Name: in vitro characterization of histone chaperones using analytical, pull-down and chaperoning assays
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The extramural grants to Dileep Vasudevan from the Science and Engineering Research Board, Government of India [CRG/2018/000695/PS] and the Department of Biotechnology, Ministry of Science and Technology, Government of India [BT/INF/22/SP33046/2019], as well as the intramural support from the Institute of Life Sciences, Bhubaneswar are greatly acknowledged.&#160;We thank Ms. Sudeshna Sen and Ms. Annapurna Sahoo for their help with histone preparation. The discussions with our colleagues Dr. Chinmayee Mohapatra, Mr. Manas Kumar Jagdev, and Dr. Shaikh Nausad Hossain are also acknowledged.

1. Analytical size-exclusion chromatography to elucidate the oligomeric status and stability of histone chaperones
<ol>
	<li>Analysis of the oligomeric status of histone chaperones
	<ol>
		<li>Equilibrate a 24 mL analytical size-exclusion chromatography (SEC) column with 1.2 column volume (CV), i.e., 28.8 mL of degassed SEC buffer [20 mM of Tris-HCl (pH 7.5), 300 of mM NaCl, and 1 mM of &#946;-mercaptoethanol (&#946;-ME)] at 4 &#176;C (see Table of Materials). 
		NOTE: Column type...

<ol>
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This work demonstrates and validates a comprehensive set of protocols for the biochemical and biophysical characterization of a putative histone chaperone. Herein, recombinantly expressed and purified NAP family proteins, AtNRP1 and AtNRP2, and the N-terminal nucleoplasmin domain of AtFKBP53 were used to demonstrate the protocols. The same set of experiments could very well be used to delineate the functional attributes of previously uncharacterized histone chaperones from any organism.
The fi...

