Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Yujin Kang; Subin Bae; Soyeong An; Ja Yil Lee

doi:10.3791/63501

JoVE Journal > Biochemistry

生物化学

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Published: March 09, 2022

doi:

10.3791/63501

Yujin Kang¹, Subin Bae¹, Soyeong An¹, Ja Yil Lee^1,2

¹Department of Biological Sciences,Ulsan National Institute of Science and Technology, ²Center for Genomic Integrity,Institute for Basic Science (IBS)

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 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.

Protocol

1. Preparation of the flow cell

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.
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.

Representative 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 (Figure 2A). To exclude the possibility of YOYO-1 hindering the interaction of DNA and histone proteins, YOYO-1 was removed from DNA with a high salt buffer before adding histone proteins. When Cy5-H3-H4 dimers were injected into the DNA curtain in the absence of Abo1, Cy5-H3-H4 did not bind to DNA, indicating a lack of spontaneous binding of H3-H4 dimers to DNA (Figure 2B). When Cy5-H3-H4 was injected with Abo1, red fluorescent puncta were seen on DNA molecules, suggesting that Abo1 loads H3-H4 dimers onto DNA (Figure 2C). The buffer flow was transiently switched off to ensure that Cy5-H3-H4 dimers did not bind to the slide surface while binding to DNA. When the flow was turned off, fluorescently labeled proteins bound to DNA disappeared because DNA molecules recoiled out of the evanescent field. In contrast, proteins adsorbed to the surface remained the same. The fluorescent signals disappeared in the absence of flow and reappeared when the flow was resumed, suggesting that H3-H4 dimers bind to DNA (Figure 2C). The binding of H3-H4 dimers to DNA was also confirmed by the kymograph, in which the fluorescence signals disappeared whenever the flow was turned off (Figure 2D). Because Abo1 is an AAA+ ATPase, the effect of ATP hydrolysis on histone loading activity of Abo1 was examined¹⁶. Few fluorescent puncta appeared in the DNA curtain either in the absence of nucleotide (Apo) or in the presence of ADP (Figure 2E), indicating that H3-H4 dimers rarely bind to DNA either in the Apo state or in the presence of ADP. Figure 2F displays quantitative analyses of Figure 2B,C, and Figure 2E, showing that the number of H3-H4 dimers bound to DNA increases in the presence of ATP. The results suggest that ATP hydrolysis is essential for H3-H4 loading onto DNA by Abo1 (Figure 2E,F).

Next, it was tested whether Abo1 preferentially loads histones to the Widom 601 sequence, which has a ten-times higher binding affinity to histones than random sequences in vitro, even though nucleosomes have no specificity for the Widom 601 sequence in vivo³⁶^,³⁷^,³⁸^,³⁹^,⁴⁰. The DNA curtain was formed with lambda DNA that contains Widom 601 repeats at one end or internally (Figure 3A,B). The binding landscape of H3-H4 dimers on each DNA construct was obtained (Figure 3C,D). The binding location of Cy5-H3-H4 was estimated from the center position of 2D Gaussian fitting for each fluorescent punctum¹⁷^,⁴¹. If the H3-H4 loading activity of Abo1 depends on the DNA sequence, then the binding distribution would be skewed toward the Widom 601 sequence. Figure 3C,D displays the binding distribution histograms of H3-H4 dimers by Abo1 on lambda DNA containing Widom 601 repeats at the end (ten repeats) and internally (five repeats), respectively. There was no preferential binding to the multiple Widom 601 repeats, and the binding distribution was random, suggesting that Abo1 does not have any preference for the Widom 601 sequence but instead loads H3-H4 onto DNA in a sequence-independent manner (Figure 3C,D).

