Chromatin Immunoprecipitation Assay for Early Zebrafish Embryos (Prot59)

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uio.no Publication date: 23 October, 2012 Last reviewed: 10 October 2012, Ferenc Müller, Institute of Biomedical Research, School of Clinical and Experimental Medicine, College of Medical and Dental

1 Chromatin Immunoprecipitation Assay for Early Zebrafish Embryos (Prot59) Leif C. Lindeman Leif C. Lindeman 1, Philippe Collas 1 1 Stem Cell Epigenetics Laboratory (Collas lab), Institute of Basic Medical Sciences, University of Oslo, PO Box 1112 Blindern, 0317 Oslo, Norway l.c.lindeman@medisin.uio.no Publication date: 23 October, 2012 Last reviewed: 10 October 2012, Ferenc Müller, Institute of Biomedical Research, School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, B1S 2TT Birmingham, UK Introduction Zebrafish (Danio rerio) is well established as a model organism to study embryogenesis. Practical advantages of using zebrafish are that hundreds of synchronized embryos can easily be collected, embryos are transparent, development is rapid and external, and its genome is sequenced. The 9 th assembly of the zebrafish genome (Zv9) reports 1.41 billion base pairs with ~24,000 protein-coding genes. Information on the Danio rerio genome assembly can be found at A unique feature of zebrafish (and of anamniote vertebrates) is a developmental period of several hours after fertilization in the quasi-absence of on-going transcription. In zebrafish, this developmental period lasts for 3.3 h during which the embryo undergoes 10 rounds of synchronous 1

2 cell divisions. Zygotic genome activation (ZGA) occurs at the ~1,000-cell stage, at the mid-blastula transition (MBT) (Tadros and Lipshitz, 2009) (see for a description of zebrafish developmental stages). This 3.3 h pre-mbt period provides a unique opportunity to identify epigenetic processes, including enrichment in post-translationally modified histones, associated with the establishment of the embryonic gene expression program (Lindeman et al., 2011). A widely used method for identifying occupancy of the genome by modified histones is chromatin immunoprecipitation (ChIP). However, a challenge with ChIP assays from early-stage zebrafish embryos is access to chromatin, due to a thick glycoprotein chorion which protects the embryo, the large amount of yolk (which supports embryo development) and the low nuclearcytoplasmic ratio in embryonic cells particularly prior to the MBT. In addition, we have found that the use of protease (pronase) to remove the chorion in order to isolate embryonic cells and prepare chromatin, as carried out in earlier protocols (Hart et al., 2007; Havis et al., 2006; Vastenhouw et al., 2010; Wardle et al., 2006), is detrimental to the efficiency of ChIP (Lindeman et al., 2009), notably by resulting in the degradation in modified histone epitopes such as H3K27me3. We have alleviated the use of protease to isolate embryonic cells, and present here our ChIP protocol for early stage zebrafish embryos (Lindeman et al., 2009). This protocol enables the examination of modified histones by quantitative PCR (ChIP-qPCR) or by hybridization of ChIP DNA to microarrays (ChIP-chip) as early as the 256-cell stage (Lindeman et al., 2011). Limitations of the protocol This protocol has been successfully used for embryos from the 256-cell stage to the 50% epiboly stage (5.3 h post-fertilization). It has however not been thoroughly tested on embryos before or after these stages pending adjustments in the cell isolation procedure. This protocol has been used for ChIP analysis of modified histones and RNA polymerase 2, but has not been tested for ChIP of other transcription factors or other non-histone DNA- bound proteins. For each ChIP (done in a single tube as described below), only up to 10 PCRs can be run (this number may vary pending on developmental stage). 2

