Chapter 23 - Springer Link

Chapter 23 Chromatin Immunoprecipitation of Mouse Embryos Anne K. Voss, Mathew P. Dixon, Tamara McLennan, Andrew J. Kueh, and Tim Thomas Abstract During prenatal development, a large number of different cell types are formed, the vast majority of which contain identical genetic material. The basis of the great variety in cell phenotype and function is the differential expression of the approximately 25,000 genes in the mammalian genome. Transcriptional activity is regulated at many levels by proteins, including members of the basal transcriptional apparatus, DNA-binding transcription factors, and chromatin-binding proteins. Importantly, chromatin structure dictates the availability of a specific genomic locus for transcriptional activation as well as the efficiency, with which transcription can occur. Chromatin immunoprecipitation (ChIP) is a method to assess if chromatin modifications or proteins are present at a specific locus. ChIP involves the cross linking of DNA and associated proteins and immunoprecipitation using specific antibodies to DNA-associated proteins followed by examination of the co-precipitated DNA sequences or proteins. In the last few years, ChIP has become an essential technique for scientists studying transcriptional regulation and chromatin structure. Using ChIP on mouse embryos, we can document the presence or absence of specific proteins and chromatin modifications at genomic loci in vivo during mammalian development. Here, we describe a ChIP technique adapted for mouse embryos. Key words: ChIP, Histones, Transcription factors, Activators, Repressors, Chromatin-modifying enzymes

1. Introduction The genetic material consists of the DNA double helix, which is wrapped 1.6 times around a protein core consisting of a (H3–H4)2 tetramer flanked on two sides by H2A–H2B dimers (1). The DNA and the eight core histones form the smallest chromatin particle, the nucleosome. Nucleosomes can be arranged in an open conformation as beads on a string or packaged in tight proximity to each

Ales Vancura (ed.), Transcriptional Regulation: Methods and Protocols, Methods in Molecular Biology, vol. 809, DOI 10.1007/978-1-61779-376-9_23, © Springer Science+Business Media, LLC 2012

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other forming 30-nm fibres. The 30-nm fibres are further organised into fibres of increasing diameter from 60 to 80 nm at interphase to 500–750 nm in metaphase chromosomes (2, 3). The open conformation, euchromatin, is permissive for transcriptional activation, whereas the closed conformation, including the highly condensed heterochromatin, generally silences gene activity, although exceptions to this rule exist (4). Whether chromatin is present in an open or closed conformation depends to a large part on posttranslational modifications to the core histone, in particular histone acetylation (5). To examine the molecular effects of certain interventions, such as mutation of a particular chromatin modifier or DNA-binding transcription factor, on their putative or known target genes in vivo, it is desirable to assess if the intervention causes changes in chromatin structure. In order to understand the changes that take place during embryonic development, it is necessary to perform chromatin immunoprecipitation (ChIP) experiments on embryonic tissues immediately after dissection. The following ChIP protocol was developed based on the detailed instructions accompanying Upstate cat#17-295, but was modified so that it could be used on mouse embryos (6). In principle, a ChIP experiment consists of two parts: (a) immunoprecipitation of DNA sequences and associated proteins and (b) analysis of the chromatin immunoprecipitate, i.e. the analysis of the precipitated DNA or proteins. Analysis of the precipitated DNA is typically performed by quantitative PCR (qPCR) or by one of the mass sequencing techniques. All analysis techniques rely on the quality of the chromatin immunoprecipitate. The protocol described here is concerned with generating high-quality ChIPDNA, which can be analysed using qPCR or mass sequencing.

2. Materials 2.1. Embryo Recovery

1. Autoclaved MT-PBS (16 mM Na2HPO4 2H2O, 4 mM NaH2PO4 H2O, 149 mM NaCl, pH to 7.4). 2. Sterile, 50-ml conical tube (Falcon). 3. Dissection instruments: Two pairs of fine forceps, a pair of fine scissors, sterilised in 95% ethanol for 3 min and air dried. 4. Stereo-dissection microscope. 5. Sterile, 10-cm bacterial Petri dishes. 6. Autoclaved 1.5-ml microcentrifuge tubes. 7. 1,000-μl micropipette. 8. Autoclaved tips for 1,000-μl micropipette.

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1. Sterile, 50-ml conical tubes. 2. MilliQ water, >18 MΩ/cm2, autoclaved. 3. Paraformaldehyde. 4. 1 N KOH (corrosive!). 5. 1.375 M glycine in autoclaved milliQ water. 6. Vortexer. 7. Rocking platform or roller mixer at room temperature. 8. Autoclaved MT-PBS. 9. Complete protease inhibitors (Roche, 11 873 580 001, EDTA free). 10. 0.5 M sodium butyrate (Sigma, B5887, histone deacetylase inhibitor). 11. Dry ice or liquid nitrogen.

