DNA curtain, a high-throughput single-molecule imaging technique, provides a platform for real-time visualization of diverse protein-DNA interactions. The present protocol utilizes the DNA curtain technique to investigate the biological role and molecular mechanism of Abo1, a Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase.
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Method Article
Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique
In This Article
Summary
Abstract
Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in response to environmental stimuli. These changes are essential for genomic integrity, epigenetic regulation, and DNA metabolic reactions such as replication, transcription, and repair. Chromatin assembly is crucial for chromatin dynamics and is catalyzed by histone chaperones. Despite extensive studies, the mechanisms by which histone chaperones enable chromatin assembly remains elusive. Moreover, the global features of nucleosomes organized by histone chaperones are poorly understood. To address these problems, this work describes a unique single-molecule imaging technique named DNA curtain, which facilitates the investigation of the molecular details of nucleosome assembly by histone chaperones. DNA curtain is a hybrid technique that combines lipid fluidity, microfluidics, and total internal reflection fluorescence microscopy (TIRFM) to provide a universal platform for real-time imaging of diverse protein-DNA interactions.Using DNA curtain, the histone chaperone function of Abo1, the Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase, is investigated, and the molecular mechanism underlying histone assembly of Abo1 is revealed. DNA curtain provides a unique approach for studying chromatin dynamics.
Introduction
Eukaryotic DNA is packaged into a higher-order structure known as chromatin1,2. Nucleosome is the fundamental unit of chromatin, which consists of approximately 147 bp DNA wrapped around the octameric core histones3,4. Chromatin plays a critical role in eukaryotic cells; for example, the compact structure protects DNA from endogenous factors and exogenous threats5. Chromatin structure changes dynamically according to the cell cycle phase and environmental stimuli, and these changes control protein access during DNA transactions such as replication, transcription, and repair6. Chromatin dynamics are also important for genomic stability and epigenetic information.
Chromatin is dynamically regulated by various factors, including histone tail modifications and chromatin organizers such as chromatin remodelers, polycomb group proteins, and histone chaperones7. Histone chaperones coordinate the assembly and disassembly of nucleosomes via deposition or detachment of core histones8,9. Defects in histone chaperones induce genome instability and cause developmental disorders and cancer9,10. Various histone chaperones do not need chemical energy consumption like ATP hydrolysis to assemble or disassemble nucleosomes9,11,12,13. Recently, researchers reported that bromodomain-containing AAA+ (ATPase associated with diverse cellular activities) ATPases play a role in chromatin dynamics as histone chaperones14,15,16,17. Human ATAD2 (ATPase family AAA domain-containing protein 2) promotes chromatin accessibility to enhance gene expression18. As a transcriptional co-regulator, ATAD2 regulates the chromatin of oncogenic transcriptional factors14, and the overexpression of ATAD2 is related to poor prognosis in many types of cancer19. Yta7, the Saccharomyces cerevisiae (S. cerevisiae) homolog of ATAD2, decreases nucleosome density in chromatin15. In contrast, Abo1, the Schizosaccharomyces pombe (S. pombe) homolog of ATAD2, increases nucleosome density16. Using a unique single-molecule imaging technique, DNA curtain, whether Abo1 contributes to nucleosome assembly or disassembly is addressed17,20.
Traditionally, the biochemical properties of biomolecules have been examined by bulk experiments such as the electrophoretic mobility shift assay (EMSA) or co-immunoprecipitation (co-IP), in which a large number of molecules are probed, and their average properties are characterized21,22. In bulk experiments, molecular sub-states are veiled by the ensemble-average effect, and probing biomolecular interactions is restricted. In contrast, single-molecule techniques circumvent the limitations of bulk experiments and enable the detailed characterization of biomolecular interactions. In particular, single-molecule imaging techniques have been widely used to study DNA-protein and protein-protein interactions23. One such technique is DNA curtain, a unique single-molecule imaging technique based on microfluidics and total internal reflection fluorescence microscopy (TIRFM)24,25. In a DNA curtain, hundreds of individual DNA molecules are anchored to the lipid bilayer, which permits the two-dimensional motion of DNA molecules due to lipid fluidity. When hydrodynamic flow is applied, DNA molecules move along the flow on the bilayer and get stuck at a diffusion barrier, where they are aligned and stretched. While DNA is stained with intercalating agents, fluorescently labeled proteins are injected, and TIRFM is used to visualize protein-DNA interactions in real-time at a single-molecule level23. The DNA curtain platform facilitates the observation of protein movements such as diffusion, translocation, and collision26,27,28. Moreover, DNA curtain can be used for protein mapping on DNA with defined positions, orientations, and topologies or applied to the study of phase separation of protein and nucleic acids29,30,31.
