Name: deciphering molecular mechanism of histone assembly by dna curtain technique
Uploaded: 2022-03-09T14:30:00.000+00:00
Duration: 6 min 32 s
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 Materials) with 10 mTorr of argon gas.</li>
		<li>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 &#176;C for 3 min.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Carve nano-trenches on the slides using sulfur tetrafluoride (SF4), tetrafluoromethane (CF4), and oxygen (O2) gases (see Table of Materials).</li>
		<li>Remove the remaining Al layer by soaking the slides in Al etchant (AZ 300 MIF developer, see Table of Materials) for 10 min.</li>
		<li>After fabrication, rinse the slides with deionized water and sonicate in acetone for 30 min using a bath-type sonicator.</li>
		<li>Clean the slides in 2% Hellmanex III solution (see Table of Materials) for at least 1 day with magnetic stirring.</li>
		<li>Sonicate the slides in acetone for 30 min and 1 M NaOH for 30 min successively.</li>
		<li>Rinse the slides with deionized water and dry with a nitrogen (N2) gas.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Excise the paper using a clean blade (Figure 1A).</li>
	<li>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).</li>
	<li>Place the assembled flow cell between two microscope slides and clip them.</li>
	<li>Bake the flow cell in a 120 &#176;C vacuum oven for 45 min.</li>
	<li>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.</li>
	<li>Connect a Luer lock syringe containing 3 mL of deionized water and wash the chamber.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Inject 0.025 mg/mL of streptavidin in 1 mL of BSA buffer with two shots and 10 min incubation per shot.</li>
	<li>Wash out the residual streptavidin with 3 mL of BSA buffer.</li>
	<li>Inject ~300 pM (30 &#181;L) of biotinylated lambda phage DNA in 1 mL of BSA buffer into the chamber with two shots and 10 min incubation per shot. 
	&#8203;NOTE: Preparation of biotinylated lambda DNA is described in a previously published report34.</li>
</ol>
2. Connecting flow cell to the microfluidic system and loading it onto the microscope
<ol>
	<li>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.</li>
	<li>Connect a syringe containing 10 mL of imaging buffer to a syringe pump and remove bubbles in all tubing lines in the microfluidic system.</li>
	<li>Couple the prepared flow cell with the microfluidic system via drop-to-drop connection to avoid bubble injection into the flow cell (Figure 1B).</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Histone assembly by Abo1 using DNA curtain
<ol>
	<li>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 &#181;L of imaging buffer. All proteins were prepared following previously published report17.</li>
	<li>Incubate the mixture on ice for 15 min. 
	NOTE: To avoid photobleaching of Cy5, the incubation must be done in the dark.</li>
	<li>Inject ~20 pM (2 &#181;L) of YOYO-1 dye in imaging buffer through a 100 &#181;L of sample loop to stain lambda DNA molecules.</li>
	<li>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.</li>
	<li>Inject 2 mL of high salt buffer (200 mM of NaCl and 40 mM of MgCl2) to eliminate YOYO-1 dye from DNA.</li>
	<li>After YOYO-1 dye has been removed, inject the pre-incubated protein sample.</li>
	<li>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.</li>
	<li>Wash out the unbound Abo1 and histone proteins for 5 min.</li>
	<li>Switch on the shut-off valve and resume the flow injection.</li>
	<li>Turn on the 637 nm laser and image the fluorescent proteins while the buffer flow is on.</li>
	<li>Transiently turn off the buffer flow to check if histone proteins are loaded onto DNA (Figure 2C).</li>
	<li>Collect DNA curtain images with an image acquisition program (see Table of Materials). 
	&#8203;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.</li>
</ol>
4. Data analysis
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Create the fluorescence intensity profile from the kymograph and fit it with multiple Gaussian functions.</li>
	<li>Collect the center coordinates of peaks from the fluorescence intensity profile. In this step, the minimum intensity of histone fluorescence can be obtained.</li>
	<li>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.</li>
	<li>Perform steps 4.2-4.5 for other DNA molecules.</li>
	<li>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.</li>
</ol>

The authors have nothing to disclose.

