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• W2000166892 abstract "BioTechniquesVol. 40, No. 1 BenchmarksOpen AccessWhole embryo chromatin immunoprecipitation protocol for the in vivo study of zebrafish developmentEmmanuelle Havis, Isabelle Anselme & Sylvie Schneider-MaunouryEmmanuelle Havis*Address correspondence to Emmanuelle Havis, Université Pierre et Marie Curie, CNRS UMR 7622, Bât. C, 7ème étage, 9, Quai Saint Bernard, 75005 Paris, France. e-mail: E-mail Address: emmanuelle.havis@snv.jussieu.frCentre National de la Recherche Scientifique UMR 7622, Université Pierre et Marie Curie, Paris, FranceSearch for more papers by this author, Isabelle AnselmeCentre National de la Recherche Scientifique UMR 7622, Université Pierre et Marie Curie, Paris, FranceSearch for more papers by this author & Sylvie Schneider-MaunouryCentre National de la Recherche Scientifique UMR 7622, Université Pierre et Marie Curie, Paris, FranceSearch for more papers by this authorPublished Online:21 May 2018https://doi.org/10.2144/000112098AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail The chromatin immunoprecipitation (ChIP) assay is a powerful technique to determine the presence of a protein on a specific DNA region, depending on the cellular context. This method is commonly used to study chromatin extracted from cell culture (1). Briefly, it is based on three major steps: (i) cell fixation with formaldehyde to crosslink proteins and DNA; (ii) chromatin fragmentation by sonication; and (iii) selective immunoprecipitation of DNA-protein complexes with a specific antibody. The immunoprecipited DNA is analyzed by PCR. Recently, the ChIP assay has been used to study in vivo the mechanisms of transcriptional regulation by thyroid hormone receptors during Xenopus laevis development (2,3).We adapted for the first time the ChIP assay to whole Danio rerio (zebrafish) embryos, in order to study the mechanisms controlling early brain patterning. The krox20 (krx20 in zebrafish) gene is involved in this process, and its expression pattern has been studied previously by in situ hybridization (4). krx20 expression is first activated at 100% epiboly [10 h postfertilization (hpf)], and the gene is expressed in two hindbrain segments until around 30 hpf. A good indicator of the activation of a gene is the hyperacetylation of its promoter (5). We investigated the level of histone acetylation on the krx20 promoter by ChIP, using an antibody directed against acetylated histone H4 (Upstate Biotechnology, Lake Placid, NY, USA) at different stages of zebrafish development, from 30% epiboly (4.7 hpf) to six-somites (12 hpf).First, to remove chorions, the embryos were incubated for 2 min at room temperature in 1 mg/mL Pronase in Embryo medium E3 (6), then transfered in E3 where the chorion was removed with forceps. To eliminate the lipid-rich vitellus and isolate nuclei, dechorionated embryos were homogenized in 1 mL of a 2.2 M sucrose, 3 mM CaCl2, 10 mM Tris-HCl, pH 7.5, 0.5% Triton® X-100, 1 mM phenylmethylsulphonyl fluoride (PMSF), 1 µg/mL aprotinin, 1 µg/mL pepstatin solution (7) and centrifuged at 100,000× g for 3 h at 4°C in a TL55 rotor (Beckman Coulter, Fullerton, CA, USA). Next, pelleted nuclei were resuspended in 1 mL of a 0.25 M sucrose, 10 mM Tris-HCl, pH 7.5, 3 mM CaCl2, 1 mM PMSF, 1 µg/mL aprotinin, 1 µg/mL pepstatin solution (2), then centrifuged at 6000× g for 5 min at 4°C, and resuspended in 360 µL of this solution. Proteins were cross-linked to DNA by the addition of 10 µL of a 37% formaldehyde solution, with a 10-min incubation on ice, then a 20-min incubation at room temperature. The nuclei were pelleted at 6000× g for 5 min at 4°C, resuspended in 400 µL sodium dodecyl sulfate (SDS) lysis buffer with anti-proteases (2) (this buffer is provided in the ChIP assay kit; Upstate Biotechnology), and incubated for 10 min on ice before sonication. The average amount of chromatin recovered per embryo was 0.4 µg at the blastula stage, 1 µg at 100% epiboly, 1.3 µg at the four-somite stage, and 1.5 µg at 24 hpf.The ChIP assay requires the segmentation of the chromatin into 200- to 800-bp immunoprecipitable fragments. We determined the optimal sonication conditions for whole embryos at different stages (Figure 1). The embryo lysate was subjected to 16 sets of 10-s pulses using a Vibra Cell sonicator 75041 (Bioblock Scientific, Illkirch, France) equipped with a 2-mm tip and set to 150 W. The lysate was left for 4 min on ice between each pulse. Two 10-µL samples were taken from the sonicated lysate after a different number of pulses. One of the two series of samples was centrifuged at 15,500× g for 10 min at 4°C to recover the supernatant. The two series were then loaded on a 1% agarose gel with ethidium bromide (Figure 1). We observed that shearing zebrafish chromatin into 200-to 800-bp fragments required 14 pulses independent of the developmental stage, from blastula to 24 hpf, for chromatin amounts between 50 and 150 µg. Neither RNase treatment nor the use of embryos from different stages modified the sonication profile (data not shown).Figure 1. Sonication profile of whole zebrafish embryo chromatin.