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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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+multimeric+assemblies+of+nucleosome+assembly+protein+and+histones+revealed+by+small-angle+X-ray+scattering+and+electron+microscopy.">Large multimeric assemblies of nucleosome assembly protein and histones revealed by small-angle X-ray scattering and electron microscopy.</a> Journal of Biological Chemistry. 287 (32), 26657-26665 (2012).</li><li>Edlich-Muth, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pentameric+nucleoplasmin+fold+is+present+in+Drosophila+FKBP39+and+a+large+number+of+chromatin-related+proteins.">The pentameric nucleoplasmin fold is present in Drosophila FKBP39 and a large number of chromatin-related proteins.</a> Journal of Molecular Biology. 427 (10), 1949-1963 (2015).</li><li>Franco, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+into+the+ability+of+nucleoplasmin+to+assemble+and+chaperone+histone+octamers+for+DNA+deposition.">Structural insights into the ability of nucleoplasmin to assemble and chaperone histone octamers for DNA deposition.</a> Scientific Reports. 9 (1), 9487 (2019).</li><li>Xiao, H., Jackson, V., Lei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+FK506-binding+protein,+Fpr4,+is+an+acidic+histone+chaperone.">The FK506-binding protein, Fpr4, is an acidic histone chaperone.</a> FEBS Letters. 580 (18), 4357-4364 (2006).</li><li>Graziano, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+hydrophobic+effect+in+the+salt-induced+dimerization+of+bovine+beta-lactoglobulin+at+pH+3.">Role of hydrophobic effect in the salt-induced dimerization of bovine beta-lactoglobulin at pH 3.</a> Biopolymers. 91 (12), 1182-1188 (2009).</li><li>Burgess, R. 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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...

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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 ...

