UNIT 21.8Analysis of Protein Co-Occupancy byQuantitative Sequential ChromatinImmunoprecipitation

BASICPROTOCOL

Chromatin immunoprecipitation (ChIP; UNIT 21.3) is a widely used and powerful methodfor assaying individual protein-DNA interactions in vivo. As discussed earlier in thischapter (UNIT 21.3), ChIP experiments provide quantitative information about the relativelevel of binding of a given protein or proteins to different genomic regions. However, whatstandard ChIP experiments do not address is whether two proteins can simultaneously co-occupy a specific genomic region. For example, with conventional ChIP, the observationthat two proteins associate with a given genomic region might reflect co-occupancy, or,alternatively, it might indicate that the two proteins associate with different populations ofDNA molecules in a mutually exclusive fashion. There is simply no way to determine theextent to which (either positively or negatively) any two proteins influence one anotherwhen it comes to binding to the specific fragment in question via conventional ChIP.

Sequential chromatin immunoprecipitation (SeqChIP), on the other hand, is a powerfultool to address precisely this type of question: do two proteins simultaneously co-occupy aspecific DNA region in vivo? In SeqChIP, protein-DNA complexes from the first ChIP aresubjected to an additional immunoprecipitation with an antibody of a different specificity,typically to a second protein that is tested for its ability to co-occupy with the initiallyimmunoprecipitated protein. The cross-links of these doubly immunoprecipitated protein-DNA complexes are then reversed, and the DNAs are analyzed by quantitative PCR inan manner analogous to conventional ChIP samples (see UNIT 21.3, Alternate Protocol 2).Occupancy values for both single and double immunoprecipitations are first calculatedand then compared for any enrichment brought about by the second immunoprecipita-tion relative to the singly immunoprecipitated sample. While the following protocol hasbeen successfully developed for and used with Saccharomyces cerevisiae samples, it issufficiently general that it should be applicable for other species with minor modifications.

Materials

100 mg/ml bovine serum albumin (BSA, Fraction V; Sigma) in water (store at−20◦C)

50 mg/ml λ phage DNA (not sheared; New England Biolabs)5 mg/ml E. coli tRNA in water (store at −20◦C)20 mg/ml glycogen, or Pellet Paint (Novagen) as DNA carrier

Additional reagents and equipment for ChIP (UNIT 21.3, Basic Protocol), growth ofSaccharomyces cerevisiae (UNITS 13.1 & 13.2), extraction and purification of DNA(UNIT 2.1A), and real-time quantitative PCR (UNIT 21.3, Alternate Protocol 2)

Cross-link protein-DNA complexes in vivo1. For each sample, grow 400 ml Saccharomyces cerevisiae to OD600 = 0.6 to 0.8

(UNITS 13.1 & 13.2).

It is necessary to use more extract for sequential immunoprecipitations (80 to 100 mlcells) than for conventional samples (20 to 40 ml cells) in order to ensure that enoughmaterial remains after the first immunoprecipitation to generate a reproducible signalfollowing the second immunoprecipitation. The actual amount of extract required for thispurpose depends on a number of factors, including cross-linking efficiency and protein

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abundance, and may have to be adjusted accordingly. This protocol doubles the culturevolume (400 ml) used as compared to the single ChIP protocol (200 ml); consequently, itis helpful to split the cultures into the equivalent of two 200-ml samples for the cell lysisand sonication steps.

2. Add 11 ml of 37% formaldehyde (1% final). Cross-link for 20 min at room temper-ature by occasionally swirling flask or shaking slowly on a platform.

3. Add 60 ml heat-sterilized 2.5 M glycine and incubate an additional 5 min at roomtemperature.

Glycine stops the cross-linking by reacting with formaldehyde.

4. Split the cultures into two equal 200-ml samples and harvest cells, isolate chromatin,and shear the DNA fragments exactly as described in UNIT 21.3, Basic Protocol, steps4 to 13.

5. Microcentrifuge 30 min at maximum speed, 4◦C. Transfer the supernatants into afresh 15-ml disposable conical tube, adjust the total sample volume to ∼4.5 ml withice-cold FA lysis buffer, and gently mix by inversion. Remove 250 µl to check DNAfragment size (see UNIT 21.3, Basic Protocol, steps 15 and 16) and freeze the remainingchromatin solution in 1-ml aliquots in liquid nitrogen up to 1 year.

Perform first immunoprecipitation6. Incubate 1 ml chromatin solution with 20 µl primary antibody against the protein or

epitope of interest and 20 µl of 50% (v/v) protein A–Sepharose beads in TBS, on anend-over-end rotator for 90 min at room temperature.

