Mapping Recombination Initiation Sites Using Chromatin Immunoprecipitation

Abstract

Genome-wide maps of recombination sites provide valuable information not only on the recombination pathway itself but also facilitate the understanding of genome dynamics and evolution. Here, we describe a chromatin immunoprecipitation (ChIP) protocol to map the sites of recombination initiation in plants with maize used as an example. ChIP is a method that allows identification of chromosomal sites occupied by specific proteins. Our protocol utilizes RAD51, a protein involved in repair of double-strand breaks (DSBs) that initiate meiotic recombination, to identify DSB formation hotspots. Chromatin is extracted from meiotic flowers, sheared and enriched in fragments bound to RAD51. Genomic location of the protein is then identified by next-generation sequencing. This protocol can also be used in other species of plants, animals, and fungi.

1 Introduction

Meiotic recombination is one of the key functions of chromosomes. It is required for faithful segregation of genetic material to the progeny and also generates genetic variation. Recombination is initiated by the formation of double-strand breaks (DSBs) in chromosomal DNA [1, 2]. The breaks are subsequently repaired into either crossovers (COs) or non-crossovers (NCOs), which include gene conversions. DSBs in most species, including plants , vastly outnumber COs [3]. In maize , roughly 500 DSBs are created in each cell during meiosis [4, 5], of which fewer than 20 become CO sites.

Recombination events in most species, including plants , are not uniformly distributed along chromosomes but form distinct hotspots [6–8]. Determining the location of recombination hotspots facilitates the understanding of genome dynamics and evolution as well as the elucidation of factors that cause specific regions of the genome to become recombination hotspots.

COs sites can be inferred by following exchanges of genetic markers (e.g., Single Nucleotide Polymorphisms or SNPs) between parental chromosomes in hybrid progeny [9]. Thus, resolution of CO mapping varies, depending on SNP density, and can be quite poor in regions of limited DNA sequence polymorphism. Sites of meiotic DSBs are most often identified using biochemical approaches [7, 10–12].

One of the most successful methods of identifying DSB hotspot locations genome-wide is finding chromosome sites associated with the RAD51 protein [7, 12] using chromatin immunoprecipitation (ChIP) . Following DSB formation, DNA ends flanking DSB sites are resected to form single-stranded DNA ends. These ends become then coated by two recombination proteins, RAD51 and DMC1 [13–16]. RAD51 catalyzes the first step of repair of meiotic DSBs and localizes to DNA segments immediately adjacent to the DSB sites [16]. The protein forms discrete foci on chromosomes during meiotic prophase I (Fig. 1). The number of foci is thought to represent the number of meiotic DSBs .

Fig. 1

The RAD51 protein localize to discrete sites on maize chromosomes during meiotic prophase I. Chromosome sites where RAD51 is located can be determined with high resolution using chromatin immunoprecipitation (ChIP). Red = chromatin. Green = RAD51. Bar = 10 μm. Modified from Pawlowski et al. [27]

To conduct ChIP experiments, chromosomal proteins are crosslinked, chromatin is extracted, and enriched in fragments containing the protein of interest using a specific antibody [17, 18]. The antibody -enriched fragments are identified using either whole-genome DNA tiling arrays or next-generation sequencing. The ChIP technique offers several advantages for mapping recombination hotspots. First, all hotspots, not only those that produce COs, can be surveyed. Second, hotspot sites can be determined at a very high resolution of a few hundred base pairs. Third, a very large number of meiocytes can be surveyed in a single experiment. Finally, the mapping resolution is independent of SNP density and hotspots can be mapped in homozygous strains.

In this chapter, we describe a ChIP protocol to map sites of meiotic recombination hotspots in maize using an antibody against RAD51. This protocol is a modification of a previously published general-use ChIP protocol [19] for specific use in mapping RAD51-marked DSB sites. The protocol is used by us to map the landscape of DSB hotspots in maize [12]. It can also be used in other species.