In this work, the DNA curtain technique is used to provide evidence for the function of chaperones through direct visualization of specific proteins. Moreover, because DNA curtain is a high-throughput platform, it facilitates an extent of data collection sufficient for statistical reliability. Here, it is described how to conduct the DNA curtain assay in detail to investigate the molecular role of S. pombe bromodomain-containing AAA+ ATPase Abo1.
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Protocol
1. Preparation of the flow cell
- Prepare a cleaned fused silica slide containing nano-trench patterns following previously published report25.
- Drill two holes with 1 mm diameter in a cleaned fused silica slide (Figure 1A) using a diamond-coated drill bit (see Table of Materials).
- Deposit 250 nm of thick aluminum (Al) on the slide using DC sputter32 (see Table of Materials) with 10 mTorr of argon gas.
- Spin-coat a 310 nm thick layer of 4% 950K poly(methyl methacrylate) (PMMA) (see Table of Materials) at 4,000 rpm for 1 min and bake on a hot plate at 180 °C for 3 min.
- Draw the nano-trench patterns on the PMMA layer between the two holes using electron beam (ebeam) lithography33 with 0.724 nA current at 80 kV.
NOTE: The architecture and the dimensions of the nano-trench patterns are shown in Figure 1A. - Remove the ebeam-exposed PMMA by soaking the slides in the developing solution (1:3 ratio of methyl isobutyl ketone and isopropanol, see Table of Materials) for 2 min.
- Etch Al layers with inductively coupled plasma-reactive ion etching (ICP-RIE) using chlorine (Cl2) and boron trichloride (BCl3) gases.
NOTE: These two gases can remove the Al from ebeam-exposed areas. - Carve nano-trenches on the slides using sulfur tetrafluoride (SF4), tetrafluoromethane (CF4), and oxygen (O2) gases (see Table of Materials).
- Remove the remaining Al layer by soaking the slides in Al etchant (AZ 300 MIF developer, see Table of Materials) for 10 min.
- After fabrication, rinse the slides with deionized water and sonicate in acetone for 30 min using a bath-type sonicator.
- Clean the slides in 2% Hellmanex III solution (see Table of Materials) for at least 1 day with magnetic stirring.
- Sonicate the slides in acetone for 30 min and 1 M NaOH for 30 min successively.
- Rinse the slides with deionized water and dry with a nitrogen (N2) gas.
- Put a clean paper (5 mm x 35 mm) on the center of double-sided tape. Attach the tape over the slide to cover the two holes and the nano-patterns with the paper.
- Excise the paper using a clean blade (Figure 1A).
- Put a glass coverslip on top of the double-sided tape and rub the coverslip using a pipette tip to form a microfluidic chamber (Figure 1A).
- Place the assembled flow cell between two microscope slides and clip them.
- Bake the flow cell in a 120 °C vacuum oven for 45 min.
- Attach the fluid connector (Nanoport) for the chip-based analyses (see Table of Materials) to each open hole using a hot glue gun and connect the two lines of Luer lock tubing.
- Connect a Luer lock syringe containing 3 mL of deionized water and wash the chamber.
- Wash the chamber with 3 mL of lipid buffer (20 mM of Tris-HCl, pH 8.0, and 100 mM of NaCl) via the drop-by-drop connection.
NOTE: Drop-by-drop connection links all syringes to the flow cell to avoid injecting air bubbles into the chamber. - Inject 1 mL of 0.04x biotinylated lipid in lipid buffer into the chamber in two shots with 5 min incubation per shot.
NOTE: Preparation of 1x biotinylated lipid stock is described in a previously published report34. - Wash the chamber with 3 mL of lipid buffer and incubate for 20 min for the lipid bilayer to be matured on the slide surface.