The amount of antibody used is double that in the Basic Protocol of UNIT 21.3, to reflect theincreased amount of protein in the SeqChIP extract. As with conventional ChIP, the optimalamount of antibody will vary with each antigen and has to be empirically determined.

Sequential immunoprecipitations should normally be performed in both the forward andreverse directions to be able to analyze results in a quantitative fashion and to unam-biguously determine the extent of co-occupancy between two proteins (see AnticipatedResults).

7. Microcentrifuge beads 1 min at 3000 rpm, room temperature. Transfer 100 µl super-natant into a 0.5-ml PCR tube labeled “INPUT.” Discard the rest of the liquid.

8. Resuspend beads by pipetting up and down in 700 µl FA lysis buffer, room temper-ature, and transfer mixture into a Spin-X centrifuge-tube filter.

The use of Spin-X filters aids in the recovery of the beads after washes and results inbetter uniformity between different samples. The procedure is also substantially fasterwith the filters, particularly when multiple samples are processed simultaneously. Alter-natively, one could use conventional microcentrifuge tubes for the washes and aspiratethe supernatant with a narrow-bore pipet tip after each spin.

9. Place the filter into a 1.5-ml microcentrifuge tube and mix sample 3 min on an end-over-end rotator. Microcentrifuge 2 min at 3000 rpm, room temperature. Discard theflowthrough liquid at the bottom of the tube.

10. Add 700 µl FA lysis buffer, room temperature, to the beads and repeat step 9 twoadditional times.

Elute protein from beads11. Wash beads for 3 min each with 700 µl FA lysis buffer/0.5 M NaCl, 700 µl ChIP

wash buffer, and finally 700 µl TE buffer.

For many polyclonal antibodies, the more stringent washes in this step result in a cleanersignal, while gentle washes frequently lead to an unacceptably high background. For some

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antibodies (e.g., monoclonal against peptide epitopes; see UNIT 21.3, Alternate Protocol 1),repeated washes with FA lysis buffer, which are gentler, might be more appropriate. ConsultUNIT 21.3 for more information.

12. Place filter unit containing the beads into a new 1.5-ml microcentrifuge tube andadd 100 µl of ChIP elution buffer. Gently pipet up and down two or three times todislodge beads from the filter. Incubate 10 min in a 65◦C water bath.

A water bath is used instead of other heating apparatuses to improve heat transfer.

13. Microcentrifuge beads 2 min at 3000 rpm, room temperature. Discard filter withbeads. Transfer 9 µl (out of 100 µl) of the eluate into a 0.5-ml PCR tube labeled“1st IP.”

Perform second immunoprecipitation14. Add 775 µl FA lysis buffer (without SDS), 50 µl of 100 mg/ml BSA, 50 µl of

50 mg/ml λ phage DNA, 5 µl of 5 mg/ml E. coli tRNA, 10 to 20 µl antibody, and 20µl of 50% protein A–Sepharose to the remaining 91 µl of eluate obtained in the step13. Incubate on an end-over-end rotator for 90 min at room temperature.

In case peptide elution was performed (see Alternate Protocol 1 in UNIT 21.3), substitute FAlysis buffer containing 0.1% SDS for the non-SDS-containing buffer recommended above.It is important to keep overall SDS concentration to ≤0.1%. BSA, λ phage DNA, and E.coli tRNA are carriers designed to mimic the protein/nucleic acid mixture in chromatinsamples and to prevent nonspecific precipitation of cross-linked protein-DNA complexes.They are also necessary for the first and second immunoprecipitations to be experimentallyequivalent (see Critical Parameters and Troubleshooting). Finally, as in the case of thefirst immunoprecipitation, the amount of antibody required to produce optimal signal withthe lowest background may have to be empirically determined.

15. Wash beads and elute protein complexes by following steps 8 to 13 (above). Transfereluate into a 0.5-ml PCR tube labeled “SeqChIP,” taking note of the order of IPs.

For gentle washes (i.e., peptide elution), follow steps of UNIT 21.3, Alternate Protocol 1.

Reverse cross-links and purify DNA16. Add 100 µl ChIP elution buffer to the tube marked “INPUT” and “1st IP,” add 100 µl

and 91 µl TE to the tubes marked “SeqChIP” and “1st IP”, respectively, and 20 µlPronase in TBS to all three tubes.

If either the first IP and/or second IP was eluted by peptide, add 100 µl ChIP elutionbuffer instead of 100 µl TE to the relevant sample(s).