2 Materials

2.1 Reagents

2.1.1 Staging and Collecting Meiotic Flowers

• 1. Maize plants grown in a controlled environment growth chamber (see Note 1 ). We use a 12 h day/12 h night photoperiod, temperature of 31 °C during the day and 22 °C at night, and light intensity of about 600 μmol/m²/s.

• 2. Acetocarmine stain: 2 % acetocarmine powder in 45 % acetic acid. Boil the solution for 6–8 h in a flask with boiling stones and an attached reflux column. Then, filter the solution through filter paper when it is still warm. Store stain in a dark bottle at room temperature.

2.1.2 Chromatin Crosslinking

• 1. Crosslinking buffer: 10 mM Tris–HCl (pH 8.0), 0.4 M sucrose, 10 mM MgCl₂, 5 mM β-mercaptoethanol, 1 % formaldehyde.

• 2. 2 M glycine in water.

2.1.3 Chromatin Extraction and Sonication

• 1. Chromatin extraction buffer A: 10 mM Tris–HCl (pH 8.0), 0.4 M sucrose, 10 mM MgCl₂, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM β-mercaptoethanol. Before use, add protease inhibitor (see Note 2 ).

• 2. Chromatin extraction buffer B: 10 mM Tris–HCl (pH 8.0), 0.25 M sucrose, 10 mM MgCl₂, 1 % Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM β-mercaptoethanol, protease inhibitor (see Note 2 ).

• 3. Chromatin extraction buffer C: 10 mM Tris–HCl (pH 8.0), 1.7 M sucrose, 2 mM MgCl₂, 0.15 % Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM β-mercaptoethanol, protease inhibitor (see Note 2 ).

• 4. Nuclei lysis buffer: 50 mM Tris–HCl (pH 8.0), 10 mM EDTA, 1 % (w/v) SDS, 1 mM phenylmethylsulfonyl fluoride (PMSF), protease inhibitor (see Note 2 ).

2.1.4 Chromatin Immunoprecipitation

• 1. Dynabeads (Invitrogen, Grand Island, NY, USA) (see Note 3 ).

• 2. ChIP dilution buffer: 16.7 mM Tris–HCl (pH 8.0), 1.2 mM EDTA, 167 mM NaCl, 1.1 % Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), protease inhibitor (see Note 2 ).

• 3. Blocking buffer: 16.7 mM Tris–HCl (pH 8.0), 1.2 mM EDTA, 167 mM NaCl, 1.1 % Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), protease inhibitor (see Note 2 ).

• 4. Low salt wash buffer: 20 mM Tris–HCl (pH 8.0), 2 mM EDTA, 150 mM NaCl, 0.1 % SDS, 1 % Triton X-100.

• 5. High salt wash buffer: 20 mM Tris–HCl (pH 8.0), 2 mM EDTA, 500 mM NaCl, 0.1 % SDS, 1 % Triton X-100.

• 6. LiCl wash buffer: 10 mM Tris–HCl (pH 8.0), 1 mM EDTA, 250 mM LiCl, 1 % NP-40, 1 % sodium deoxycholate.

• 7. TE buffer: 10 mM Tris–HCl (pH 8.0), 1 mM EDTA.

• 8. Elution buffer: 50 mM Tris–HCl (pH 8.0), 10 mM EDTA, 200 mM NaCl, 1 % SDS.

• 9. 10 mg/mL RNase.

• 10. 20 mg/mL Proteinase K.

• 11. PCR purification kit.

• 12. High-sensitivity DNA quantification kit (see Note 4 ).

2.1.5 ChIP -seq Library Construction and Quality Control

• 1. ChIP -seq DNA Sample Prep Kit (Illumina, San Diego, CA, USA) (see Note 5 ).

• 2. PCR purification kit, such as Qiagen’s MinElute or QIAquick.

• 3. Real-Time PCR mix, such as iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA).

