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, buffer composition, and buffer pH may be selected based on the protein of interest. The sample injection volume should not exceed 500 &#181;L for a 24 mL column. Also, the column pressure needs to be maintained below 5 MPa.</li>
		<li>From a higher concentration protein stock solution, prepare 500 &#181;L of 0.5 mg/mL protein sample in degassed SEC buffer and inject it into the pre-equilibrated column using a 500 &#181;L injection loop. Allow the chromatography run to proceed at an isocratic flow rate of 0.2-0.3 mL/min with the SEC buffer at 4 &#176;C.</li>
		<li>Monitor the elution profile of the protein by measuring absorbance at a wavelength of 280 nm. When dealing with proteins lacking aromatic residues, measure the absorbance at 214 nm.</li>
		<li>Use the elution volume of the protein to calculate its approximate molecular weight in kDa using the standard calibration curve21. 
		NOTE: Calibration curve is prepared by plotting the retention volume of known molecular weight proteins against the log of their respective molecular weights (log Mr), eluted using the same column.</li>
	</ol>
	</li>
	<li>Analysis of the thermal stability of the histone chaperones
	<ol>
		<li>Take 500 &#181;L of 0.5 mg/mL of the protein sample prepared in degassed SEC buffer (same as used in 1.1.1) in individual microcentrifuge tubes and heat each tube to a particular temperature ranging between 20 &#176;C and 90 &#176;C (20 &#176;C, 40 &#176;C, 60 &#176;C, and 90 &#176;C) for 10 min in a water bath.</li>
		<li>Subsequently, centrifuge the heat-treated samples at 16,200 x g for 10 min at 4 &#176;C, collect the supernatant with a micropipette, and inject each sample individually using a 500 &#181;L injection loop into the analytical column, pre-equilibrated with the SEC buffer at 4 &#176;C.</li>
		<li>Allow the chromatography run to proceed at an isocratic flow rate of 0.2-0.3 mL/min with the SEC buffer at 4 &#176;C.</li>
		<li>Observe the position and height of the elution peaks and look for the appearance of additional peaks for the different samples.</li>
	</ol>
	</li>
	<li>Analysis of the chemical stability of the histone chaperones
	<ol>
		<li>To examine the salt stability of histone chaperones, incubate 500 &#181;L of 0.5 mg/mL protein sample prepared in a Tris buffer [20 mM of Tris-HCl (pH 7.5), and 1 mM of &#946;-ME] supplemented with increasing concentrations of NaCl (300 mM, 600 mM, 1 M, 1.5 M, and 2 M) in separate microcentrifuge tubes for 30 min at 4 &#176;C. Centrifuge the samples at 16,200 x g for 10 min at 4 &#176;C and retain the supernatant.</li>
		<li>Next, load the protein samples in different NaCl concentrations individually, using a 500 &#181;L injection loop into the analytical column pre-equilibrated with 1.2 CV (28.8 mL) of the respective buffer containing increasing NaCl concentrations at 4 &#176;C.</li>
		<li>Allow the chromatography run to proceed at an isocratic flow rate of 0.2-0.3 mL/min with 1 CV (24 mL) of the respective buffer at 4 &#176;C.</li>
		<li>Observe the position and height of elution peaks and look for the appearance of additional peaks for the different samples.</li>
		<li>Similarly, for urea stability analysis, incubate 500 &#181;L of 0.5 mg/mL protein sample prepared in a Tris buffer [20 mM of Tris-HCl (pH 7.5), and 1 mM of &#946;-ME]&#160;supplemented with increasing urea concentrations (1 M, 2 M, 3 M, 4 M, and 5 M) in separate microcentrifuge tubes for 16 h at room temperature. Centrifuge the samples at 16,200 x g for 10 min at room temperature and retain the supernatant.</li>
		<li>Next, load the urea-treated protein samples individually using a 500 &#181;L injection loop into the analytical column pre-equilibrated with 1.2 CV (28.8 mL) of the corresponding buffer containing different urea concentrations at room temperature.</li>
		<li>Allow the chromatography run to proceed at an isocratic flow rate of 0.2-0.3 mL/min with 1 CV (24 mL) of the respective buffer at room temperature. 
		CAUTION: Do not perform the experiments with buffer containing urea at a lower temperature as urea tends to crystallize and damage the column.</li>
		<li>Observe the position and height of the elution peaks and look for the appearance of additional peaks for the different samples.</li>
	</ol>
	</li>
</ol>
2. Salt gradient-based pull-down assays to understand the type of interactions contributing to the complex formation between histone oligomers and a histone chaperone
<ol>
	<li>For each reaction of pull-down assay, pipette 40 &#181;L of Ni-NTA resin into a spin column and wash with sterile double-distilled water. Subsequently, equilibrate the resin with 100 CV (4 mL) of equilibration buffer [20 mM of Tris-HCl (pH 7.5), 300 mM of NaCl, 10 mM of imidazole, 10 &#181;g/mL of BSA, and 1 mM of &#946;-ME] (see Table of Materials). 
	NOTE: Pull-down can also be performed in a 1.5 mL microcentrifuge tube.</li>
	<li>Prepare the sample by mixing 5 &#181;M of the His-tagged histone chaperone with either 20 &#181;M of histone H2A/H2B dimer or H3/H4 tetramer in the equilibration buffer. Incubate the sample on ice for 1 h. 
	NOTE: H2A/H2B dimer and H3/H4 tetramer are prepared from recombinant human histones21, and the integrity of the oligomers is confirmed based on the estimated molecular masses by analytical ultracentrifugation (AUC). The same histone oligomers have been used for all experiments mentioned below.</li>