2.2.1. Preparation of Fresh 18.5% Formaldehyde

Safety warning: This is a potentially hazardous procedure. Read material safety instructions for all reagents before proceeding. Formaldehyde should be handled in a fume hood. Wear full-face shield, gloves, mask, and lab coat. In case of contact with eyes, flush immediately and continually for 15 min. Follow protocol exactly and with caution. Full-face shield is essential for the heating step. 1. Add 4.8 ml of autoclaved milliQ water to a 50-ml conical tube. 2. Add 0.925 g of paraformaldehyde. 3. Add 35 μl of 1 N KOH. Full-face shield is required for heating step. Use fume hood with shield down. 4. Cap tightly and place in a 400–500-ml glass beaker containing 200 ml of water. 5. Microwave until the water in the beaker begins to boil. Do not allow the water to boil vigorously. Overheating may cause the tube to leak. Remove the tube with caution. 6. Vortex paraformaldehyde solution. 7. Repeat heating and vortexing until paraformaldehyde is in solution. 8. Cool and retain solution on ice; use within 2 h.

2.2.2. Preparation of 100× Protease Inhibitors

1. Dissolve 1 tablet of complete protease inhibitors (Roche, 11 873 580 001, EDTA free) in 400 μl of autoclaved water.

2.2.3. Preparation of MT-PBS with Fresh Inhibitors

1. 38.8 ml MT-PBS. 2. 400 μl of 100× protease inhibitors (1× final concentration).

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3. 800 μl of 0.5 M sodium butyrate (10 mM final concentration, when aiming to detect histone acetylation status). 4. Add protease inhibitors just before use. 2.3. Fragmentation of the Chromatin

1. Autoclaved milliQ water. 2. 0.5 M EDTA, pH 8.0. 3. 1 M Tris–HCl, pH 8.0. 4. 10% (w/v) SDS in autoclaved milliQ water. 5. Autoclaved 5 M NaCl. 6. 10% (v/v) Triton X-100 in autoclaved milliQ water. 7. SDS lysis buffer: 1% SDS, 10 mM EDTA, 50 mM Tris–HCl, pH 8.1. 8. SDS lysis buffer with fresh inhibitors (see 2.3.1). 9. Cooling tube rack. 10. Branson 250 sonifier with a stepped microtip or equivalent. 11. Autoclaved 1.5-ml microcentrifuge tubes (Eppendorf). 12. 1-ml cuvettes for spectrophotometry at 260 and 280 nm. 13. Autoclaved 2-ml microcentrifuge tubes (Eppendorf). 14. ChIP dilution buffer: 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl, pH 8.1, 167 mM NaCl. 15. RNase A, 10 mg/ml in H2O (Sigma); DNase inactivated at 95°C for 10 min.

2.3.1. Preparation of SDS Lysis Buffer with Fresh Inhibitors

1. 38.8 ml SDS lysis buffer. 2. 400 μl of 100× protease inhibitors (1× final concentration). 3. 800 μl of 0.5 M sodium butyrate (10 mM final concentration, when aiming to detect histone acetylation status). 4. Add protease inhibitors just before use.

2.3.2. Preparation of ChIP Dilution Buffer with Fresh Inhibitors

1. 38.8 ml ChIP dilution buffer. 2. 400 μl of 100× protease inhibitors (1× final concentration). 3. 800 μl of 0.5 M sodium butyrate (10 mM final concentration, when aiming to detect histone acetylation status). 4. Add protease inhibitors just before use.

2.4. Chromatin Immunoprecipitation

1. TE buffer: 10 mM Tris–HCl, pH 8.0, 1 mM EDTA, pH 8.0. 2. 50% slurry of pre-absorbed protein A agarose beads/salmon sperm DNA/BSA (protein A for rabbit polyclonal ChIP antibodies; note relative affinity of protein A versus G to antibodies of different species and isotypes).

