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Molecular & Biochemical Parasitology 166 (2009) 77–80

Contents lists available at ScienceDirect

Molecular & Biochemical Parasitology

hort technical report

uantitative chromatin immunoprecipitation (Q-ChIP) applied tochistosoma mansoni

téphanie Cabya,b,c, Raymond J. Piercea,b,c,∗

Inserm, U547, Lille, FranceUniversité Lille 2, Lille, FranceInstitut Pasteur de Lille, IFR 142, 1 rue du Professeur A. Calmette, 59019 – Lille, France

r t i c l e i n f o

rticle history:eceived 6 January 2009eceived in revised form 17 February 2009ccepted 18 February 2009

a b s t r a c t

The life-cycle of the platyhelminth parasite Schistosoma mansoni is characterized by marked morpho-logical changes between the various stages that are the result of a complex developmental program. Inorder to study the role of epigenetic mechanisms in regulating this program, and more particularly therole of changes in histone modifications in the control of the transcription of key genes, we have adapted

vailable online 9 March 2009

eywords:hromatin immunoprecipitation (ChIP)chistosoma mansoniistonepigenetics

the technique of quantitative chromatin immunoprecipitation (Q-ChIP) to larval stages and adult worms.We have used the classical method involving formaldehyde-induced cross-linking of DNA-associated pro-teins, followed by ultrasonication to fragment the DNA before immunoprecipitation and have establisheda protocol for use with schistosomes. We show, using antibodies directed against acetylated histone H4,that the technique is applicable to the parasite and allows the quantification and comparison of the levelsof modified histone at gene promoters at different life-cycle stages.

ranscription

. Introduction

Chromatin exhibits numerous types of ‘epigenetic’ modifica-ions (of histones or of DNA) that may be associated with theontrol of transcription [1]. The importance of these modifica-ions in regulating diverse developmental programs has led uso study their importance in the development and differenti-tion of schistosomes throughout their complex life-cycle. Oneuch modification, the methylation of DNA, is absent in schis-osomes [2], indicating that modifications of histones representhe primary epigenetic transcriptional control mechanism in theselatyhelminths. The N-terminal domains of histones can undergoumerous post-translational modifications including acetylation,ethylation, phosphorylation, ubiquitinylation and SUMOylation

hat have the effect of either repressing or activating transcription1]. The acetylation of lysine residues, particularly on histones H3nd H4, is generally associated with transcriptional activation, andheir deacetylation with repression. Recently, we and others have

haracterized some of the enzymes involved in histone acetyla-ion and deacetylation [3–5] in Schistosoma mansoni. In order totudy in more detail the role of histone acetylation in the activa-ion of transcription in schistosomes it was necessary to adapt the

∗ Corresponding author at: Inserm U 547, Institut Pasteur de Lille, 1 rue du Pro-essor A. Calmette, 59019 – Lille, France. Tel.: +33 320877783; fax: +33 320877888.

E-mail address: Raymond.Pierce@pasteur-lille.fr (R.J. Pierce).

166-6851/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.molbiopara.2009.02.014

© 2009 Elsevier B.V. All rights reserved.

methodology of chromatin immunoprecipitation (ChIP) to the par-asite. ChIP has become a very useful technique for the location ofmodified histones, transcription factors and non-histone chromo-somal proteins. Moreover, it is an in situ technique that allows theestablishment of a physiological representation of nuclear eventsinvolved in the processing of DNA [6]. It is most usually applied tocell cultures but we have succeeded in applying the technique towhole organisms. Although this has already been done with wholeCaenorhabditis elegans worms [7], our challenge was to work withdifferent developmental stages of the parasite. Here we describe indetail the quantitative ChIP protocol that we have developed thatallows the investigation of the attachment of modified histones,or any other protein including transcription factors and cofactors,that are bound to defined DNA sequences. We illustrate this pro-tocol by its use to determine the levels of acetylated histone H4at the promoters of the nuclear receptor SmFtz-F1 [8] and histonedeacetylase 1 (SmHDAC1) [5] genes at two developmental stages,miracidia and cercariae. In this assay, parasites were incubated witha cross-linker to prevent the separation of DNA-associated proteinsfrom their target DNA sequence. Ultrasonication was used to dis-rupt the parasites and fragment the DNA. The sheared DNA–proteincomplex was then immunoprecipitated using antibodies specific

for the histone modification under study. An aliquot of shearedDNA before immunoprecipitation was used as a reference sample(input). The protein–DNA complexes from reference and ChIP sam-ples were reverse cross-linked and the DNA was purified for use inquantitative real-time PCR.