17. To reverse cross-links, place tubes into a PCR machine. Incubate 2 hr at 42◦C,followed by 6 hr at 65◦C. Store samples at 4◦C until used.

The incubation at 42◦C allows for Pronase digestion of cross-linked polypeptides, whilethe 65◦C-incubation results in a reversal of the formaldehyde cross-links.

The samples may be stored up to several days at 4◦C.

18. Transfer samples to new 1.5-ml microcentrifuge tubes, add 20 µl of 4 M LiCl, andpurify by extracting with 25:24:1 phenol/chloroform/isoamyl alcohol, followed byextraction with chloroform (UNIT 2.1A). Add 2 µl of 20 mg/ml glycogen (or PelletPaint; Novagen) as carrier, and 2.5 vol ethanol. Vortex briefly and precipitate theDNA for 1 hr at −20◦C (see UNIT 2.1A).

19. Microcentrifuge 30 min at maximum speed, 4◦C. Discard aqueous phase and washwith 1 ml of 70% ethanol, room temperature, by gently inverting samples severaltimes.

20. Microcentrifuge 5 min at maximum speed, room temperature. Aspirate supernatantand air dry pellet for ≤5 min.

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21. Resuspend in 150 µl TE buffer and store at –20◦C.

DNA stored in this fashion should be stable for years.

22. Analyze samples with real-time quantitative PCR (UNIT 21.3).

As signals from sequential immunoprecipitations are considerably weaker than from con-ventional ChIPs, it is recommended that the total number of amplification cycles beincreased from 35 to 40. In some extreme circumstances where the amount of DNA tobe amplified is minute, it might be beneficial to go beyond 40 cycles. Other parametersshould be identical to those described in UNIT 21.3.

COMMENTARY

Background InformationSequential chromatin immunoprecipita-

tion, a natural extention of conventional ChIP,arose due to the inability of conventional ChIPto determine whether two different proteinscan simultaneously co-occupy a given stretchof DNA in vivo. As described in this unit, thetechnique simply involves an additional im-munoprecipitation with antibody to an unre-lated protein, with subsequent steps essentiallyidentical to conventional ChIP (UNIT 21.3). Theresulting material can then be analyzed byeither conventional (UNIT 21.3) or real-timeQPCR (UNIT 21.3, Alternate Protocol 2), withreal-time QPCR clearly being the preferredmethod due to its superior linear range, whichpermits simultaneous analysis of both singlyand doubly immunoprecipitated samples.

SeqChIP has been successfully applied inthe analysis of wide-ranging biological phe-nomena in a number of different organismsand cell types. For example, SeqChIP wasused to help elucidate the mechanism ofPPRE-dependent RXR homodimer signaling(Ijpenberg et al., 2004), to demonstrate TBPbinding to nucleosomal DNA (Soutoglou andTalianidis, 2002), to identify the various pro-tein complexes involved in mediating tran-scriptional activation by the estrogen recep-tor alpha (Metivier et al., 2003), and to studyhyperosmotic stress response at the transcrip-tional level in yeast (Proft and Struhl, 2002).The procedure outlined in the Basic Proto-col, with some modifications, can be readilyadapted for use on non–Saccharomyces cere-visiae chromatin.

Critical Parameters andTroubleshooting

Sequential IP analysis requires substan-tially greater quantities of starting material (ex-tract) than normally used for single ChIP ex-periments. This is due to a number of factors,including low cross-linking efficiencies (typ-ically <5%, depending on both protein and

DNA region) and the necessity to do two IPs in-stead of one. The problem becomes especiallyacute if the proteins in question are poorlycross-linked to DNA, are low in abundance,and exhibit no or partial co-occupancy. In theBasic Protocol, the amount of extract (and thecorresponding purified DNA) used is four- tofive-fold greater than typically used for singleIPs, and is likely to be sufficient for most Seq-ChIP applications in yeast. Additional scale-upmay be desirable in some specific instances.

Of special concern is the need to ensurethat the second IP behaves identically to thefirst IP from a quantitative standpoint. The ad-dition of BSA, E. coli tRNA, and λ phageDNA to the eluate from the first ChIP servesto ensure that the two IPs behave in a simi-lar manner irrespective of actual order. BSA,E. coli tRNA, and λ phage DNA mimic theconcentrated cross-linked S. cerevisiae chro-matin that is present in the sample prior to thefirst IP, but which is effectively absent from thepost-elution complexes. Alternatively, cross-linked chromatin from an unrelated yeast suchas Kluyveromyces lactis, or even E. coli chro-matin, can be used as a carrier in place ofthe combination of BSA/tRNA/λ DNA. Anadded benefit of using non–S. cerevisiae yeast(or E. coli) chromatin is that these organismscan be engineered to serve as internal con-trols for the efficiency of the second immuno-precipitation. However, the actual strain con-struction and preparation of cross-linked chro-matin from these organisms is time-consumingand provides no appreciable benefit (relative toBSA/tRNA/λ DNA) when it comes controllingvariability between 1st and 2nd IPs.