Nucleosomes composed of DNA and histone proteins form the structural unit of chromatin and regulate several critical cellular events. Nucleosomes are dynamically repositioned and remodeled to make DNA accessible to various processes such as replication, transcription, and translation1,2. Histones that are highly basic either tend to interact with acidic proteins in the cellular milieu or undergo aggregation, thus leading to various cellular defects3,4,5. A group of dedicated proteins called histone chaperones aid the transport of histones from the cytoplasm to the nucleus and prevent aberrant histone-DNA aggregation events6,7. Fundamentally, most histone chaperones store and transfer histones onto DNA at physiological ionic strength, thereby aiding the formation of nucleosomes8,9. Some histone chaperones have a definite preference for the histone oligomers H2A/H2B or H3/H410.
Histone chaperones are characterized based on their ability to assemble nucleosomes dependent or independent of DNA synthesis11. For example, chromatin assembly factor-1 (CAF-1) is dependent while histone regulator A (HIRA) is independent of DNA synthesis12,13. Similarly, the nucleoplasmin family of histone chaperones is involved in sperm chromatin decondensation and nucleosome assembly14. The nucleosome assembly protein (NAP) family members facilitate the formation of nucleosome-like structures in vitro and are involved in the shuttling of histones between cytoplasm and nucleus15. Nucleoplasmins and NAP family proteins are both functional histone chaperones but do not share any structural features. Essentially, no single structural feature allows the classification of a protein as a histone chaperone16. The usage of functional and biophysical assays along with structural studies work best in characterizing histone chaperones.
This work describes biochemical and biophysical methods to characterize a protein as a histone chaperone that aids nucleosome assembly. First, analytical size-exclusion chromatography was carried out to analyze the oligomeric status and stability of histone chaperones. Next, a pull-down assay was performed to determine the driving forces and the competitive nature of histone chaperone-histone interactions. However, the hydrodynamic parameters of these interactions could not be accurately calculated using analytical size-exclusion chromatography because of the protein&#39;s shape and its complexes that impact their migration through the column. Therefore, analytical ultracentrifugation was used, which provides in-solution macromolecular properties that include accurate molecular weight, the stoichiometry of interaction, and the shape of the biological molecules. Past studies have extensively used in vitro histone chaperoning assay to functionally characterize histone chaperones such as yScS11617, DmACF18, ScRTT106p19, HsNPM120. Histone chaperoning assay was also used to functionally characterize the proteins as histone chaperones.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic acid (glacial)</td>
			<td>Sigma</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>MP Biomedicals</td>
			<td>814326</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>MP Biomedicals</td>
			<td>193983</td>
			<td></td>
		</tr>
		<tr>
			<td>AKTA Pure 25M FPLC</td>
			<td>Cytiva</td>
			<td>29018226</td>
			<td>Instrument for protein purification</td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma</td>
			<td>A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>An-60Ti rotor</td>
			<td>Beckman Coulter</td>
			<td>361964</td>
			<td>Rotor for analytical ultracentrifugation</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>C2432</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie brilliant blue R 250</td>
			<td>Sigma</td>
			<td>1.15444</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing (7 kDa cut-off)</td>
			<td>Thermo Fisher</td>
			<td>68700</td>
			<td>For dialysing protein samples</td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>MP Biomedicals</td>
			<td>100597</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Loading Dye</td>
			<td>New England Biolabs</td>
			<td>B7025S</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA disodium salt</td>
			<td>MP Biomedicals</td>
			<td>194822</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>Shimadzu</td>
			<td>ATX224R</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium bromide (EtBr)</td>
			<td>Sigma</td>
			<td>E8751</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Doc System</td>
			<td>Bio-Rad</td>
			<td>12009077</td>
			<td>For imaging gels after staining</td>
		</tr>
		<tr>
			<td>Horizontal gel electrophoresis apparatus</td>
			<td>Bio-Rad</td>
			<td>1704405</td>
			<td>Instrument for agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Sigma</td>
			<td>320331</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>MP Biomedicals</td>
			<td>102033</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2)</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td>Eppendorf</td>
			<td>Z683779</td>
			<td>For pipetting of micro-volumes</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN electrophoresis system</td>
			<td>Bio-Rad</td>
			<td>1658000</td>
			<td>Instrument for SDS-PAGE</td>
		</tr>
		<tr>
			<td>N,N-methylene-bis-acrylamide</td>
			<td>MP Biomedicals</td>
			<td>800172</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano drop</td>
			<td>Thermo Fisher</td>
			<td>ND-2000</td>
			<td>For measurement of protein and DNA concentrations</td>
		</tr>
		<tr>
			<td>Ni-NTA agarose</td>
			<td>Invitrogen</td>
			<td>R901-15</td>
			<td>Resin beads for pull-down assay</td>
		</tr>
		<tr>
			<td>Optima AUC analytical ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>B86437</td>
			<td>Instrument for analytical ultracentrifugation</td>
		</tr>
		<tr>
			<td>pH Meter</td>
			<td>Mettler Toledo</td>
			<td>MT30130863</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Sigma</td>
			<td>P4557</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid isolation kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma-Aldrich</td>
			<td>1.07393</td>
			<td></td>
		</tr>
		<tr>
			<td>pUC19</td>
			<td>Thermo Fisher</td>
			<td>SD0061</td>
			<td>Plasmid for supercoiling assay</td>
		</tr>
		<tr>
			<td>Refrigerated high-speed centrifuge</td>
			<td>Thermo Fisher</td>
			<td>75002402</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE protein marker</td>
			<td>Bio-Rad</td>
			<td>1610317</td>
			<td></td>
		</tr>
		<tr>
			<td>SEDFIT</td>
			<td></td>
			<td></td>
			<td>Free software program for analytical ultracentrifugation data analysis</td>
		</tr>
		<tr>
			<td>SEDNTERP</td>
			<td></td>
			<td></td>
			<td>Free software program to estimate viscosity and density of buffer and partial specific volume of a protein</td>
		</tr>
		<tr>
			<td>SigmaPrep Spin Columns</td>
			<td>Sigma</td>
			<td>SC1000</td>
			<td>For pull-down assay</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Merck</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>MP Biomedicals</td>
			<td>102918</td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex 200 Increase 10/300 GL</td>
			<td>Cytiva</td>
			<td>28990944</td>
			<td>Column for analytical size-exclusion chromatography</td>
		</tr>
		<tr>
			<td>Superdex 75 Increase 10/300 GL</td>
			<td>Cytiva</td>
			<td>29148721</td>
			<td>Column for analytical size-exclusion chromatography</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Sigma</td>
			<td>1.10732</td>
			<td></td>
		</tr>
		<tr>
			<td>Topoisomerase I</td>
			<td>Inspiralis</td>
			<td>WGT102</td>
			<td>Enzyme used in plasmid supercoiling assay</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Merck</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>MP Biomedicals</td>
			<td>191450</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>N&#252;ve</td>
			<td>NB 5</td>
			<td>For heat treatment of protein samples</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol (&#946;-ME)</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The recombinant N-terminal nucleoplasmin domain of the protein FKBP53 from Arabidopsis thaliana was subjected to analytical SEC. The elution peak volume was plotted against the standard curve to identify its oligomeric state. The analytical SEC results revealed that the domain exists as a pentamer in solution, with an approximate molecular mass of 58 kDa (Figure 1A,B). Further, the nucleoplasmin domain was analyzed for thermal and chemical stability in conjunction w...

Watch this Scientific Journal Video about In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays at JoVE.com

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.

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 ...

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