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3. Low-salt, immune complex wash buffer: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.1, 150 mM NaCl (no inhibitors). 4. High-salt, immune complex wash buffer: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.1, 500 mM NaCl (no inhibitors). 5. 10% Nonidet P-40 in autoclaved milliQ water (or IGEPALCA630). 6. 10% sodium deoxycholate in autoclaved milliQ water. 7. LiCl immune complex wash buffer: 0.25 M LiCl, 1% IGEPALCA630, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris– HCl, pH 8.1 (no inhibitors). 8. Freshly prepared elution buffer: 1% SDS, 0.1 M NaHCO3 (no inhibitors). 9. Rocking platform or roller mixer at 4°C. 10. Rocking platform or roller mixer at room temperature. 11. Autoclaved 2-ml microcentrifuge tubes (Eppendorf). 2.4.1. Pre-absorbed Protein A Agarose Beads

Prepare a day in advance to allow the BSA and DNA to absorb to the resin. 1. Centrifuge 1.5-ml vial containing 500 μl of compact with protein A agarose beads at 2,500 rpm in a Heraeus Biofuge Pico, rotor #3325 [593 ´ g] for 1 min in a microcentrifuge. 2. Remove and discard supernatant. 3. Wash beads with autoclaved MT-PBS pipetting with a widebore tip in order not to damage the beads. 4. Centrifuge vial with beads at 2,500 rpm [593 ´ g] for 1 min in a microcentrifuge. 5. Remove and discard supernatant. 6. Wash beads with autoclaved MT-PBS. 7. Centrifuge vial with beads at 2,500 rpm [593 ´ g] for 1 min as above. 8. Remove and discard supernatant. 9. Wash beads with autoclaved TE, pH 8.0. 10. Remove and discard supernatant. 11. Add autoclaved TE, pH 8.0, to beads 1:1 (v/v; i.e. 50% slurry). 12. Add 500 μg BSA (purified BSA 100×, NEB, B90015, stock 10 mg/ml) per 500 μl of compact bead volume. 13. Add 200 μg sonicated salmon sperm DNA per 500 μl of compact bead volume (stock 10 mg/ml, Stratagene). 14. Incubate overnight on roller at 4°C to allow the BSA and DNA to absorb to the resin.

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2.5. Reversal of Cross Linking and DNA Recovery

1. Autoclaved 5 M NaCl. 2. Autoclaved 0.5 M EDTA, pH 8.0. 3. Autoclaved 1 M Tris–HCl, pH 6.5. 4. Proteinase K 10 mg/ml in autoclaved milliQ water. 5. Phenol, equilibrated to pH 6.5. 6. Chloroform. 7. Isoamyl alcohol. 8. Phenol/chloroform/isoamyl alcohol (50:49:1, v/v/v). 9. Chloroform/isoamyl alcohol (49:1, v/v). 10. Autoclaved 3 M sodium acetate, pH 5.2, in milliQ water. 11. 100% ethanol. 12. 70% ethanol in autoclaved milliQ water. 13. Autoclaved 10 mM Tris–HCl, pH 7.4. 14. Shaking heating block, 45 and 65°C. Safety warning: Read material safety instructions for all reagents before proceeding. Avoid inhalation of fumes and contact with phenol and chloroform. Wear eye protection, lab coat, and gloves. Handle in a fume hood with care. Discard phenol/chloroform according to local regulations.

3. Methods Consideration of appropriate controls. Consideration should be given to the appropriate controls for specific experimental settings. ChIP is commonly conducted to determine the presence of a particular protein, a DNA-binding transcription factor, or a chromatinbinding transcriptional regulator at a specific locus or to determine the genome-wide distribution of such proteins. Commonly used controls are (a) an isotype control, (b) anti-RNA polymerase II control, (c) no-antibody control, and (d) input control. The isotype control is a control antibody of the same immunoglobulin isotype as the specific antibody, but not detecting an antigen in the sample material. This is a negative control detecting non-specific binding of the control antibody to chromatin-associated proteins. It does not necessarily produce the same non-specific component as the experimental antibody. The anti-RNA polymerase II antibody serves as a positive control and is expected to precipitate transcriptionally active genes and within these the transcription start site with preference. The no-antibody control yields chromatin that is precipitated non-specifically by the protein A agarose beads. The input control is derived from the same sample, but has not undergone the precipitation step. Comparison of abundance of genomic regions between input and precipitate (provided the same total amount of

