Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Podsumowanie

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

Streszczenie

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Wprowadzenie

Mapping the binding sites of transcription factors (TFs), chromatin remodelers, and other chromatin-associated regulatory factors is key to understanding all chromatin-based processes. While chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq) approaches have been used to gain many important insights into genome biology, they have notable limitations. We recently introduced an alternative method, termed chromatin endogenous cleavage and high-throughput sequencing (ChEC-seq)¹, to circumvent these drawbacks.

ChIP-seq is most often performed with an initial formaldehyde crosslinking step (X-ChIP-seq) to preserve protein-DNA interactions. However, a number of recent studies have indicated that X-ChIP-seq captures transient or nonspecific protein-DNA interactions^{2,3,4,5,6,7,8}, giving rise to false positive binding sites. In addition, sonication, commonly used to fragment chromatin in X-ChIP-seq experiments, preferentially shears regions of open chromatin, leading to biased recovery of fragments from these regions^9,10. Sonication also yields a heterogeneous mixture of fragment lengths, ultimately limiting binding site resolution, though the addition of an exonuclease digestion step can greatly improve resolution^11,12. Native ChIP methods such as occupied regions of genomes from affinity-purified naturally isolated chromatin (ORGANIC)¹³ do not use crosslinking and fragment chromatin with micrococcal nuclease (MNase), alleviating potential biases associated with formaldehyde crosslinking and sonication. However, the solubility of many chromatin-bound proteins under the relatively mild conditions required for native chromatin extraction is poor, potentially leading to reduced dynamic range and/or false negatives¹⁴.

While various iterations of ChIP-seq are most commonly used for genome-wide mapping of protein-DNA interactions, several mapping techniques based on fusion of proteins of interest to various DNA-modifying enzymes have also been implemented. One such approach is DNA adenine methyltransferase identification (DamID)¹⁵, wherein a chromatin-binding protein of interest is genetically fused to Dam and this fusion is expressed in cells or animals, resulting in methylation of GATC sequences proximal to binding sites for the protein. DamID is advantageous in that it does not rely upon immunoprecipitation and so avoids crosslinking, antibodies, or chromatin solubilization. It is also performed in vivo. However, the resolution of DamID is limited to the kilobase scale and the methylating activity of the Dam fusion protein is constitutive. A second method based on enzymatic fusion is Calling Card-seq¹⁶, which employs fusion of a factor of interest to a transposase, directing site-specific integration of transposons. Like DamID, Calling Card-seq is not based on immunoprecipitation and thus has similar advantages, with the added benefit of increased resolution. However, Calling Card-seq may be limited by sequence biases of transposases and is also reliant on the presence of restriction sites close to transposon insertion sites.

A third enzymatic fusion method, developed in the Laemmli lab, is chromatin endogenous cleavage (ChEC)¹⁷. In ChEC, a fusion between a chromatin-associated protein and MNase is expressed in cells, and upon calcium addition to activate MNase, DNA is cleaved proximal to binding sites for the tagged factor (Figure 1). In conjunction with Southern blotting, ChEC has been used to characterize chromatin structure and protein binding at a number of individual loci in yeast^17,18, and has been combined with low-resolution microarray analysis to probe the interaction of nuclear pore components with the yeast genome¹⁹. ChEC offers benefits similar to DamID and Calling Card-seq, and its resolution is nearly single-base pair when analyzed by primer extension¹⁹. ChEC is also controllable: robust DNA cleavage by MNase depends on the addition of millimolar calcium, ensuring that MNase is inactive at the low free calcium concentrations observed in live yeast cells²⁰.

Previously, we postulated that combining ChEC with high-throughput sequencing (ChEC-seq) would provide high-resolution maps of TF binding sites. Indeed, ChEC-seq generated high-resolution maps of the budding yeast general regulatory factors (GRFs) Abf1, Rap1, and Reb1 across the genome¹. We have also successfully applied ChEC-seq to the modular Mediator complex, a conserved, essential global transcriptional coactivator²¹, expanding the applicability of ChEC-seq to megadalton-size complexes that do not directly contact DNA and may be difficult to map by ChIP-based methods. ChEC-seq is a powerful method both for independent validation of ChIP-seq results and generation of new insights into the regulation of chromatin-resident processes. Here, we present a step-by-step protocol for the implementation of this method in budding yeast.

