gcg gag cct tag

ctg cac aa, DHFR 30: tac cag cct tca cgc tag ga, b-actin50: acc gag cgt ggc tac agc tt, b-actin 30: tgg ccg tca ggc

agc tca ta, GAPDH 50: cca atg tgt ccg tcg tgg atc t, and

GAPDH 30: gtt gaa gtc gca gga gac acc. The linearity of

analysis was confirmed by using serial sample dilutions

(data not shown). All PCRs gave a unique product of

expected size, as confirmed by agarose gel analysis. For

each sample the value of the signal was calculated as a

percentage of input. Then the signal to noise ratio wascalculated taking the value of a GFP sample as a non-

specific background (noise).

Results

Description of the pBBH and CMV.BBH vectors

The principle of in vivo biotinylation is based on

coexpression of the biotinylation enzyme BirA from

Escherichia coli together with the protein of interest

fused to a short peptide, the biotin acceptor domain

(BAD), shown to be an efficient target for BirA [8].

Fig. 1 shows a map of the retroviral vector pBBH that

was generated in our laboratory to express proteins

biotinylated in vivo.The principal feature of our design is the placing of

the BirA ORF on the same vector downstream of the

gene of interest. Its expression from the same mRNA is

ensured by an internal ribosome entrance sequence

(IRES). The rationale for this particular design is to

provide for a high local concentration of the BirA en-

zyme in the vicinity of the protein of interest, which

would ensure efficient biotinylation with minimalamounts of the BirA enzyme. To minimize the vector

size, we used as an IRES a short sequence (designated

ShIRES) containing a 9-nt sequence complementary to

18S rRNA [9].

We generated versions of the vector that can be used

for either N-terminal or C-terminal tagging. We also

added a His tag for double-epitope tag purification. In

addition to the pBBHN and pBBHC vectors for gen-eration of stable cell lines based on the retroviral

MMLV backbone, we constructed vectors for transient

transfection, with transcription driven by a strong CMV

promoter (CMV.BBHN and CMV.BBHC, for N- and

C-terminal tagging correspondingly).

An additional feature of our vectors is the third ORF

encoding a selection marker, IL2R [10], which is under

control of a second IRES (EMC IRES). This tricistronicdesign allows us to minimize the number of steps and

reagents necessary to generate a required cell line.

100% efficiency of biotinylation of a target protein

To determine how efficiently a target protein could be

biotinylated in our system, we cloned the eGFP gene

(Clontech) in the retroviral vector pBBHN and gener-ated a HeLa cell line stably transduced with this con-

struct. As a control to test the requirement for BirA

enzyme, we also generated HeLa cells transduced with

pBH.GFP (this vector expresses the BAD.GFP fusion,

but it does not encode BirA). The state of the GFP in

these cells was monitored by Western blotting with anti-

GFP antibodies. As seen from Fig. 2A (lane 2 versus

lane 1), when BirA is expressed in the cells (pBBH.GFPtransduced cells), the BAD.GFP fusion protein migrates

noticeably slower compared to the same protein from

the cells that do not express BirA (pBH.GFP transduced

cells). To confirm that this change in migration is due to

biotinylation of the protein, we took advantage of the

fact that streptavidin–biotin binding resists conditions

of SDS–PAGE electrophoresis. Preincubation of the

sample with 1 lg of streptavidin before SDS–PAGEcaused a further, more dramatic shift in the GFP mo-

bility (lane 3). Since no residual fast-migrating band

remained after the streptavidin supershift, we conclude

that 100% of the GFP is biotinylated under these con-

ditions. Addition of streptavidin to the sample from the

pBH.GFP transduced cells (not expressing BirA) does

not cause a shift in mobility (not shown). The biotiny-

lated status of GFP from the pBBH.GFP transducedcells was also confirmed by probing the Western blot

with a peroxydase-conjugated streptavidin (Fig. 2B).

Similar tests with a GFP fusion having the BAD

domain at its C terminus also show 100% biotinylation

(Fig. 2A, lanes 5 and 6). The CMV.BBH vectors for

transient transfection were tested and gave 100% bioti-

nylated GFP (lanes 7–9 for CMV.BBHN; CMV.BBHC

not shown).

