Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Summary

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.

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 chromatin^1,2. Nucleosome is the fundamental unit of chromatin, which consists of approximately 147 bp DNA wrapped around the octameric core histones^3,4. 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 histones^8,9. Defects in histone chaperones induce genome instability and cause developmental disorders and cancer^9,10. Various histone chaperones do not need chemical energy consumption like ATP hydrolysis to assemble or disassemble nucleosomes^9,11,12,13. Recently, researchers reported that bromodomain-containing AAA+ (ATPase associated with diverse cellular activities) ATPases play a role in chromatin dynamics as histone chaperones^14,15,16,17. 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 addressed^17,20.

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 characterized^21,22. 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 collision^26,27,28. 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 acids^29,30,31.

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.

Protocol

1. Preparation of the flow cell

1. Prepare a cleaned fused silica slide containing nano-trench patterns following previously published report²⁵.
   1. 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).
   2. Deposit 250 nm of thick aluminum (Al) on the slide using DC sputter³² (see Table of Materials) with 10 mTorr of argon gas.
   3. 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.
   4. 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.
   5. 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.
   6. 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.
   7. Carve nano-trenches on the slides using sulfur tetrafluoride (SF₄), tetrafluoromethane (CF₄), and oxygen (O₂) gases (see Table of Materials).
   8. Remove the remaining Al layer by soaking the slides in Al etchant (AZ 300 MIF developer, see Table of Materials) for 10 min.
   9. After fabrication, rinse the slides with deionized water and sonicate in acetone for 30 min using a bath-type sonicator.
   10. Clean the slides in 2% Hellmanex III solution (see Table of Materials) for at least 1 day with magnetic stirring.
   11. Sonicate the slides in acetone for 30 min and 1 M NaOH for 30 min successively.
   12. Rinse the slides with deionized water and dry with a nitrogen (N₂) gas.
2. 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.
3. Excise the paper using a clean blade (Figure 1A).
4. 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).
5. Place the assembled flow cell between two microscope slides and clip them.
6. Bake the flow cell in a 120 °C vacuum oven for 45 min.
7. 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.
8. Connect a Luer lock syringe containing 3 mL of deionized water and wash the chamber.
9. 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.
10. 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.
11. 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.
12. 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.
13. Inject 0.025 mg/mL of streptavidin in 1 mL of BSA buffer with two shots and 10 min incubation per shot.
14. Wash out the residual streptavidin with 3 mL of BSA buffer.
15. 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

1. 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.
2. Connect a syringe containing 10 mL of imaging buffer to a syringe pump and remove bubbles in all tubing lines in the microfluidic system.
3. Couple the prepared flow cell with the microfluidic system via drop-to-drop connection to avoid bubble injection into the flow cell (Figure 1B).
4. 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.
5. 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

1. 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.
2. Incubate the mixture on ice for 15 min.
   NOTE: To avoid photobleaching of Cy5, the incubation must be done in the dark.
3. Inject ~20 pM (2 µL) of YOYO-1 dye in imaging buffer through a 100 µL of sample loop to stain lambda DNA molecules.
4. 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.
5. Inject 2 mL of high salt buffer (200 mM of NaCl and 40 mM of MgCl₂) to eliminate YOYO-1 dye from DNA.
6. After YOYO-1 dye has been removed, inject the pre-incubated protein sample.
7. 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.
8. Wash out the unbound Abo1 and histone proteins for 5 min.
9. Switch on the shut-off valve and resume the flow injection.
10. Turn on the 637 nm laser and image the fluorescent proteins while the buffer flow is on.
11. Transiently turn off the buffer flow to check if histone proteins are loaded onto DNA (Figure 2C).
12. 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

1. 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.
2. 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.
3. Create the fluorescence intensity profile from the kymograph and fit it with multiple Gaussian functions.
4. Collect the center coordinates of peaks from the fluorescence intensity profile. In this step, the minimum intensity of histone fluorescence can be obtained.
5. 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.
6. Perform steps 4.2-4.5 for other DNA molecules.
7. 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.

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 (

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...

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