Protokół

1. Generation of Yeast Strains

1. Generate a yeast strain bearing the factor of interest tagged with MNase.
   1. PCR amplify the MNase tagging cassette from the desired vector (Table 1) using the specified reaction mixture (Table 2) and cycling conditions (Table 3). Mix 5 µL of the PCR reaction and 1 µL of 6X DNA loading dye. Run each PCR aliquot on a 0.8% agarose gel at 120 V for 40 min. The expected product size is ~2.3 kb.
   2. Transform the amplified tagging cassette into the strain of choice using standard lithium acetate transformation²² and perform selection.
   3. Verify correct integration of the tagging cassette with colony PCR.
      1. Prepare the specified reaction mixture (Table 4) for the number of colonies to be screened. Keep on ice until needed.
      2. Pick a portion of each colony to be tested into the bottom of a PCR tube using a sterile toothpick or pipet tip.
      3. Microwave the picked colonies at high power for 1 min.
      4. Add 50 µL of PCR mix (Table 4) to each microwaved colony and pipet up and down to mix. Perform PCR using the specified cycling conditions (Table 5).
      5. Mix the 50 µL of PCR reaction and 10 µL of 6X DNA loading dye directly in each PCR tube. Run 10 µL of each sample on a 1% agarose gel at 120 V for 40 min. The expected product size is ~ 700 bp.
         NOTE: If desired, verify appropriate expression of the MNase fusion protein via western blotting. A ~ 20.6 kilodalton shift in the molecular weight of the tagged protein is expected.
2. Generate a free MNase strain by homology-based cloning of the promoter of the gene of interest into pGZ136 (Table 1) and transformation and selection as in step 1.1.2.
   1. Alternatively, assemble the promoter of the gene of interest and 3xFLAG-MNase-SV40 NLS into a plasmid vector, transform and select as in step 1.1.2, and grow the strain in the appropriate selective condition for maintenance of the plasmid.

2. ChEC

1. In the afternoon/evening of the day prior to the ChEC experiment, inoculate 3 mL yeast-peptone-dextrose (YPD) or appropriate selective medium with a single colony of the strain bearing the MNase-tagged factor. Incubate this culture overnight (180 RPM shaking, 30 °C).
2. In the morning of the day of the ChEC experiment, dilute the overnight culture into 50 mL of medium to an optical density at 600 nm (OD₆₀₀) of 0.2 - 0.3. Incubate this culture (180 RPM shaking, 30 °C) until it reaches an OD₆₀₀ of 0.5-0.7. As the culture approaches the appropriate OD₆₀₀, set heat block or water bath to 30 °C and start thawing Buffer A additives (Table 6).
3. Prepare 5 mL of Buffer A plus additives (Table 6) per culture. Keep buffer A at RT during the procedure.
4. Prepare microfuge tubes containing 90 µL of stop solution (Table 7) and 10 µL of 10% SDS for each time point to be collected. For each new factor analyzed, take samples at 0 s, 30 s, 1 min, 2.5 min, 5 min, and 10 min.
5. Decant culture into a 50 mL conical tube and centrifuge at 1,500 x g and room temperature (RT) for 1 min.
6. Thoroughly resuspend cells in 1 mL of Buffer A and transfer the resuspended cells to a microfuge tube. Pellet resuspended cells in a microfuge at 1,500 x g and RT for 30 s.
7. Aspirate supernatant and thoroughly resuspend cells in 1 mL of Buffer A by pipetting up and down. Pellet resuspended cells in a microfuge at 1,500 x g and RT for 30 s. Repeat this step once.
8. Thoroughly resuspend cells in 570 µL of Buffer A. Add 30 µL of 2% digitonin (0.1% final concentration) to facilitate permeabilization of the cells, and invert to mix.
9. Permeabilize cells in heat block or water bath at 30 °C for 5 min.
10. Pipet the reaction up and down to ensure an even distribution of cells. Remove a 100 µL aliquot of permeabilized cells as a negative control to a microfuge tube containing stop solution and SDS (step 2.4) and vortex briefly to mix.
11. Add 1.1 µL of 1 M CaCl₂ (2 mM final concentration) to permeabilized cells to activate MNase and invert several times quickly or vortex briefly to mix. Immediately return the mixed reaction at 30 °C and start a timer.
12. At each time point to be collected, pipet the reaction up and down to ensure an even distribution of cells, then remove a 100 µL aliquot of permeabilized cells to a microfuge tube containing stop solution and SDS and vortex briefly to mix. For each new factor analyzed, take 0 s (no CaCl₂ added), 30 s, 1 min, 2.5 min, 5 min, and 10 min time points.
    NOTE: ChEC experiments with factors that rapidly cleave DNA at 30°C may be performed at lower temperatures to slow MNase cleavage kinetics.
13. Once all time points have been collected, add 4 µL of 20 mg/mL proteinase K to each sample, vortex briefly to mix, and incubate at 55 °C for 30 min.
14. Add 200 µL of 25:24:1 phenol:chloroform:isoamyl alcohol to each sample, vortex vigorously to mix, and centrifuge at maximum speed and RT in a microfuge for 5 min.
15. Remove each aqueous phase (~ 150 µL) to a new tube. Add 30 µg of glycogen and 500 µL of 100% ethanol. Vortex vigorously to mix and precipitate on dry ice for 10 min or until solution is viscous.
16. Pellet precipitated DNA at maximum speed and 4 °C in a microfuge for 10 min.
17. Decant supernatants and wash pellets with 1 mL of RT 70% ethanol.
18. Decant ethanol, removing the remainder by inverting and gently tapping the tubes on a paper towel. Briefly spin samples using a benchtop centrifuge and use a pipet to remove residual ethanol, taking care not to disturb the pellet. Air-dry pellets for at RT for 5 min.
19. While pellets are drying, make a master mix to resuspend dried pellets in 29 µL of Tris, pH 8.0 + 1 µL of 10 mg/mL RNase A each. Digest RNA at 37 °C for 20 min.
20. Mix 1 µL of 6X DNA loading dye with 5 µL of each sample and run on a 1.5% agarose gel at 120 V for 40 min.

