This protocol presents a locus-specific chromatin isolation method based on site-specific recombination to purify a single-copy gene locus of interest in its native chromatin context from budding yeast, Saccharomyces cerevisiae.
The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone octamer. Chromatin is defined as the entity of NCPs and numerous other protein complexes, including transcription factors, chromatin remodeling and modifying enzymes. It is still unclear how these protein-DNA interactions are orchestrated at the level of specific genomic loci during different stages of the cell cycle. This is mainly due to the current technical limitations, which make it challenging to obtain precise measurements of such dynamic interactions. Here, we describe an improved method combining site-specific recombination with an efficient single-step affinity purification protocol to isolate a single-copy gene locus of interest in its native chromatin state. The method allows for the robust enrichment of the target locus over genomic chromatin, making this technique an effective strategy for identifying and quantifying protein interactions in an unbiased and systematic manner, for example by mass spectrometry. Further to such compositional analyses, native chromatin purified by this method likely reflects the in vivo situation regarding nucleosome positioning and histone modifications and is, therefore, amenable to further structural and biochemical analyses of chromatin derived from virtually any genomic locus in yeast.
The dynamic organization of eukaryotic genomes into chromatin compacts the DNA to fit within the confines of the nucleus while ensuring sufficient dynamics for gene expression and accessibility for regulatory factors. In part, this versatility is mediated by the nucleosome, the basic unit of chromatin, which comprises a core particle with 147 bp of DNA wrapped ~1.7 times around the histone octamer1. The nucleosome is a highly dynamic structure with respect to its composition, with numerous histone variants and posttranslational modifications (PTMs) on the N- and C-terminal histone tails. Furthermore, eukaryotic chromatin interacts with a multitude of other essential components, such as transcription factors, DNA and RNA processing machinery, architectural proteins, enzymes involved in chromatin remodeling and modification, and RNA molecules associated with chromatin. These crucial machineries involved in transcription, replication, and repair all require access to chromatin, which serves as the natural substrate for these processes. Consequently, comprehending the molecular mechanisms underlying these DNA transactions necessitates a precise definition of the collective alterations in chromatin structure at the specific genomic regions where these machineries converge and facilitate biological reactions.
Despite the identification of numerous chromatin factors through genetics and protein-protein interaction studies, performing direct, unbiased, and comprehensive analyses of chromatin interactions at particular genomic sites has remained a significant obstacle2,3. Initially, only highly abundant regions of the genome (i.e., repetitive loci) or multi-copy plasmids could be isolated in sufficient amounts and purity for mass spectrometric identification of the associated proteins4,5,6,7. A series of new approaches based on the direct hybridization of capture probes to chromatinized DNA, proximity biotinylation using the CRISPR-dCas9 system, or the binding of sequence-specific adapter proteins to the locus of interest have started to unravel the proteome of single-copy loci from yeast and mammalian genomes8,9,10. However, all these methods require formaldehyde crosslinking to stabilize the protein-DNA interactions and sonication to solubilize the chromatin for subsequent purification. Together, both manipulations exclude the possibility of subsequent structural and functional studies of the purified chromatin.
To overcome these limitations, we previously devised a methodology that employs site-specific recombination to extract targeted chromosomal domains from yeast11,12. In essence, the genomic region of interest is surrounded by recognition sites (RS) for the site-specific R-recombinase from Zygosaccharomyces rouxi while simultaneously incorporating a group of three DNA binding sites for the prokaryotic transcriptional repressor LexA protein (LexA) within the same region. The yeast cells contain an expression cassette for the simultaneous expression of R-recombinase and a LexA protein fused to a tandem affinity purification (TAP) tag. After the induction of R-recombinase, the enzyme efficiently excises the targeted region from the chromosome in the form of a circular chromatin domain. This domain can be purified via the LexA-TAP adapter protein, which binds to the LexA DNA binding sites, as well as to an affinity support. This method has been recently used to isolate distinct chromatin domains containing selected replication origins of yeast chromosome III13.
One major advantage of this ex vivo approach is that it allows for functional analyses of the isolated material. For example, replication origin domains purified with this method can be subjected to in vitro replication assays to assess the efficiency of origin firing in a test tube from native in vivo assembled chromatin templates. Ultimately, the biochemical and functional characterization of the isolated material may allow the reconstitution of nuclear processes using purified proteins together with the native chromatin template. In summary, this methodology opens an exciting avenue in chromatin research, as it will be possible to follow the collective compositional and structural chromatin changes of a specific genomic region undergoing a certain chromosomal transaction.
