Name: reconstitution of nucleosomes with differentially isotope-labeled sister histones
Uploaded: 2017-03-26T13:00:00
Description: Watch this Scientific Journal Video about Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones at JoVE.com

The author thanks Dr. Philipp Selenko (FMP-Berlin) for providing wet-lab space and infrastructure to perform experiments and Deutsche Forschungsgemeinschaft (DFG) for funding the work through a research grant (LI 2402/2-1).

1. Reconstitution of Nucleosomes with Differentially Isotope-labeled (and Asymmetrically Modified) Sister Histones
NOTE: The current protocol describes the reconstitution of nucleosomes with differentially isotope-labeled histone H3. To this end, two pools of histone H3 were used; one was 15N-labeled and contained a 6xHis-tag at the N-terminus and the second was 13C-labeled and contained the Strep peptide (WSHPQFEK) fused at the N-terminus. Both tags were separated f...

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Chem. Int. Ed. 55 (29), 8262-8265 (2016).</li><li>St&#252;tzer, 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=Modulations+of+DNA+Contacts+by+Linker+Histones+and+Post-translational+Modifications+Determine+the+Mobility+and+Modifiability+of+Nucleosomal+H3+Tails.">Modulations of DNA Contacts by Linker Histones and Post-translational Modifications Determine the Mobility and Modifiability of Nucleosomal H3 Tails.</a> Mol. Cell. 61 (2), 247-259 (2016).</li><li>Zhou, B. 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=Histone+H4+K16Q+Mutation,+an+Acetylation+Mimic,+Causes+Structural+Disorder+of+Its+N-terminal+Basic+Patch+in+the+Nucleosome.">Histone H4 K16Q Mutation, an Acetylation Mimic, Causes Structural Disorder of Its N-terminal Basic Patch in the Nucleosome.</a> J. Mol. 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For nucleosome reconstitution, the current protocol utilizes a 165 bp long DNA template containing the 601-Widom nucleosome positioning sequence20, but similar performance is expected using various lengths of DNA templates. The protocol was designed and employed using asymmetric types of histone H3. With the same principle, the method can be applied also for the other core histones and additionally can be used to reconstitute complexes carrying two distinct core histones with different isotope lab...

