Method Article

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

DOI:

10.3791/63218

⸱

December 29th, 2021

 ,  ,  ,  ,  ,  ,  ,  ,  , 
1Institute of Life Sciences, 2Regional Centre for Biotechnology, 3School of Biotechnology, KIIT University, 4Department of Molecular Biophysics and Biochemistry, Yale University, 5Department of Pharmacology, University of Vermont College of Medicine

* These authors contributed equally

In This Article

Summary

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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.

Abstract

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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.

Introduction

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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'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.

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Protocol

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1. Analytical size-exclusion chromatography to elucidate the oligomeric status and stability of histone chaperones

  1. Analysis of the oligomeric status of histone chaperones
    1. 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 β-mercaptoethanol (β-ME)] at 4 °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 µL for a 24 mL column. Also, the column pressure needs to be maintained below 5 MPa.
    2. From a higher concentration protein stock solution, prepare 500 µL of 0.5 mg/mL protein sample in degassed SEC buffer and inject it into the pre-equilibrated column using a 500 µL 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 °C.
    3. 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.
    4. 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.
  2. Analysis of the thermal stability of the histone chaperones
    1. Take 500 µL 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 °C and 90 °C (20 °C, 40 °C, 60 °C, and 90 °C) for 10 min in a water bath.
    2. Subsequently, centrifuge the heat-treated samples at 16,200 x g for 10 min at 4 °C, collect the supernatant with a micropipette, and inject each sample individually using a 500 µL injection loop into the analytical column, pre-equilibrated with the SEC buffer at 4 °C.
    3. Allow the chromatography run to proceed at an isocratic flow rate of 0.2-0.3 mL/min with the SEC buffer at 4 °C.
    4. Observe the position and height of the elution peaks and look for the appearance of additional peaks for the different samples.
  3. Analysis of the chemical stability of the histone chaperones
    1. To examine the salt stability of histone chaperones, incubate 500 µL of 0.5 mg/mL protein sample prepared in a Tris buffer [20 mM of Tris-HCl (pH 7.5), and 1 mM of β-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 °C. Centrifuge the samples at 16,200 x g for 10 min at 4 °C and retain the supernatant.
    2. Next, load the protein samples in different NaCl concentrations individually, using a 500 µL 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 °C.
    3. 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 °C.
    4. Observe the position and height of elution peaks and look for the appearance of additional peaks for the different samples.
    5. Similarly, for urea stability analysis, incubate 500 µL of 0.5 mg/mL protein sample prepared in a Tris buffer [20 mM of Tris-HCl (pH 7.5), and 1 mM of β-ME] 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.
    6. Next, load the urea-treated protein samples individually using a 500 µL 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.
    7. 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.
    8. Observe the position and height of the elution peaks and look for the appearance of additional peaks for the different samples.

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

  1. For each reaction of pull-down assay, pipette 40 µL 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 µg/mL of BSA, and 1 mM of β-ME] (see Table of Materials).
    NOTE: Pull-down can also be performed in a 1.5 mL microcentrifuge tube.
  2. Prepare the sample by mixing 5 µM of the His-tagged histone chaperone with either 20 µM 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.
  3. 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 °C. Centrifuge the columns at 1000 x g for 1 min.
  4. 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 β-ME] containing different salt concentrations (i.e., 300 mM, 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.
  5. After the salt washing step, elute the protein from the different columns using 100 µL of elution buffer [20 mM of Tris-HCl (pH 7.5), 300 mM of NaCl, 300 mM of imidazole, and 1 mM of β-ME].
  6. 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.

3. Competitive pull-down assay to identify the preference of a histone chaperone for H2A/H2B or H3/H4

  1. Prepare spin column as described in step 2.1
  2. Incubate 5 µM of the histone chaperone with 20 µM of H2A/H2B dimer in 300 µL 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.
  3. Centrifuge the histone chaperone-H2A/H2B complex at 16,200 x g for 5 min at 4 °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 °C.
  4. 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 β-ME] to remove excess H2A/H2B dimer. Next, mix the histone chaperone-H2A/H2B complex with 20-60 µM of H3/H4 tetramer and incubate for 30 min on ice.
  5. 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.

4. Analytical ultracentrifugation - sedimentation velocity (AUC-SV) experiments to analyze the binding stoichiometry between histone chaperones and histones

  1. Sample preparation for AUC
    1. 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 β-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.
    2. 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.
  2. Sample loading for AUC
    1. Mix the purified proteins in a final volume of 450 µL 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.
    2. 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.
    3. Fill 400 µL of the sample solution and 420 µL 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.
    4. 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 °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's molecular mass; smaller proteins require larger time intervals between the scans. The rotor speed is also set according to the protein's expected molecular mass, and the experiment is conducted at 20 °C. The absorbance data is monitored at 280 nm.
    5. To obtain the exact stoichiometry, keep the histone chaperone concentration constant and incubate with increasing concentrations of histone oligomers to attain saturation.
  3. AUC data analysis
    1. 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.
    2. 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.
    3. 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.
    4. 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.
    5. Estimate the molecular masses of the peaks by choosing the option: show peak "Mw in c(s)" in the display function of the main toolbar, which will provide information about the 's' of the molecule/complex.

