SHORT COMMUNICATION

Optimization of a Method for Chromatin ImmunoprecipitationAssays in the Marine Invertebrate Chordate Ciona

Hitoshi Aihara & Lavanya Katikala & Robert W. Zeller &

Anna Di Gregorio & Yutaka Nibu

Received: 11 December 2012 /Accepted: 3 March 2013 /Published online: 17 April 2013# Springer Science+Business Media New York 2013

Abstract Chromatin immunoprecipitation (ChIP) assays al-low the efficient characterization of the in vivo occupancy ofgenomic regions by DNA-binding proteins and thus facilitatethe prediction of cis-regulatory sequences in silico and guidetheir validation in vivo. For these reasons, these assays andtheir permutations (e.g., ChIP-on-chip and ChIP-sequencing)are currently being extended to several non-mainstreammodelorganisms, as the availability of specific antibodies increases.Here, we describe the development of a polyclonal antibodyagainst the Brachyury protein of the marine invertebrate chor-date Ciona intestinalis and provide a detailed ChIP protocolthat should be easily adaptable to other marine organisms.

Keywords Ascidian . Brachyury . Chromatinimmunoprecipitation .Ciona . cis-regulatory module .

Enhancer . Transcription factor

Abbreviationsbp Base pair(s)BSA Bovine serum albumincDNA Complementary DNAChIP Chromatin immunoprecipitationCRM cis-regulatory moduleDAPI 4′,6-Diamidino-2-phenylindole

PBS Phosphate-buffered salinePCR Polymerase chain reactionqPCR Quantitative PCRwt Wild type

Introduction

After serving as the model organisms of choice for embryolog-ical studies for well over a century (Chabry 1887; Conklin1905), ascidians have rapidly entered their post-genomic eraafter the genomes of Ciona intestinalis and Ciona savignyiwerefully sequenced (Dehal et al. 2002; Vinson et al. 2005).Currently, there is a growing interest in studying the in vivooccupancy of genomic sequences that might work as enhancers,or cis-regulatory modules (CRMs), by transcription factors, bothat the level of individual Ciona CRMs (e.g., Kanda et al. 2009;Dunn and Di Gregorio 2009) or on a genome-wide scale (Kuboet al. 2010). Such studies, which mainly rely upon chromatinimmunoprecipitation (ChIP), are instrumental for the reconstruc-tion of the gene regulatory networks controlling the developmentof these simple chordates, and are informative for more complexorganisms in this phylum (Delsuc et al. 2006; Davidson andChristiaen 2006; Lemaire 2009). For these reasons, we haveoptimized in C. intestinalis the following ChIP protocol, whichshould provide a useful reference for similar assays in otherascidian species and in related marine embryos. The method isused here in combination with a polyclonal antibody that wasraised against the Ciona Brachyury (Ci-Bra) protein.

Materials and Methods

Animal Husbandry and Embryo Culturing

Adult C. intestinalis (species A; Caputi et al. 2007) werepurchased from Marine Research and Educational Products

Hitoshi Aihara and Lavanya Katikala contributed equally to this work(as first authors).

Anna Di Gregorio and Yutaka Nibu contributed equally to this work(as senior authors).

H. Aihara : L. Katikala :A. Di Gregorio (*) :Y. Nibu (*)Department of Cell and Developmental Biology, Weill MedicalCollege of Cornell University, 1300 York Avenue, Box 60,New York, NY 10065, USAe-mail: and2015@med.cornell.edue-mail: yun2001@med.cornell.edu

R. W. ZellerDepartment of Biology, San Diego State University, 5500Campanile Drive, San Diego, CA 92182, USA

Mar Biotechnol (2013) 15:520–525DOI 10.1007/s10126-013-9504-5

(Carlsbad, CA) andmaintained in a refrigerated aquariumwithrecirculating artificial seawater at a temperature of 18 °C.Detailed protocols describing fertilization, dechorionation,and culturing of Ciona embryos have been published previ-ously (Zeller 2004; Christiaen et al. 2009). DechorionatedC. intestinalis embryos were grown in Petri dishes in filteredartificial seawater (FSW) until the desired stage(s) wasreached, usually at temperatures ranging from 15 to 21 °C(Whittaker 1977).