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><tbody><tr><td>1 mL luer-lock syringe</td><td>BecktonDickinson</td><td>301321</td><td></td></tr><tr><td>1&#039; x 3&#039; 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 (&amp;Delta;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&#039;-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 (BCl&lt;sub&gt;3&lt;/sub&gt;)</td><td>UNIONGAS</td><td></td><td>Purity: &gt;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 (Cl&lt;sub&gt;2&lt;/sub&gt;)</td><td>UNIONGAS</td><td></td><td>Purity: &gt;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 &quot;PFA Tubing Natural&quot; 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 &quot;PFA Tubing Natural&quot; 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 &amp;beta;-D-1-thiogalactopyranoside (IPTG)</td><td>Glentham Life Sciences</td><td>GC6586-100g</td><td>Used for induction of &amp;beta;-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&amp;deg;, 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 (O&lt;sub&gt;2&lt;/sub&gt;)</td><td>NOBLEGAS, South Korea</td><td></td><td>Purity: &gt;99.99%</td></tr><tr><td>PFA tubing natural</td><td>IDEX</td><td>1512L</td><td>It is connected with &quot;Fingertight Fittings&quot; 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 (SF&lt;sub&gt;4&lt;/sub&gt;)</td><td>NOBLEGAS, South Korea</td><td></td><td>Purity: &gt;99.99%</td></tr><tr><td>Syringe pump</td><td>KD Scientific</td><td>78-8210</td><td></td></tr><tr><td>Tetrafluoromethane (CF&lt;sub&gt;4&lt;/sub&gt;)</td><td>NOBLEGAS, South Korea</td><td></td><td>Purity: &gt;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>&amp;beta;-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 ...

deciphering-molecular-mechanism-histone-assembly-dna-curtain

Research

JoVE Journal

Biochemistry

1.7K Views.  Ulsan National Institute of Science and Technology. 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.

Video: Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, such as transcription, DNA replication, and DNA repair. HMGB1 binds to structurally distorted DNA with higher affinity than to canonical B-DNA. For example, we found that HMGB1 binds to DNA interstrand crosslinks (ICLs), which covalently link the two strands of the DNA, cause distortion of the helix, and if left unrepaired can cause cell death. Due to their cytotoxic potential, several ICL-inducing agents are currently used as chemotherapeutic agents in the clinic. While ICL-forming agents show preferences for certain base sequences (e.g., 5&#39;-TA-3&#39; is the preferred crosslinking site for psoralen), they largely induce DNA damage in an indiscriminate fashion. However, by covalently coupling the ICL-inducing agent to a triplex-forming oligonucleotide (TFO), which binds to DNA in a sequence-specific manner, targeted DNA damage can be achieved. Here, we use a TFO covalently conjugated on the 5&#39; end to a 4&#39;-hydroxymethyl-4,5&#39;,8-trimethylpsoralen (HMT) psoralen to generate a site-specific ICL on a mutation-reporter plasmid to use as a tool to study the architectural modification, processing, and repair of complex DNA lesions by HMGB1 in human cells. We describe experimental techniques to prepare TFO-directed ICLs on reporter plasmids, and to interrogate the association of HMGB1 with the TFO-directed ICLs in a cellular context using chromatin immunoprecipitation assays. In addition, we describe DNA supercoiling assays to assess specific architectural modification of the damaged DNA by measuring the amount of superhelical turns introduced on the psoralen-crosslinked plasmid by HMGB1. These techniques can be used to study the roles of other proteins involved in the processing and repair of TFO-directed ICLs or other targeted DNA damage in any cell line of interest.

Targeted DNA damage can be achieved by tethering a DNA damaging agent to a triplex-forming oligonucleotide (TFO). Using modified TFOs, DNA damage-specific protein association, and DNA topology modification can be studied in human cells by the utilization of modified chromatin immunoprecipitation assays and DNA supercoiling assays described herein.

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, ...