• 4. 10 mg/mL ethidium bromide solution in water.

• 5. TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8.0).

• 6. Gel loading buffer (50 mM Tris pH 8.0, 40 mM EDTA, 40 % (w/v) sucrose) (see Note 6 ).

2.2 Supplies and Equipment

2.2.1 Staging and Collecting Meiotic Flowers

• 1. Glass scintillation vials or 15 mL plastic tubes to collect flowers for staging.

• 2. Razor blade.

• 3. Tweezers with fine tips.

• 4. Dissecting needle.

• 5. Rusty nail (see Note 7 ).

• 6. Glass microscope slides and cover slips.

• 7. Dissecting stereoscope.

• 8. Bright-field microscope.

2.2.2 Chromatin Crosslinking

• 1. 50 mL conical tubes.

• 2. Miracloth.

• 3. Liquid nitrogen.

• 4. Vacuum desiccator.

2.2.3 Chromatin Extraction and Sonication

• 1. Small ceramic mortar and pestle.

• 2. Liquid nitrogen.

• 3. Miracloth.

• 4. Probe sonicator.

• 5. Microcentrifuge.

• 6. Refrigerated centrifuge.

• 7. Tabletop shaker.

2.2.4 Chromatin Immunoprecipitation

• 1. Magnetic separation stands for bead removal.

• 2. Tube rotator for mixing tube contents.

2.2.5 ChIP -seq Library Construction and Quality Control

• 1. Thermocycler.

• 2. Real-time PCR machine.

3 Methods

3.1 Staging and Collecting Maize Meiotic Flowers

• 1. For DSB mapping , male flowers containing anthers at the zygotene stage of meiotic prophase I should be used (see Note 8 ). At this stage, the maize tassel is still inside the stalk. The presence of the tassel can be felt just below the top node of the plant by gently squeezing the leaf whorl. After establishing that the tassel is large enough to be felt, make a small longitudinal incision with a razor blade through the leaves to the tassel, just below the top node.

• 2. Remove several flowers with needle-nosed forceps. Dissect anthers from the collected flowers on a microscope slide under a stereo dissecting microscope (see Note 9 ).

• 3. Add a drop of acetocarmine solution for staining. Mix anthers with the stain using a dissecting needle or a rusty nail over gentle heat/flame until the color of the stain turns from deep red to purple without boiling the stain solution. Place a cover slip over the anthers and gently press to break the anthers and release meiocytes. Determine the stage of meiosis under a bright-field compound microscope.

• 4. If the anthers are not yet at the desired meiosis stage, tape over the incision with masking tape and repeat the staging procedure in a day or two.

• 5. When the tassel is found to contain anthers at the zygotene stage of prophase I, collect the entire plant by cutting it at several nodes below the tassel.

• 6. Gently remove leaves surrounding the tassel. To prevent the tassel from drying out during dissection, place it on wet paper towels in a tray and put more wet paper towels on top of it.

• 7. Identify the tassel sections containing anthers at zygotene. Collect individual flowers from at least 20 tassels into 50 mL conical tube (see Note 10 ).

3.2 Chromatin Crosslinking

• 1. Add 37 mL of crosslinking buffer (see Note 11 ) to the 50 mL conical tube containing the collected flowers. Cap the tube with Miracloth to prevent the tissue from floating on the surface.

• 2. Vacuum infiltrate the solution for 10 min.

• 3. Release vacuum slowly and remove Miracloth. Stop the crosslinking reaction by adding 2.5 mL of 2 M glycine. Vacuum infiltrate for 5 min.

• 4. Decant supernatant and wash the tissue three times with 40 mL of distilled water. After the third wash, dry the tissue between paper towels.

• 5. Transfer the dry tissue into a new 50 mL conical tube. Snap-freeze in liquid nitrogen and store at −80 °C.

3.3 Chromatin Extraction and Sonication

• 1. Grind the tissue to a fine power with a mortar and pestle in liquid nitrogen.