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8 S. Caby, R.J. Pierce / Molecular & Bio

A Puerto–Rican strain of S. mansoni was maintained by pas-age through Biomphalaria glabrata snails and golden hamstersMesocricetus auratus). Cercariae were released from infected snailsnd harvested on ice. They were washed three times by resuspen-ion in 30 ml of Hank’s Balanced Salt Solution (Gibco-BRL) in a corexube (Corning) and centrifugation for 10 min at 1500 × g. Schisto-omula were obtained in vitro [9] and were maintained in cultureor 3 h. Adult worms were obtained by whole-body perfusion of-week infected hamsters [10]. Eggs were obtained from the liv-rs of infected hamsters and hatched out under light to obtainiracidia [11]. The ChIP protocol, based on [6] was adapted to S.ansoni as follows. We used 25,000 larvae (cercariae, schistoso-ula or miracidia) or 50 adult worms for the input and for each

mmunoprecipitation. Parasites were treated with 1% formalde-yde (final concentration in 10 mM sodium phosphate buffer pH.0 containing 0.15 M NaCl (PBS)) for 10 min at room tempera-ure to cross-link bound proteins to DNA. Glycine (0.125 M finaloncentration) was added for 5 min at room temperature to stophe reaction. The parasite pellet was washed twice with PBS andesuspended in 500 �l lysis buffer (10 mM EDTA, 50 mM Tris–HClH 8, 1% SDS and protease inhibitors (Sigma)). In the case ofdult worms only, a Dounce homogeniser was used before theltrasonication step to disrupt the parasites. The suspension wasonicated on ice using a Branson Digital 400w s-450D Sonifierith a 3 mm microprobe to shear DNA into 100–1000 pb frag-ents. Optimised conditions using this apparatus are given in the

egend to Fig. 1, and the figure shows typical results of DNA frag-entation obtained. However, it must be emphasized that these

onditions should be experimentally determined for each typef sonifier and probe used. Between each pulse, ultrasonicationas stopped for 1 min to cool the suspension and avoid denatu-

ation of proteins. The sonicate was centrifuged at 13,000 rpm for0 min at 4 ◦C and the supernatant was diluted 10-fold in immuno-recipitation buffer (2 mM EDTA, 100 mM NaCl, 20 mM Tris–HClH 8.1, 0.5% Triton X-100 and protease inhibitors). It should beoted that, in the case of investigations of histone acetylation,he addition of sodium butyrate (20 mM), an inhibitor of his-one deacetylases, to all buffers has been recommended [12] butn our hands and specifically using an antibody directed againsteveral acetylated lysine residues in histone H4 (see below) we

ave not found that this addition affects the results obtained. The

nput, the same number of parasites treated by ultrasonication butot subjected to immunoprecipitation, was stored at −20 ◦C untilsed in the PCR assays. To reduce non-specific background, theiluted sonicate was pre-cleared with 10 �g salmon sperm DNA,

ig. 1. Fragmentation of schistosome DNA by ultrasonication. Formaldehyde-fixed ptal Sonifier. The conditions (total duration of pulses/duration of individual pulses/aercariae—3 min/30 s/30%; schistosomula—3 min/30 s/10%; adult worms—4 min/30 s/10%ion. The samples were extracted using phenol/chloroform and separated on Tris–acetatekb plus DNA ladder) are shown on the left-hand lanes.