Figure 1: Preparation of flow cell. (A) Schematics of flow cell assembly and DNA curtain system. The microfluidic chamber can be formed between a fused-silica slide and a glass coverslip stuck together with double-sided tape. Under the hydrodynamic flow in the chamber, a large amount of lambda DNA molecules are aligned at a diffusion barrier (a nano-trench here) because of the fluidity of the lipid bilayer and stretched like a curtain. In the enlarged view of the diffusion barrier, the nano-trench has saw-tooth patterns with 1.5 µm pitch, 350 nm width, and 1.4 µm depth. (B) Photograph of the microfluidic system consisting of a syringe pump, a shut-off valve, and a 6-way sample injection valve with a sample loop. (C) Pictures of slide holder components (left), the assembly of holders and flow cell (middle), and the fully assembled flow cell mounted on a custom-made TIRF microscope (right). Please click here to view a larger version of this figure.

Figure 2: Visualization of histone loading by Abo1 using single-molecule DNA curtain. (A) Image of DNA curtain, where DNA molecules stained with YOYO-1 (green) are well-aligned at a diffusion barrier. (B) Images of Cy5-H3-H4 (red) without Abo1. (C) Images of Cy5-H3-H4 (red) loaded by Abo1 in the presence (top) and absence (bottom) of buffer flow. (D) Kymograph extracted from a single DNA molecule from (C). When the flow is transiently turned off, the Cy5 fluorescence signals disappear, indicating that Cy5-H3-H4 binds to DNA but not to the slide surface. (E) Image of Cy5-H3-H4 loaded by Abo1 without nucleotide (Apo) (top). Image of Cy5-H3-H4 loaded by Abo1 in the presence of ADP (bottom). (F) Quantification of Cy5-H3-H4 loaded by Abo1 according to nucleotides. Error bars depicts the standard deviation in triplicate. Each experiment involved the analysis of 100-200 molecules. Please click here to view a larger version of this figure.

Figure 3: Sequence-independent loading of H3-H4 by Abo1. (A) One end of the lambda DNA is anchored to biotinylated lipid via streptavidin, and the other end contains ten repeats of the Widom 601 sequence (top). The other lambda DNA contains five repeats of the Widom 601 sequence inside lambda DNA (bottom). (B) Schematic of DNA curtain assay with lambda DNA containing Widom 601 repeats. (C,D) Binding distribution histograms of Cy5-H3-H4 on lambda DNA containing Widom 601 repeats at the end (C) and internally (D). There is no sequence specificity for DNA when H3-H4 is assembled by Abo1. Error bars are obtained by bootstrapping⁴² with a 70% confidence interval. Total events are 312 and 252 for (C) and (D), respectively. Please click here to view a larger version of this figure.

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 along with DNA, and protein-protein collision on DNA¹⁷^,²⁰^,²⁶. Additionally, the high-throughput nature of the DNA curtain enables the collection of enough data to ensure statistical reliability. DNA curtain facilitates the investigation of the bio-physicochemical properties of proteins, including kinetic parameters, diffusion coefficient, speed, and processivity¹⁷^,²⁶^,²⁷^,³⁰. Importantly, DNA curtain can be used to determine the binding landscape of nucleosome deposition, which indicates the intrinsic sequence preference of nucleosomes³⁰.

Several points need to be considered to obtain high-quality data from the DNA curtain assay. First, the lipid bilayer must be appropriately formed on the slide surface. The fluidity of the lipid bilayer allows DNA molecules to move on the slide and be aligned at a diffusion barrier in the presence of hydrodynamic flow. The lipid bilayer also passivates the surface of the microfluidic chamber to prevent nonspecific adsorption of proteins to the surface. Because the passivation by the lipid bilayer is not complete, fluorescently labeled proteins are adsorbed nonspecifically to the surface, leading to incorrect interpretation of the results. However, the surface-stuck proteins can be distinguished from DNA-bound ones by turning the transient flow on and off because DNA-bound proteins disappear when the flow stops. Second, photobleaching of fluorophores needs to be suppressed. Many single-molecule fluorescence imaging methods adopt an oxygen scavenging system to reduce photobleaching. Several oxygen scavenging systems have been developed, the most popular of which is gloxy. Gloxy, consisting of glucose oxidase and catalase, enzymatically reduces molecular oxygens in solution but lowers the pH⁴⁴^,⁴⁵^,⁴⁶. Low pH is not compatible with physiological conditions and reduces lipid fluidity. To delay the decrease in pH, (1) the imaging buffer needs to be prepared with degassed deionized water, (2) the buffer must be immediately used, and (3) the buffer must be sealed and stored on ice until use.

