Name: deciphering molecular mechanism of histone assembly by dna curtain technique
Uploaded: 2022-03-09T14:30:00
Description: Watch this Scientific Journal Video about Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique at JoVE.com

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).

1. Preparation of the flow cell
<ol>
	<li>Prepare a cleaned fused silica slide containing nano-trench patterns following previously published report25.
	<ol>
		<li>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).</li>
		<li>Deposit 250 nm of thick aluminum (Al) on the slide using DC sputter32 (see Table of Materia...

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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL luer-lock syringe</td>
			<td>BecktonDickinson</td>
			<td>301321</td>
			<td></td>
		</tr>
		<tr>
			<td>1&#39; x 3&#39; fused-silca slide glass</td>
			<td>G. Finkenbeiner</td>
			<td></td>
			<td>1 inch x 3 inch rectangular and 1 mm thickness</td>
		</tr>
		<tr>
			<td>10 mL luer-lock syringe</td>
			<td>BecktonDickinson</td>
			<td>302149</td>
			<td></td>
		</tr>
		<tr>
			<td>18:1 (&#916;9-Cis) PC (DOPC)</td>
			<td>Avanti</td>
			<td>850375</td>
			<td>This is a component of biotinylated lipid stock</td>
		</tr>
		<tr>
			<td>18:1 Biotinyl cap PE</td>
			<td>Avanti</td>
			<td>870273</td>
			<td>This is a component of biotinylated lipid stock</td>
		</tr>
		<tr>
			<td>18:1 PEG2000 PE</td>
			<td>Avanti</td>
			<td>880130</td>
			<td>This is a component of biotinylated lipid stock</td>
		</tr>
		<tr>
			<td>3 mL luer-lock syringe</td>
			<td>BecktonDickinson</td>
			<td>302832</td>
			<td></td>
		</tr>
		<tr>
			<td>6-way sample injection valve</td>
			<td>IDEX</td>
			<td>MX series II</td>
			<td></td>
		</tr>
		<tr>
			<td>950K PMMA</td>
			<td>All-resist</td>
			<td>671.04</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>SAMCHUN</td>
			<td>A1759</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#39;-triphosphate disodium salt hydrate (ATP)</td>
			<td>Sigma</td>
			<td>A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum (Al)</td>
			<td>TASCO, South Korea</td>
			<td>LT50AI414</td>
			<td>Diameter 4 inch, thickness 1/4 inch</td>
		</tr>
		<tr>
			<td>Amicon Ultra centrifugal filter, MWCO 10 kDa</td>
			<td>Millipore</td>
			<td>Z648027</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Mbcell</td>
			<td>MB-A4128</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>AZ 300 MIF developer</td>
			<td>Merck</td>
			<td>10454110521</td>
			<td>Used for removing aluminum</td>
		</tr>
		<tr>
			<td>Blade</td>
			<td>DORCO</td>
			<td>DN52</td>
			<td>12 mm x 6 m</td>
		</tr>
		<tr>
			<td>Boron trichloride (BCl3)</td>
			<td>UNIONGAS</td>
			<td></td>
			<td>Purity: &#62;99.99%</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma</td>
			<td>C40-1g</td>
			<td>This is a component of 100x gloxy stock</td>
		</tr>
		<tr>
			<td>Chlorine (Cl2)</td>
			<td>UNIONGAS</td>
			<td></td>
			<td>Purity: &#62;99.99%</td>
		</tr>
		<tr>
			<td>Clear double-sided tape</td>
			<td>3M</td>
			<td>313770</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-glucose</td>
			<td>Sigma</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>DC sputter</td>
			<td>Sorona</td>
			<td>SRM-120</td>
			<td>Used for deposition aluminum on a slide</td>
		</tr>
		<tr>
			<td>Diamond-coated drill bit</td>
			<td>Eurotool</td>
			<td>DIB-211.00</td>
			<td>Used for making holes in a fusced silica slide</td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr>
			<td>Dove-prism</td>
			<td>Korea Electro-Optics Co. Ltd.</td>
			<td>1906-106</td>
			<td>Custom-made fused-silica dove prism with anti-reflection coating</td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>Dremel</td>
			<td>Dremel 3000</td>
			<td>Used for making holes in a fusced silica slide</td>
		</tr>
		<tr>
			<td>Electron Bean Lithography</td>
			<td>Nanobeam Ltd.</td>
			<td>NB3</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene-diamine-tetraacetic acid (EDTA)</td>
			<td>Sigma</td>
			<td>EDS-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Fingertight fittings</td>
			<td>IDEX</td>
			<td>F-300</td>
			<td>It is connected with &#34;PFA Tubing Natural&#34; to form luer-lock tubing</td>
		</tr>
		<tr>
			<td>Flangeless male nut</td>
			<td>IDEX</td>
			<td>P-235</td>
			<td>It is connected with &#34;PFA Tubing Natural&#34; to form luer-lock tubing</td>
		</tr>
		<tr>
			<td>Freeze Dryer, HyperCOOL</td>
			<td>Labogene</td>
			<td>HC3110</td>
			<td>Used for lyophilizing liquid proteins</td>
		</tr>
		<tr>
			<td>Glucose oxidase</td>
			<td>Sigma</td>
			<td>G2133-50KU</td>
			<td>This is a component of 100x gloxy stock</td>
		</tr>
		<tr>
			<td>Guanidinium hydrochloride</td>
			<td>Acros Organics</td>
			<td>364790025</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton Company</td>
			<td>80065</td>
			<td>This syringe is used for sample injection</td>
		</tr>
		<tr>
			<td>Hellmanex III</td>
			<td>Sigma</td>
			<td>Z805939</td>
			<td></td>
		</tr>
		<tr>