Eukaryotic DNA is tightly packaged within cell nuclei into chromatin. The fundamental building block of chromatin is the nucleosome core particle that contains ~147 bp of DNA wrapped around an octameric complex made up of two copies each of the four core histones (H3, H4, H2A, H2B). Histone proteins harbor a plethora of Post-translational Modifications (PTMs). These covalent substitutions induce alterations in chromatin structure, both directly by affecting the physical chemistry of the system and indirectly by recruiting chromatin-remodeling activities1,2,3. By those means, histone PTMs control chromatin accessibility and, hence, regulate all DNA-based cellular functions4.
PTMs are installed by histone-modifying enzyme systems mainly on the unstructured N-terminal segments (tails) of nucleosome-incorporated core histones. Due to the many modification sites on the relatively short sequence of histone tails, PTMs influence each other by inducing or blocking subsequent modification reactions, an effect known as modification cross-talk5. Because of the overall symmetric architecture of the nucleosome, modification reactions and crosstalk mechanisms were thought to occur similarly onto the two copies of each nucleosomal histone (sister histones). This concept was recently challenged and subsequently disproved. Particularly, in vitro enzymatic assays on free histone H3 tail peptides and on nucleosomes demonstrated that a set of H3 kinases introduced phosphorylation in an asymmetric manner6. Additionally, affinity-purification-based LC-MS/MS analysis revealed the existence of asymmetrically H3-methylated nucleosomes in several types of eukaryotic cells7. Thus, asymmetrically modified nucleosomes constitute novel species, and tools are needed to uncover the mechanisms that control their formation and to analyze the crosstalk effects that this asymmetry might exert.
Commonly, Western Blotting (WB) and Mass Spectrometry (MS) analysis have been used to detect histone PTMs. Despite its easy application, WB suffers from specificity/cross-reactivity problems. On top of that, it is incapable of performing simultaneous multi-PTM analysis and direct quantification of the modification reactions8. On the other hand, MS analysis employs sophisticated instrumentation that requires high-level training, but provides high specificity as well as simultaneous mapping and quantification of multiple PTMs9.&#160;However, both methods are disruptive and the nucleosomal complexes are dissociated before analysis, giving rise to a mixture of histones and/or histone-derived peptides. This manipulation removes the ability to distinguish independent modification reactions that occur on each of the two sister histones and to report the copy-specific modification status of the nucleosomal histones.
Nuclear Magnetic Resonance (NMR) spectroscopy evolved as an alternative method to map PTM reactions. NMR is nondisruptive and thus allows the monitoring of PTM events in a real-time manner in reconstituted mixtures, and even in intact cells10,11. The development of routines for fast data acquisition as well as for high-resolution mapping based on 2D hetero-nuclear correlation methods of isotope-labeled (15N and/or 13C) samples12 allowed the simultaneous mapping of different types of PTMs, such as serine/threonine/tyrosine phosphorylation, lysine acetylation/methylation, and arginine methylation13. Depending on the PTM under investigation, 15N- or 13C-labeling protocols can be employed to mark the protein functional group that serves as a modification reporter. Consequently, PTM mapping can be performed by following the characteristic chemical shift displacement of the corresponding functional group &#39;sensing&#39; the alteration on the chemical environment. In most cases, both N-H and C-H chemical groups can be used to report the evolution of the PTM of interest.
The current protocol describes the generation of nucleosomes containing differentially isotope-labeled sister histones. It combines the flexibility of NMR spectroscopy to map PTMs using both 1H-15N and 1H-13C correlation spectra with the utilization of different protein affinity tags for purification of the selected reconstituted histone complexes. Notably, the protocol employs two different pools of a particular histone for nucleosome reconstitution. These pools are differentially isotope-labeled (one with 15N, the other with 13C), and they are fused to a polyhistidine and a streptavidin affinity tag, respectively. A tandem affinity purification scheme with Ni-NTA and streptavidin-based chromatography initially used by Voigt et al.7&#160;is&#160;employed to purify asymmetric species from symmetric counterparts (Figure 1A). Asymmetric histone octamers are used subsequently to reconstitute equivalent nucleosomal complexes (Figure 1B), using the standard salt dialysis method14. Additionally, through the same procedure and by having one of the histone pools pre-modified, a PTM can be incorporated asymmetrically onto the resulting nucleosomes. The reaction of these substrates with histone-modifying enzymes and subsequent NMR-mapping of modification events enable the characterization of crosstalk mechanisms both in-cis (premodified histone copy) and in-trans (unmodified histone copy) (Figure 1C).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Unfolding Buffer</td>
			<td>7M guanidinium HCl</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>20mM Tris pH7.5</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>10mM DTT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Refolding Buffer</td>
			<td>10mM Tris pH7.5</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2M NaCl</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>1mM EDTA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2mM DTT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Assay Buffer</td>
			<td>25mM NaxHxPO4 pH6.8</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>25mM NaCl</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2mM DTT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Guanidinium HCl</td>
			<td>Applichem</td>
			<td>A14199</td>
			<td>Use high quality Gu-HCl</td>
		</tr>
		<tr>
			<td>Tris&#160;</td>
			<td>Roth</td>
			<td>4855.2</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Applichem</td>
			<td>A1101</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>VWR chemicals</td>
			<td>27810.364</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Roth</td>
			<td>8043.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Applichem</td>
			<td>A1046</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Applichem</td>
			<td>A3902</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Applichem</td>
			<td>A1073</td>
			<td></td>
		</tr>
		<tr>
			<td>d-Desthiobiotin</td>
			<td>Sigma</td>
			<td>D1411</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Superflow</td>
			<td>Qiagen</td>
			<td>1034557</td>
			<td></td>
		</tr>
		<tr>
			<td>Strep-Tactin Superflow</td>
			<td>IBA</td>
			<td>2-1207-001</td>
			<td></td>
		</tr>
		<tr>
			<td>His-probe antibody</td>
			<td>Santa Cruz</td>
			<td>sc-8036</td>
			<td></td>
		</tr>
		<tr>
			<td>Strep-tactin conjugated HRP</td>
			<td>IBA</td>
			<td>2-1502-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hi-Load 16/600 Superdex 200pg</td>
			<td>GE Healthcare</td>
			<td>28-9893-35</td>
			<td></td>
		</tr>
		<tr>
			<td>6-8kDa dialysis membrane</td>
			<td>Spectrumlabs</td>
			<td>spectra/por 1, 132650</td>
			<td></td>
		</tr>
		<tr>
			<td>50kDa dialysis membrane</td>
			<td>Spectrumlabs</td>
			<td>spectra/por 7, 132129</td>
			<td></td>
		</tr>
		<tr>
			<td>10kDa centrigugal filter unit</td>
			<td>Merck Millipore</td>
			<td>UFC901024</td>
			<td></td>
		</tr>
		<tr>
			<td>30kDa centrigugal filter unit</td>
			<td>Merck Millipore</td>
			<td>UFC903024</td>
			<td></td>
		</tr>
		<tr>
			<td>Solution-state NMR spectrometer&#160;</td>
			<td></td>
			<td></td>
			<td>at least 500 MHz operating frequency, equipped with a triple-resonance cryoprobe</td>
		</tr>
	</tbody>
</table>

reconstitution of nucleosomes with differentially isotope-labeled sister histones

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.