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DNA is used for quantitation of both) allows assessment of the enrichment of specific genomic regions by precipitation with an antibody directed against a particular chromatin-associated protein. If specifically modified histone residues are the primary interest, an antibody precipitating all modified and unmodifed versions of the same histone is often used as a control. When starting material from different experimental groups is used, for example mouse embryos that are either wild type or homozygous for a loss-offunction mutation, wild-type material serves as a control for the mutant material. When analysing the genomic material in the chromatin immunoprecipitate by qPCR, further controls can be introduced. These aim to assess (a) the efficiency of the qPCR reaction and (b) the overall efficiency of the ChIP. The efficiency of the PCR reaction can be assessed by analysing fivefold or tenfold dilution standard curves for each pair of PCR amplification primers and within sample by assessing the accumulation of the PCR product during each PCR cycle (7, 8). The overall efficiency of the ChIP can be assessed if the effects of the experimental variable are expected to result in pronounced differences, e.g. treatment with histone deacetylase inhibitors in the case of detecting specific, acetylated histone residues. Similarly, mutation of loci encoding a histone-modifying enzyme can be expected to result in detectable differences in histone acetylation (6). The presence of unaffected or mildly affected loci in the precipitate can be quantified and can serve as normaliser loci for the affected loci (8). Calibrator samples can serve as controls for experimental groups (8). For example, if wild-type mouse embryos serve as controls for embryos that are mutant for a particular DNAbinding transcription factor X, then a known target gene Y that is downregulated in the embryos mutant for X would be an affected locus and would be expected to exhibit a reduction in transcriptionally active chromatin marks. In contrast, an unrelated housekeeping gene that is expressed at normal levels in both wild-type and mutant embryos would be expected to have normal levels of transcriptionally active chromatin marks and can serve as a normaliser control. The great advantage of such a normaliser control is that it is a within-sample control and allows controlling for variability during the immunoprecipitation procedure, which none of the other types of controls addresses. The most critical aspects of the method. We found that three aspects of the procedure were most essential for success: (a) consistency of chromatin fragmentation, (b) avoiding loss of material during the procedure, and (c) the specificity of the antibody. Chromatin fragmentation can be achieved by sonication and enzymatic cleavage. In Notes 1–3, sonication using two different sonication apparatuses and one enzymatic method are discussed. The optimal chromatin fragment size depends on the aim of the experiment. If the survey of a large region of DNA by qPCR is planned, less-frequent cleavage of chromatin may be desirable. A fragment peak size of 1,000 bp

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would allow the assessment of an average of 1 kb per qPCR primer pair, whereas fragment size of 150 bp would require seven primer pairs to cover the same genomic region. Moreover, transcription factor-binding sites are largely depleted of nucleosomes (9). There fore, fragmentation between most nucleosomes may disrupt a particular target sequence and so impair its detection by PCR. If, however, the aim is to examine chromatin modifications or protein association with a specific, short DNA sequence or mass sequencing of the precipitated DNA is planned, then short chromatin fragments may be preferable. The primer pairs are typically designed to amplify approximately 100 bp in both cases. Avoiding loss of material throughout the procedure is critical if a precipitate-internal control is impractical. A growing number of ChIP-grade antibodies are commercially available. However, good-quality antibodies that display high specificity in immunofluorescence and immunoblotting, but have not been tested in ChIP, may also be used successfully. 3.1. Embryo Recovery

The following procedure is generally subject to animal ethics regulations and a permit specifying the procedure must be obtained prior to performing the experiment. Mouse embryos from E8.5 to E11.5 have been used successfully in this protocol. The total DNA yield in the immunoprecipitate depends on the number of genomic sites occupied by the protein of interest and the developmental stage of the embryo. An E8.5 embryo precipitated with an antibody directed against acetylated histone 3 lysine 14 yields sufficient DNA to perform assessment of six genomic sites in triplicate qPCR repeats. 1. Prepare fresh 18.5% paraformaldehyde for Subheading 3.2. 2. Sacrifice pregnant dam by cervical dislocation. 3. Dissect uterus. 4. Place uterus into 50-ml conical tube containing sterile MT-PBS. 5. Transfer uterus to 10-cm Petri dish with sterile MT-PBS. 6. Dissect embryos. 7. If required, retain yolk sac in additional labelled 1.5-ml tube for genotyping. 8. Transfer E8.5–E10.5 embryos into labelled 1.5-ml tubes. 9. Place embryo containing tubes on ice. 10. Proceed immediately to Subheading 3.2.

3.2. Cross Linking of DNA and Associated Proteins

1. Add 1 ml of MT-PBS to each embryo. 2. Keep embryos on ice. 3. Gently triturate approximately ten times to break up the embryo using a 1,000-μl micropipette. 4. Add 56 μl of fresh 18.5% formaldehyde (final concentration 1%) and invert five times to mix.