Anticipated Results

Predicted outcomes of SeqChIP experimentsSeqChIP experiments have three possi-

ble outcomes: complete co-occupancy, no co-occupancy, and partial co-occupancy (Fig.21.8.1). Complete co-occupancy describes thescenario when two proteins, A and B, are

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Figure 21.8.1 Schematic depiction of the three possible outcomes of a SeqChIP between proteins A and B: completeco-occupancy (top left), no co-occupancy (top right), and partial co-occupancy (bottom panel). Partial co-occupancy canbe further subdivided into two categories: A is required (bottom left) or not required (bottom right) for the binding of B.

always present in unison at any given DNAmolecule. No A/B co-occupancy takes placewhen both A and B bind to the DNA region inquestion, yet are confined to non-overlappingsubsets of molecules and are never found at theDNA fragments at the same time. Partial co-occupancy takes place when some of the DNAmolecules are bound by both A and B, whileothers are bound by only A and/or only B.

Partial co-occupancy can be further subdi-vided into two distinct subgroups, differenti-ated by the ability or inability of one protein(B) to bind DNA independently of the other (A)as illustrated in Figure 21.8.1. In one scenario,DNA binding of protein B always occurs incombination with (and is completely depen-dent of the presence of) protein A, whereasprotein A can associate with DNA in the ab-sence of protein B. An example of this factor-

dependent DNA binding can be found in theinteractions between the yeast TATA-box bind-ing protein (TBP) and TBP-associated factors(TAFs): promoter binding by TAFs is criticallydependent on TBP, yet TBP can be found atmany promoters that lack TAFs (Kuras et al.,2000; Li et al., 2000). In the second scenario, Aand B can bind independently of one another toa given genomic region, yet the proteins mayco-occupy the same stretch of DNA in some(but not all) of the cases. This scenario is likelyto be valid for two DNA-binding proteins thatdo not interact with one another and bind to dif-ferent target sequences in an enhancer or otherregulatory region of a eukaryotic promoter.

Quantitative analysis of SeqChIP resultsFold-enrichment over background for both

individual ChIPs and SeqChIPs can be

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calculated relative to a control genomic loca-tion as described in UNIT 21.3. Let A, B, andAB represent the fold-enrichments for the first,second, and sequential IPs, respectively. Then,in cases of complete co-occupancy (e.g., com-ponents of the general RNA polymerase IItranscription machinery), the fold-enrichmentof individual ChIPs should be equal to theproduct of the fold-enrichment of the indi-vidual ChIPs (A × B = AB; Geisberg andStruhl, 2004). In cases of no co-occupancy,no enrichment is seen over that obtainedby the first immunoprecipitation (AB ≈ A).For partial co-occupancy, the fold-enrichmenttypically lies somewhere in between A (noco-occupancy) and AB (full co-occupancy).Mathematically, efficiency of co-occupancy(C, in percent) can be defined as follows:

C = 100(AB − A)/(A × B − A)

In cases of complete co-occupancy, AB typi-cally equals the product of A and B, and C =100. If the value of AB = A, then there is noenrichment over the first immunoprecipitationand C = 0. For cases of partial co-occupancy,typical experimentally obtained C values rangebetween >0 to significantly <100. It is im-portant to note that in order for C values tobe meaningful, fold-occupancies for individ-ual ChIPs must be substantially above back-ground (typically greater than two-fold), andSeqChIP enrichment (the ratio AB/A) must bereproducibly above background as well (AB/A>2 works well). Of course, the definition ofwhat represents experimental background issomewhat arbitrary and will vary dependingon experimental setup, the number of repeti-tions, as well as other factors.

Order of IPs may influence SeqChIPoutcomes

An interesting (and predicted) outcome insome SeqChIP experiments is that the order ofsequential IPs makes a difference in cases ofpartial co-occupancy. In a typical experiment,there are three classes of DNA molecules ofinterest—those bound to A alone, B alone, orboth A and B. If X is defined as the fraction ofA-DNA molecules that also contain B, and Yis defined as the fraction of B-DNA that alsocontain A, then the expected fold-enrichmentsfor the three different classes of molecules aregiven by:

(1 − X)A for the A-alone DNA molecules(1 − Y)B for the B-alone DNA molecules(XA)(YB) for the A + B + DNA molecules

It should be noted that X and Y are independentof one another, as A and B may have differ-ent occupancy properties. Using the equationsabove, it follows that:

when A ChIP is first, the fold-enrichment(AB) for the SeqChIP = (1 − X)A +(XA)(YB)

when B ChIP is first, the fold-enrichment(BA) for the SeqChIP = (1 − Y)B +(XA)(YB).