(A) Ten-microliter samples of a chromatin solution from 60 zebrafish embryos at the four-somite stage, taken at a different number of sonication pulses, were loaded on agarose gel. The size of the chromatin fragments decreases progressively with the number of pulses. (B) Because the chromatin immunoprecipitation (ChIP) assay was performed after centrifugation of the sonicated chromatin solution, we centrifuged samples before loading the supernanant on an agarose gel. The size of chromatin fragments decreased progressively with the number of pulses, and high molecular weight fragments were pelleted. Fourteen pulses were necessary to obtain fragments of the appropriate size.We then carried out the ChIP assay to determine the acetylation level of the krx20 promoter during zebrafish development. Each immunoprecipitation and input (chromatin isolated before immunoprecipitation) required 10 µg chromatin. The IP protocol was done using the ChIP assay kit mentioned above, in the conditions recommended by the supplier. After the ChIP protocol, the recovered DNA was resuspended in 20 µL water for ChIP samples and 40 µL for the input. Eight micrograms of anti-acetylated histone H4 antiserum were added to 1 mL chromatin solution, from 30% epiboly (4.7 hpf), one-somite stage (11 hpf), four-somite stage (11.5 hpf), and six-somite stage (12 hpf) embryos. All PCRs were performed in 50 µL with 25 µL PCR Master Mix (AB gene, Courtaboeuf, France), 2 µL each primer (2 µM each; MWG-Biotech, Roissy, France), and 2 µL of the immunoprecipitated DNA sample. After a first denaturation of 5 min at 94°C, between 35 and 40 cycles were performed as follows: 1 min at 94°C, 1 min at annealing temperature, and 1 min at 72°C. A final elongation step of 5 min at 72°C followed. PCR analysis was performed using primers framing the -425 to -115 region of the krx20 promoter (Table 1). Forty cycles of PCR were performed, with an annealing temperature of 56°C. We observed that histone H4 on the krx20 promoter was not acetylated at 30% epiboly (Figure 2A). Acetylation was observed from the one-somite stage onward and persisted at the four- and six-somite stages (Figure 2A). These results are consistent with the krx20 expression pattern observed by in situ hybridization (4). To demonstrate that the procedure does not preferentially isolate nuclei from one tissue or cell type relative to others, we studied the acetylation of histone H4 in six-somite stage embryos on the promoters of myoD and keratin8, genes that are specifically expressed, respectively, in somitic mesoderm and in the enveloping layer of the embryo (8,9). For primers framing the -121 to +9 region of the myoD promoter (Table 1), 35 cycles were performed with an annealing temperature of 55 °C. For primers framing the -471 to -255 region of the keratin8 promoter (Table 1), 40 cycles were performed, with an annealing temperature of 56°C. Histone H4 was found to be acetylated on the promoters of these two genes (Figure 2A).Figure 2. Chromatin immunoprecipitation (ChIP) analysis of histone H4 acetylation on the krx20, keratin8, and myoD promoters and recruitment of Meis1.1 on the hoxb2a promoter, in whole zebrafish embryos.(A) ChIP assay performed on embryos, at different early stages of development with an H4-acetylated antiserum. Hyperacetylation of histones H4 on the krx20 promoter was observed from the one-somite stage onward, but not at 30% epiboly. Hyperacetylation was observed at the six-somite stage on keratin8 and myoD promoters. (B) A ChIP assay on four-somite stage embryos injected with a Myc-Meis1.1 RNA using an anti-Myc antibody. Myc-Meis1.1 binds to the hoxb2a promoter, in the -1164 to -861 region, which contains the consensus Meis binding site. Myc-Meis1.1 does not bind on the -654 to -417 region of the ZPC promoter, nor on the-471 to -255 region of the keratin8 promoter, which do not contain Meis binding sites. (C) A ChIP assay on non-injected four-somite stage embryos with a Myc-Meis1.1 RNA using an anti-Myc antibody. “Input” corresponds to chromatin isolated before