• 2. Resuspend the powder in 40 mL of ice-cold chromatin extraction buffer A. Incubate for 20 min at 4 °C with gentle shaking.

• 3. Filter the solution into a new 50 mL conical tube through two layers of Miracloth placed in a plastic funnel.

• 4. Centrifuge at 1250 × g for 20 min at 4 °C.

• 5. Pour out the supernatant and resuspend the pellet in 1 mL of ice-cold extraction buffer B by gently pipetting up and down with a 1000 μL automatic pipette. Transfer the suspension to a 1.5 mL microcentrifuge tube. Incubate on ice for 15 min with occasional agitation.

• 6. Centrifuge at 20,000 × g in a microcentrifuge for 10 min at 4 °C. Discard the supernatant and resuspend the pellet in 500 μL of ice-cold extraction buffer C by gently pipetting up and down with a 1000 μL automatic pipette (see Note 12 ).

• 7. In a clean 1.5 mL microcentrifuge tube, add 500 μL of extraction buffer C. Layer the resuspended pellet from step 6 on top of this “cushion.”

• 8. Centrifuge at 20,000 × g in a microcentrifuge for 1 h at 4 °C.

• 9. Discard the supernatant and resuspend the nuclei pellet in 500 μL of ice-cold nuclei lysis buffer.

• 10. Sonicate the extracted chromatin on ice using several pulses into fragments of average length of 200–400 bp using eight sonicator pulses, 5 s each, lasting for 5 s each (see Notes 13 and 14 ).

• 11. Centrifuge the chromatin solution at 20,000 × g in a microcentrifuge for 5 min at 4 °C to pellet tissue debris. Transfer the supernatant containing the chromatin fragments to a new tube.

3.4 Chromatin Immunoprecipitation

3.4.1 Blocking Dynabeads (See Note 15 )

• 1. For each ChIP sample, take 100 μL of Dynabeads slurry into a 1.5 mL microcentrifuge tube.

• 2. Separate the beads on a magnetic separation stand for 1 min. Without disturbing the beads, pipette out the supernatant.

• 3. Wash beads twice with 1 mL of ChIP dilution buffer. For each wash, add the buffer and vortex the beads briefly to break coagulates. Then, remove the buffer using the magnet as described in Subheading 3.4.1, step 2.

• 4. Resuspend the beads in 1 mL of blocking buffer. Incubate at 4 °C with gentle shaking for at least 2 h.

• 5. Wash the beads three times with 1 mL of ChIP dilution buffer as described in Subheading 3.4.1, step 3.

• 6. Add ChIP dilution buffer back to the original bead volume from Subheading 3.4.1, step 1.

3.4.2 Immunoprecipitation and Washes

• 1. Take a 10 μL aliquot of the sonicated chromatin sample to use as an input control sample for ChIP product sequencing (see Note 16 ).

• 2. Split the chromatin sample from Subheading 3.3, step 11 (approx. 450 μL) into three 1.5 mL tubes of equal volume (150 μL in each tube) and dilute the chromatin sample in each tube tenfold by adding 1350 μL of ChIP dilution buffer (see Note 17 ).

• 3. Preclear each chromatin sample by mixing with 40 μL of Dynabeads beads for 3 h with gentle rotation of the tubes on a tube rotator at 4 °C.

• 4. Separate the beads on the magnetic separation stand.

• 5. Transfer the supernatant from each tube into a new tube. The first tube will serve as the “no-antibody ” control. Add 10 μg of preimmune or normal rabbit IgG to the second tube to use an “IgG control.” And 10 μg of your target antibody to the third tube (see Note 18 ).

• 6. Incubate the chromatin samples overnight with rotating at 4 °C.

• 7. Capture the protein–DNA complexes by adding 40 μL of coated beads and rotating the tubes on a tube rotator for 2.5 h at 4 °C. Separate beads on the magnetic separation stand and remove the supernatant.