For cases of complete co-occupancy (X =1, Y = 1), the fold-enrichment is the same ir-respective of IP order, as the equations abovesimplify to AB = BA. Likewise, the order ofIPs makes no difference in cases of no co-occupancy (X = 0, Y = 0), as AB = BA = 0.By contrast, the order of IPs does make a dif-ference in cases of partial co-occupancy. Forexample, when X = 1 and Y is small (i.e., Aalways co-occupies with B, but B rarely co-occupies with A), meaningful co-occupancyis likely to be observed in only one direction.In specific terms, if X = 1, Y = 0.1, A =10, and B = 50, then the fold-enrichmentsare 50 when A ChIP is first and 95 when BChIP is first. Clearly, these two values are notequal (AB �= BA). More importantly, whenA ChIP is performed first, the SeqChIP valueof 50 represents a five-fold enrichment rela-tive to the value of the single IP, although itis also well below the value for predicted fullco-occupancy (A × B = 500). By contrast,when B ChIP is performed first, the SeqChIPvalue of 95 is less than two-fold greater than thevalue of the single IP (B = 50), and typicallyfalls within the cut-off for experimental error.The theoretically predicted (and experimen-tally confirmed) IP-order-dependent nature ofSeqChIP values requires careful interpretationof so-called negative (no co-occupancy) re-sults: definitive demonstration of lack of co-occupancy between two factors requires thatthe SeqChIP be performed in both directionswith identical results of no co-occupancy. Onthe other hand, partial (or full) co-occupancycan be effectively demonstrated if observed inone direction.

Time ConsiderationsCell growth, formaldehyde cross-linking,

and chromatin isolation/purification can becompleted in 2 to 3 days. Sequential immuno-precipitation requires a total of 7 to 8 hr: 2 hrfor antibody binding, 1 hr for washes, and30 min for elution for each IP, with 30 minset-up time in between the two immunoprecip-itations. Complexes eluted from the first IP can

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be conveniently frozen at −20◦C and the sec-ond immunoprecipitation can be performed ata later time for added convenience. Cross-linkreversal takes ≥6 hr and is best performed ina thermal cycler programmed to run overnight(see UNIT 21.3, Alternate Protocol 2). DNA pu-rification takes 2 hr and quantitative PCR takes≤2 to 3 hr, depending on instrument and cy-cling parameters.

Literature CitedGeisberg, J.V. and Struhl, K. 2004. Quantitative

sequential chromatin immunoprecipitation, amethod for analyzing co-occupancy of proteinsat genomic regions in vivo. Submitted.

Ijpenberg, A., Tan, N.S., Gelman, L., Kersten, S.,Seydoux, J., Xu, J., Metzger, D., Canaple, L.,Chambon, P., Wahli, W., and Desvergne, B.2004. In vivo activation of PPAR target genesby RXR homodimers. EMBO J. 23:2083-2091.

Kuras, L., Kosa, P., Mencia, M., and Struhl, K. 2000.TAF-containing and TAF-independent forms oftranscriptionally active TBP in vivo. Science288:1244-1248.

Li, X.-Y., Bhaumik, S.R., and Green, M.R. 2000.Distinct classes of yeast promoters revealed bydifferential TAF recruitment. Science 288:1242-1244.

Metivier, R., Penot, G., Hubner, M.R., Reid, G.,Brand, H., Kos, M., and Gannon, F. 2003. Es-trogen receptor-alpha directs ordered, cyclical,and combinatorial recruitment of cofactors on anatural target promoter. Cell 115:751-763.

Proft, M. and Struhl, K. 2002. Hog1 kinase convertsthe Sko1-Cyc8-Tup1 repressor complex into anactivator that recruits SAGA and SWI/SNF inresponse to osmotic stress. Mol. Cell 9:1307-1317.

Soutoglou, E. and Talianidis, I. 2002. Coordinationof PIC assembly and chromatin remodeling dur-ing differentiation-induced gene activation. Sci-ence 295:1901-1904.

Contributed by Joseph V. Geisberg andKevin Struhl

Harvard Medical SchoolBoston, Massachusetts