See the Table of Materials for details related to all the materials and instruments used in this protocol. See Table 1 for a list of the solutions, buffers, and media used.
1. Recombinant yeast strain construction
2. Coupling IgG antibodies to epoxy-activated magnetic beads
NOTE: Couple IgG antibodies to epoxy-activated magnetic beads according to the following published protocol11.
3. Yeast cell culture and harvesting
4. Chromatin locus purification
NOTE: See Figure 2 for a schematic overview of the steps involved in this locus-specific chromatin purification protocol.
5. TEV protease-mediated elution
6. Denaturation elution
7. DNA and protein analysis
The purification of the ~1.4 kb ARS316 chromatin domain was mediated by the constitutively expressed LexA-TAP adapter protein. To serve as a negative control, we conducted purifications using an isogenic strain that expresses LexA-TAP but does not contain integrated RS and LexA-binding sites. Figure 3 illustrates the DNA analysis outcome from a standard purification experiment performed on both the control and a recombination-competent strain targeting the ARS316 locus. After DNA isolation and restriction enzyme digestion to linearize the circular DNA molecules, qPCR analysis was performed to assess the purification efficiency of the ARS316 target locus over the unrelated genomic control locus PDC1.
Furthermore, the enrichment of the ARS316 locus over the total chromatin was quantified (Figure 3A,B). The negative control strain showed no recovery of the ARS316 or PDC1 loci in any of the fractions except for the crude cell extract, input, and flow-through, suggesting that no specific enrichment could be observed in the TEV and denaturing elutions. In contrast, the ARS316 locus was quantitatively depleted in the flow-through in the recombination strain and could be recovered on the beads after TEV elution and in the denaturing elution. After TEV elution, the beads showed a higher recovery percentage of the ARS316 locus as the TEV cleavage efficiency was not 100%. This resulted in a fraction of chromatin rings remaining bound to the beads. The TEV protease cleavage efficiency can be improved by increasing the elution volume above 100 µL. In contrast, the denaturing elution showed 80%-90% recovery of the ARS316 locus in the recombination strain (Figure 3A), corresponding to the high enrichment of ARS316 molecules when factoring in the size of the yeast genome (Figure 3B).
Southern blot was additionally performed to validate the qPCR results. Due to its high sensitivity, it can detect relatively lower concentrations of target DNA, thus allowing for quantitative analysis of the samples. The results obtained from the southern blot analysis using radioactively labeled probe confirmed the specific enrichment of the ARS316 locus in the fractions collected from the recombination strain but not in the control strain fractions (Figure 3C). In the recombination strain, higher enrichment of the ARS316 locus was observed based on the signal intensity in the TEV beads and the denaturation elution samples, concordant with the qPCR results. Similarly, the weak signals observed in the TEV elution and denaturation beads samples are concordant with the observed low enrichment of the ARS316 locus in these fractions (Figure 3C).
The LexA-TAP cleavage and pulldown efficiencies were also assessed by western blot analysis. The LexA-TAP protein has a molecular weight of ~80 kDa (Figure 4A). In both the recombination and control strains, western blot analysis was performed using the PAP antibody, which targets the protein A moiety of LexA-TAP in the purification samples. As anticipated, the results demonstrated near-total depletion of LexA-TAP in the flow-through, with recovery observed in the final bead and elution fractions (Figure 4B[i]). The PAP antibody also detects the small protein A fragment of ~15 kDa that remains on the beads after TEV elution. The same western blot was stripped and immunostained with an antibody against the LexA moiety of LexA-TAP (Figure 4B[ii]), showing complementary results (Figure 4B[ii]). The strong band at ~50 kDa in both blots indicates the IgG heavy chain coupled to the magnetic beads, which cross-hybridizes with the primary/secondary antibody conjugates (Figure 4B[ii]).
Figure 1: Schematics of yeast strain construction. A modified yeast strain with integrated LexA and recombination sites on chromosome III is transformed with a plasmid (K238) with expression cassettes for the constitutive expression of the LexA-TAP fusion protein using the TEF2 promoter and the galactose-inducible expression cassette for R recombinase (RecR) expression using a GAL10 promoter. The plasmid is linearized by SbfI restriction digestion, thereby creating homologous recombination arms for the integration of the expression cassette into an intergenic location on chromosome I. Competent cells are selected based on the LEU2 selection marker on the growth medium lacking leucine. Please click here to view a larger version of this figure.