• 8. Wash the beads five times with 1 mL of each of the following buffers (1) low salt wash buffer, (2) high salt wash buffer, (3) LiCl wash buffer, and twice in TE buffer. To conduct the washes, rotate the tubes for 5 min at 4 °C and remove the buffer as described in Subheading 3.4.1, step 2. After the final wash, make sure to remove all TE.

• 9. Add 200 μL of freshly prepared elution buffer. Resuspend beads by vortexing and incubate at room temperature for 30 min with occasional agitation.

• 10. Centrifuge at 2000 × g for 1 min and collect the supernatant into a new tube.

• 11. Conduct a second immunoprecipitation (see Note 19 ) by repeating steps 5–8 of Subheading 3.4.2.

• 12. Add 200 μL of freshly prepared elution buffer. Resuspend beads by vortexing and incubate at 65 °C for 30 min with occasional agitation.

• 13. Centrifuge at 2000 × g for 1 min and collect the supernatant into a new tube.

3.4.3 Decrosslinking

• 1. Add 4 μL of 10 mg/mL RNase to the supernatants from Subheading 3.4.2, step 13, and incubate at 37 °C for 1.5 h.

• 2. Add 4 μL of 20 mg/mL Proteinase K and incubate at 45 °C for 2 h.

• 3. Reverse crosslink at 65 °C for 8 h or overnight.

3.4.4 DNA Recovery

• 1. Purify DNA from each sample from Subheading 3.4.3, step 3 using a PCR purification kit, eluting in 30 μL of H₂O.

• 2. Measure the DNA concentration with a fluorometer following manufacturer’s instructions.

3.5 ChIP -seq Library Construction and Sequencing

• 1. Follow the current Illumina protocol to construct a sequencing library using the ChIP -seq DNA Sample Prep Kit (Illumina, San Diego, CA, USA) with the exception that DNA size selection should be done after the PCR step [7].

• 2. Perform a quality control experiment to validate the sequencing library. To do this, design a pair of PCR primers for a genome region known to be a recombination hotspot and a pair of primers for a random region, such as the Ubiquitin locus. Conduct three independent quantitative PCR reactions using the iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) following manufacturer’s instructions. Use an average of the three experiments to calculate hotspots enrichment. First, normalize the ChIP data using enrichment of a known hotspot region in the ChIP sample to that in the input sample. Then, normalize to the Ubiquitin gene region using the following equation: 2^{[Ct(Hotspot region_ChIP)−Ct(Hotspot region_Input)]}/2^{[Ct(Ubiquitin region_ChIP)−Ct(Ubiquitin region_Input)]}.

3.6 Computational Analyses

3.6.1 Processing and Mapping Illumina Reads to the Genome Scaffold

• 1. Perform base calling and read quality control using the standard Illumina protocol.

• 2. Align reads that passed quality control to the reference genome sequence using the Burrows-Wheeler Aligner (BWA) [20].

• 3. Trim reads progressively at the 3′ termini 1 bp at a time until they can be mapped to the genome scaffold with no more than two mismatches. Only reads that are longer than 40 bp after trimming should be aligned.

• 4. To identify recombination hotspots, conduct a peak detection analysis using MACS [21]. We use the following parameters for RAD51 ChIP peak detection: bandwidth = 800 bp, shift size = 400 bp, MACS mode off, and q-value cutoff = 0.01.

• 5. Use several control datasets to identify regions of RAD51 ChIP enrichment, such as: (1) input chromatin (from Subheading 3.4.2, step 1), (2) ChIP conducted using preimmune or normal rabbit IgG on meiotic chromatin (from Subheading 3.4.2, step 5), and (3) ChIP conducted with the anti-RAD51 antibody using chromatin extracted from young seedlings.

• 6. The Integrative Genomics Viewer (IGV) [22] can be used to visualize high-resolution DSB hotspot maps.