- Add 1 mL of BSA buffer (40 mM of Tris-HCl, pH 8.0, 50 mM of NaCl, 2 mM of MgCl2, and 0.2 mg/mL of BSA) and incubate for 5 min to passivate the slide surface.
NOTE: This step can reduce the nonspecific binding of proteins to the slide surface. - Inject 0.025 mg/mL of streptavidin in 1 mL of BSA buffer with two shots and 10 min incubation per shot.
- Wash out the residual streptavidin with 3 mL of BSA buffer.
- Inject ~300 pM (30 µL) of biotinylated lambda phage DNA in 1 mL of BSA buffer into the chamber with two shots and 10 min incubation per shot.
NOTE: Preparation of biotinylated lambda DNA is described in a previously published report34.
2. Connecting flow cell to the microfluidic system and loading it onto the microscope
- Prepare Abo1 imaging buffer (50 mM of Tris-HCl, pH 8.0, 100 mM of NaCl, 1 mM of DTT, 1 mM of ATP, 2 mM of MgCl2, 1.6% glucose, and 0.1x of gloxy, see Table of Materials).
NOTE: Gloxy is an oxygen scavenging system that reduces the photobleaching of fluorescent dyes. Preparation of 100x gloxy stock with glucose oxidase and catalase is described in Reference35. - Connect a syringe containing 10 mL of imaging buffer to a syringe pump and remove bubbles in all tubing lines in the microfluidic system.
- Couple the prepared flow cell with the microfluidic system via drop-to-drop connection to avoid bubble injection into the flow cell (Figure 1B).
- Assemble the flow cell and flow cell holders and mount the assembly on a custom-built TIRF microscope (Figure 1C).
CAUTION: When the flow cell is put under the microscope, carefully set the height of the objective lens. The flow cell must not touch the objective lens. This can damage the lens. - Drop index matching oil on the center of the flow cell and put a custom-made dove prism (see Table of Materials) on the oil drop.
3. Histone assembly by Abo1 using DNA curtain
- Mix 5 nM of Abo1 and 12.5 nM of Cy5-labeled H3-H4 histone dimer (Cy5-H3-H4) (see Table of Materials) in 150 µL of imaging buffer. All proteins were prepared following previously published report17.
- Incubate the mixture on ice for 15 min.
NOTE: To avoid photobleaching of Cy5, the incubation must be done in the dark. - Inject ~20 pM (2 µL) of YOYO-1 dye in imaging buffer through a 100 µL of sample loop to stain lambda DNA molecules.
- Run imaging software (see Table of Materials) and turn on the 488 nm laser to check whether DNA curtains are well-formed.
NOTE: If DNA curtains are well-formed, the DNA molecules, which are stained with YOYO-1, are shown as aligned lines at a barrier in the presence of flow. The stretched lines recoil and diffuse away from the barrier when the flow is turned off. - Inject 2 mL of high salt buffer (200 mM of NaCl and 40 mM of MgCl2) to eliminate YOYO-1 dye from DNA.
- After YOYO-1 dye has been removed, inject the pre-incubated protein sample.
- When the proteins reach the DNA curtain, turn off the syringe pump, switch off the shut-off valve, and incubate 15 min for histone loading by Abo1.
- Wash out the unbound Abo1 and histone proteins for 5 min.
- Switch on the shut-off valve and resume the flow injection.
- Turn on the 637 nm laser and image the fluorescent proteins while the buffer flow is on.
- Transiently turn off the buffer flow to check if histone proteins are loaded onto DNA (Figure 2C).
- Collect DNA curtain images with an image acquisition program (see Table of Materials).
NOTE: When the buffer flow transiently stops, DNA recoils out of the evanescent field of total internal reflection, and fluorescently labeled proteins bound to DNA disappear.
4. Data analysis
- Transform the images taken by the image acquisition program into TIFF format as image sequences.
NOTE: All data were analyzed using Image J (NIH) as described in the Reference20. - Pick a single DNA molecule from the image sequences and draw a kymograph.
NOTE: The kymograph can show the change in fluorescence intensity of each histone protein bound to a single DNA molecule as a function of time. - Create the fluorescence intensity profile from the kymograph and fit it with multiple Gaussian functions.
- Collect the center coordinates of peaks from the fluorescence intensity profile. In this step, the minimum intensity of histone fluorescence can be obtained.