3. Size Selection

NOTE: The goal of size selection is to remove multi-kilobase fragments of genomic DNA from the sample to be sequenced and enrich fragments of ~150 bp (approximately the size of nucleosomal DNA) or smaller. In sequencing data, fragments <400 bp are enriched, with a notable peak around the size of nucleosomal DNA (~150 bp) and a peak or broad distribution of subnucleosomal fragments.

1. To each sample, add 75 µL of solid-phase reversible immobilization (SPRI) beads in a polyethylene glycol (PEG) solution²³ and pipet up and down 10 times to mix. Incubate the bead:DNA mixture at RT for 5 min.
2. During the SPRI bead incubation, prepare microfuge tubes containing 96 µL of 10 mM Tris, pH 8.0 and 4 µL of 5 M NaCl for each sample.
3. Place tubes in a magnetic rack to collect beads on the side of the tube. Allow the beads to collect on the side of the tube for 2 min.
4. Transfer each supernatant to a new tube containing Tris and NaCl (step 3.2).
5. Add 200 µL of 25:24:1 phenol:chloroform:isoamyl alcohol to each sample, vortex to mix, and centrifuge at maximum speed and RT in a microfuge for 5 min.
6. Remove each aqueous phase to a new tube. Add 30 µg of glycogen and 500 µL of 100% ethanol. Precipitate DNA on dry ice for 10 min or until solution is viscous.
7. Pellet precipitated DNA at maximum speed and 4 °C in a microfuge for 10 min.
8. Decant supernatants and wash pellets with 1 mL of 70% ethanol.
9. Decant ethanol, removing the remainder by inverting and gently tapping the tubes on a paper towel. Briefly spin samples using a benchtop centrifuge and use a pipet to remove residual ethanol, taking care not to disturb the pellet. Air-dry pellets for at RT for 5 min.
10. Resuspend dried pellets in 25 µL of Tris, pH 8.0. Determine sample concentration using a high-sensitivity assay. For TF ChEC experiments, 10-100 ng DNA is routinely recovered after size selection.
11. Prepare sequencing libraries. Library preparation protocols that do not rely upon size selection, as previously described²⁴, are desirable to allow sequencing of a large range of fragment sizes (25-500 bp).
12. Sequence libraries in paired-end mode. A minimum of 25 bases of sequence on each end is needed for high quality read mapping. Between 1 and 2 million reads provides sufficient coverage for a ChEC sample.
    NOTE: Analysis of ChEC-seq data is a complex procedure and beyond the scope of this article. Detailed information on data analysis can be found in previous publications^1,21, and the custom scripts used for analysis are available on GitHub (https://github.com/zentnerlab/chec-seq). HOMER²⁵ (http://homer.salk.edu) can also be used for command-line analysis of ChEC-seq data.

Wyniki

In the case of a successful ChEC experiment, analysis of DNA by agarose gel electrophoresis will reveal a calcium-dependent increase in DNA fragmentation over-time, as indicated by smearing and eventual complete digestion of genomic DNA. In some cases, a ladder of bands similar to that seen with a traditional MNase digestion is observed after extended digestion. This is the case for ChEC analysis of Reb1, a general regulatory factor that binds nucleosome-depleted regions (NDRs) (

Dyskusje

We have shown that ChEC can map diverse classes of yeast proteins on chromatin, and anticipate that it will be broadly applicable to different families of TFs and other chromatin-binding factors in yeast. ChEC-seq is advantageous in that it does not require crosslinking, chromatin solubilization, or antibodies. Thus, ChEC avoids artifacts potentially present in X-ChIP-seq, such as the hyper-ChIPable artifact^3,4, and native ChIP, such as false negatives due to inc...

Materiały

Name	Company	Catalog Number	Comments
dNTPs	NEB	N0447
Q5 high-fidelity DNA polymerase	NEB	M0491L	Other high-fidelity DNA polymerases, such as Phusion, may be used for cassette amplification.
TrackIt 1 Kb Plus DNA ladder	ThermoFisher Scientific	10488085
Taq DNA polymerase	NEB	M0273L
cOmplete Mini EDTA-free protease inhibitor cocktail	Sigma-Aldrich	11836170001	It is important that an EDTA-free protease inhibitor mix is used, so as not to inhibit MNase cleavage by chelation of Ca2+.
PMSF	ACROS Organics	AC215740010
Digitonin, High Purity	EMD Millipore	300410-250MG	Make a 2% stock by dissolving 20 mg digitonin in 1 mL DMSO with vigorous vortexing.
Proteinase K, 20 mg/mL	Invitrogen	25530049
RNase A, 10 mg/mL	ThermoFisher Scientific	EN0531
Ampure XP beads	Beckman Coulter	A63880	Ampure-like beads can be generated using a published protocol (ref 24).
MagneSphere magnetic rack	Promega	Z5342