	<li>Load the samples into separate pre-equilibrated spin columns with Ni-NTA resin from step 2.1, each labeled for a particular salt concentration, and keep the columns for 30 min at 4 &#176;C. Centrifuge the columns at 1000 x g for 1 min.</li>
	<li>Next, wash the columns with 100 CV (4 mL) of wash buffer [20 mM of Tris-HCl (pH 7.5), 50 mM of imidazole, 0.2% of Tween-20, and 1 mM of &#946;-ME] containing different salt concentrations (i.e., 300 mM,&#160;500 mM, 600 mM, 700 mM, 800 mM, 900 mM and 1 M NaCl). Wash each column with a buffer having a particular salt concentration.</li>
	<li>After the salt washing step, elute the protein from the different columns using 100 &#181;L of elution buffer [20 mM of Tris-HCl (pH 7.5), 300 mM of NaCl, 300 mM of imidazole, and 1 mM of &#946;-ME].</li>
	<li>Subsequently, subject the eluted samples to 18% SDS-PAGE22 and visualize the gel after staining with Coomassie Brilliant Blue R250 (see Table of Materials). Alternatively, you may directly load the resin onto the SDS-PAGE gel instead of eluting the bound protein from the Ni-NTA resin. 
	NOTE: Equilibration, wash, and elution buffer compositions and pH may be modified depending upon the protein of interest.</li>
</ol>
3. Competitive pull-down assay to identify the preference of a histone chaperone for H2A/H2B or H3/H4
<ol>
	<li>Prepare spin column as described in step 2.1</li>
	<li>Incubate 5 &#181;M of the histone chaperone with 20 &#181;M of H2A/H2B dimer in 300 &#181;L of equilibration buffer (prepared in step 2.1) for 30 min on ice. 
	NOTE: The ratio of histone oligomer to histone chaperone in the reaction can be chosen based on known binding stoichiometry data; use five-fold excess histone if no information is available.</li>
	<li>Centrifuge the histone chaperone-H2A/H2B complex at 16,200 x g for 5 min at 4 &#176;C to remove any precipitate. Next, load the sample on the spin column pre-equilibrated with equilibration buffer (prepared in step 2.1) and incubate for 30 min at 4 &#176;C.</li>
	<li>Wash the column with 100 CV (4 mL) of wash buffer [20 mM of Tris-HCl (pH 7.5), 300 mM of NaCl, 50 mM of imidazole, 0.2% of Tween-20, and 1 mM of &#946;-ME] to remove excess H2A/H2B dimer. Next, mix the histone chaperone-H2A/H2B complex with 20-60 &#181;M of H3/H4 tetramer and incubate for 30 min on ice.</li>
	<li>Rewash the column with 100 CV (4 mL) of wash buffer (prepared in step 3.4) to remove any unbound H3/H4 tetramer and elute the sample using elution buffer (prepared in step 2.5). Subject the eluted samples to 18% SDS-PAGE and visualize after staining with Coomassie Brilliant Blue R250. 
	NOTE: The assay could be reversed wherein, first, H3/H4 tetramer can be mixed with the chaperone, the complex allowed to bind to Ni-NTA beads, and the complex then be incubated with varying concentrations of H2A/H2B dimer.</li>
</ol>
4. Analytical ultracentrifugation - sedimentation velocity (AUC-SV) experiments to analyze the binding stoichiometry between histone chaperones and histones
<ol>
	<li>Sample preparation for AUC
	<ol>
		<li>Dialyze the reconstituted histone H2A/H2B dimer, H3/H4 tetramer, and the histone chaperone separately through a 7 kDa cut-off dialysis tubing23, against a dialysis buffer [20 mM of Tris (pH 7.5), 300 mM of NaCl, and 1 mM of &#946;-ME] (see Table of Materials). To minimize background error due to buffer mismatch, perform dialysis extensively against the dialysis buffer, preferably three times over a period of 24 h. 
		NOTE: The initial OD280 of the protein samples should have a two to a three-fold higher value to achieve a final OD280 of 0.3-0.5. This is essentially done to nullify the effects of dilution.</li>
		<li>Purify H2A/H2B dimer, H3/H4 tetramer, and the histone chaperone individually with the dialysis buffer, using analytical size-exclusion chromatography (as mentioned in step 1). Save the buffer from the run to prepare further dilutions later on and to use as a reference in the AUC cell.</li>
	</ol>
	</li>
	<li>Sample loading for AUC
	<ol>
		<li>Mix the purified proteins in a final volume of 450 &#181;L using dialysis buffer from step 4.1.1 to reach an OD280 of 0.3-0.6. Mix the histone chaperone with H2A/H2B dimer or H3/H4 tetramer for complex formation in separate reaction tubes. Incubate the protein mixtures for 2-3 h. 
		NOTE: Alternatively, sedimentation data can be acquired with an interference optical scanning system in the analytical ultracentrifuge. Separately, for mixing purified proteins, fix the histone chaperone concentration and incubate it with increasing concentrations of the histone oligomers to obtain the exact stoichiometry.</li>
		<li>Assemble the cell with a double sector centerpiece and quartz windows for the AUC-SV experiment using an absorbance detector of the analytical ultracentrifuge as described previously in detail24.</li>
		<li>Fill 400 &#181;L of the sample solution and 420 &#181;L of dialysis buffer into the two sectors (sample and reference sectors, respectively) of the cell. 
		NOTE: A larger volume of buffer is used in the reference sector to keep the reference meniscus above the meniscus of the sample. However, while using an optical interference system, fill the two sectors with equal volume.</li>
		<li>Weigh and accurately balance the cells and load them into a four-place titanium rotor (see Table of Materials). Align the cells using the marks provided at the bottom of the cells and the rotor. Load the rotor in the centrifuge, close the lid and allow to develop a vacuum until the pressure drops to less than 15 microns of Hg and the rotor temperature stabilizes to 20 &#176;C (usually takes 2-2.5 h). 