Our protocol describes how techniques like pull-down assays analytical size exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used to confirm whether a protein is a histone chaperone. The techniques covered here, when using tandem, could be applied directly to characterize a putative chaperone. his could go along with the structural biology technique.The techniques are applicable to the field of chromatin research. However, the individual methods could be applied to any other protein of interest, as required. Demonstrating the procedure will be Mr.Surajit Gandhi, a PhD student from my laboratory, for the complicated pull-down assay;Ms.Archana Samal, a PhD student, along with Mr.Ruchir C.Bobde for the analytical ultra-centrifugation experiment;Mr. Somanath Baral, a PhD student;along with Mr.Ketul Saharan for the plasmid super-coiling assay First, mix five-micromolar histone chaperones with 20-micromolar H2A/H2B dimers in 300 microliters of equilibration buffer. Centrifuge the histone chaperone H2A/H2B complex to remove any precipitate.Load the sample on the spin column pre-equilibrated with equilibration buffer. Wash the column with 100 column volumes or four milliliters of wash buffer to remove excess H2A/H2B dimer. Next, mix the histone chaperone H2A/H2B complex with 20-to 60-micromolar H3/H4 tetramer.Rewash the column with 100 column volumes or four milliliters of wash buffer to remove any unbound H3/H4 tetramer, then elute the samples with elution buffer. Subject the eluted samples to 18%SDS-PAGE. Then, stain with Coomassie brilliant blue R250 and visualize the gel.Purify the H2A/H2B dimer, H3/H4 tetramer in the histone chaperone individually with the dialysis buffer using analytical size exclusion chromatography. Save the buffer from the run to prepare further dilutions later on and to use as a reference in the AUC cell. For AUC samples, mix the purified chaperones with dimer or tetramer in separate tubes to a final volume of 450 microliters using the saved dialysis buffer to reach an OD280 of 0.3 to 0.6.Assemble the cell with a double-sector centerpiece and quartz windows provided with an analytical ultra centrifuge with an absorbance detector. Fill 400 microliters of the sample solution and 420 microliters of dialysis buffer into the reference and sample sectors of the cell, respectively. Weigh and accurately balance the cells and load them into a four-hole titanium rotor.Align the cells using the marks at the bottom of the cells and the rotor. Load the rotor in the centrifuge, close the lid, and start the run, which allows a vacuum to develop and the temperature to stabilize. For data analysis, calculate the density and viscosity for the proteins buffer components and partial specific volume based on the amino acid composition of the protein using the program SEDNTERP.Load the data from the AUC machine into the program SEDFIT. Define the meniscus, red line, the cell bottom, blue line, and data analysis boundaries green lines. Choose Continuous CS distribution as a model.In the parameters section, set resolution maximum up to 100. Next, set sedimentation coefficient minimum to zero and maximum to 10 to 15. Set the frictional ratio to 1.2 initially and opt to float to derive the ratio from the data.Set the F-ratio to 0.68 for one sigma regularization. Set partial specific volume, buffer density, and viscosity as obtained from the SEDNTURP. Press Run to allow the software to solve the lam equation.Press Fit to refine. Choose Show Peak Mw in CS in the display function of the main toolbar to estimate molecular masses. Mix two-micromolar H3/H4 tetramer and four-micromolar H2A/H2B dimer with increasing concentrations of histone chaperone, ranging from one-to six-micromolar, in an assembly buffer to a final volume of 50 microliters.Incubate the mixture at four degrees Celsius for 30 minutes. Simultaneously, pretreat 500 nanograms of the negatively super-coiled PUC-19 plasmid with one microgram of topoisomerase I enzyme in the assembly buffer in a final volume at 50 microliters. Next, combine the pre-mixed solution containing tetramer, dimer, and histone chaperone with the relaxed plasma DNA.Add 100 microliters of 2X stop buffer and incubate to end the assembly reaction by deproteinizing the plasmid DNA. Add an equal volume of Tris-saturated phenol in the tube containing the reaction mixture and mix the contents of the reaction mixture well. Then, centrifuge the sample.Gently collect the upper aqueous phase containing the plasmid DNA with a micropipette and mix an equal volume of chloroform. Vortex the mixture. Next, collect the upper aqueous phase, add a 1/10 volume of three-molar sodium acetate of pH 5.5, and 2.5 volumes of ice-cold ethanol.Mix the solution well by inverting the tube three to four times. Centrifuge the sample at 16, 200 G for 10 minutes and gently discard the supernatant. Keep the tubes open at room temperature until even trace amounts of ethanol evaporate, leaving the precipitated plasmid DNA in the tube.The analytical SEC results revealed that the recombinant N-terminal nucleoplasmin domain of the Arabidopsis thaliana protein FKBP53 is a pentamer of 65 kilodaltons in solution. The nucleoplasmin sample subjected to heat treatment showed that the domain is highly thermo-stable. The nucleoplasmin domain displayed salt stability up to two molars of sodium chloride and urea stability up to four molars.A pull-down assay revealed that the interaction of the nucleoplasmin domain with H2A/H2B dimer was stable up to 0.4-molar sodium chloride, whereas the association with H3/H4 was stable up to 0.7 molars. The chaperone binds H2A/H2B dimer and H3/H4 tetramer simultaneously, irrespective of the order in which they are added to the chaperone, indicating separate interaction sites for the oligomers. The estimated molecular masses through AUC-SV reveal a 1:1 stoichiometry of nucleoplasmin domain with the histone oligomers.In the plasmid super-coiling assay, recombinant plant histone chaperones AtNRP1 and AtNRP2 increased the amount of super-coiled plasmid, suggesting it could deposit histones onto the DNA to form nucleosomes, causing DNA super coiling. Plasmid super-coiling assay needs special care and possible optimization for histone DNA and chaperone characterization. Isomeric characterization telemetry can be used as a follow-up to analytical size-exclusion experiments to confirm the binding stability.

in-vitro-characterization-histone-chaperones-using-analytical-pull

Research

JoVE Journal

Biochemistry

2.6K visualizações.  Institute of Life Sciences. Nosso protocolo descreve como técnicas como cromatografia analítica de exclusão de tamanho de ensaios pull-down, ultracentrifugação analítica e ensaio de chaperoning de histonas podem ser usadas para confirmar se uma proteína é uma chaperona de histonas. As técnicas aqui abordadas, ao utilizar o tandem, poderiam ser aplicadas diretamente para caracterizar um suposto acompanhante. ele poderia ir junto com a técnica de biologia estrutural.As técnicas são aplicáveis ao campo ...