Intracellular localization of proteins is not generally

disturbed by their biotinylation in vivo

Epitope-tagging of proteins can lead to artifacts, as in

some cases it can disturb the protein�s properties [11]. Todetermine whether, in addition to this general concern,

biotinylation presents any specific problem for all pro-teins, we tested the intracellular localization of several

biotinylated proteins with a known distribution in the

nucleus.

We transiently transfected NIH3T3 cells with vectors

expressing biotinylated HP1a, HP1b, and HP1c, the

mammalian homologues of the Drosophila heterochro-

matin protein HP1. To follow the nuclear localization of

Fig. 1. Vectors for in vivo biotinylation. (A) Map of the retroviral vector pBBHN. The principal components (BAD domain, BirA, IL2R, and

IRESes) and restriction sites are shown. (B) Sequences of the BAD domain, the 6XHis tag, and the multiple cloning sites for the N-terminal fusion

and C-terminal fusion vectors, correspondingly. The amino acid sequence of the tags and the restriction sites are underlined.


these proteins, we used two complementary approaches:

staining with streptavidin–FITC to detect biotinylated

proteins and staining with monoclonal antibodies spe-

cific for different HP1 proteins. All HP1s showed clear

colocalization with heterochromatin by both kinds of

staining (Fig. 3A). Intriguingly, HP1c, believed to belocalized in a different manner, showed a pattern un-

distinguishable from those of the other HP1s, even in

untransfected cells (Fig. 3B). On the other hand, the

staining pattern of HP1a was dependent on the antibody

used (Fig. 3B, compare 2HP-269 and 2HP-1H5, see

Discussion).

To rule out the unlikely possibility that all biotiny-

lated proteins are recruited into heterochromatin, we

tested several proteins known to show a different local-

ization pattern. Two examples presented here are

SUMO1 and HMG14. As seen from Fig. 3C, they show

staining distinct from that of HP1 proteins. Thus, bio-tinylation does not target every protein to heterochro-

matin, and the localization of proteins by our technique

should reflect their physiological localization.

Thus, at least by the criterion of intracellular locali-

zation, in vivo biotinylation does not appear to disturb

protein function. Admittedly, one cannot exclude the

Fig. 2. 100% efficiency of GFP biotinylation. (A) Western analysis with anti-GFP antibody of extracts from the cells expressing N-terminal (lanes 1–

3, 8 and 9) or C-terminal (lanes 5 and 6) fusions of eGFP to the BAD domain. Lanes 1–3, 5 and 6 correspond to stable cell lines generated via

retroviral transduction, lanes 8 and 9 correspond to transient transfection; U.T, extract from untranfected or untransduced cells. Streptavidin was

added before SDS–PAGE in lanes 3, 6, and 9. Lane 1 corresponds to the cells expressing BAD-GFP fusion in absence of BirA (transduced with

pBHN.GFP vector). (B) Western analysis with streptavidin–HPO of extracts from the cells transduced with pBHN.GFP vector (lane 1) and

pBBHN.GFP (lane 2).

Fig. 3. Localization of biotinylated proteins. (A) NIH T3 transiently transfected with pBBHN.HP1a, b, or c (top, middle, and bottom, respectively).

Left panels correspond to chromatin staining with Hoechst, center panels to the staining with native antibodies against HP1s, and right panels to

staining with streptavidin–FITC. (B) Untransfected NIH 3T3 cells stained with antibodies against HP1a (top) and HP1c (bottom). The clone names

of different anti HP1a antibodies are indicated. (C) NIH T3 cells transiently transfected with pBBHC.HMG14 and pBBHBN.SUMO (top and

bottom, respectively). Left panels correspond to chromatin staining with Hoechst and right panels to streptavidin–FITC staining.


possibility of an artifact for any particular protein,

however, in this regard biotinylation does not appear to

create more problems than does any other epitope tag.

Biotin–streptavidin interaction allows for more stringent

washing conditions in chromatin immunoprecipitation

experiments

We next tested whether our system provides an

advantage for the chromatin immunoprecipitation

procedure. In particular, one might expect that, due to

the strength of the streptavidin–biotin interaction, it

would tolerate washing conditions that are more strin-

gent than those employed in the regular ChIP protocol,resulting in a reduced background (nonspecific chro-

matin binding).