Generation of a Ciona Brachyury Polyclonal Antibody

The full-length 1,324-bp Ci-Bra cDNA (NCBI accession no.NM_001032487; Corbo et al. 1997) was PCR amplified usingas a template RNA extracted from mid-tailbud embryos, aspreviously described (Oda-Ishii and Di Gregorio 2007), wascloned first into the pGEM-T vector (Promega, Madison, WI,USA), and then transferred to the BglII and EcoRI sites of thepRSET-B vector (Invitrogen, Carlsbad, CA, USA) in framewith a Histidine tag. The His-Ci-Bra protein was induced inbacteria at 25 °C using 0.5 mM IPTG and purified essentiallyas previously published (Gazdoiu et al. 2005). The purifiedprotein was run on a 10 % sodium dodecyl sulfate (SDS)-PAGE gel and stained with Coomassie blue (Bio-Rad,Hercules, CA, USA).

The size of the purified tagged protein was ~53.5 kDa, aspredicted (data not shown). Approximately 2 mg were sentto Covance, Inc. (Princeton, NJ, USA) for the generation ofpolyclonal antibodies in rabbits.

In parallel, we also generated a GST-Ci-Bra protein by clon-ing the sequence encoding the C-terminal half of the Ci-Braprotein in the pGEX2T vector. We used this protein to purifythe anti-His-Bra antibody from the immune sera by attaching itcovalently to agarose-glutathione beads, using the GSTOrientation kit (Thermo Scientific, Rockford, IL, USA). Theaffinity resin that was reacted with the immune sera was washedtwice with 0.5 M KCl-HEG buffer (0.5 M KCl, 25 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-KOH pH 7.5, 0.5 mM EDTA pH 8.0, 0.1 % NP-40, 10 %glycerol), then extensively washed with wash buffer (20 mMTris–HCl pH 7.5, 1 M NaCl, 1 % Triton X-100). The antibodywas eluted by adding elution buffer (0.2Mglycine pH 2.2, 0.5MNaCl), and the eluate was rapidly neutralized with 1MTris-HCl,pH 8.0. The purified antibody was concentrated by ammoniumsulfate precipitation and subsequently dialyzed against 50 mMKCl-HEG overnight, then quantified against bovine serumalbumin (BSA) standards using a spectrophotometer.

Immunohistochemistry

Dechorionated C. intestinalis embryos were fixed at roomtemperature for 50 min in 4 % paraformaldehyde/phosphate-buffered saline (PBS), washed twice in PBS for 5 min, then

washed for 20 min in 0.25 % Triton X-100, 0.1 % Tween-20in PBS, and washed again once in PBS before being incubatedovernight in PBS/1 % BSA at 4 °C in the presence of the Ci-Bra-specific antibody, as described in Zega et al. (2008). Aftera series of washes in PBS, the embryos were incubated over-night at 4 °C, in the dark and with gentle rocking, with a goatanti-rabbit Alexa Fluor 546 fluorescent secondary antibody inPBS (Invitrogen, Carlsbad, CA, USA). The following day, theembryos were washed five to six times in PBS for a totalof ~1 h, mounted with mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vectashield; VectorLaboratories, Burlingame, CA, USA), and photographedusing a Leica DMR fluorescent microscope.

Chromatin Immunoprecipitation

This method has been adapted to Ciona embryos using previ-ously published protocols as a reference (Lee et al. 2006;Nelson et al. 2006). The ChIP protocol detailed here has beenused for embryos at the mid-tailbud stage, grown at 15 °C for15 h, and has been employed successfully on bothwild-type andtransgenic embryos. For reference, a 100-mm-diameter Petridish containing 25 mL FSW holds about 5,000 embryos andprovides a ~25-μL pellet, corresponding to roughly 107 cells.

Cross-Linking

Embryos were fixed for 10 min at room temperature on arotating platform after adding fresh formaldehyde(Polysciences, Warrington, PA, USA) to a final concentrationof 1 % to the Petri dishes. After cross-linking was quenchedwith 2.5 M glycine for 5 min, embryos were transferred tofresh Petri dishes containing PBS pH 7.4 (Life Technologies,catalog no. 10010-023) and washed for 10 min with gentlerotation, then transferred to 1.5-mLmicrofuge tubes, collectedby brief centrifugation, and washed once more in PBS.

Note that at this point, after carefully removing as muchPBS as possible, embryos can be stored at −80 °C as apellet, if necessary.