			<td>HiLoad 26/600 SuperdexTM 200 pg</td>
			<td>Cytiva</td>
			<td>28-9893-36</td>
			<td>Used for FPLC (size exclusion)</td>
		</tr>
		<tr>
			<td>Hot plate stirrer</td>
			<td>Corning</td>
			<td>PC-420D</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma</td>
			<td>H1759</td>
			<td>Used for Tris-HCl</td>
		</tr>
		<tr>
			<td>Index matching oil</td>
			<td>ZEISS</td>
			<td>444970-9000-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Inductively coupled plasma-reactive ion etching</td>
			<td>Top Technology Ltd.</td>
			<td>FabStar</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG)</td>
			<td>Glentham Life Sciences</td>
			<td>GC6586-100g</td>
			<td>Used for induction of &#946;-galactosidase activity</td>
		</tr>
		<tr>
			<td>Lambda phage DNA</td>
			<td>NEB</td>
			<td>N0311</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>BD difco</td>
			<td>244610</td>
			<td>Media for E.coli cell growth</td>
		</tr>
		<tr>
			<td>Luer adapter 10-32</td>
			<td>IDEX</td>
			<td>P-659</td>
			<td>This connects luer-lock syringe and tubing</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>fisher bioreagents</td>
			<td>BP214</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl isobutyl ketone (MIBK)</td>
			<td>KAYAKU ADVANCED MATERIALS</td>
			<td></td>
			<td>Used for developing solution</td>
		</tr>
		<tr>
			<td>Microscope (Eclipse Ti2)</td>
			<td>Nikon</td>
			<td>Eclipse Ti2</td>
			<td>Inverted fluorescence microscope</td>
		</tr>
		<tr>
			<td>Microscope glass coverslip</td>
			<td>MARIENFELD</td>
			<td>101142</td>
			<td>22 x 50 mm (No. 1)</td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>DURAN GROUP</td>
			<td>DU.2355013</td>
			<td>Slide glass ground edge 45&#176;, plain 26 x 76 mm</td>
		</tr>
		<tr>
			<td>Nanoport</td>
			<td>IDEX</td>
			<td>N-333-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective lens</td>
			<td>Nikon</td>
			<td>CFI Plan Apochromat VC 60XC WI</td>
			<td>Immersion type: water, magnification: 60x, correction: 18, working distance: 0.29 (0.31-0.28)</td>
		</tr>
		<tr>
			<td>One Shot BL21 (DE3)pLysS Chemically Competent E. coli</td>
			<td>Thermo Fisher Scientific</td>
			<td>C6060-03</td>
			<td>Competent cell for overexpressing proteins</td>
		</tr>
		<tr>
			<td>Oxygen (O2)</td>
			<td>NOBLEGAS, South Korea</td>
			<td></td>
			<td>Purity: &#62;99.99%</td>
		</tr>
		<tr>
			<td>PFA tubing natural</td>
			<td>IDEX</td>
			<td>1512L</td>
			<td>It is connected with &#34;Fingertight Fittings&#34; to form luer-lock tubing</td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>Roche</td>
			<td>11359061001</td>
			<td>Protease inhibitor</td>
		</tr>
		<tr>
			<td>Sephacryl S-200 High Resolution</td>
			<td>Cytiva</td>
			<td>17-0584-01</td>
			<td>Used for FPLC (size exclusion)</td>
		</tr>
		<tr>
			<td>Shut-off valve</td>
			<td>IDEX</td>
			<td>P-732</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma</td>
			<td>791741</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma</td>
			<td>s5881</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectra/Por molecularporous membrane tubing, MWCO 6-8 kDa</td>
			<td>Spectrum laboratories</td>
			<td>132660</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>S888</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfur tetralfluoride (SF4)</td>
			<td>NOBLEGAS, South Korea</td>
			<td></td>
			<td>Purity: &#62;99.99%</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>KD Scientific</td>
			<td>78-8210</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrafluoromethane (CF4)</td>
			<td>NOBLEGAS, South Korea</td>
			<td></td>
			<td>Purity: &#62;99.99%</td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Sigma</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td>Used for Tris-HCl</td>
		</tr>
		<tr>
			<td>TSKgel SP-5PW</td>
			<td>TOSOH</td>
			<td>14715</td>
			<td>Used for FPLC (ion exchange)</td>
		</tr>
		<tr>
			<td>Union assembly</td>
			<td>IDEX</td>
			<td>P-760</td>
			<td>This connects tubings</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma</td>
			<td>U5378</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum oven</td>
			<td>Jeio Tech</td>
			<td>OV-11</td>
			<td></td>
		</tr>
		<tr>
			<td>YOYO-1</td>
			<td>Thermo Fisher Scientific</td>
			<td>Y3601</td>
			<td>This intercalation dye is diluted in DMSO</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol (BME)</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td></td>
		</tr>
	</tbody>
</table>

deciphering molecular mechanism of histone assembly by dna curtain technique

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.

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 (<strong class="xfi...

Watch this Scientific Journal Video about Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique at JoVE.com

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

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.

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in ...

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