		NOTE: AUC operating parameters include experimental temperature, rotor speed, the interval between scans, and the number of scans to be collected. In the case of SV experiments, the scan interval is given according to the protein&#39;s molecular mass; smaller proteins require larger time intervals between the scans. The rotor speed is also set according to the protein&#39;s expected molecular mass, and the experiment is conducted at 20 &#176;C. The absorbance data is monitored at 280 nm.</li>
		<li>To obtain the exact stoichiometry, keep the histone chaperone concentration constant and incubate with increasing concentrations of histone oligomers to attain saturation.</li>
	</ol>
	</li>
	<li>AUC data analysis
	<ol>
		<li>Perform the data analysis as previously described25. Briefly, calculate the density and viscosity for the buffer components using the program SEDNTERP26 (see Table of Materials). Similarly, calculate the partial specific volume of the protein based on its amino acid composition, also using SEDNTERP.</li>
		<li>Load the data from the AUC machine into the program SEDFIT27 and define the meniscus (red line), the cell bottom (blue line), and data analysis boundaries (green lines). Choose continuous C(s) distribution as a model.</li>
		<li>Next, set resolution maximum up to 100; set sedimentation coefficient (s), s min: 0 and s max: 10-15; set frictional ratio to 1.2 initially and opt to float to derive the ratio from the data; set confidence level (F-ratio; which determines the magnitude of regularization) to 0.68 for 1 sigma regularization; set partial specific volume, buffer density and buffer viscosity values as obtained from SEDNTERP.</li>
		<li>Press RUN to allow the software to solve the Lamm equation27. Adjust the parameters if there is a significant data mismatch. After adjusting the parameters, press FIT to refine all parameters. Assess the quality of fit based on the root-mean-square deviation (RMSD) value, which should be less than 0.01 signal units.</li>
		<li>Estimate the molecular masses of the peaks by choosing the option: show peak &#34;Mw in c(s)&#34; in the display function of the main toolbar, which will provide information about the &#39;s&#39; of the molecule/complex.</li>
	</ol>
	</li>
</ol>
5. Plasmid supercoiling assay to confirm histone chaperoning function
<ol>
	<li>Nucleosome assembly reaction
	<ol>
		<li>Mix 2 &#181;M of H3/H4 tetramer and 4 &#181;M of H2A/H2B dimer with increasing concentrations of the histone chaperone (1-6 &#181;M) in an assembly buffer [20 mM of Tris HCl (pH 7.5), 1 mM of DTT, 1 mM of MgCl2, 0.1 mg/mL of BSA, and 100 mM of NaCl] to a final volume of 50 &#181;L. Incubate the mixture at 4 &#176;C for 30 min.</li>
		<li>Simultaneously, in a separate reaction, pretreat 500 ng of the negatively supercoiled pUC19 plasmid with 1 &#181;g of topoisomerase I enzyme (see Table of Materials) in the assembly buffer in a final volume of 50 &#181;L and incubate at 30 &#176;C for 30 min. 
		NOTE: Topoisomerase I relaxes the supercoiled double-stranded plasmid DNA by generating a single-stranded nick. A topoisomerase I enzyme of eukaryotic origin, such as the commercially available wheat germ topoisomerase I or recombinantly expressed Drosophila melanogaster topoisomerase I, could be used.</li>
		<li>Next, combine the H3/H4 tetramer, H2A/H2B dimer, histone chaperone mixture (from step 5.1.1), the relaxed plasmid DNA reaction mixture (from step 5.1.2), and incubate further at 30 &#176;C for 90 min. 
		NOTE: Set up two control reactions for the assay; one having the histone chaperone and the relaxed plasmid DNA (but not the histones) and the other having the histone oligomers and the relaxed plasmid DNA (but not the histone chaperone).</li>
		<li>Stop the assembly reaction by adding 100 &#181;L of 2x stop buffer (40 mM of EDTA, 2% of SDS, and 0.4 mg/mL of proteinase K) and incubate at 37 &#176;C for 30 min. 
		NOTE: Stop buffer deproteinizes the plasmid DNA by denaturation and proteolysis of bound histones.</li>
	</ol>
	</li>
	<li>Phenol-chloroform extraction and ethanol precipitation
	<ol>
		<li>Add an equal volume of Tris-saturated phenol in the tube containing the reaction mixture from step 5.1.4 and mix well, followed by centrifugation at 16,200 x g for 10 min at room temperature.</li>
		<li>Gently collect the upper aqueous phase having the plasmid DNA with a micropipette and mix with an equal volume of chloroform. Vortex the mixture and centrifuge at 16,200 x g for 10 min at room temperature. 
		NOTE: Isoamyl alcohol could be included at this step to avoid a fuzzy interface between the aqueous and organic phases.</li>
		<li>Next, collect the upper aqueous phase, add 1/10th volume of 3 M sodium acetate (pH 5.5) and 2.5 volumes of ice-cold ethanol (see Table of Materials). Mix the solution well by inverting the tube 3-4 times and keep the mixture in a -20 &#176;C freezer for 30 min for complete precipitation of the plasmid DNA.</li>
		<li>Centrifuge the sample from step 5.2.3 at 16200 x g for 10 min 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.</li>
	</ol>
	</li>
	<li>Perform the agarose gel electrophoresis to observe the plasmid supercoiling effect.
	<ol>
		<li>Dissolve the precipitated plasmid DNA from step 5.2.4 in sterile double-distilled water.</li>
		<li>Resolve the samples on a 1% agarose gel in 1x Tris-acetate-EDTA (TAE) buffer (40 mM of Tris, 20 mM of acetic acid, and 1 mM of EDTA) (see Table of Materials).</li>
		<li>Stain the gel with 0.2-0.5 &#181;g/mL concentration of ethidium bromide and observe under UV to visualize the DNA bands on the gel.</li>
	</ol>
	</li>
</ol>