- Divide all peak intensities collected from the profiles by minimum intensity. The number of H3-H4 dimers bound to DNA is estimated in this step.
- Perform steps 4.2-4.5 for other DNA molecules.
- Create a histogram for the binding distribution of H3-H4 dimers with 1 kbp bin size. The number of analyzed molecules is at least 300.
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Results
This work describes the procedure for flow cell preparation for the DNA curtain assay (Figure 1A). The DNA curtain assay facilitated the study of histone H3-H4 dimer assembly on DNA by Abo1. First, DNA curtain formation was checked by staining DNA molecules with YOYO-1, an intercalating dye. Green lines were shown in parallel arrays, indicating that YOYO-1 intercalated into DNA molecules, which were well-aligned and stretched at a diffusion barrier under hydrodynamic flow (
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Discussion
As a single-molecule imaging technique, DNA curtain has been used extensively to probe DNA metabolic reactions43. DNA curtain is a hybrid system that concatenates lipid fluidity, microfluidics, and TIRFM. Unlike other single-molecule techniques, DNA curtain enables high-throughput real-time visualization of protein-DNA interactions. Therefore, the DNA curtain technique is suitable for probing the mechanism behind molecular interactions, including sequence-specific association, protein movement alo...
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Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors appreciate the kind support for Abo1 and Cy5-H3-H4 by Professor Ji-Joon Song, Carol Cho, Ph.D., and Juwon Jang, Ph.D., in KAIST, South Korea. This work is supported by the National Research Foundation Grant (NRF-2020R1A2B5B01001792), intramural research fund (1.210115.01) of Ulsan National Institute of Science and Technology, and the Institute for Basic Science (IBS-R022-D1).
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 1 mL luer-lock syringe | BecktonDickinson | 301321 | |
| 1' x 3' fused-silca slide glass | G. Finkenbeiner | 1 inch x 3 inch rectangular and 1 mm thickness | |
| 10 mL luer-lock syringe | BecktonDickinson | 302149 | |
| 18:1 (Δ9-Cis) PC (DOPC) | Avanti | 850375 | This is a component of biotinylated lipid stock |
| 18:1 Biotinyl cap PE | Avanti | 870273 | This is a component of biotinylated lipid stock |
| 18:1 PEG2000 PE | Avanti | 880130 | This is a component of biotinylated lipid stock |
| 3 mL luer-lock syringe | BecktonDickinson | 302832 | |
| 6-way sample injection valve | IDEX | MX series II | |
| 950K PMMA | All-resist | 671.04 | |
| Acetone | SAMCHUN | A1759 | |
| Adenosine 5'-triphosphate disodium salt hydrate (ATP) | Sigma | A2383 | |
| Aluminum (Al) | TASCO, South Korea | LT50AI414 | Diameter 4 inch, thickness 1/4 inch |
| Amicon Ultra centrifugal filter, MWCO 10 kDa | Millipore | Z648027 | |
| Ampicillin | Mbcell | MB-A4128 | Antibiotics |
| AZ 300 MIF developer | Merck | 10454110521 | Used for removing aluminum |
| Blade | DORCO | DN52 | 12 mm x 6 m |
| Boron trichloride (BCl3) | UNIONGAS | Purity: >99.99% | |
| Bovine serum albumin (BSA) | Sigma | A7030 | |
| Catalase | Sigma | C40-1g | This is a component of 100x gloxy stock |
| Chlorine (Cl2) | UNIONGAS | Purity: >99.99% | |
| Clear double-sided tape | 3M | 313770 | |
| D-(+)-glucose | Sigma | G7528 | |
| DC sputter | Sorona | SRM-120 | Used for deposition aluminum on a slide |
| Diamond-coated drill bit | Eurotool | DIB-211.00 | Used for making holes in a fusced silica slide |
| DL-Dithiothreitol (DTT) | Sigma | D0632 | |