ical Parasitology 166 (2009) 77–80

20 �l non-immunised rabbit serum, 1 mg/ml BSA and 5 mg pro-tein A-Sepharose (Sigma) for 2 h at 4 ◦C with rotation. At this andsubsequent steps, the material was transferred to a clean tubebefore proceeding with the next step in order to minimize back-ground due to DNA adsorbed onto the tubes. The agarose waspelleted by brief centrifugation and the supernatant was then incu-bated with rotation overnight at 4 ◦C with 10 �g salmon spermDNA, 1 mg/ml BSA, 5 mg protein A-agarose and antibody (non-immunised rabbit serum, or rabbit anti-acetyl histone H4 serum;ChIP-grade, Upstate). The extreme conservation of S. mansoniH4 (Sm06454; http://www.genedb.org/genedb/smansoni/) com-pared to the human orthologue (100% peptide sequence identity),including the epitope recognized by the commercial antibody (notshown), allows the use of this antiserum. However, we further veri-fied the specificity of the antibody by western blotting (not shown),confirming that it indeed recognizes acetylated S. mansoni H4. Theagarose was again pelleted and subjected to: 2 washes in buffer 1(2 mM EDTA, 20 mM Tris–HCl pH 8, 150 mM NaCl, 0.1% SDS and 1%Triton X-100); 2 washes in buffer 2 (2 mM EDTA, 20 mM Tris–HCl pH8, 500 mM NaCl, 0.1% SDS and 1% Triton X-100); 2 washes in buffer3 (1 mM EDTA, 10 mM Tris–HCl pH 8, 0.1% Nonidet, 0.25 M LiCl and1% sodium deoxycholate) and 2 final washes in TE (1 mM EDTA,10 mM Tris–HCl pH 8). The histone–DNA complex was eluted fromthe protein A-agarose by adding 250 �l freshly prepared elutionbuffer (1% SDS, 0.1 M NaHCO3) mixing briefly (vortex) and incubat-ing at room temperature for 15 min with rotation. The supernatantwas transferred to a new tube and the elution step was repeated.The histone–DNA cross-linking was reversed by heating both theimmunoprecipitated complexes and the input at 65 ◦C overnight.Both samples were then incubated at 45 ◦C for 1 h with 20 �g pro-teinase K. Finally, DNA was purified using a Wizard® SV Gel andPCR Clean-Up System (Promega) according to the manufacturer’sinstructions.

The input and immunoprecipitated DNA were initially usedas a template for conventional PCR amplification of thepromoter regions of the SmHDAC1 gene (the region imme-diately upstream of the transcription start site: nucleotides7411–7824 of Smp contig001596; http://www.sanger.ac.uk/cgi-bin/blast/submitblast/s mansoni) and SmFtz-F1 gene ([8], acces-sion number AY028787; nucleotides 850–1160). Oligonucleotide

primers used are given in the legend to Fig. 2. Results shown in Fig. 2demonstrate that fragments of the expected size were amplifiedfrom cercarial and miracidial chromatin immunoprecipitated withthe anti-acetylated H4 antibodies, but not with non-immunizedrabbit serum. Controls carried out using a rabbit antiserum against

arasites were subjected to ultrasonication using a Branson 400w s-450D Dig-mplitude) were optimised for each life-cycle stage: miracidia—3 min/10 s/10%;. Adult worms were disrupted using a Dounce homogeniser prior to ultrasonica-1% agarose gels stained with ethidium bromide. Molecular size markers (Invitrogen

S. Caby, R.J. Pierce / Molecular & Biochemical Parasitology 166 (2009) 77–80 79

Fig. 2. PCR amplification of promoter regions of the SmHDAC1 (lanes 1–6) and SmFtz-F1 (lanes 7–12) genes from immunoprecipitated chromatin from miracidia (lanes 1–3and 7–9) and cercariae (lanes 4–6 and 10–12) using oligonucleotides: SmHD1P2F1, 5′-ATG ACG CGG GAA AAT GTA GTG AA-3′; SmHD1P2R1, 5′-TT GGA GGC TTG AGA CGGT , 5′-Gp 4, 7, 10s H4 anS m the

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AT GG-3′; PromoFF1F, 5′-CAA TAA AAT CTT ATG GTA AGG GCT CAA-3′; PromoFF1Rolymerase (Invitrogen) for 38 cycles at 95 ◦C 30 s, 55 ◦C 30 s and 72 ◦C 45 s. Lanes 1,erum; lanes 3, 6, 9, 12: material immunoprecipitated with anti-acetylated histoneeparate gels are shown for PCR products amplified from the two promoters but fro

n irrelevant antigen (human lactoferrin; Sigma) gave identicalesults (not shown).