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A., Ghirlando, R., Schuck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+current+methods+in+sedimentation+velocity+and+sedimentation+equilibrium+analytical+ultracentrifugation.">Overview of current methods in sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation.</a> Current Protocols in Protein Science. , (2013).</li><li>Schuck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-distribution+analysis+of+macromolecules+by+sedimentation+velocity+ultracentrifugation+and+Lamm+equation+modelling.">Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modelling.</a> Biophysical Journal. 78 (3), 1606-1619 (2000).</li><li>Kumar, A., Kumar Singh, A., Chandrakant Bob de, R., Vasudevan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+characterization+of+Arabidopsis+thaliana+NAP1-related+protein+2+(AtNRP2)+and+comparison+with+its+homolog+AtNRP1.">Structural characterization of Arabidopsis thaliana NAP1-related protein 2 (AtNRP2) and comparison with its homolog AtNRP1.</a> Molecules. 24 (12), 2258 (2019).</li><li>Liu, W. H., Roemer, S. C., Port, A. M., Churchill, M. E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAF-1-induced+oligomerization+of+histones+H3/H4+and+mutually+exclusive+interactions+with+Asf1+guide+H3/H4+transitions+among+histone+chaperones+and+DNA.">CAF-1-induced oligomerization of histones H3/H4 and mutually exclusive interactions with Asf1 guide H3/H4 transitions among histone chaperones and DNA.</a> Nucleic Acids Research. 45 (16), 9809 (2017).</li><li>Bowman, 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=The+histone+chaperones+Vps75+and+Nap1+form+ring-like,+tetrameric+structures+in+solution.">The histone chaperones Vps75 and Nap1 form ring-like, tetrameric structures in solution.</a> Nucleic Acids Research. 42 (9), 6038-6051 (2014).</li><li>Newman, E. 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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No conflict of interest was declared.