immunoprecipitation and shows that experiments were done with equal chromatin amounts. “No DNA” corresponds to a PCR performed on water, to show that there is no PCR contamination. “No antibody” corresponds to a ChIP experiment performed without an antibody, to show that there is no nonspecific binding of chromatin on the agarose beads.Table 1. PCR PrimersTo illustrate the feasibility of ChIP assay on whole zebrafish embryos to study transcription factor binding on promoters, we investigated the binding of the Meis 1.1 protein on the hoxb2a promoter. Indeed, hoxb2a and meis1.1 are both expressed during zebrafish hindbrain segmentation (10). Moreover, in mouse embryos, Meis proteins bind to consensus sites of the hoxb2 promoter and mediate its expression in the hindbrain (11). In zebrafish, the characterization of the hoxb2a promoter led to the identification of consensus binding sites for Meis proteins (12). Because no antibody against Meis1.1 is available, we overexpressed a Myc-tagged Meis1.1 protein in zebrafish embryos by RNA injection at the two-cell stage. Consequently, the ChIP assay was performed by adding 8 µg of an antibody directed against Myc (Ozyme, St. Quentin en Yvelines, France) in a 1-mL solution containing 10 µg chromatin. PCR primers were chosen framing the -1164 to -861 region (Table 1), which contains the Meis binding site. We performed 40 cycles of PCR with an annealing temperature of 50°C. As expected, we could observe the in vivo binding of Meis1.1 on the hoxb2a promoter from Myc-Meis 1.1-injected embryos, whereas in the same experimental conditions, no binding was observed on chromatin from uninjected embryos (Figure 2, B vs. C).As a negative control, we showed the absence of binding of Myc-Meis 1.1 on the promoter of ZPC, a gene expressed only in the oocytes of mature females (13), and on the keratin8 promoter, where no Meis consensus binding site was identified and therefore it should not bind (Figure 2B). The PCR conditions were, for the ZPC promoter, 40 cycles with an annealing temperature of 53°C, and for the keratin8 promoter, 40 cycles with an annealing temperature of56°C.A very promising application for the ChIP assay is the identification of new target genes of transcription factors. Indeed, ChIP assay products can be cloned and sequenced or spotted on genomic microarrays in order to determine on which DNA sequences the immunoprecipitated protein was fixed (14). Our ChIP protocol adapted to zebrafish embryos will be helpful to supplement knowledge in developmental biology, physiology, or drug discovery, all disciplines for which zebrafish is a model of particular interest (15).AcknowledgmentsThis work was supported by the Centre National de la Recherche Scientifique, the Université Pierre et Marie Curie (Paris 6), the Association pour la Recherche sur le Cancer, and a postdoctoral fellowship from the Fondation pour la Recherche Médicale (toE.H.).Competing Interests StatementThe authors declare no competing interests.References1. Das, P.M., K. Ramachandran, J. vanWert, and R. Singal. 2004. Chromatin immunopre-cipitation assay. BioTechniques 37:961–969.Link, CAS, Google Scholar2. Sachs, L.M. and Y.B. Shi. 2000. Targeted chromatin binding and histone acetylation in vivo by thyroid hormone receptor during amphibian development. Proc. Natl. Acad. Sci. USA 97:13138–13143.Crossref, Medline, CAS, Google Scholar3. Havis, E., L.M. Sachs, and B.A. Demeneix. 2003. Metamorphic T3-response genes have specific co-regulator requirements. EMBO Rep. 4:883–888.Crossref, Medline, CAS, Google Scholar4. Oxtoby, E. and T. Jowett. 1993. Cloning of the zebrafish krox-20 gene (krx-20) and its expression during hindbrain development. Nucleic Acids Res. 21:1087–1095.Crossref, Medline, CAS, Google Scholar5. Sterner, D.E. and S.L. Berger. 2000. Acetylation of histones and transcription-related factors. Microbiol. Mol. Biol. Rev. 64:435–459.Crossref, Medline, CAS, Google Scholar6. Nüsslein-Volhard, C. and R. Dahm. 2002. Zebrafish. Practical Approach. Oxford University Press, New York.Google Scholar7. Almouzni, G., S. Khochbin, S. Dimitrov, and A.P. Wolffe. 1994. Histone acetylation influences both gene expression and development of Xenopus laevis. Dev. Biol. 165:654–669.Crossref, Medline, CAS, Google Scholar8. Weinberg, E.S., M.L. Allende, C.S. Kelly, A. Abdelhamid, T. Murakami, P. Andermann, O.G. Doerre, and D.J. Grunwald. 