We prepared chromatin from NIH3T3 cells ex-

pressing biotinylated H2A.Z, a replacement histone

shown to be enriched in active chromatin, but

also present in other parts of the genome [12]. This

Fig. 4. Chromatin immunoprecipitation I. H2A.Z interaction with total genome of NIH3T3 cells. Cells were transiently transfected with

pBBHN.H2AZ vector. Comparison of regular washing conditions (top) with stringent washing conditions (bottom). (Left) Quantitative PCR

analysis of genomic DNA present in the immunoprecipitates from cells transiently expressing biotinylated H2A.Z or eGFP. Shown are the results of

one of four representative experiments which were performed independently starting from transfection and gave very similar results. (Right) Ratio

between amounts of DNA pulled down from H2A.Z transfected sample and eGFP transfected sample (nonspecific background).


chromatin was used for a chromatin immunoprecipi-

tation experiment with immobilized streptavidin.

Washing conditions of various stringency were used

(see Materials and methods). To follow the presence of

mouse genomic DNA in our samples, we used quan-titative PCR to amplify B1 sequences. These short re-

petitive sequences are very abundant (about 106 copies

per genome) and are distributed almost randomly

throughout the genome; therefore, the PCR with B1

primers allows for very sensitive detection of mouse

genomic DNA in a sample [13]. Fig. 4 shows a com-

parison between the standard washing conditions [7]

and the more stringent conditions (2% SDS) found tobe the best of several tested (2% SDS, 100mM NaOH,

3M guanidin–Cl, 8M urea, 0.5% SDS+100mM

NaOH, not shown). Both washing conditions show a

difference between experimental sample (chromatin

with biotinylated H2A.Z) and control (chromatin from

cells transfected with GFP). However, the addition of a

2% SDS washing step (Fig. 4, bottom) significantly and

reproducibly reduced the presence of background ge-nomic DNA compared to the standard wash (by ap-

proximately one order of magnitude).

Detection of interaction of a unique DNA target with a

transcriptional factor

To test whether we can detect interaction of a unique

DNA target with a protein less abundant than H2A.Z inthis system, we expressed biotinylated transcription

factor DP1 in NIH3T3 cells and tested it for interaction

with a known target of this protein, the DHFR pro-

moter. As seen in Fig. 5A (top), the experimental sample

gave a higher signal than the nonspecific background

(GFP). The normal washes showed significantly less

difference between control and experimental samples

compared to the stringent wash that included a 2% SDSstep (Fig. 5A, bottom). Importantly, there was no en-

richment of irrelevant promoter sequences in the same

precipitated samples (actin, GAPDH, Fig. 5B), con-

firming the specificity of the observed interaction.

Importantly, the amounts of chromatin used in the

previous experiments were one order of magnitude lower

than commonly used in chromatin IP (50 lg vs 500 lg).To compare further our method to the ChIP with regularantibody, we expressed HA-tagged DP1 in NIH3T3 cells

and performed immunoprecipitation using high-affinity

Fig. 5. Chromatin immunoprecipitation II. (A) DP1 interaction with DHFR promoter. NIH 3T3 cells were transiently transfected with pBBHN.DP1

vector. Shown is the ratio between amounts of DNA pulled down from DP1 transfected sample and eGFP transfected sample, considered as a

nonspecific background (mean of three experiments). Top histogram shows results after stringent washing and the bottom histogram after regular

washing. (B) DP1 interaction with irrelevant GAPDH (top) and b-actin (bottom) promoters. Immunoprecipitate from the same experiment as in A

was tested with the GAPDH- and b-actin-specific primers. (C) Interaction of HA-tagged DP1 and DHFR promoter. DP1 ORF was cloned into

pOZFHN vector [14]; 500lg (top histogram) or 50lg (bottom histogram) of chromatin were used for the ChIP.


HA antibody. As seen in Fig. 5C (top), when we use

500 lg of chromatin, we can detect a DP1-DHFR in-

teraction in NIH3T3 cells. However, when low amounts

of chromatin (50 lg) are used (Fig. 5C, bottom), the re-

sults become highly irreproducible and the signal to noise

ratio approaches 1. Moreover, after more stringent

washing (including 2% SDS) the amounts of material

bound to beads become undetectable (not shown).