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 first part of the protocol section&#160;involves investigating the oligomeric state and stability of a histone chaperone. Several reports indicate that histone chaperones exhibit considerable diversity in their oligomeric state. For example, human CAF-1 exists as a monomer29. NAP family members exist as dimer or tetramer29,30,31. Nucleoplasmins reveal pentameric and often decameric oligomeric states32,33. An analytical SEC experiment can determine the oligomeric state of a histone chaperone, and AUC-SV experiments can confirm the same. Several of the histone chaperones are known to be highly stable under various thermal and chemical stress conditions33,34. The thermal and chemical stability features of histone chaperones could also be explored in conjunction with analytical SEC. Further, circular dichroism spectroscopy could be effectively used for in-depth analysis of the changes in the secondary structure of the chaperone when subjected to increasing temperatures or higher concentrations of a chemical agent.
The second part of the protocol section covers pull-down assays that could examine the fundamental interactions that aid the association of histone oligomers with the chaperone by using a salt-gradient approach and a competitive pull-down assay to identify the histone oligomer preference of a chaperone. If the complex falls apart with a slight increase in salt concentration, that would suggest a major contribution of electrostatic interactions in stabilizing the complex. An intact complex in high salt would suggest a significant role for hydrophobicity in stabilizing the complex35. The competitive pull-down assay could be easily employed to determine the specificity or preference of a histone chaperone to a specific histone oligomer class. Based on their preference towards histone oligomers, histone chaperones can be classified into three categories such as H2A/H2B chaperones, H3/H4 chaperones, and H2A/H2B-H3/H4 chaperones10,36. In addition, if necessary, isothermal titration calorimetry (ITC) could be used to understand the histone oligomer specificity of a given chaperone and&#160;the thermodynamic characteristics of their interactions.
The third part of the protocol section covers the investigation of the interaction stoichiometry between a histone chaperone and the histone oligomers. In general, the different families of histone chaperones differ considerably for the stoichiometry of their association with H2A/H2B or/and H3/H421,28,37,38. AUC-SV experiment aids in obtaining sedimentation coefficient (s) and molecular mass of a protein or its complex, which becomes very useful in accurately estimating the stoichiometry in the complex formation. Alternatively, ITC can also be used to examine stoichiometry.
The fourth part of the protocol section focuses on investigating the nucleosome assembly function of histone chaperones. Histone chaperones play a crucial role in nucleosome assembly, which regulates vital cellular processes such as replication, transcription, and DNA repair39. Plasmid supercoiling assay that is typically employed for the in vitro assessment of histone chaperoning activity of histone chaperones is&#160;elaborated in this section.
It may be noted that not all histone chaperones are fully structured. Few are known to have intrinsically disordered regions40,41. Therefore, thermal and chemical stability assays may not be suitable for such proteins. Further, histone chaperones from different organisms have different oligomeric states and differential abilities to bind to histones. Therefore, this protocol may be a good starting point but would entail modifications as necessary.