Figure 2: Purification of the ARS316 chromatin locus from the yeast Saccharomyces cerevisiae. The ARS316 locus is excised in the form of chromatin rings from its genomic location in yeast cells by galactose-induced site-specific recombination. These chromatin rings are isolated from the crude cell extract using the LexA-TAP affinity tag bound to IgG-coupled magnetic beads. Chromatin rings can be released from the beads by two methods: i) TEV protease-mediated cleavage, or ii) denaturation elution using ammonium solution. The dotted arrow represents the possibility of performing denaturation elution after TEV protease-mediated elution to release the remaining bead-bound chromatin rings, as the TEV protease cleavage efficiency is not 100%. The outlined boxes represent the samples obtained at different steps of the purification for protein and DNA analyses. Abbreviation: RS = recombination sites. Please click here to view a larger version of this figure.
Figure 3: DNA analysis to evaluate the ARS316 locus purification. (A) Bar graph showing the purification of the ARS316 locus and an unrelated region, PDC1, in a series of DNA samples collected during ARS316 chromatin locus purification from the recombination yeast strain. (B) Bar graph showing the enrichment of the ARS316 locus over total chromatin in a series of DNA samples collected during ARS316 chromatin locus purification from control and recombination yeast strains. (C) Southern blot image showing the enrichment of the ARS316 locus in a series of DNA samples collected during ARS316 chromatin locus purification from control and recombination yeast strains. Please click here to view a larger version of this figure.
Figure 4: Protein analysis to evaluate LexA-TAP pull-down and cleavage efficiency. (A) Cartoon representing the different protein domains of the LexA-TAP fusion protein, along with the size of each domain. (B) Western blot images showing immunostaining against (i) PAP and (ii) LexA in a series of protein samples collected during ARS316 chromatin locus purification from control and recombination yeast strains. Please click here to view a larger version of this figure.
Figure 5: Flow diagram highlighting the potential downstream applications of chromatin domains purified using LexA-TAP affinity purification. Please click here to view a larger version of this figure.
Table 1: A list of the solutions and media used in this protocol. Please click here to download this Table.
Table 2: Quantitative PCR program. Please click here to download this Table.
The identification of the factors and chromatin landscape of a specific target genomic region continues to pose a major challenge in chromatin research18. This protocol describes an efficient system to specifically excise and purify distinct chromatin domains from yeast chromosomes. To our knowledge, the purity and yield of this single-step purification overcome many of the limitations of locus-specific chromatin purification methods, thus allowing for the achievement of very high enrichment of the targeted loci compared to other unrelated genomic regions.
Due to the additional excision step using site-specific R-recombinase, another advantage is that one can exactly determine which genomic region is purified depending on the precise location of the recombination sites in the genome. In contrast, other methods are reliant on the shearing of the DNA by sonication, which results in heterogeneous molecule lengths; this heterogeneity makes those methods less precise in terms of the purification of a well-defined target locus.
Finally, this purification strategy utilizes native conditions without the inclusion of chemical crosslinkers like formaldehyde. Thus, the isolated material obtained by this approach is amenable to structural and functional analyses. A critical step in the purification protocol is the efficiency of the TEV elution, which strongly depends on several parameters, including the activity of the TEV protease, the reaction conditions, and the reaction volumes. For the experiment shown, the elution volume was set to a minimum, which affected the efficiency of elution. However, increasing the volume and amount of TEV protease could significantly improve the elution efficiency. Despite having low efficiency, TEV elution yields highly specific cleavage and recovery of the desired chromatin domains. In comparison, denaturation elution has an elution efficiency of more than 90% but might elute some non-specific DNA or protein molecules from the beads. Therefore, the method of choice depends upon the desired downstream application of the isolated material. For instance, denaturing elution has been demonstrated to allow for the mass spectrometric identification of the associated chromatin factors, whereas native TEV elution is preferred for downstream functional assays such as in vitro transcription or replication assays. Figure 5 summarizes the potential downstream applications of the described purification protocol. In summary, this improved workflow and highly efficient method for studying the chromatin composition of single loci addresses a long-standing challenge in chromatin research.
The authors have nothing to disclose.
Work in the S.H. laboratory was supported by the DFG through SFB1064 (project ID 213249687), the European Research Council (ERC Starting Grant 852798 ConflictResolution), and the Helmholtz Gesellschaft.