3 Procedure Set up zebrafish breeding and collect embryos at developmental time point(s) selected for your analysis according to your laboratory procedures. 1. Cross-linking DNA and proteins 1.1 Using a transfer pipette, transfer 500 embryos (see note 1) in PBS containing 20 mm Na-butyrate and protease inhibitors (1:100 dilutions) into a 5 ml syringe fitted with a 21G needle (see note 2) 1.2 Let embryos sink to the bottom of the syringe and remove the PBS, leaving ~0.5 ml above the embryos 1.3 Force embryos through the needle into a 1.5 ml tube; this is usually sufficient to dissociate all cells 1.4 Add formaldehyde from a 36.5% stock to 1% final concentration, vortex and incubate for 8 min at room temperature 1.5 Add glycine from a 1.25 M stock to M final concentration to quench excess formaldehyde; vortex and incubate on ice for 5 min 1.6 Centrifuge at 470 g for 10 min at 4 o C in a table top centrifuge; discard the supernatant 1.7 Add 500 µl ice-cold PBS supplemented with protease inhibitors diluted 1:100; resuspend cells by vortexing, centrifuge as in step 1.6 and discard the supernatant 1.8 Repeat step 1.7 once 1.9 At this stage cells can be stored as a pellet at -80 o C for several weeks or months (see note 3); cells can also be shipped in dry ice 2. Preparation of antibody-magnetic bead complexes 2.1 Wash Dynabeads twice in RIPA buffer using a magnetic rack to collect them 2.2 Prepare a 1:1 (vol:vol) Dynabeads slurry in RIPA buffer 2.3 For each ChIP, add in a 200 µl tube: 90 µl RIPA buffer, 10 µl Dynabeads slurry, and ChIP antibody (we use 2.4 µg anti-histone ChIP-grade antibody) (see note 4) 2.4 For negative controls, proceed as above without adding antibody or with an irrelevant antibody 2.5 Incubate on a rotator at 40 rpm, 2 h, 4 o C 3

4 3. Chromatin preparation 3.1 To a tube containing cross-linked cells, add lysis buffer to a total volume 300 µl and resuspend cells with a 1 ml pipette from which the tip has been cut to enlarge the opening; incubate on ice for 5-10 min 3.2 Sonicate the sample on ice using a sonication regime that results in all cells and nuclei being lysed (when setting up the procedure, lysis should be monitored by removing a drop to a microscope slide) (see note 5) 3.3 Centrifuge at 12,000 g, 10 min, 4 o C in a table-top centrifuge; transfer the supernatant (chromatin) into a 1.5 ml tube 3.4 Vortex, remove 2 µl of chromatin to measure absorbance at 260 nm (A 260 ) with a Nanodrop using lysis buffer with additives as blank 3.5 Dilute chromatin to 0.25 U A 260 in RIPA ChIP buffer and mix by vortexing); chromatin can be stored at -80 o C (see note 6) 4. Immunoprecipitation and washes of the ChIP material 4.1 Spin tubes containing antibody-bead complexes in a minicentrifuge for 1-2 sec to collect any beads trapped in the lid; capture beads with a chilled magnetic rack and remove the buffer 4.2 Remove the tube strips from the magnetic rack and add 100 µl chromatin to each tube, including the negative control(s), and mix; also add 100 µl chromatin to be used as the input fraction in a 1.5 ml tube. The input fraction should be kept on ice until elution and de-crosslinking 4.3 Incubate on rotator at 40 rpm, 2 h, 4 o C 4.4 Spin the tubes in a minicentrifuge, capture the beads, discard the supernatant, add 100 µl cold RIPA buffer to each tube and release the beads into the buffer; resuspend by tapping the tubes and place tubes on rotator at 40 rpm, 4 min, 4 o C 4.5 Repeat step 4.4 twice 4.6 Spin the tubes in minicentrifuge, capture the beads, remove the supernatant, add 100 µl TE buffer and incubate on rotator at 40 rpm, 4 min, 4 o C 4.7 Spin the tubes in minicentrifuge and transfer the entire content of the tubes into clean 200 µl tubes; capture the beads and discard the TE buffer; this step results in the 4