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><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 (MgCl&lt;sub&gt;2&lt;/sub&gt;)</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&amp;uuml;ve</td><td>NB 5</td><td>For heat treatment of protein samples</td></tr><tr><td>&amp;beta;-mercaptoethanol (&amp;beta;-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 with analytical SEC. The nucleoplasmin sample subjected to heat-treatment up to 90 &#176;C displayed no apparent shift in the elution volume and the peak height compared to the samples maintained at 20 &#176;C, suggesting that the domain is highly thermostable (Figure 1C). Likewise, the nucleoplasmin domain displayed salt stability up to 2 M of NaCl (Figure 1D) and urea stability up to 4 M (Figure 1E). However, the nucleoplasmin pentamer started dissociating in higher urea concentrations.
A pull-down assay was performed to determine the type of interactions contributing to the complex formation between the histone chaperone (nucleoplasmin domain of AtFKBP53) and the histone oligomers H2A/H2B dimer and H3/H4 tetramer using a gradient salt wash. The interaction of the nucleoplasmin domain with H2A/H2B dimer was stable up to a salt concentration of 0.4 M NaCl (Figure 2A). In comparison, the association with H3/H4 was reasonably stable up to 0.7 M NaCl (Figure 2B). The ability of the chaperone-histone complexes to withstand high salt concentration suggests the role of hydrophobic interactions in stabilizing the complexes. The chaperone complex with H3/H4 being stable even in high salt concentrations suggests a predominant role of hydrophobic interactions in the complex formation. The lower stability of the H2A/H2B-chaperone complex in high salt concentrations reveals a significant role for electrostatic interactions in the complex formation. In another experiment, the pull-down assay was used to examine whether the chaperone prefers either H2A/H2B dimer or H3/H4 tetramer. The results revealed that the chaperone binds to H2A/H2B dimer and H3/H4 tetramer simultaneously and irrespective of the order in which they are added to the chaperone (Figure 2C,D). This indicated that the chaperone possesses separate sites for its interaction with the histone oligomers.
AUC-SV experiments (Figure 3) were performed to study the stoichiometry of interaction between histone oligomers and chaperones. AUC-SV data analysis provided a sedimentation coefficient (s) value of 5.40 S for the AtFKBP53 nucleoplasmin domain in complex with H2A/H2B that corresponded to a molecular mass of 104 kDa. The complex of the nucleoplasmin domain with H3/H4 gave a sedimentation coefficient value of 7.35 S, corresponding to 129 kDa. The estimated molecular mass of the complexes reveals that the pentameric nucleoplasmin forms complex with both H2A/H2B dimer and H3/H4 tetramer in a 1:1 stoichiometry.
It is essential to show that the protein can deposit histone oligomers onto DNA to confirm that it is a histone chaperone. Towards this end, a plasmid supercoiling assay was adopted (Figure 4). The relaxed circular plasmid was incubated with the histone oligomers H2A/H2B and H3/H4 with the recombinant plant histone chaperones of the NAP family - AtNRP1 and AtNRP228. The presence of the chaperone increased the amount of supercoiled plasmid, suggesting it could deposit histones onto the DNA to form nucleosomes, causing DNA supercoiling.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63218/63218fig01.jpg" /> 
Figure 1: Oligomeric state and stability of the nucleoplasmin domain of AtFBP53. (A) Analytical size-exclusion chromatography profile of the AtFKBP53 nucleoplasmin domain. (B) Calibration curve obtained using globular proteins of known molecular mass. The blue dots represent the molecular mass of the known proteins, whereas the red dot represents the AtFKBP53 nucleoplasmin domain. (440 kDa - ferritin, 158 kDa-aldolase, 75 kDa-con albumin, 44 kDa-ovalbumin, 6.5 kDa-aprotinin). (C)&#160;Analytical size-exclusion chromatogram of 500 &#181;L of 0.5 mg/mL AtFKBP53 nucleoplasmin domain subjected to heat treatment at different temperatures: 20 &#176;C (green),&#160;40 &#176;C (orange), 60 &#176;C (black), 90 &#176;C (light blue). (D) Analytical size-exclusion chromatogram of 500 &#181;L of 0.5 mg/mL AtFKBP53 nucleoplasmin domain in buffers containing different NaCl concentrations: 0.3 M (purple), 0.6 M (red), 1.0 M (light blue), 1.5 M (green), 2.0 M (black). (E) Analytical&#160;size-exclusion chromatogram of the AtFKBP53 nucleoplasmin domain in buffers with different urea concentrations: 0 M (control; light blue), 1.0 M (pink), 2.0 M (black), 3.0 M (dark blue), 4.0 M (green), 5.0 M (brown). The nucleoplasmin pentamer shows high stability to thermal and chemical stress conditions. The figure is adapted from Reference21. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63218/63218fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63218/63218fig02.jpg" /> 
Figure 2: Pull-down assays for the interaction of the nucleoplasmin domain of AtFKBP53 with histone oligomers. 18% SDS-PAGE images of the elution fractions from the assays are presented here. Pull-down assay for (A) 20 &#181;M H2A/H2B dimer and (B) 20 &#181;M H3/H4 tetramer with 5 &#181;M AtFKBP53 nucleoplasmin domain in increasing concentrations of NaCl in the range of 0.3 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, and 1.0 M. 5 &#181;M AtFKBP53 FKBD was used as a negative control here. For the competitive binding experiment, (C) a mixture of 5 &#181;M AtFKBP53 nucleoplasmin domain and 20 &#181;M H3/H4 tetramer incubated with a range of 20-60 &#181;M H2A/H2B dimer and (D) a mixture of 5 &#181;M AtFKBP53 nucleoplasmin domain and 20 &#181;M H2A/H2B dimer incubated with a range of 20-60 &#181;M H3/H4 tetramer has been used. The label AtFKBP53 corresponds to the nucleoplasmin domain of AtFKBP53. Elution fractions show simultaneous binding of both the histone oligomers to the nucleoplasmin. The figure is adapted from Reference21. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63218/63218fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63218/63218fig03.jpg" /> 
Figure 3: Analytical ultracentrifugation - sedimentation velocity (AUC-SV) experiment of histone oligomers, the nucleoplasmin domain of AtFKBP53, and their complexes. The AUC distance distribution vs. sedimentation coefficient (S) plot. The obtained sedimentation coefficient (s) values and molecular masses are also provided. The label AtFKBP53 corresponds to the nucleoplasmin domain of AtFKBP53. The estimated molecular masses reveal a 1:1 stoichiometry for the AtFKBP53 nucleoplasmin domain with the histone oligomers H2A/H2B dimer and H3/H4 tetramer. 450 &#181;L of all the protein samples having an OD280 of 0.3-0.5 were used for the AUC-SV experiments.&#160;The figure is adapted from Reference21. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63218/63218fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63218/63218fig04.jpg" /> 
Figure 4: Plasmid supercoiling assay. Plasmid supercoiling assay for the histone chaperones AtNRP1 and AtNRP2. 500 ng of pUC19 plasmid DNA was pretreated with 1 &#181;g of Topoisomerase I for the experiment. 4 &#181;M AtNRP1, 4 &#181;M AtNRP2, and a mixture of 4 &#181;M H2A/H2B dimer and 2 &#181;M H3/H4 tetramer were as control that shows no supercoiling activity when incubated with the pretreated pUC19 DNA. The lanes with a mixture of 4 &#181;M H2A/H2B of dimer and 2 &#181;M H3/H4 of tetramer and 4 &#181;M each of AtNRP1 and AtNRP2 show the formation of supercoiled DNA upon incubation with the pretreated pUC19 DNA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63218/63218fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