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5. Incubate on rocking platform for 15 min at room temperature. 6. Add 106 μl of 1.375 M glycine to stop cross-linking reaction and invert five times to mix. 7. Incubate on rocking platform for 5 min. 8. Centrifuge for 4 min at 5,000 rpm [2,375 ´ g] in microcentrifuge. 9. Aspirate and discard supernatant. (At each step, remove as much medium as possible without disturbing the cell pellet). 10. Wash cells using ice-cold MT-PBS with fresh inhibitors (complete protease inhibitors and, in addition for detection of histone acetylation, histone deacetylase inhibitor sodium butyrate). 11. Centrifuge for 4 min at 5,000 rpm [2,375´g] in microcentrifuge. 12. Aspirate and discard supernatant. 13. Wash cells using ice-cold MT-PBS with inhibitors. 14. Centrifuge for 4 min at 5,000 rpm [2,375´g] in microcentrifuge. 15. Aspirate and discard supernatant. 16. Snap freeze on dry ice or in liquid nitrogen. 17. Store at −80°C. 3.3. Fragmentation of the Chromatin

1. Defrost cross-linked cell pellet on ice. 2. Resuspend cell pellet in 200 μl of SDS lysis buffer with inhibitors (add protease inhibitors to PBS just prior to use). 3. Incubate for 10 min on ice (any SDS that precipitates during this time is dissolved in the next step). 4. Sonicate lysate on ice two times at 10% power for 11 pulses on 90% duty cycle with the stepped microprobe (see Notes 1–3 for details of sonication, alternative methods, and optimisation of chromatin fragmentation). Safety warning: Ear protection must be worn during sonication. 5. Rest on ice while processing other samples. 6. Repeat steps 4 and 5 two times resulting in a total of 6× 11 pulses of sonication per sample. 7. Centrifuge samples for 10 min at 13,000 rpm [16,060 ´ g] at 4°C in microcentrifuge. 8. Transfer the supernatant to a new 1.5-ml microcentrifuge tube and discard pellet. 9. Withdraw 20 μl of each sample and dilute 1:50 with autoclaved milliQ water. 10. Measure the absorbance of a 1/50 dilution at 260 and 280. 11. Use the OD260 reading to adjust concentrations (see Note 4; adjusting the chromatin material based on optical density). 12. Transfer 200 μl of sonicated and concentration adjusted material to a fresh 2-ml microcentrifuge tube.

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13. Add 1.8 ml of ChIP dilution buffer with fresh inhibitors. 14. Mix. 15. Proceed immediately to Subheading 3.4. 16. Retain 1–5% (20–100 μl, larger volumes from younger embryos) of sonicated, concentration-adjusted material as “input”, i.e. starting material before precipitation at −20°C (see Note 5; uses and processing of input material). 3.4. Chromatin Immunoprecipitation

Steps 1–4 are aimed to reduce non-specific background (“preclearing”). 1. To 2 ml of sonicated, diluted supernatant (from Subheading 3.3, step 14 above), add 75 μl of protein A agarose beads/salmon sperm DNA/BSA (50% slurry). 2. Incubate for 30 min at 4°C with agitation on rocking platform. 3. Centrifuge at 4,000 rpm [1,520 ´ g] for 1 min to pellet agarose beads. 4. Transfer the supernatant fraction to a new 2-ml microcentrifuge tube and discard pelleted beads. 5. Add the immunoprecipitating antibody to the 2-ml supernatant fraction and incubate overnight at 4°C with agitation on rocking platform. The amount of antibody to be added needs to be determined empirically. A good starting amount is between 2 and 10 μg. 6. Add 60 μl of protein A agarose beads/salmon sperm DNA/ BSA (50% slurry). 7. Incubate for 1 h at 4°C with agitation on rocking platform to bind the antibody/chromatin complex. 8. Gently centrifuge at 2,500 rpm [593 ´ g] at 4°C for 1 min to pellet the agarose bead-bound antibody/chromatin complex. 9. Carefully remove the supernatant, which contains unbound chromatin. Retain the pellet for the next step. The unbound chromatin fraction can be retained for comparison of precipitated versus non-precipitated chromatin (see Note 6; uses of unbound chromatin fraction). Sequentially wash the protein A agarose beads/antibody/chromatin complex in the following manner. 10. Wash the protein A agarose beads/antibody/chromatin complex for 5 min on a roller mixer at 4°C with 1 ml of low-salt, immune complex wash buffer. 11. Gently centrifuge at 2,500 rpm [593 ´ g] at 4°C for 1 min to pellet the agarose bead-bound antibody/chromatin complex. 12. Carefully remove and discard the supernatant.