5. Plasmid supercoiling assay to confirm histone chaperoning function

  1. Nucleosome assembly reaction
    1. Mix 2 µM of H3/H4 tetramer and 4 µM of H2A/H2B dimer with increasing concentrations of the histone chaperone (1-6 µM) 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 µL. Incubate the mixture at 4 °C for 30 min.
    2. Simultaneously, in a separate reaction, pretreat 500 ng of the negatively supercoiled pUC19 plasmid with 1 µg of topoisomerase I enzyme (see Table of Materials) in the assembly buffer in a final volume of 50 µL and incubate at 30 °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.
    3. 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 °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).
    4. Stop the assembly reaction by adding 100 µL of 2x stop buffer (40 mM of EDTA, 2% of SDS, and 0.4 mg/mL of proteinase K) and incubate at 37 °C for 30 min.
      NOTE: Stop buffer deproteinizes the plasmid DNA by denaturation and proteolysis of bound histones.
  2. Phenol-chloroform extraction and ethanol precipitation
    1. 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.
    2. 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.
    3. 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 °C freezer for 30 min for complete precipitation of the plasmid DNA.
    4. 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.
  3. Perform the agarose gel electrophoresis to observe the plasmid supercoiling effect.
    1. Dissolve the precipitated plasmid DNA from step 5.2.4 in sterile double-distilled water.
    2. 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).
    3. Stain the gel with 0.2-0.5 µg/mL concentration of ethidium bromide and observe under UV to visualize the DNA bands on the gel.

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Results

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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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Discussion

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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...

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Disclosures

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No conflict of interest was declared.

Acknowledgements

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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. 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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Acetic acid (glacial)SigmaA6283
AcrylamideMP Biomedicals814326
AgaroseMP Biomedicals193983
AKTA Pure 25M FPLCCytiva29018226Instrument for protein purification
Ammonium persulfate (APS)SigmaA3678
An-60Ti rotorBeckman Coulter361964Rotor for analytical ultracentrifugation
Bovine serum albumin (BSA)SigmaA7030
ChloroformSigmaC2432
Coomassie brilliant blue R 250Sigma1.15444
Dialysis tubing (7 kDa cut-off)Thermo Fisher68700For dialysing protein samples
Dithiothreitol (DTT)MP Biomedicals100597
DNA Loading DyeNew England BiolabsB7025S
EDTA disodium saltMP Biomedicals194822
Electronic balanceShimadzuATX224R
EthanolSigmaE7023
Ethidium bromide (EtBr)SigmaE8751
Gel Doc SystemBio-Rad12009077For imaging gels after staining
Horizontal gel electrophoresis apparatusBio-Rad1704405Instrument for agarose gel electrophoresis
Hydrochloric acid (HCl)Sigma320331
ImidazoleMP Biomedicals102033
Magnesium chloride (MgCl2)SigmaM8266
MicropipettesEppendorfZ683779For pipetting of micro-volumes
Mini-PROTEAN electrophoresis systemBio-Rad1658000Instrument for SDS-PAGE
N,N-methylene-bis-acrylamideMP Biomedicals800172
Nano dropThermo FisherND-2000For measurement of protein and DNA concentrations
Ni-NTA agaroseInvitrogenR901-15Resin beads for pull-down assay
Optima AUC analytical ultracentrifugeBeckman CoulterB86437Instrument for analytical ultracentrifugation
pH MeterMettler ToledoMT30130863
PhenolSigmaP4557
Plasmid isolation kitQiagen27104
Proteinase KSigma-Aldrich1.07393
pUC19Thermo FisherSD0061Plasmid for supercoiling assay
Refrigerated high-speed centrifugeThermo Fisher75002402
SDS-PAGE protein markerBio-Rad1610317
SEDFITFree software program for analytical ultracentrifugation data analysis
SEDNTERPFree software program to estimate viscosity and density of buffer and partial specific volume of a protein
SigmaPrep Spin ColumnsSigmaSC1000For pull-down assay
Sodium acetateSigmaS2889
Sodium chloride (NaCl)MerckS9888
Sodium dodecyl sulfate (SDS)MP Biomedicals102918
Superdex 200 Increase 10/300 GLCytiva28990944Column for analytical size-exclusion chromatography
Superdex 75 Increase 10/300 GLCytiva29148721Column for analytical size-exclusion chromatography
TEMEDSigma1.10732
Topoisomerase IInspiralisWGT102Enzyme used in plasmid supercoiling assay
Tris baseMerckT1503
Tween-20SigmaP1379
UreaMP Biomedicals191450
Water bathNüveNB 5For heat treatment of protein samples
β-mercaptoethanol (β-ME)SigmaM6250

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Histone ChaperonesChromatin AssemblyPull Down AssayAnalytical UltracentrifugationSize Exclusion ChromatographyPlasmid Super CoilingNucleosome AssemblyH2A H2B DimerH3 H4 TetramerProtein DNA Interaction

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