In order to quantify the levels of acetylated H4 at the pro-oters at different life-cycle stages, we next carried out real-time

uantitative PCR on the immunoprecipitated DNA and the inputs.he MESA Green qPCR Master Mix Plus (Eurogentec) and the ABIrism 7000 sequence detection system (Applied Biosystems) weresed. Primers specific for S. mansoni HDAC1 and SmFTZ-F1 pro-oters were designed (within the regions amplified in the initial

CR experiment, see legend to Fig. 3) by the Primer Express Pro-ram (Applied Biosystems) and used for amplification in triplicatessays. PCR efficiencies were determined for each primer pair usingtandard curves as previously described [13]. The primers used allhowed very high and comparable efficiency rates and the iden-ity of PCR products was checked by sequencing. PCR was carriedut on 1/100 and 1/1000 dilutions of the input and immunopre-ipitated samples were diluted 1/25 before analysis. Results for themmunoprecipitated fragments were calculated compared to the Ctalues obtained for the input samples in each case as described [12]nd are expressed as a percentage of the input. Results of a typicalxperiment (Fig. 3) show that both promoters have a relatively highevel (around 10% of input) of acetylated H4 in both miracidia and

ercariae. Using parasites treated with the HDAC inhibitor, tricho-tatin A (manuscript in preparation) we obtained a maximum levelf around 20% of input using the antibody against acetylated H4.ackground levels, obtained using non-immunised rabbit serum inhe immunoprecipitation step, were always lower than 0.25% input.

ig. 3. Quantitative real-time PCR of promoter regions of SmHDAC1 and SmFtz-F1enes. Primers used to amplify the SmHDAC1 and SmFTz-F1 promoter regions were,espectively: SmHDAC1P2F2, 5′-TCA GCA ACT AAC AAA AAG GTC TGC-3′; SmH-AC1P2R2, 5′-CAA AGG ACC CAA ATA CAT AAC TTC AC-3′; FTZF1prom1F, 5′-CTC GCTTG AAA AGC AGT GCT-3′; FTZF1promR, TGA TCT TCG CTA CTA ACG AGC CTT AT-3′ .esults of a representative experiment are expressed as the percentage of the input

mmunoprecipitated by anti-acetylated histone H4 antiserum for miracidia (Mir)nd cercariae (Cerc). The level obtained with the material immunoprecipitated withon-immunized rabbit serum never exceeded 0.25% of input.

AC CTT CCA AAG TGA CCT TCG AA-3′ . PCR was carried out using recombinant Taq: input; lanes 2, 5, 8, 11: material immunoprecipitated with non-immunized rabbittiserum. PCR products were separated on 1% agarose gels as in the legend to Fig. 1.same ChIP experiment.

The comparison of miracidia and cercariae indicates that the formerhave a higher level of acetylation of both the SmHDAC1 and SmFtz-F1 promoters. This may be correlated with the increased transcriptlevels of both genes in miracidia compared to cercariae [5,13] butwe need to investigate more specific histone marks, such as acety-lated H4K16 or H3K9 before drawing any conclusions. Moreover,our results emphasize that it is necessary to carry out real-timequantitative PCR in order to evaluate the levels of modified histonesat different stages and on different promoters.

The method we describe is relatively simple to execute andis reproducible. In comparison with the native-ChIP methoddescribed by Cosseau et al. [14] in this journal, our method requiresmore starting material due to loss during the ultrasonication step,although the numbers of parasites (25,000 larvae, 50 adult worms)could certainly be reduced. The formaldehyde fixation of proteinsbound to DNA, however, is an advantage for the investigation of pro-teins (transcription factors, cofactors) that are more loosely boundthan are histones and particularly those only bound indirectly inmulti-protein complexes.

Acknowledgements

The work was supported by Inserm (U547), the Institut Pasteurde Lille, the CNRS and the ANR (grant number ANR-07-BLAN-0119-02 2008-10).

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