2.7K Views.  Institute of Life Sciences. Ons protocol beschrijft hoe technieken zoals pull-down assays analytical size exclusion chromatography, analytical ultra-centrifugation en histone chaperoning assay kunnen worden gebruikt om te bevestigen of een eiwit een histon chaperone is. De technieken die hier worden behandeld, kunnen bij het gebruik van tandem direct worden toegepast om een vermeende chaperonne te karakteriseren. hij kon meegaan met de structurele biologietechniek.De technieken zijn toepasbaar op het gebied van c...

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

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous nature of most mammalian chromatin-modifying complexes. In order to overcome these hurdles, we have adapted a sequential salt extraction (SSE) assay for evaluating the relative binding affinities of chromatin-bound complexes. This easy and straightforward assay can be used by non-experts to evaluate the relative difference in binding affinity of two related complexes, the changes in affinity of a complex when a subunit is lost or an individual domain is inactivated, and the change in binding affinity after alterations to the chromatin landscape. By sequentially re-suspending bulk chromatin in increasing amounts of salt, we are able to profile the elution of a particular protein from chromatin. Using these profiles, we are able to determine how alterations in a chromatin-modifying complex or alterations to the chromatin environment affect binding interactions. Coupling SSE with other in vitro and in vivo assays, we can determine the roles of individual domains and proteins on the functionality of a complex in a variety of chromatin environments.

Sequential salt extraction of chromatin bound proteins is a useful tool for determining the binding properties of large protein complexes. This method can be employed to evaluate the role of individual subunits or domains in the overall affinity of a protein complex to bulk chromatin.

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous ...

Genetics

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

Medicine

Assays for Validating Histone Acetyltransferase Inhibitors

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene expression. KATs, such as CBP/p300, are under intense investigation as therapeutic targets due to their critical role in tumorigenesis of diverse cancers. The development of novel small molecule inhibitors targeting the histone acetyltransferase (HAT) function of KATs is challenging and requires robust assays that can validate the specificity and potency of potential inhibitors.
This article outlines a pipeline of three methods that provide rigorous in vitro validation for novel HAT inhibitors (HATi). These methods include a test tube HAT assay, Chromatin Hyperacetylation Inhibition (ChHAI) assay, and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). In the HAT assay, recombinant HATs are incubated with histones in a test tube reaction, allowing for acetylation of specific lysine residues on the histone tails. This reaction can be blocked by a HATi and the relative levels of site-specific histone acetylation can be measured via immunoblotting. Inhibitors identified in the HAT assay need to be confirmed in the cellular environment.
The ChHAI assay uses immunoblotting to screen for novel HATi that attenuate the robust hyperacetylation of histones induced by a histone deacetylase inhibitor (HDACi). The addition of an HDACi is helpful because basal levels of histone acetylation can be difficult to detect via immunoblotting.
The HAT and ChHAI assays measure global changes in histone acetylation, but do not provide information regarding acetylation at specific genomic regions. Therefore, ChIP-qPCR is used to investigate the effects of HATi on histone acetylation levels at gene regulatory elements. This is accomplished through selective immunoprecipitation of histone-DNA complexes and analysis of the purified DNA through qPCR. Together, these three assays allow for the careful validation of the specificity, potency, and mechanism of action of novel HATi.

Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene ...

Cancer Research

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular processes. The conserved MEEVD pentapeptide at the C-terminus of Hsp90 is responsible for the interaction with the tetratricopeptide repeat (TPR) motif of co-chaperones. FK506-binding protein (FKBP) 51 and FKBP52 are two similar TPR-motif co-chaperones involved in steroid hormone-dependent diseases with different functions. Therefore, identifying molecules specifically blocking interactions between Hsp90 and FKBP51 or FKBP52 provides a promising therapeutic potential for several human diseases. Here, we describe the protocol for an amplified luminescent proximity homogenous assay to probe interactions between Hsp90 and its partner co-chaperones FKBP51 and FKBP52. First, we have purified the TPR motif-containing proteins FKBP51 and FKBP52 in glutathione S-transferase (GST)-tagged form. Using the glutathione-linked donor beads with GST-fused TPR-motif proteins and the acceptor beads coupled with a 10-mer C-terminal peptide of Hsp90, we have probed protein-protein interactions in a homogeneous environment. We have used this assay to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

This protocol presents a technique for probing protein-protein interactions using glutathione-linked donor beads with GST-fused TPR-motif co-chaperones and acceptor beads coupled with an Hsp90-derived peptide. We have used this technique to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular ...