4 Notes

• 1. Plants for DSB mapping experiments should be grown in controlled environment growth chambers as temperature is known to affect both the number and distribution of recombination events (see ref. 23, 24).

• 2. We use one tablet of cOmplete Protease Inhibitor (Roche Applied Science, Indianapolis, IN, USA) per 50 mL of buffer. However, similar products from other manufacturers can be used instead.

• 3. Similar products from other manufacturers can be used instead but should be tested in pilot experiments. Use magnetic beads coupled to Protein A or Protein G, depending on the animal species in which the antibody of choice was produced.

• 4. We use Quant-IT dsDNA HS Assay Kit (Invitrogen, Grand Island, NY, USA). However, similar products from other manufacturers can be used instead.

• 5. Equivalent kits from other manufacturers, such as NEB (Ipswich, MA, USA) or homemade kits can also be used.

• 6. Any homemade or commercially available gel loading buffer can be used.

• 7. Iron oxide that leaches from the rusty nail enhances the staining reaction. Too little iron oxide will result in weak staining.

• 8. DSBs in plants are generated very early in meiosis , most likely before the onset of leptotene (see ref. 25, 26). However, RAD51 foci are present on chromosomes from late leptotene to mid-pachytene but exhibit their peak at mid-zygotene (see ref. 4, 27).

• 9. If flower samples need to be transported, they can be collected into glass scintillation vials or tubes containing Farmer’s fixative (three volumes of 100 % ethanol, one volume of glacial acetic acid).

• 10. Using anthers instead of whole flowers as input material might help reduce experimental background but anthers are much more time consuming to collect than whole flowers.

• 11. The length of crosslinking and the formaldehyde concentration need to be optimized for each tissue type. Insufficient crosslinking may result in decreased binding of the ChIP antibody while excessive crosslinking may lead to nonspecific binding.

• 12. Avoid introducing air bubbles or forming froth on the surface as this may lead to protein degradation in subsequent steps.

• 13. To produce desired fragment sizes, sonication conditions need to be optimized for every sonicator type and tissue type. Over-sonication will lead to DNA degradation, whereas insufficient sonication will lead to nonspecific antibody binding and decrease ChIP yield.

• 14. Heat generated during sonication may cause protein degradation. To avoid it, keep samples on ice during the entire procedure and allow at least 30 s between each sonicator pulse to let the samples cool down.

• 15. The bead blocking step can be carried out before starting the ChIP experiment. After blocking, beads can be stored at 4 °C. Blocking the beads decreases nonspecific binding of the antibody . We strongly recommend including this step, even though it is not always suggested in published ChIP protocols. Do not use DNA as a blocking reagent if the ChIP DNA product will be analyzed by sequencing. Otherwise, most of the sequence reads will represent carrier DNA.

• 16. The sonicated chromatin sample needs to be decrosslinked before DNA extraction. To do this, add 140 μL of TE buffer, 5 μL of 5 M NaCl, and 10 μL of 10 % SDS to a 10 μL aliquot of sonicated chromatin. Reverse crosslink overnight at 65 °C. Purify DNA using a PCR purification kit. To check sonication efficiency, electrophorese an aliquot of the extracted DNA in 2 % agarose gel.

• 17. From this step on, use low-retention microcentrifuge tubes.

• 18. Using antibodies with high specificity and affinity to native proteins is very important. Performance of an antibody in immunolocalization and/or western blot experiments may not be a predictor of its suitability for ChIP . ChIP experiments, with appropriate negative controls, should be carried out to determine antibody ’s performance. The amount of antibody that should be used in a ChIP experiment depends on the affinity between the antibody and the antigen, which varies from one antibody to another, and should be optimized for each antibody .

• 19. The second round of immunoprecipitation is used to increase specificity. It should be performed in the same manner as the first immunoprecipitation, except the final elution step, which is carried out at 65 °C.