5 elimination of any antibody-unbound chromatin from tube walls and has been shown to enhance specificity (Dahl and Collas, 2008) 4.8 To each tube, add 150 µl ChIP elution buffer and incubate on thermomixer at 1,300 rpm for 2 h at 68 o C; this step reverses the formaldehyde-induced cross-links 4.9 Spin the tubes, capture the beads, and transfer the eluate from each tube into a clean 1.5 ml tube 4.10 Add 150 µl ChIP elution buffer to the remaining beads and incubate on thermomixer at 1,300 rpm for 15 min at 68 o C 4.11 Spin the tubes, capture the beads, remove the eluate and pool it with the first eluate; collected fractions contain the ChIP ed proteins with associated DNA 4.12 To the pooled eluate (300 µl total), add 200 µl ChIP elution buffer and proceed with DNA isolation as described in Section In parallel to step 4.8 above, add proteinase K to 2 mg/ml to the input chromatin sample and incubate on thermomixer at 1,300 rpm for 15 min at 68 o C 5. Isolation of ChIP and input DNA We purify ChIP DNA by phenol:chloroform:isoamylalcohol extraction. DNA purification kits can also be used although they tend to result in a loss of DNA which can be significant when starting from low numbers of cells for ChIP. They mays also lead to eluated ChIP DNA at a concentration too low for qpcr or processing for microarray hybridization. 5.1 Add 500 µl phenol:chloroform:isoamylalcohol, vortex, and centrifuge at 15,000 g for 5 min; transfer 450 µl of the upper (aqueous) phase to a new tube 5.2 To this aqueous phase, add 450 µl chloroform:isoamylalcohol, vortex and centrifuge at 15,000 g for 5 min; transfer 400 µl of the upper (aqueous) phase to a clean 1.5 ml tube 5.3 To this aqueous phase, add 10 µl acrylamide carrier, 40 µl of a 3 M NaAc stock solution, and 1 ml of 96% or 100% ethanol; mix by vortexing and inversion and place tubes at - 80 o C for 2 h 5.4 Centrifuge at 20,000 g for 20 min at 4 o C 5.5 Discard the supernatant, wash the pellet with 1 ml 70% ethanol, and let the DNA detach from the tube wall; centrifuge at 20,000 g for 10 min, 4 o C and remove the ethanol 5.6 Repeat step 5.5; let the DNA pellet dry 5

6 5.7 Add 50 µl TE buffer and allow DNA to dissolve overnight at 4 o C. At this stage, ChIP DNA can be stored at -20 o C and analyzed by quantitative PCR (see note 7) Notes 1. Number of embryos: we routinely prepare chromatin from batches of 500 embryos at the 256-cell to 50% epiboly stages. The amount of chromatin recovered from later stage embryos is evidently higher than for earlier stage embryos; thus to standardize the amount of chromatin in ChIP assays between developmental stages, a greater dilution of chromatin from later stage embryos is required (see step 3.6). We find that a pool of 500 late MBTstage embryos at the 1,000-2,000-cell state provide enough chromatin for ~50 ChIPs. 2. Na-butyrate is a histone deacetylase inhibitor which is recommended if histone acetylation is to be examined from the chromatin batch to be prepared. If not, it can be omitted. Protease inhibitors always refer to a protease inhibitor cocktail and 1 mm PMSF added freshly just before use. PMSF is added from a 100 mm stock. 3. In our hands, starting with a frozen or fresh cross-linked cell pellet has no noticeable influence on ChIP results. 4. We use one 200-µl tube per ChIP; we find it convenient to use those in an 8-tube strip format (e.g. from Axygen) to enable parallel ChIPs to be done conveniently. We carry out replicate ChIPs in replicate tubes in the strip. A ChIP from one tube usually provides enough DNA for up to 10 qpcrs. For more PCRs, pool DNA from several parallel ChIPs. For ChIP-chip experiments, we usually pool DNA from 5 ChIPs; note that this number may vary with the ChIP antibody used. 5. Sonication: we have used a Sartorius Labsonic M tip sonicator with a 3-mm diameter probe, and a sonication regime of 8 times 30 sec on ice, with 30 sec pauses on ice between sonication rounds. Other sonicators can be used, including bath sonicators (e.g. a Bioruptor from Diagenode); sonication conditions must be tested in this case. 6. Dilution of chromatin to 0.25 U A 260 enables, in a 100 µl reaction volume per ChIP, ten qpcr (see step 5.7). To increase the number of PCRs, or for application to ChIP-chip, several ChIPs can be done in parallel in 200-µl tubes as described in this protocol, or ChIPs can be done in a larger volume with more embryos (e.g. prepare 2,000 embryos in 15 ml 6