Chromatin Fragmentation

To fractionate chromatin, approximately 100 μL of fixedembryos (i.e., ~20,000 embryos, collected from four 100-mm Petri dishes) were sonicated in a final volume of 1 mLin IP buffer (Table 1) containing a protease inhibitor cocktail(Roche Applied Science, no. 1873580).

Note that as an alternative to the pre-made cocktail, wealso used successfully a combination of protease inhibitorscontaining, per milliliter, 2.5 μL of 200 mM PMSF (finalconcentration 0.5 mM), 1 μL of 10 μg/μL leupeptin (finalconcentration 10 μg/mL), and 1 μL of 10 μg/μL aprotinin(final concentration 10 μg/mL).

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Sonication was carried out on ice in a 1.5-mL microfugetube, 20 times for 12 s each, using a Branson 250 sonicatorat a power setting of 4 and 30 % duty cycle.

Note that sonication is a key step in this protocol. Theoptimal conditions required to obtain the desired fragmentsize may vary depending upon the sonicator employed andshould be determined by monitoring the fragmentationachieved using different conditions (e.g., 14, 16, 18 times,etc.). As an alternative to sonication, the chromatin can bedigested with micrococcal nuclease (Gaffney et al. 2012).

Reverse Cross-Linking and DNA Purification of the InputSample

After sonication, 30 μL of the sonicated sample [whole cellextract (WCE)] were transferred to a 2-mL microfuge tube forreverse cross-linking to be used as the WCE input sample,while the remainder (970 μL) was stored at −80 °C. The WCEinput sample was first mixed with 210μL of DNase-free waterand 60 μg of RNase A (from a 10 mg/mL stock) and incubatedat 37 °C for 30 min to remove RNA that might interfere withthe visualization of short DNA fragments on agarose gel. Thechromatin was then reverse cross-linked by incubating over-night at 65 °C in the presence of 30 μg of proteinase K (from a

10 mg/mL stock), 125 mM NaHCO3, and 1.25 % SDS. Afteraddition of 1.5 mL PB buffer (Qiagen, Valencia, CA, USA;catalog no. 27104), the sample was incubated for 1 h at roomtemperature on a nutator, then purified using the QIAprep SpinMiniprep columns (Qiagen). The WCE input sample wasapplied to a column, then centrifuged at 13,200 rpm for1 min. The column was then washed first with 0.6 mL of PBbuffer and subsequently with 0.75 mL of PE buffer (Qiagen),followed by centrifugation as described above. The purifiedDNAwas subsequently eluted by incubating twice with 50 μLof Elution Buffer (Qiagen) for 10 min, followed by centrifu-gation at 13,200 rpm for 1 min. Ten microliters of the purifiedWCE input was used to check the DNA fragmentation, whilethe remaining amount was stored at −20 °C, to be used later forquantitative PCR (qPCR). The size of the DNA fragments wasassessed by electrophoresis using a 1.5 % agarose gel andstaining with ethidium bromide to a final concentration of0.5 μg/mL. If the fragment size was found to range between200 and 800 bp, then the 970-μLWCE sample(s), previouslystored at −80 °C, was used for immunoprecipitation.

Immunoprecipitation

For the immunoprecipitation, 50 μL of Dynabeads Protein A(Invitrogen, Carlsbad, CA, USA) were pretreated by washingwith 950 μL IP buffer (Table 1), and then by incubating with950 μL of IP buffer containing 1 mg BSA and 2 μg sonicatedsalmon sperm DNA for 3 h at 4 °C with rotation.

Note that salmon sperm DNA or other carrier DNAsshould not be used in the blocking solution if ChIP-Seqexperiments are planned, in order to avoid contaminationin the sequencing steps.

After the 3-h incubation, the beads were separated fromthe IP buffer using the Dynal magnetic rack (Invitrogen),according to the manufacturer’s instructions, then quicklywashed twice with fresh IP buffer at 4 °C.

The 970-μL WCE samples were thawed on ice, thencentrifuged at 13,200 rpm for 15 min at 4 °C, and thesupernatant was transferred to fresh microfuge tubes. Forpreclearing nonspecific background, the samples were incu-bated with 50 μL of the pretreated beads and 0.5 μg normalrabbit IgG (Jackson ImmunoResearch, West Grove, PA,USA) for 6 h at 4 °C on a nutator.

Note that after the first 3 h of incubation, pretreatment oftwo additional 50-μL aliquots of beads in 1.5-mL microfugetubes should begin, as described above.