Yeast strains | |||
Control Strain: MATa; ura3Δ0; leu2Δ0; his3Δ1; met15Δ0; bar1::kanMX4; Chr I 212kb::LEU2 pTEF2-LEXA-TAP pGAL1-10 RecR | Section 1, see references 13 and 14 | ||
Recombination Strain: MATa; ura3Δ0; leu2Δ0; his3Δ1; met15Δ0; bar1::kanMX4; RS_LEXA_NS-3_ARS316_NS+3_RS; Chr I 212kb::LEU2 pTEF2-LEXA-TAP pGAL1-10 RecR | Section 1, see reference 13 and 14 | ||
Plasmid | |||
K238 plasmid | Section 1, see reference 13 Storage: Store at -20 °C |
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K071 Spike-in plasmid DNA | Section 7.1, see reference 13 Storage: Store at -20 °C |
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Reagents | |||
Acetone | Carl Roth | 5025.1 | Section 2 Storage: Store at room temperature |
Ammonium acetate (NH4Ac) | Sigma Aldrich | A7262 | Section 6 and 7.1 Storage: Store at room temperature |
Ammonium solution (NH4OH) 25% | Merck Millipore | 533003 | Section 6 Storage: Store at room temperature |
Ammonium sulfate | Santa Cruz | Sc-29085 | Section 2 Storage: Store at room temperature |
Bacto agar | BD (VWR) | 90000-760 | Section 3 Storage: Store at room temperature |
Bacto peptone | BD (VWR) | 211820 | Section 3 Storage: Store at room temperature |
β-Mercaptoethanol | Sigma Aldrich | 07604 | Section 7.2 Storage: Store at 4 °C |
Chemiluminescent substrate kit | ThermoFisher | 34580 | Section 7.2 Storage: Store at 4 °C |
Di-Sodium Hydrogen phosphate dodecahydrate | Merck | 1.06579.1000 | Section 2 and 7.1 Storage: Store at room temperature |
Dithiothreitol (DTT) | ThermoFisher | 15508013 | Section 4 Storage: Store at 4 °C |
Ethanol | Merck | 100983 | Section 7.1 Storage: Store at room temperature |
Ethylenediaminetetraacetic acid (EDTA) | Sigma Aldrich | ED | Section 7.1 Storage: Store at room temperature |
Galactose (20% (w/v) stock) | Sigma Aldrich | G0625-1KG / 5KG | Section 3 Storage: Store at room temperature |
Gel loading dye (6x) | BioLabs | B7024A | Section 7.1 Storage: Store at -20 °C |
Glusose | Sigma-Aldrich | G8270 | Section Storage: Store at room temperature |
Glycine | Carl Roth | .0079.4 | Section 2 Storage: Store at room temperature |
Glycogen (5 mg/mL) | Invitrogen | AM9510 | Section 7.1 Storage: Store at -20 °C |
Hydrochloric acid (HCl) | PanReac AppliChem | 182109.1211 | Section 2, 4 and 7.1 Storage: Store at room temperature |
Magnesium Acetate (MgAc) | Bernd Kraft | 15274.2600/C035 | Section 4 Storage: Store at room temperature |
Magnesium chloride (MgCl2) | Sigma Aldrich | M8266 | Section 6 Storage: Store at room temperature |
Nu PAGE LDS sample buffer (4x) | Invitrogen | 2399549 | Section 7.2 Storage: Store at room temperature |
Phenol/Chloroform/Isoamyl alcohol (25:24:1 v/v) | Invitrogen | 15593-031 | Section 7.1 Storage: Store at 4 °C |
Potassium chloride (KCl) | Sigma | P9541 | Section 4 Storage: Store at room temperature |
Radioactively labeled α-32P dATP (3,000 Ci/mmol, 10 mCi/mL) | Hartmann Analytic | SRP-203 | Section 7.1 Storage: Store at 4 °C |
RadPrime labeling system | ThermoFisher | 18428-011 | Section 7.1 Storage: Store at -20 °C |
Raffinose (20% (w/v) stock) | SERVA | 34140.03 | Section 3 Storage: Store at room temperature |
Sodium chloride (NaCl) | Merck | K53710504142 | Section 7.1 Storage: Store at room temperature |
Sodium citrate (Na3C6H5O7) | Sigma-Aldrich | 71402 | Section 7.1 Storage: Store at room temperature |
Sodium hydroxide (NaOH) | Sigma Aldrich | S5881 | Section 7.1 Storage: Store at room temperature |
Sodium n-dodecyl sulfate (SDS) (5% stock (w/v) ) | Alfa Aesar | A11183 | Section 7.1 Storage: Store at room temperature |