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13. Wash the protein A agarose beads/antibody/chromatin complex for 5 min on a roller mixer at 4°C with 1 ml of high-salt, immune complex wash buffer. 14. Centrifuge at 2,500 rpm [593 ´ g] at 4°C for 1 min to pellet the agarose bead-bound antibody/chromatin complex. 15. Remove and discard the supernatant. 16. Wash the protein A agarose beads/antibody/chromatin complex for 5 min on a roller mixer at 4°C with 1 ml of LiCl immune complex wash buffer. 17. Centrifuge at 2,500 rpm [593 ´ g] at 4°C for 1 min to pellet the agarose bead-bound antibody/chromatin complex. 18. Remove and discard the supernatant. 19. Wash the protein A agarose bead-bound antibody/chromatin complex for 5 min on a roller mixer at 4°C with 1 ml of TE buffer. 20. Centrifuge at 2,500 rpm [593 ´ g] at 4°C for 1 min to pellet the agarose bead-bound antibody/chromatin complex. 21. Remove and discard the supernatant. 22. Wash the protein A agarose bead-bound antibody/chromatin complex for 5 min on a roller mixer at 4°C with 1 ml of TE buffer. 23. Centrifuge at 2,500 rpm [593 ´ g] at 4°C for 1 min to pellet the agarose bead-bound antibody/chromatin complex. 24. Remove and discard the supernatant. This bead-bound fraction could be used directly for the analysis of chromatin-precipitated proteins or as outlined in the following steps to recover the chromatin-precipitated DNA and associated proteins. 25. Add 250 μl of freshly prepared elution buffer to the pelleted protein A agarose beads/antibody/chromatin complex. 26. Invert several times and vortex briefly to mix. 27. Incubate at room temperature for 15 min on the roller mixer with regular inversion. 28. Centrifuge at 4,000 rpm [1,520 ´ g] for 1 min in microcentrifuge. 29. Carefully transfer the supernatant fraction (eluate) to a fresh tube. 30. To the pelleted protein A agarose beads, add 250 μl elution buffer to elute a second time. 31. Invert several times and vortex briefly to mix. 32. Incubate at room temperature for 15 min on the roller mixer with regular inversion. 33. Centrifuge at 4,000 rpm [1,520 ´ g] for 1 min as above.

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34. Carefully transfer the supernatant fraction (eluate) to combine eluates (total volume = ~500 μl) and discard beads. 35. Centrifuge combined eluates at 4,000 rpm [1,520 ´ g] for 2 min to pellet any residual beads. 36. Carefully transfer the combined eluates to a new tube and discard beads (see Note 7; alternative precipitation method). 3.5. Reversal of Cross Linking and DNA Extraction

1. Add 20 μl of 5 M NaCl to the combined eluates (500 μl). 2. Mix by vortexing. 3. Heat at 65°C for 4 h to reverse histone-DNA cross linking. 4. Optional: Store sample at −20°C and continue the next day. 5. Add 10 μl of 0.5 M EDTA, 20 μl of 1 M Tris–HCl, pH 6.5, and 2 μl of 10 mg/ml proteinase K to the combined eluates. 6. Mix by vortexing. 7. Incubate for 1 h at 45°C. 8. Add an equal volume of phenol/chloroform/isoamyl alcohol (50:49:1). 9. Mix by vortexing. 10. Centrifuge at 13,000 rpm [16,060´g] for 5 min in microcentrifuge. 11. Recover aqueous top phase into a fresh 2-ml microcentrifuge tube. Do not disturb interface. 12. Add an equal volume of chloroform/isoamyl alcohol (49:1). 13. Centrifuge at 13,000 rpm [16,060 ´ g] for 5 min as above. 14. Recover aqueous top phase into a fresh 2-ml microcentrifuge tube. Do not disturb interface. 15. Add 1/10 volume of 3 M sodium acetate, pH 5.2 (55 μl). 16. Mix by vortexing. 17. Optional: Add 20 μg glycogen (1 μl of 20 mg/ml). Mix by vortexing (see Note 8). 18. Add 2.2 volumes of 100% ethanol (1,320 μl). 19. Mix by vortexing. 20. Incubate at −20°C overnight. 21. Centrifuge at 13,000 rpm [16,060 ´ g] for 15 min. 22. Remove and discard supernatant without touching the pellet. 23. Wash pellet and tube with 1 ml 70% ethanol. 24. Centrifuge at 13,000 rpm [16,060 ´ g] for 10 min. 25. Remove and discard supernatant without touching the pellet. 26. Air dry pellet briefly. 27. Resuspend in 20–100 μl of 10 mM Tris–HCl, pH 7.4, depending on the age of the embryo and the expected abundance of chromatin- or DNA-binding sites of the precipitation target.

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28. Mix by vortexing. 29. Inspect visually for dissolution. 30. Store at −20°C. This DNA material is suitable for quantitation by a variety of methods, including qPCR.