After preclearing, the WCE samples were separated fromthe beads using the magnetic rack, and the beads werediscarded. Precleared WCE samples were then dividedequally into the two tubes containing the 50-μL aliquots ofpretreated beads and incubated overnight at 4 °C on anutator with 0.25 μg of normal rabbit IgG and the specificantibody of choice, respectively.

Table 1 ChIP buffers composition

IP buffer

150 mM NaCl

50 mM Tris-Cl, pH 7.5

5 mM EDTA

0.5 % NP-40

1 % Triton X-100

RIPA buffer

50 mM HEPES, pH 7.5

0.5 M LiCl

1 mM EDTA

1 % NP-40

0.5 % sodium deoxycholic acid

Bicarbonate/SDS buffer

0.1 M NaHCO3

1 % SDS

TE buffer

10 mM Tris-HCl, pH 8.0

1 mM EDTA

Tris/acetic acid/EDTA (TAE) buffer (1×)

40 mM Tris

20 mM acetic acid

1 mM EDTA

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Note that the beads should not be allowed to dry out; forthis reason, we recommend removing the IP buffer justbefore adding the precleared WCE samples to the beads.

We have used this protocol with the His-Ci-Bra antibodyon wild-type embryos (Fig. 1), and with green fluorescentprotein (GFP) antibodies on transgenic embryos carryingthe Ci-Bra>Ci-Bra/GFP transgene (data not shown).

Note that to minimize background signal, we recommendpurifying the antibody (e.g., as described above).

Dynabeads bound to the immunoprecipitated DNA werethen washed five times for 10 min on a nutator at 4 °C with1.5 mL of ice-cold radioimmunoprecipitation assay (RIPA)buffer (Table 1), followed by two 2-min washes with 1.5 mLof ice-cold Tris–EDTA (TE) buffer (Table 1). After trans-ferring the tubes to the magnetic rack, the supernatant wascarefully aspirated, and the tubes were removed from therack. Subsequently, the beads were incubated in 300 μL of

TE buffer containing 60 μg of RNase A at 37 °C for 30 min,then washed again with 1.5 mL of TE buffer.

Note that at this point, most of the supernatant should becarefully removed.

Reverse Cross-Linking and DNA Purificationof the Immunoprecipitated Sample

The immunoprecipitated DNAwas eluted by incubating twicefor 45 min with 150 μL of freshly made bicarbonate/SDSbuffer (Table 1), at room temperature. The eluted sample(300 μL) was reverse cross-linked by addition of 30 μg ofproteinase K and overnight incubation at 65 °C, purified asdescribed above, then subjected to PCR and/or qPCR inparallel with the WCE input sample.

Note that proteinase K incubation can be shortenedto 2 h.

Fig. 1 Use of the Ciona Brachyury antibody for immunofluorescenceand ChIP assays. a–c C. intestinalis mid-tailbud embryo immunostainedwith the Ci-Bra antibody. a Counterstaining with DAPI visualizes nuclei.b, c The Ci-Bra antibody specifically stains the nuclei of the 40 notochordcells. d, e Results of ChIP assays on the Ci-tune notochord CRM, asdetected by PCR (d) and qPCR (e). In d, lanes were loaded as follows:Lane 1 DNA ladder (1-kb Plus DNA Ladder, Invitrogen, cat no. 10787-018). Lane 2 PCR product (134 bp) obtained from chromatinimmunoprecipitated with 50 μg of Ci-Bra antibody (red arrow). Primers:Ci-tune forward 5′-GTGTTGCGTACACACTCAAAGTCAG-3′ and Ci-tune reverse 5′-GCAGGGCAGTTCTGATAAACACGTTGT-3′. Lane 3negative control; PCR product of Ciona chromatin immunoprecipitated

with 50 μg of pre-immune serum. Lane 4 134-bp PCR product obtainedby amplifying 10 μL of a 50 % dilution (~200 ng) of the WCE inputsample (red arrow). In e, ChIP was performed on two biological repli-cates, and qPCR was performed on 10 ng of each sample, in triplicate,using SYBRGreen in a Prism 7700 Real-Time qPCR thermocycler (ABI,Carlsbad, CA, USA). To obtain standard curves, we used samplescontaining 20, 2, 0.2, and 0.02 ng/μL of Ciona genomic DNA, each induplicate. The percent input and standard deviation were calculated fromthe average of triplicate immunoprecipitated/input WCE scores. Onerepresentative biological dataset is shown in the figure. The p value(p<0.001) was calculated using a two-tailed Student’s t test