A unique platform was developed to improve the DNA curtain system in which carved nano-trenches serve as diffusion barriers instead of chromium nano-patterns²⁵. Since these nano-trenches are more robust under harsh cleaning conditions with strong solvents, they allow repeatable, clearer imaging. However, the DNA curtain technique has several limitations. Under continuous laser illumination, the fluorophores that label proteins are photobleached, making long-time measurements challenging. DNA curtain does not work at low pH (lower than ~6) because the lipid bilayer is not fluidic. In addition, the fluorescence background and nonspecific binding of proteins to the slide surface disturb single-molecule imaging when protein concentration is high. Another drawback of the DNA curtain is that the spatial resolution of the DNA curtain is ~1 kbp/pixel, so the movement of proteins at less than 1 kbp cannot be observed. In addition, DNA curtain continuously applies hydrodynamic force to proteins on DNA. But the force by flow is weak (less than 1 pN), and hence it is challenging that histones or nucleosomes move along DNA in the curtain. It was also reported that nucleosomes rarely slide along DNA without chromatin remodelers⁴⁷. If histones or nucleosomes slide along DNA, most of them will stay at the end region of DNA in the curtain. However, we did not see the biased distribution. On the other hand, if histones or nucleosomes runoff from the DNA end, they would be depleted at the end of DNA. We did not observe this either.

This work demonstrates that the DNA curtain assay is a single-molecule imaging platform ideal for investigating chromatin dynamics. DNA curtain can be applied to study the process by which histone chaperones such as bromodomain-containing AAA+ ATPases assemble chromatin. The biological function of bromodomain-containing AAA+ ATPases is controversial. Lack of human ATAD2 or S. cerevisiae Yta7 downregulates gene expression via chromatin condensation¹⁸. In contrast, S. pombe Abo1 increases nucleosome density¹⁶. The single-molecule studies show that Abo1 catalyzes histone H3-H4 loading onto DNA. It is shown that the histone loading activity of Abo1 is dependent on ATP hydrolysis (Figure 2C,E,F). Moreover, the binding distribution of H3-H4 dimers shows that H3-H4 dimers are loaded onto DNA by Abo1 in a sequence-independent manner (Figure 3). In conclusion, the DNA curtain can be used to unravel the biological role of Abo1 as a histone chaperone in histone assembly. Using the DNA curtain technique, nucleosome formation by Abo1 and other histone chaperones such as CAF-1 and FACT will be studied in the future.

Based on this protocol, DNA curtain assay can be used to observe chromatin reorganization by remodeling complexes that translocate and rearrange nucleosomes.

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).