Sodium phosphate monobasic | Sigma-Aldrich | 71496 | Section 2 and 7.1 Storage: Store at room temperature |
Sodium azide | Santa Cruz Biotechnology | sc-208393 | Section 2 Storage: Store at -20 °C |
Triethylamine | Sigma Aldrich | 90340 | Section 2 Storage: Store at room temperature |
Tris base | Chem Cruz | SC-3715B | Section 2 and 4 Storage: Store at room temperature |
Triton X-100 | Sigma Aldrich | X100 | Section 2 and 4 Storage: Store at room temperature |
Tween-20 | Bernd Kraft | 18014332 | Section 4 Storage: Store at room temperature |
Yeast extract | BD (VWR) | 212720 | Section 3 Storage: Store at room temperature |
Yeast mating factor alpha (1 µg/mL stock ) | Biomol | Y2016.5 | Section 3 Storage: Store at -20 °C |
Yeast Synthetic Drop-out medium Supplements without LEUCINE | Sigma Aldrich | Y1376 | Section 1, see reference 14 |
Enzymes | |||
HpaI restriction enzyme (5,000 U/mL) | NEB | R0105S | Section 7.1 Storage: Store at -20 °C |
Protease and Phosphatase Inhibitor Cocktail (100x) | ThermoFisher Scientific | 78446 | Section 4 Storage: Store at4 °C |
Proteinase K (10 mg/mL) | SERVA | 33756 | Section 7.1 Storage: Store at -20 °C |
RNase A (10 mg/mL) | ThermoFisher | EN0531 | Section 7.1 Storage: Store at -20 °C |
TEV protease (10000 U/µL) | NEB | P8112S | Section 5 Storage: Store at -20 °C |
Materials | |||
BcMag Epoxy-Activated Magnetic Beads | Bioclone Inc. | FC-102 | Section 2 Storage: Store at 4 °C |
Dry ice | Section 4 | ||
Low-binding centrifuge tubes 2.0 mL | Eppendorf | 22431102 | Section 4 |
Microspin G-25 Columns | Cytiva | 27-5325-01 | Section 7.1 Storage: Store at room temperature |
Parafilm | Merck | P7793 | Section 4 |
Positive nylon membrane | Biozol | 11MEMP0001 | Section 7.1 Storage: Store at room temperature |
PVDF transfer membrane | Immobilon-Merck Millipore | IPVH00010 | Section 7.2 Storage: Store at room temperature |
SDS-PAGE gel 4-12% bis-tris (15 well, 1.5 mm) | Invitrogen | NP0336BOX | Section 7.2 Storage: Store at 4 °C |
Syringe (25 mL) with luer fitting | Henke Sass Wolf | 4200-000V0 | Section 3 |
Whatman paper (Grade 3MM CHR Cellulose Western Blotting Paper Sheet) | Cytiva | 3030-917 | Section 7.1 Storage: Store at room temperature |
Antibodies | |||
Anti-LexA, rabbit polyclonal IgG, DNA binding region antibody | Merck Millipore | 06-719 | Section 7.2 Storage: Store at -20 °C |
Goat Anti-Rabbit IgG (H+L), Horseradish peroxidase conjugate | Invitrogen | G21234 | Section 7.2 Storage: Store at -20 °C |
Peroxidase Anti-Peroxidase (PAP) antibody produced in rabbit for the detection of TAP-tagged proteins | Sigma Aldrich | P1291-500UL | Section 7.2 Storage: Store at -20 °C |
Rabbit IgG antibodies | Sigma | I5006-100MG | Section 2 Storage: Store at 4 °C |
Primers (10 µM) | |||
ARS316: fwd 5'- CGGCATTATCGTACACAACCT, rev 5'- GTTCTTCGTTGCCTACATTTTCT | Section 7.1 | ||
K071 Spike-in plasmid DNA: fwd: 5'-TTTTCGCTGCTTGTCCTTTT, rev 5'- CATTTTCGTCCTCCCAACAT | Section 7.1 | ||
PCR fragment from yeast genomic DNA as a template for ARS316 amplification (for southern blot): fwd 5’- AAATTCTGCCCTTGATTCGT rev 5’- TTTGTTTATCTCATCACTAAT | Section 7.1 | ||
PDC1: fwd 5'- CATGATCAGATGGGGCTTCA, rev 5'-ACCGGTGGTAGCGACTCTGT | Section 7.1 | ||
Equipment | |||
Coffee grinder | Gastroback | 42601 | Section 4 |
Dewar flask | NAL GENE | 4150-2000 | Section 3 |
DynaMag TM-2 magnetic rack | Invitrogen | 12321D | Section 4, 5 and 6 |
Hybridization oven | Hybaid Mini10 | Ri418 | Section 2 |
Microcentrifuge | Eppendorf | 5424R | Section 4 and 7.1 |
UV-crosslinker | Analytikjena | 95-0174-02 | Section 7.1 |