1996. Developmental regulation of zebrafish MyoD in wild-type, no tail and spadetail embryos. Development 122:2711–280.Crossref, Google Scholar9. Gong, Z., B. Ju, X. Wang, J. He, H. Wan, P.M. Sudha, and T. Yan. 2002. Green fluo rescent protein in germ-line transmitted transgenic zebrafish under a stratified epithelial promoter from keratin8. Dev. Dyn. 223:204–215.Crossref, Medline, CAS, Google Scholar10. Moens, C. and V.E. Prince. 2002. Constructing the hindbrain: insights from the zebrafish. Dev. Dyn. 224:1–17.Crossref, Medline, Google Scholar11. Jacobs, Y., C.A. Schnabel, and M.L. Cleary. 1999. Trimeric association of Hox and TALE homeodomain proteins mediates Hoxb2 hindbrain enhancer activity. Mol. Cell. Biol. 19:5134–5142.Crossref, Medline, CAS, Google Scholar12. Scemama, J.L., M. Hunter, J. McCallum, V. Prince, and E. Stellwag. 2002. Evolutionary divergence of vertebrate Hoxb2 expression patterns and transcriptional regulatory loci. J. Exp. Zool. 294:285–299.Crossref, Medline, CAS, Google Scholar13. Onichtchouk, D., K. Aduroja, H.G. Belting, L. Gnügge, and W. Driver. 2003. Transgene driving GFP expression from the promoter of the zona pellucida gene zpc is expressed in oocytes and provides an early marker for gonad differenciation in zebrafish. Dev. Dyn. 228:393–404.Crossref, Medline, CAS, Google Scholar14. Weinmann, A.S. and P.J. Farnham. 2002. Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation. Methods 26:37–47.Crossref, Medline, CAS, Google Scholar15. Zon, L.I. and R.T. Peterson. 2005. In vivo drug discovery in the zebrafish. Nat. Rev. Drug Discov. 4:35–44.Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByTMEM8C-mediated fusion is regionalized and regulated by NOTCH signalling during foetal myogenesis10 January 2022 | DevelopmentChromatin Immunoprecipitation3 August 2020 | Cold Spring Harbor Protocols, Vol. 2020, No. 8Bloody Zebrafish: Novel Methods in Normal and Malignant Hematopoiesis15 October 2018 | Frontiers in Cell and Developmental Biology, Vol. 6Egr1 deficiency induces browning of inguinal subcutaneous white adipose tissue in mice23 November 2017 | Scientific Reports, Vol. 7, No. 1Developmentally arrested Austrofundulus limnaeus embryos have changes in post-translational modifications of histone H3Journal of Experimental Biology, Vol. 23The RNA-binding protein Rbm24 is transiently expressed in myoblasts and is required for myogenic differentiation during vertebrate developmentMechanisms of Development, Vol. 134Bcl6a function is required during optic cup formation to prevent p53-dependent apoptosis and colobomata12 May 2013 | Human Molecular Genetics, Vol. 22, No. 17Sim2 prevents entry into the myogenic program by repressing MyoD transcription during limb embryonic myogenesisDevelopment, Vol. 139, No. 11ISL1 Directly Regulates FGF10 Transcription during Human Cardiac Outflow Formation27 January 2012 | PLoS ONE, Vol. 7, No. 1Analysis of In Vivo Transcription Factor Recruitment by Chromatin Immunoprecipitation of Mouse Embryonic Kidney19 January 2012An evolutionarily conserved kernel of gata5, gata6, otx2 and prdm1a operates in the formation of endoderm in zebrafishDevelopmental Biology, Vol. 357, No. 2EGR1 and EGR2 Involvement in Vertebrate Tendon DifferentiationJournal of Biological Chemistry, Vol. 286, No. 7Integration of Telencephalic Wnt and Hedgehog Signaling Center Activities by Foxg1Developmental Cell, Vol. 16, No. 4A functional interaction between Irx and Meis patterns the anterior hindbrain and activates krox20 expression in rhombomere 3Developmental Biology, Vol. 327, No. 2Functional analysis of the evolutionarily conserved cis-regulatory elements on the sox17 gene in zebrafishDevelopmental Biology, Vol. 326, No. 2Fish’n ChIPs: Chromatin Immunoprecipitation in the Zebrafish Embryo26 June 2009Rostral hindbrain patterning involves the direct activation of a Krox20 transcriptional enhancer by Hox/Pbx and Meis factorsDevelopment, Vol. 135, No. 20Receptor-Mediated Mechanisms of Toxicity10 September 2010Xenobiotic- and vitamin D-responsive induction of the steroid/bile acid-sulfotransferase Sult2A1 in young and old mice: The role of a gene enhancer in the liver chromatinGene, Vol. 386, No. 1-2 Vol. 40, No. 1 Follow us on social media for the latest updates Metrics History Received 5 September 2005 Accepted 9 November 2005 Published online 21 May 2018 Published in print January 2006 Information© 2006 Author(s)AcknowledgmentsThis work was supported by the Centre National de la Recherche Scientifique, the Université Pierre et Marie Curie (Paris 6), the Association pour la Recherche sur le Cancer, and a postdoctoral fellowship from the Fondation pour la Recherche Médicale (toE.H.).Competing Interests StatementThe authors declare no competing interests.PDF download" @default.
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