Materials

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

References

Woodcock, C. L., Ghosh, R. P. Chromatin higher-order structure and dynamics. Cold Spring Harbor Perspectives in Biology. 2 (5), 000596 (2010).
Kim, K., Eom, J., Jung, I. Characterization of structural variations in the context of 3D chromatin structure. Molecules and Cells. 42 (7), 512-522 (2019).
Kornberg, R. D. Chromatin structure: a repeating unit of histones and DNA. Science. 184 (4139), 868-871 (1974).
McGhee, J. D., Felsenfeld, G. Nucleosome structure. Annual Review of Biochemistry. 49, 1115-1156 (1980).
Takata, H., et al. Chromatin compaction protects genomic DNA from radiation damage. PLOS One. 8 (10), 75622 (2013).
Ehrenhofer-Murray, A. E. Chromatin dynamics at DNA replication, transcription and repair. European Journal of Biochemistry. 271 (12), 2335-2349 (2004).
Peterson, C. L., Almouzni, G. Nucleosome dynamics as modular systems that integrate DNA damage and repair. Cold Spring Harbor Perspectives in Biology. 5 (9), 012658 (2013).
Torigoe, S. E., Urwin, D. L., Ishii, H., Smith, D. E., Kadonaga, J. T. Identification of a rapidly formed nonnucleosomal histone-DNA intermediate that is converted into chromatin by ACF. Molecules and Cells. 43 (4), 638-648 (2011).
Gurard-Levin, Z. A., Quivy, J. P., Almouzni, G. Histone chaperones: assisting histone traffic and nucleosome dynamics. Annual Review of Biochemistry. 83, 487-517 (2014).
Burgess, R. J., Zhang, Z. Histone chaperones in nucleosome assembly and human disease. Nature Structural & Molecular Biology. 20 (1), 14-22 (2013).
Das, C., Tyler, J. K., Churchill, M. E. The histone shuffle: histone chaperones in an energetic dance. Trends in Biochemical Sciences. 35 (9), 476-489 (2010).
Hammond, C. M., Stromme, C. B., Huang, H., Patel, D. J., Groth, A. Histone chaperone networks shaping chromatin function. Nature Reviews Molecular Cell Biology. 18 (3), 141-158 (2017).
De Koning, L., Corpet, A., Haber, J. E., Almouzni, G. Histone chaperones: an escort network regulating histone traffic. Nature Structural & Molecular Biology. 14 (11), 997-1007 (2007).
Zou, J. X., Revenko, A. S., Li, L. B., Gemo, A. T., Chen, H. W. ANCCA, an estrogen-regulated AAA+ ATPase coactivator for ERalpha, is required for co-regulator occupancy and chromatin modification. Proceedings of the National Academy of Sciences of the United States of America. 104 (46), 18067-18072 (2007).
Lombardi, L. M., Davis, M. D., Rine, J. Maintenance of nucleosomal balance in cis by conserved AAA-ATPase Yta7. 遗传学. 199 (1), 105-116 (2015).
Gal, C., et al. Abo1, a conserved bromodomain AAA-ATPase, maintains global nucleosome occupancy and organisation. EMBO Reports. 17 (1), 79-93 (2016).
Cho, C., et al. Structural basis of nucleosome assembly by the Abo1 AAA+ ATPase histone chaperone. Nature Communications. 10 (1), 5764 (2019).
Morozumi, Y., et al. Atad2 is a generalist facilitator of chromatin dynamics in embryonic stem cells. Journal of Molecular Cell Biology. 8 (4), 349-362 (2016).
Zhang, M., Zhang, C., Du, W., Yang, X., Chen, Z. ATAD2 is overexpressed in gastric cancer and serves as an independent poor prognostic biomarker. Clinical and Translational Oncology. 18 (8), 776-781 (2016).
Kang, Y., Cho, C., Lee, K. S., Song, J. J., Lee, J. Y. Single-molecule imaging reveals the mechanism underlying histone loading of schizosaccharomyces pombe AAA+ ATPase Abo1. Molecules and Cells. 44 (2), 79-87 (2021).
Fried, M. G. Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay. Electrophoresis. 10 (5-6), 366-376 (1989).
Kessler, S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. Journal of Immunology. 115 (6), 1617-1624 (1975).
Lu, H. P. Single-molecule study of protein-protein and protein-DNA interaction dynamics. Methods in Molecular Biology. 305, 385-414 (2005).
Fazio, T., Visnapuu, M. L., Wind, S., Greene, E. C. DNA curtains and nanoscale curtain rods: high-throughput tools for single molecule imaging. Langmuir. 24 (18), 10524-10531 (2008).