Genetics

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription to take place in eukaryotic cells. The dynamics of nucleosomes provides access to the DNA by replication and transcription machineries, and critically contributes to the molecular mechanisms underlying chromatin functions. Single-molecule studies such as atomic force microscopy (AFM) imaging have contributed significantly to our current understanding of the role of nucleosome structure and dynamics. The current protocol describes the steps enabling high-resolution AFM imaging techniques to study the structural and dynamic properties of nucleosomes. The protocol is illustrated by AFM data obtained for the centromere nucleosomes in which H3 histone is replaced with its counterpart centromere protein A (CENP-A). The protocol starts with the assembly of mono-nucleosomes using a continuous dilution method. The preparation of the mica substrate functionalized with aminopropyl silatrane (APS-mica) that is used for the nucleosome imaging is critical for the AFM visualization of nucleosomes described and the procedure to prepare the substrate is provided. Nucleosomes deposited on the APS-mica surface are first imaged using static AFM, which captures a snapshot of the nucleosome population. From analyses of these images, such parameters as the size of DNA wrapped around the nucleosomes can be measured and this process is also detailed. The time-lapse AFM imaging procedure in the liquid is described for the high-speed time-lapse AFM that can capture several frames of nucleosome dynamics per second. Finally, the analysis of nucleosome dynamics enabling the quantitative characterization of the dynamic processes is described and illustrated.

Here, we present a protocol to characterize nucleosome particles at the single-molecule level using static and time-lapse atomic force microscopy (AFM) imaging techniques. The surface functionalization method described allows for the capture of the structure and dynamics of nucleosomes in high-resolution at the nanoscale.

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription ...

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in HR are closely associated with a loss of genome integrity and oncogenic transformation in human cells. HR involves coordinated actions of a complex set of proteins, many of which remain poorly understood. The key aspect of the research described here is a technology called "DNA curtains", a technique which allows for the assembly of aligned DNA molecules on the surface of a microfluidic sample chamber. They can then be visualized by total internal reflection fluorescence microscopy (TIRFM). DNA curtains was pioneered by our laboratory and allows for direct access to spatiotemporal information at millisecond time scales and nanometer scale resolution, which cannot be easily revealed through other methodologies. A major advantage of DNA curtains is that it simplifies the collection of statistically relevant data from single molecule experiments. This research continues to yield new insights into how cells regulate and preserve genome integrity.

DNA curtains present a novel method for visualizing hundreds or even thousands of DNA-binding proteins in real-time as they interact with DNA molecules aligned on the surface of a microfluidic sample chamber.

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in ...

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi pyrrolysine tRNA-synthetase (MmAcKRS1) and its cognate tRNA (tRNAPyl) to assemble reconstituted mononucleosomes with site specific acetylated histones. MmAcKRS1 and tRNAPyl deliver AcK at an amber mutation site in the mRNA of choice during translation in Escherichia coli. This technique has been used extensively to incorporate AcK at H3 lysine sites. Pyrrolysyl-tRNA synthetase (PylRS) can also be easily evolved to incorporate other noncanonical amino acids (ncAAs) for site specific protein modification or functionalization. Here we detail a method to incorporate AcK using the MmAcKRS1 system into histone H3 and integrate acetylated H3 proteins into reconstituted mononucleosomes. Acetylated reconstituted mononucleosomes can be used in biochemical and binding assays, structure determination, and more. Obtaining modified mononucleosomes is crucial for designing experiments related to discovering new interactions and understanding epigenetics.

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Here we present a method to express acetylated histone proteins using genetic code expansion and assemble reconstituted nucleosomes in vitro.

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

This protocol describes a battery of methods that includes analytical size-exclusion chromatography to study histone chaperone oligomerization and stability, pull-down assay to unravel histone chaperone-histone interactions, AUC to analyze the stoichiometry of the protein complexes, and histone chaperoning assay to functionally characterize a putative histone chaperone in vitro.

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting ...