4. Notes 1. Chromatin fragmentation using a probe sonicator. Sonication generates a considerable amount of heat, which can lead to disruption of protein–DNA interactions and to degradation, if the samples are not kept cool. Sonication is carried out with the tubes held in a Perspex tube rack containing an ice water bath. This keeps the samples cool while allowing the samples to be viewed from the side. The probe is positioned into the sample tube below the surface of the liquid close to the bottom of the tube, but without touching the tube. Operation of the sonication probe too close to the surface of the liquid should be avoided, as this can lead to foaming of the sample, which can lead to loss of material. Wash sonicator probe between samples by sonicating autoclaved milliQ water in a 50-ml conical tube. These conditions work well using the Branson 250 sonifier with a stepped microtip (Fig. 1). Other cell types or sonicators require optimisation (see Note 3). 2. Other chromatin fragmentation methods. We have conducted several tests using a sonication bath (Bioruptor) instead of a sonicator probe inserted into the sample. While the sonication bath yielded excellent results when using small numbers of cells 100 bp

1 kb

6 cycles

Fig. 1. E10.5 embryos dissociated mechanically, cross-linked, and fragmented using the Branson 250 sonifier with a stepped microtip for the number of 11-pulse cycles indicated above the lanes. Material from one-tenth of an E10.5 embryo loaded per lane. 100 bp and 1 kb indicate 100 bp and 1 kb ladder.

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in single-cell suspension, the chromatin of whole, dissociated E10.5 embryos was not fragmented consistently in the sonication bath (Figs. 2–4). A whole E10.5 embryo may yield too much material for efficient and consistent fragmentation in the sonication bath. We have successfully used an enzymatic chromatin fragmentation method (Millipore, 17–375) on E8.5 mouse embryos (Fig. 5). The majority of DNA fragments yielded using either of these alternative methods were approximately 150 bp, suggesting that the chromatin was cleaved between most nucleosomes. Chromatin fragment size was easily controlled varying the number of cycles using the Branson 250 sonifier with a stepped microtip. The optimal fragment size depends on the aim of the project (see discussion in Subheading 3, Methods). 3. Optimisation of chromatin fragmentation. Optimisation of chromatin fragmentation is conducted by comparing different sonication conditions, for example vary the number of cycles of sonication, cycle length, power setting, etc. (a) Follow procedures from Subheadings 3.1 and 3.2. 100 bp

30 cycles

Fig. 2. E10.5 embryos dissociated mechanically (pipetting) and subjected to chromatin fragmentation using a sonication bath set to maximal power for thirty 30 s on/30 s off (1 min) cycles. Material from one E10.5 embryo loaded per lane. Note the inconsistent range of fragment sizes and inconsistent recovery of material. 100 bp indicates 100 bp ladder.

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100 bp

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30

40

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Fig. 3. E10.5 embryos dissociated mechanically, cross-linked, and fragmented using a sonication bath for the number of 30 s on/30 s off (1 min) cycles indicated above. Material from one E10.5 embryo loaded per lane. 100 bp indicates 100 bp ladder.

100 bp

30 cycles

40 cycles

50 cycles

Fig. 4. E10.5 embryos dissociated enzymatically (trypsin) and subjected to chromatin fragmentation using a sonication bath set to maximal power for the number of 30 s on/30 s off (1 min) cycles as indicated above the lanes (one embryo per sample). Note narrow peak of fragment size around 300 bp, the inconsistent fragmentation of the three samples processed for 40 cycles, and the inconsistency in material recovery within each treatment group. Material from one E10.5 embryo loaded per lane. 100 bp indicates 100 bp ladder.

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Fig. 5. E8.5 embryos subjected to an enzymatic chromatin preparation kit (Upstate, Millipore, 17–375) according to the manufacturer’s instructions. The E8.5 embryos in the three lanes from left to right were increasingly developmentally advanced and, therefore, yielded increasing amounts of material. Material from one-third of an E8.5 embryo loaded per lane. 100 bp and 1 kb indicate 100 bp and 1 kb ladder.

(b) Retain two samples as an unsheared DNA control. Resuspend these in 200 μl of SDS lysis buffer. (c) With the remaining samples, follow procedure from Subheading 3.3, steps 1–8, but vary sonication conditions. Use two samples for each sonication condition as replicates. (d) Reverse cross linking as in procedure from Subheading 3.5, steps 1–3. (e) Add 1 μl of DNase-free RNase A and incubate at 37°C for 30 min. (f) Extract DNA as in procedure from Subheading 3.5, steps 5–7, 15, 16, and 18–29 (i.e. without phenol/chloroform/ isoamyl alcohol extraction and without glycogen addition). (g) Separate DNA fragments on a 1% agarose/ethidium bromide gel alongside 100 bp to 5 kb DNA fragment size marker. (h) Photograph ethidium bromide-stained DNA. (i) Examine fragment sizes. Fragment sizes ranging from 200 bp to 2 kb with a peak fragment size of 1 kb are suitable for many applications and allow the sampling of the genome in tiles of an average size of 1 kb. For other applications, smaller or larger fragment sizes may be desirable and these can be produced by varying the sonication conditions. 4. Adjusting the chromatin material based on optical density. The optical density is an approximate assessment of the relative