7 tubes and use 1.5 ml tubes for immunoprecipitations). If the enriched DNA is intended for microarray analysis (ChIP-chip), RNAse should be added before phenol extraction. 7. Prepare a standard curve with fragmented genomic DNA, using, e.g., ng/µl DNA to cover the range of ChIP DNA samples. We use 5 µl of DNA in each of duplicate quantitative PCRs per ChIP and per genomic site examined. Materials and reagents Materials 1. Zebrafish and facility to produce zebrafish embryos (see Zebrafish International Resource Center; 2. Thermo Plate with adjustable temperature 3. Magnetic rack suited for 200 µl tube strips (Diagenode) µl PCR tubes in eight-tube strip format (Axygen) and 1.5 ml Eppendorf tubes 6. Magnetic holder for 1.5 ml tubes 7. Tip or bath sonicator (see note 5) 8. Rotator in the form of a rotating wheel (e.g., Science Lab Stuart SB3) placed at 4 o C 9. Thermomixer (e.g., Eppendorf) Reagents % formaldehyde 2. Glycine: 1.25 M stock solution prepared in phosphate buffered saline 3. Protease inhibitor mix (Sigma-Aldrich) 4. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich) 5. Na-butyrate: 1 M stock solution in MilliQ water 6. Proteinase K: 20 mg/ml solution in MilliQ water 7. Acrylamide carrier (Sigma-Aldrich) 8. Phenol:chloroform:isoamylalcohol (25:24:1) and chloroform:isoamylalcohol (24:1) 7

8 9. Dynabeads-Protein A or Protein G (Invitrogen). Beads should be well suspended before pipetting. Use Dynabeads-Protein A beads with rabbit IgGs and Dynabeads-Protein G with mouse IgGs. 10. Antibodies to proteins to be immunoprecipitated, preferably ChIP grade. Solutions 1. Lysis buffer: 50 mm Tris HCl, ph 8.0, 10 mm EDTA, 1% (wt/vol) SDS, protease inhibitor mix (1:100 dilution from stock; Sigma-Aldrich), 1 mm PMSF, 20 mm Na-butyrate (optional; see note 2). Protease inhibitor mix, PMSF and Na-butyrate should be added before use. 2. RIPA buffer: 10 mm Tris HCl, ph 7.5, 140 mm NaCl, 1 mm EDTA, 0.5 mm EGTA, 1% Triton X-100, 0.1% SDS, 0.1% (wt/vol) Na-deoxycholate. 3. RIPA ChIP buffer: 10 mm Tris HCl, ph 7.5, 140 mm NaCl, 1 mm EDTA, 0.5 mm EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, protease inhibitor mix (1:100 dilution from stock), 1 mm PMSF. Protease inhibitor mix and PMSF should be added before use. 4. TE buffer: 10 mm Tris HCl, ph 8.0, 10 mm EDTA 5. ChIP elution buffer: 20 mm Tris HCl, ph7.5, 5 mm EDTA, 50 mm NaCl, 1% SDS, 50 µg/ml proteinase K References Dahl JA and Collas P (2008) MicroChIP A rapid micro chromatin immunoprecipitation assay for small cell samples and biopsies. Nucleic Acids Res 36: e15 Hart DO, Raha T, Lawson ND, Green MR (2007) Initiation of zebrafish haematopoiesis by the TATA-box-binding protein-related factor Trf3. Nature 450: Havis E, Anselme I, Schneider-Maunoury S (2006) Whole embryo chromatin immunoprecipitation protocol for the in vivo study of zebrafish development. Biotechniques 40: 34, 36, 38 8

9 Lindeman LC, Andersen IS, Reiner AH, Li N, Aanes H, Østrup O, Winata CL, Mathavan S, Müller F, Aleström P, Collas P (2011) Pre-patterning of developmental gene expression by modified histones before zygotic genome activation. Dev Cell 21: Lindeman LC, Vogt-Kielland LT, Alestrom P, Collas P (2009) Fish'n ChIPs: chromatin immunoprecipitation in the zebrafish embryo. Methods Mol Biol 567: Tadros W and Lipshitz HD (2009) The maternal-to-zygotic transition: a play in two acts. Development 136: Vastenhouw NL, Zhang Y, Woods IG, Imam F, Regev A, Liu XS, Rinn J, Schier AF (2010) Chromatin signature of embryonic pluripotency is established during genome activation. Nature 464: Wardle FC, Odom DT, Bell GW, Yuan B, Danford TW, Wiellette EL, Herbolsheimer E, Sive HL, Young RA, Smith JC (2006) Zebrafish promoter microarrays identify actively transcribed embryonic genes. Genome Biol 7: R71 Reviewer's comments: The reviewer's comments have been incorporated into the protocol. Key words: ChIP; chromatin immunoprecipitation; DNA isolation; embryo; histone modification; mid-blastula transition; sonication; zebrafish 9

1. Cross-linking and cell harvesting

1. Cross-linking and cell harvesting ChIP is a powerful tool that allows the specific matching of proteins or histone modifications to regions of the genome. Chromatin is isolated and antibodies to the antigen of interest are used to determine