In Situ Nucleosome Reconstitution for Single-Molecule Studies

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding their biophysical properties and interactions with chromatin-binding proteins. Nucleosome reconstitution on DNA for these studies typically involves a salt dialysis procedure that provides precise control over the placement and number of nucleosomes formed along a DNA tether. However, this protocol is time-consuming and requires a substantial amount of DNA and histone octamers as inputs. To offer an alternative strategy, an in situ nucleosome reconstitution method for single-molecule force and fluorescence microscopy that utilizes the histone chaperone Nap1 is described. This method enables users to assemble nucleosomes on any DNA template without the need for strong nucleosome positioning sequences, adjust nucleosome density on demand, and use fewer reagents. In situ nucleosome formation occurs within seconds, offering a simpler experimental workflow and a convenient transition into single-molecule measurements. Examples of two downstream assays for probing nucleosome mechanics and visualizing the behavior of individual proteins on chromatin are further described.

Author Spotlight: Efficient Nucleosome Reconstitution for Single-Molecule Techniques

This article presents a detailed experimental procedure for reconstituting nucleosome-containing DNA tethers for single-molecule correlative force and fluorescence microscopy. It further describes several downstream experiments that can be conducted to visualize the binding behavior of chromatin-interacting proteins and analyze changes in the physical properties of nucleosomes.

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding ...

Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

Core histone octamers that are repetitively spaced along a DNA molecule are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. The latter method has the benefit of allowing for the assembly of a more compositionally uniform and precisely positioned nucleosomal array. Sedimentation velocity experiments in the analytical ultracentrifuge yield information about the size and shape of macromolecules by analyzing the rate at which they migrate through solution under centrifugal force. This technique, along with atomic force microscopy, can be used for quality control, ensuring that the majority of DNA templates are saturated with nucleosomes after reconstitution. Here we describe the protocols necessary to reconstitute milligram quantities of length and compositionally defined nucleosomal arrays suitable for biochemical and biophysical studies of chromatin structure and function.

A method is presented for the reconstitution of model nucleosomal arrays from recombinant core histones and tandemly repeated nucleosome positioning DNA. We also describe how sedimentation velocity experiments in the analytical ultracentrifuge, and atomic force microscopy (AFM) are used to monitor the extent of nucleosomal array saturation after reconstitution.

Core histone octamers that are repetitively spaced along a DNA molecule  are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two  ...

Biology

High-Speed Time-Lapse Atomic Force Microscopy to Capture Nucleosome Dynamics

Source: Stumme-Diers, M. P., et al., <a href="https://app.jove.com/v/58820">Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging.</a>&#160;J. Vis. Exp.&#160;(2019)
In this video, we demonstrate high-speed time-lapse atomic force microscopy, AFM, imaging techniques to study the dynamics of nucleosome complexes. As the AFM tip scans across the surface of the nucleosome, it detects alterations in the nucleosome height and records the DNA unwrapping over time.

Source: Stumme-Diers, M. P., et al., Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging.&#160;J. Vis. ...

JoVE Encyclopedia of Experiments

Biological Techniques

Microscopy Techniques

The Nucleosome

Human DNA is almost two meters long. However, it is compressed inside a tiny nucleus measuring only a few microns in diameter. To make this degree of compaction possible, DNA is organized into several sequential levels so that it can fit into such a tiny space. The most compact form of DNA is a chromosome that can be seen under a microscope in a dividing cell.
In a chromosome, DNA is wound twice around a protein complex called a histone octamer core, which consists of 8 histone proteins. This DNA and histone protein complex together forms the nucleosome, the fundamental and functional unit of DNA compaction. Nucleosomes can further coil around themselves into higher-order compact structures.
Histones are highly conserved proteins.
The amino acid sequences of core histone proteins are highly conserved even between distantly related species. For example, the amino acid sequence of the H3 histone between a&#160; calf thymus and a pea plant has only four amino acid differences.
Non-histone proteins
The nucleosome complex is also bound by a small proportion of non-histone proteins, which help maintain the compaction and organize long chromatin loops. Non-histone proteins are also involved in regulating DNA replication and RNA synthesis.

Human DNA is almost two meters long. However, it is compressed inside a tiny nucleus measuring only a few microns in diameter. To make this degree of ...

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Summary

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Chapters in this video

Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

High-Speed Time-Lapse Atomic Force Microscopy to Capture Nucleosome Dynamics

The Nucleosome