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concentration of material between samples. As the samples contain cross-linked DNA and protein, more exact measurements are not practical. Use the OD260 reading to adjust concentrations such that equal amounts and concentration of material can be used in the immunoprecipitation step. Make up differences in volume with SDS lysis buffer as required. Mix samples by inverting. Correct all samples to the level of the most dilute sample. However, if one sample is significantly lower than all others, it may be preferable to discard this sample rather than diluting the other samples excessively. 5. Uses and processing of the input material. One half each of the input material can be used to determine (a) the success of chromatin fragmentation by gel electrophoresis (see Notes 1–3 and Figs. 1–5) and (b) enrichment of genomic loci in the immunoprecipitate over the input material (total genome). For assessments of chromatin fragmentation, the cross linking has to be reversed, as in Note 3, (d) to (i), but with reduced volumes, e.g. 20 μl of input made up to 250 μl with elution buffer. For determining enrichment of genomic loci in the immunoprecipitate over input, the cross linking has to be reversed, the DNA in the input material has to be purified, as in Subheading 3.5. To facilitate processing of input samples, dilute in elution buffer. Typically, input samples are made to 250 μl with elution buffer and processed as half the volumes stated in Subheading 3.5. If enrichment over input is to be determined, both input and precipitate require spectrophotometric assessment of DNA concentration. 6. Uses and processing the unbound chromatin fraction. Like the input sample above, the unbound fraction has to undergo reversal of cross linking and DNA recovery. The unbound chromatin fraction is expected to be depleted of proteins and DNA fragments that are specifically enriched in the precipitate. 7. Alternative precipitation method. In young embryos (E8.5), the liquid volumes that cannot be retrieved from between the beads in the protein A/G–agarose bead precipitation method can substantially reduce the yield. We have used magnetic beads (Millipore, 16–661) as a successful alternative, if the starting material was limiting. 8. Effects of the addition of glycogen during the sodium acetate/ ethanol precipitation step on subsequent quantitation of DNA by qPCR. Addition of glycogen to the material after reversal of the cross linking can improve the yield during DNA precipitation (Fig. 6).

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Log(10) Hoxb4 TSS ± stdev

3.5 3 2.5 2 1.5 1 0.5 0

Without glycogen

With glycogen

Fig. 6. Q-PCR detection of the transcription start site of the Hoxb4 gene in anti-acetylated histone H3 lysine 9 (H3K9ac) ChIP material subjected to DNA precipitation in the presence or absence of glycogen. In this case, glycogen neither appear to improve yield dramatically nor to affect the efficiency of the qPCR reaction significantly.

References 1. Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., and Richmond, T. J. (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution, Nature 389, 251–260. 2. Kireeva, N., Lakonishok, M., Kireev, I., Hirano, T., and Belmont, A. S. (2004) Visualization of early chromosome condensation: a hierarchical folding, axial glue model of chromosome structure, J Cell Biol 166, 775–785. 3. Tremethick, D. J. (2007) Higher-order structures of chromatin: the elusive 30 nm fiber, Cell 128, 651–654. 4. Schones, D. E., and Zhao, K. (2008) Genomewide approaches to studying chromatin modifications, Nat Rev Genet 9, 179–191. 5. Shogren-Knaak, M., Ishii, H., Sun, J. M., Pazin, M. J., Davie, J. R., and Peterson, C. L. (2006) Histone H4-K16 acetylation controls

6.

7.

8.

9.

chromatin structure and protein interactions, Science 311, 844–847. Voss, A. K., Collin, C., Dixon, M. P., and Thomas, T. (2009) Moz and retinoic acid coordinately regulate H3K9 acetylation, Hox gene expression, and segment identity, Dev Cell 17, 674–686. Pfaffl, M. W. (2004) Quantification strategies in real-time PCR, in A-Z of quantitative PCR (Bustin, S. A., Ed.), International University Line (IUL), La Jolla, CA, USA. Taneyhill, L. A., and Adams, M. S. (2008) Investigating regulatory factors and their DNA binding affinities through real time quantitative PCR (RT-QPCR) and chromatin immunoprecipitation (ChIP) assays, Methods Cell Biol 87, 367–389. Henikoff, S. (2008) Nucleosome destabilization in the epigenetic regulation of gene expression, Nat Rev Genet 9, 15–26.