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Results and Discussion

Ci-Bra encodes a sequence-specific transcription factor thatin Ciona and other ascidians, such as Halocynthia, is spe-cifically expressed in the notochord (Corbo et al. 1997;Yasuo and Satoh 1993). Brachyury plays a pivotal role innotochord formation in all chordates analyzed so far and isevolutionarily conserved not only across the chordate phy-lum, but also among other deuterostomes and in proto-stomes, such as the fruit fly Drosophila, where theBrachyury ortholog, Brachyenteron, is expressed in thehindgut and is required for the morphogenesis of this struc-ture (Singer et al. 1996; Swalla 2006). In addition to being amajor developmental regulator, Brachyury is also a specificmarker and a causative agent of chordoma, a notochord-derived tumor (Vujovic et al. 2006; Yang et al. 2009). Forthese reasons, the identification of genes targeted by thistranscription factor is of particular interest.

We first tried to raise polyclonal antibodies directed againsta short synthetic peptide custom designed from the Ci-Braprotein sequence; however, this approach was unsuccessfulbecause the resulting antiserum produced mostly backgroundsignal in preliminary tests (data not shown). We then tried thebacterial expression, recovery, and purification of full-lengthand truncated versions of Ci-Bra, and the most satisfactoryresults were obtained with the full-length His-tagged Ci-Bra.

Figure 1 shows both the results of an immunofluorescenceexperiment and the detection of ChIP results. The antibodyraised against the full-length His-tagged Ci-Bra protein wasfirst tested by immunohistochemistry (Fig. 1a–c) and by west-ern blot (data not shown). The same antibodywas then affinitypurified (see “Materials and Methods”) and used for ChIPassays with primers specifically designed to amplify a 134-bpfragment of the Ci-tune notochord CRM. The Ci-tune noto-chord CRM is part of a Ciona genomic region that containstwo Ci-Bra-binding sites, which we have previously shown tobe bound by Ci-Bra via electrophoretic mobility shift assays(Passamaneck et al. 2009). Thus, the Ci-tune notochord CRMprovided us with a reliable positive control. We obtainedsimilar results when the Ci-tropomyosin-like notochordCRM, another direct Ci-Bra target (Di Gregorio and Levine1999), was used as a positive control (data not shown). TheChIP results were first visualized by agarose gel electropho-resis followed by ethidium bromide staining (Fig. 1d).Subsequently, qPCR experiments (Fig. 1e) allowed us toobtain an accurate quantification of the results.

Conclusions

The straightforward protocol provided here and the avail-ability of an ascidian Brachyury antibody pave the way forstudies of the genes regulated by this transcription factor in

Ciona, and possibly in other ascidian species, and shouldaid the application of ChIP assays in other marine organ-isms. In Ciona, numerous early target genes of Ci-Bra havebeen identified through genome-wide ChIP-chip analysesby employing anti-GFP antibodies on embryos expressinga Ci-Bra/GFP fusion (Kubo et al. 2010). This approach hasbeen very successful in early Ciona embryos and hasyielded numerous target genes not only for Ci-Bra but alsofor several other transcription factors (Kubo et al. 2010).The availability of a specific Ci-Bra antibody now allowsthe identification of notochord genes controlled by thisfactor at later developmental stages. In addition, the combi-nation of this specific antibody and of the step-by-stepprotocol described here facilitates the validation of Ci-Braoccupancy at the level of individual genomic loci of interest,as well as the prediction of late-acting, Ci-Bra-dependentnotochord cis-regulatory elements.

Acknowledgments We are indebted to Prof. Fiorenza De Bernardi(University of Milan, Italy) for the immunofluorescence protocol. Thiswork was supported by grant NIH/NIGMS GM100466 along withsupplemental funding from the American Recovery and ReinvestmentAct award R01HD050704-05S1 to ADG and by grants from theAmerican Cancer Society (RSG-08-042-01-DDC) and the Charles A.Frueauff Foundation to YN. HA was supported in part by a postdoc-toral fellowship from the Japan Society for the Promotion of Science(JSPS).

Conflict of Interest The authors declare no conflict of interest.

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