Discussion

We describe here a system designed to express a bio-

tinylated version of a protein of interest in mammalian

cells in vivo. Similar systems, one commercially avail-

able, have been described recently [15], (and Invitrogen

Bioease vectors), although their application for chro-

matin immunoprecipitation has not been reported.

Compared to these systems, our approach uses a much

shorter biotin acceptor domain (only 17 amino acids

long). Its small size is less likely to disturb protein

function, and it therefore should be preferable for mostapplications. Unlike the larger biotin acceptor domains,

however, the short tag cannot be a target of endogenous

biotinylation enzymes in mammalian cells. Thus, the

biotinylation enzyme BirA has to be coexpressed in the

same cell with the protein of interest. Our vectors are

designed to express both BirA and the target protein


from the same mRNA. Under this cis arrangement, theenzyme is synthesized in close physical proximity to its

target, ensuring efficient biotinylation of the protein of

interest. Our attempts to coexpress BirA and the target

protein from different promoters (trans-biotinylation)

never led to 100% biotinylation (data not shown).

As demonstrated by immunofluorescence experi-

ments, in vivo biotinylation does not change the intra-

cellular localization of the proteins. Thus, in this respect,it does not appear to create any additional artifacts as

compared to other epitope tags. In the course of these

experiments, we also unexpectedly observed that HP1c,previously reported to be localized in euchromatin [16],

was distributed in NIH3T3 cells in a manner undistin-

guishable from that of HP1b and HP1a. This was the

case even with endogenous HP1c in untransfected cells

detected by antibody against the native protein. Sur-prisingly, we also found that the two different anti-HP1aantibodies that we tested (2HP-1H5 and 2HP-269) differ

dramatically in the immunofluorescence pattern ob-

tained. Their specificity was confirmed in independent

experiments with siRNA knockdowns. We believe that

the euchromatic staining of HP1a seen in our study with

the 2HP-269 antibody, or that of HP1c seen in previous

studies [16], most probably reflects the masking of theepitope recognized by particular antibodies in the con-

text of heterochromatin, rather than a true localization

to the euchromatic compartment.

The principal result of this work is the demonstration

that in vivo biotinylation allows the use of more strin-

gent washing conditions in chromatin immunoprecipi-

tation, compared to those employed in a regular ChIP

protocol. This significantly improves the signal to noiseratio, as judged by quantitative PCR analysis. As a re-

sult, our system should help to streamline the ChIP

procedure and make it more robust and economical.

Accordingly, with our system, chromatin from only

5� 106 transiently transfected cells was sufficient for two

to four immunoprecipitation experiments yielding a re-

liable difference between signal and background.

It is important to note that, due to the overexpressionof proteins during transient transfection, this particular

method might not adequately reflect the distribution of

native protein throughout the genome. Therefore, it is

not advisable to use transient transfection for identifi-

cation of the DNA targets of a particular protein. Stable

cell lines with proven low expression levels of the bio-

tinylated protein (5–10% of the endogenous levels)

should be preferred. Nevertheless, the transient trans-fection could still be useful in addressing many other

questions concerning protein–DNA interactions, mostly

concerning the requirements for binding of a protein to

a given genome site. In particular, one can compare

different mutated versions of a protein in their binding

to a given genomic target and determine which domains

and particular residues play an important role in the

interaction. Alternatively, one can probe the accessibil-ity of a particular chromatin site to a given protein

under different physiological conditions. Unlike other

methods used to study protein–DNA interactions, for

instance EMSA, the binding is detected in vivo, in the

context of chromatin and the nuclear environment, and

therefore the information obtained can be considered

more physiologically relevant.

Compared to other epitope-tagging systems, bioti-nylation offers additional advantages, such as the wide

variety of commercially available reagents for detection

and purification of biotinylated macromolecules. These

reagents provide different choices of purification strat-

egies; for example, use of immobilized monomeric av-

idin allows for a gentle elution of biotinylated material,

compared to immobilized avidin or streptavidin. Thus,

the biotinylation system is also more flexible than otherepitope-tagging systems and should find many appli-

cations for the study of protein–DNA interaction

in vivo.

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Use of protein biotinylation in vivo for chromatin immunoprecipitation