Kang, Y., et al. High-throughput single-molecule imaging system using nanofabricated trenches and fluorescent DNA-binding proteins. Biotechnology and Bioengineering. 117 (6), 1640-1648 (2020).
Cheon, N. Y., Kim, H. S., Yeo, J. E., Scharer, O. D., Lee, J. Y. Single-molecule visualization reveals the damage search mechanism for the human NER protein XPC-RAD23B. Nucleic Acids Research. 47 (16), 8337-8347 (2019).
Lee, J. Y., Finkelstein, I. J., Arciszewska, L. K., Sherratt, D. J., Greene, E. C. Single-molecule imaging of FtsK translocation reveals mechanistic features of protein-protein collisions on DNA. Molecules and Cells. 54 (5), 832-843 (2014).
Kang, H. J., et al. TonEBP recognizes R-loops and initiates m6A RNA methylation for R-loop resolution. Nucleic Acids Research. 49 (1), 269-284 (2021).
Zhou, H., et al. Mechanism of DNA-induced phase separation for transcriptional repressor VRN1. Angewandte Chemie International Edition. 58 (15), 4858-4862 (2019).
Visnapuu, M. L., Greene, E. C. Single-molecule imaging of DNA curtains reveals intrinsic energy landscapes for nucleosome deposition. Nature Structural & Molecular Biology. 16 (10), 1056-1062 (2009).
Stigler, J., Camdere, G. O., Koshland, D. E., Greene, E. C. Single-molecule imaging reveals a collapsed conformational state for DNA-bound cohesin. Cell Reports. 15 (5), 988-998 (2016).
Thornton, J. A. Sputter Coating- Its Principles and Potential. SAE Transactions. 82, 1787-1805 (1973).
Grigorescu, A. E., Hagen, C. W. Resists for sub-20-nm electron beam lithography with a focus on HSQ: state of the art. Nanotechnology. 20 (29), 292001 (2009).
Meir, A., Kong, M., Xue, C., Greene, E. C. DNA curtains shed light on complex molecular systems during homologous recombination. Journal of Visualized Experiments. (160), e61320 (2020).
Cold Spring Harbor Protocols. Gloxy. Vol. 2. Cold Spring Harbor Protocols. , (2007).
Gracey, L. E., et al. An in vitro-identified high-affinity nucleosome-positioning signal is capable of transiently positioning a nucleosome in vivo. Epigenetics Chromatin. 3 (1), 13 (2010).
Subtil-Rodriguez, A., Reyes, J. C. BRG1 helps RNA polymerase II to overcome a nucleosomal barrier during elongation, in vivo. EMBO Reports. 11 (10), 751-757 (2010).
Lancrey, A., et al. Nucleosome positioning on large tandem DNA repeats of the ‘601’ sequence engineered in Saccharomyces cerevisiae. bioRxiv. , (2021).
Perales, R., Zhang, L., Bentley, D. Histone occupancy in vivo at the 601 nucleosome binding element is determined by transcriptional history. Molecular and Cellular Biology. 31 (16), 3485-3496 (2011).
Lowary, P. T., Widom, J. New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning. Journal of Molecular Biology. 276 (1), 19-42 (1998).
Rossmann, K. Point spread-function, line spread-function, and modulation transfer function. Tools for the study of imaging systems. Radiology. 93 (2), 257-272 (1969).
Blainey, P. C., et al. Nonspecifically bound proteins spin while diffusing along DNA. Nature Structural & Molecular Biology. 16 (12), 1224-1229 (2009).
Collins, B. E., Ye, L. F., Duzdevich, D., Greene, E. C. DNA curtains: novel tools for imaging protein-nucleic acid interactions at the single-molecule level. Methods in Cell Biology. 123, 217-234 (2014).
Shi, X., Lim, J., Ha, T. Acidification of the oxygen scavenging system in single-molecule fluorescence studies: in situ sensing with a ratiometric dual-emission probe. Analytical Chemistry. 82 (14), 6132-6138 (2010).
Rasnik, I., McKinney, S. A., Ha, T. Nonblinking and long-lasting single-molecule fluorescence imaging. Nature Methods. 3 (11), 891-893 (2006).
Aitken, C. E., Marshall, R. A., Puglisi, J. D. An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophysical Journal. 94 (5), 1826-1835 (2008).
Teif, V. B., Rippe, K. Nucleosome mediated crosstalk between transcription factors at eukaryotic enhancers. Physical Biology. 8 (4), 044001 (2011).

Play Video

PDF

DOI

DOWNLOAD MATERIALS LIST

Cite This Article

Kang, Y., Bae, S., An, S., Lee, J. Y. Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique. J. Vis. Exp. (181), e63501, doi:10.3791/63501 (2022).