| Dove-prism | Korea Electro-Optics Co. Ltd. | 1906-106 | Custom-made fused-silica dove prism with anti-reflection coating |
| Drill | Dremel | Dremel 3000 | Used for making holes in a fusced silica slide |
| Electron Bean Lithography | Nanobeam Ltd. | NB3 | |
| Ethylene-diamine-tetraacetic acid (EDTA) | Sigma | EDS-1KG | |
| Fingertight fittings | IDEX | F-300 | It is connected with "PFA Tubing Natural" to form luer-lock tubing |
| Flangeless male nut | IDEX | P-235 | It is connected with "PFA Tubing Natural" to form luer-lock tubing |
| Freeze Dryer, HyperCOOL | Labogene | HC3110 | Used for lyophilizing liquid proteins |
| Glucose oxidase | Sigma | G2133-50KU | This is a component of 100x gloxy stock |
| Guanidinium hydrochloride | Acros Organics | 364790025 | |
| Hamilton syringe | Hamilton Company | 80065 | This syringe is used for sample injection |
| Hellmanex III | Sigma | Z805939 | |
| HiLoad 26/600 SuperdexTM 200 pg | Cytiva | 28-9893-36 | Used for FPLC (size exclusion) |
| Hot plate stirrer | Corning | PC-420D | |
| Hydrochloric acid | Sigma | H1759 | Used for Tris-HCl |
| Index matching oil | ZEISS | 444970-9000-000 | |
| Inductively coupled plasma-reactive ion etching | Top Technology Ltd. | FabStar | |
| Isopropyl β-D-1-thiogalactopyranoside (IPTG) | Glentham Life Sciences | GC6586-100g | Used for induction of β-galactosidase activity |
| Lambda phage DNA | NEB | N0311 | |
| LB broth | BD difco | 244610 | Media for E.coli cell growth |
| Luer adapter 10-32 | IDEX | P-659 | This connects luer-lock syringe and tubing |
| Magnesium chloride hexahydrate | fisher bioreagents | BP214 | |
| Methyl isobutyl ketone (MIBK) | KAYAKU ADVANCED MATERIALS | Used for developing solution | |
| Microscope (Eclipse Ti2) | Nikon | Eclipse Ti2 | Inverted fluorescence microscope |
| Microscope glass coverslip | MARIENFELD | 101142 | 22 x 50 mm (No. 1) |
| Microscope slide | DURAN GROUP | DU.2355013 | Slide glass ground edge 45°, plain 26 x 76 mm |
| Nanoport | IDEX | N-333-01 | |
| Objective lens | Nikon | CFI Plan Apochromat VC 60XC WI | Immersion type: water, magnification: 60x, correction: 18, working distance: 0.29 (0.31-0.28) |
| One Shot BL21 (DE3)pLysS Chemically Competent E. coli | Thermo Fisher Scientific | C6060-03 | Competent cell for overexpressing proteins |
| Oxygen (O2) | NOBLEGAS, South Korea | Purity: >99.99% | |
| PFA tubing natural | IDEX | 1512L | It is connected with "Fingertight Fittings" to form luer-lock tubing |
| Phenylmethylsulfonyl fluoride (PMSF) | Roche | 11359061001 | Protease inhibitor |
| Sephacryl S-200 High Resolution | Cytiva | 17-0584-01 | Used for FPLC (size exclusion) |
| Shut-off valve | IDEX | P-732 | |
| Sodium acetate | Sigma | 791741 | |
| Sodium chloride (NaCl) | Sigma | S3014 | |
| Sodium hydroxide (NaOH) | Sigma | s5881 | |
| Spectra/Por molecularporous membrane tubing, MWCO 6-8 kDa | Spectrum laboratories | 132660 | |
| Streptavidin | Thermo Fisher Scientific | S888 | |
| Sulfur tetralfluoride (SF4) | NOBLEGAS, South Korea | Purity: >99.99% | |
| Syringe pump | KD Scientific | 78-8210 | |
| Tetrafluoromethane (CF4) | NOBLEGAS, South Korea | Purity: >99.99% | |
| TritonX-100 | Sigma | T9284 | |
| Trizma base | Sigma | T1503 | Used for Tris-HCl |
| TSKgel SP-5PW | TOSOH | 14715 | Used for FPLC (ion exchange) |
| Union assembly | IDEX | P-760 | This connects tubings |
| Urea | Sigma | U5378 | |
| Vacuum oven | Jeio Tech | OV-11 | |
| YOYO-1 | Thermo Fisher Scientific | Y3601 | This intercalation dye is diluted in DMSO |
| β-mercaptoethanol (BME) | Sigma | M6250 |
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