Histone Proteomic Analysis and Post-Translational Modification Characterization

Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as posttranslational modifications (PTMs). Identifying the position and nature of each PTM or pattern of PTMs in reference to external or genetic factors allows this information to be statistically correlated with biological responses such as DNA transcription, replication, or repair. In the present work, a high-throughput analytical protocol for the detection of histone PTMs from biological samples is described. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) enables the separation and PTM assignment of the most biologically relevant modifications in a single analysis. The described approach takes advantage of recent developments in dependent data acquisition (DDA) using parallel accumulation in the mobility trap, followed by sequential fragmentation and collision-induced dissociation. Histone PTMs are confidently assigned based on their retention time, mobility, and fragmentation pattern.

Author Spotlight: Enhanced Histone PTM Isomer Identification Through LC-TIMS-ToF MS/MS and PASEF

An analytical workflow based on liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) for high confidence and highly reproducible "bottom-up" analysis of histone modifications and identification based on principal parameters (retention time [RT], collision cross section [CCS], and accurate mass-to-charge [m/z] ratio).

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as ...

Chemistry

Intracellular Refolding Assay

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds with inhibitory/stimulating activity. Molecular chaperones are proteins involved in regulation of protein folding1 and have a crucial role in promoting cell survival upon stress insults like heat shock2, nutrient starvation and exposure to chemicals/poisons3. For this reason chaperones are found to be involved in events like tumor development, chemioresistance of cancer cells4 as well as neurodegeneration5. Design of small molecules able to inhibit or stimulate the activity of these enzymes is therefore one of the most studied strategies for cancer therapy7 and neurodegenerative disorders9. The assay here described offers the possibility to measure the refolding activity of a particular molecular chaperone and to study the effect of compounds on its activity. In this method the gene of the molecular chaperone investigated is transfected together with an expression vector encoding for the firefly luciferase gene. It has been already described that denaturated firefly luciferase can be refolded by molecular chaperones10,11. As normalizing transfection control, a vector encoding for the renilla luciferase gene is transfected. All transfections described in this protocol are performed with X-treme Gene 11 (Roche) in HEK-293 cells. In the first step, protein synthesis is inhibited by treating the cells with cycloheximide. Thereafter protein unfolding is induced by heat shock at 45&deg;C for 30 minutes. Upon recovery at 37&deg;C, proteins are re-folded into their active conformation and the activity of the firefly luciferase is used as read-out: the more light will be produced, the more protein will have re-gained the original conformation. Non-heat shocked cells are set as reference (100% of refolded luciferase).

In this protocol a method to measure intracellular protein refolding after heat shock is described. This method can be used to study foldases like molecular chaperones and their co-factors or compounds able to influence their activity. Firefly luciferase activity is used as reporter to measure chaperone refolding activity.

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds ...

Biology

Purification of H3 and H4 Histone Proteins and the Quantification of Acetylated Histone Marks in Cells and Brain Tissue

In all eukaryotic organisms, chromatin, the physiological template of all genetic information, is essential for heredity. Chromatin is subject to an array of diverse posttranslational modifications (PTMs) that mostly occur in the amino termini of histone proteins (i.e., histone tail) and regulate the accessibility and functional state of the underlying DNA. Histone tails extend from the core of the nucleosome and are subject to the addition of acetyl groups by histone acetyltransferases (HATs) and the removal of acetyl groups by histone deacetylases (HDACs) during cellular growth and differentiation. Specific acetylation patterns on lysine (K) residues on histone tails determine a dynamic homeostasis between transcriptionally active or transcriptionally repressed chromatin by (1) influencing the core histone assembly and (2) recruiting synergistic or antagonistic chromatin-associated proteins to the transcription site. The fundamental regulatory mechanism of the complex nature of histone tail PTMs influences the majority of chromatin-templated processes and results in changes in cell maturation and differentiation in both normal and pathological development. The goal of the current report is to provide novices with an efficient method to purify core histone proteins from cells and brain tissue and to reliably quantify acetylation marks on histones H3 and H4.

The purpose of this article is to provide a comprehensive, systematic guide to the efficient purification of histones H3 and H4 and the quantification of acetylated histone residues.

In all eukaryotic organisms, chromatin, the physiological template of all genetic information, is essential for heredity. Chromatin is subject to an array ...

Neuroscience

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

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

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