Properly refolded octameric species are isolated after a run of the reconstitution mixture through a gel filtration column (Figure 2). The reconstituted octameric pool containing the three different types of octamers is subjected to the tandem affinity purification scheme. Samples are collected from all the steps and analyzed by SDS-PAGE and subsequently WB. Verification of the correct execution of the protocol that results in the isolation of asymmetric species is achiev...

Watch this Scientific Journal Video about Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones at JoVE.com

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

This protocol describes the reconstitution of nucleosomes containing differentially isotope-labeled sister histones. At the same time, asymmetrically post-translationally modified nucleosomes can be generated after using a premodified histone copy. These preparations can be readily used to study modification crosstalk mechanisms, simultaneously on both sister histones, by using high-resolution NMR spectroscopy.

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

The overall goal of this biochemical protocol is to generate nucleosomes with differentially isotope-labeled sister histones. For use in mapping post translational modification reactions simultaneously on both sister histones. The overall goal of this biochem protocol is to generate nucleosomes with differentially isotope-labeled sister histones for use in mapping post translational modifications simultaneously on both sister histones.This method in combination with high resolution techniques to map post translational modifications such as in mass spectroscopy addresses key questions in chromatin biology like what mechanisms contribute to the formation of a symmetrically modified nucleosomes? The main advantage of this technique is that it can be easily established in a biochemical laboratory because it combines standard nucleosome reconstitution protocols with common protein purification methodologies. Two pools of histone H3 will be used for octamer reconstitution.Nitrogen 15 labeled H3 that contains a polyhistidine-tag at the amino terminus, and carbon 13 labeled H3 that contains a streptavidin affinity tag at the amino terminus. Begin this procedure by dissolving the lyophilized histone aliquots including the two types of histone H3 in unfolding buffer. To each tube that contains about five milligrams of histones add one milliliter of unfolding buffer and mix by pipetting, but do not vortex.Keep the tubes on ice for 30 minutes to allow complete unfolding. Next, measure the absorbance at 280 nanometers to determine the exact concentration of each histone. Mix the core histones to equally molar ratios, keeping in mind to include 50%each of the two histone H3 pools.Use unfolding buffer to adjust the final protein concentration to approximately one milligram per milliliter. Transfer the mixture to a six kilodalton cutoff dialysis membrane. And dialyze against one to two liters of chilled refolding buffer at four degrees Celsius.On the following day, collect the dialyzed material and remove any precipitates by centrifugation in a microfuge for five minutes at four degrees Celsius on maximum speed. After determining the octamer concentration, use a 10 kilodalton cutoff centrifugal filter unit to concentrate the octamer to a final volume of about two milliliters. An octamer concentration between 50 to 100 micromolar should be obtained.Prepare for gel filtration by connecting the gel filtration column to an FPLC system. Equilibrate with two column volumes, or CV of 0.22 micrometer filtered and degassed refolding buffer. Inject the concentrated material at a flow rate of one milliliter per minute and collect one to 1.5 milliliter fractions.To begin this procedure, pool the relevant fractions containing pure octamers and determine the concentration as described previously. Connect a one milliliter nickel nitrilotriacetic acid or nickel NTA column to the FPLC system. And equilibrate with 10 CV of 0.22 micrometer filtered and degassed refolding buffer at a flow rate of one milliliter per minute.Pass the purified octamer through the nickel NTA using a flow rate of one milliliter per minute. Collect the flow through. Elute with 10 CV of refolding buffer containing 250 millimolar imidazole using a flow rate of one milliliter per minute.Collect the nickel NTA elution. The streptavidin based purification should ideally be performed in a cold room. Equilibrate a one milliliter affinity column of a commercial streptavidin with 10 CV refolding buffer containing 250 millimolar imidazole using a batch gravity flow setup.Pass the nickel NTA elution through the streptavidin affinity column. Collect the flow through. Elute with 10 CV of refolding buffer containing 2.5 millimolar desthiobiotin.Collect the elution. After measuring the octamer concentration in the elution, add 10 times less TEV protease and let the reaction proceed overnight at four degrees Celsius. On the following day, transfer the mixture to a 50 kilodalton cutoff dialysis membrane and dialyze against refolding buffer overnight at four degrees Celsius.Concentrate the purified asymmetric octamer using a 10 kilodalton cutoff centrifugal filter unit to a volume of one to two milliliters. The expected octamer concentration is 20 to 50 micromolar. Nucleosome reconstitution requires DNA containing a nucleosome positioning sequence.Adjust the DNA salt concentration to two molar sodium chloride using a five molar sodium chloride stock solution. Mix the DNA with octamer and refolding buffer using the optimum octamer to DNA ratio determined from a small scale test. Add the octamer last to prevent any possibility of mixing the octamer and the DNA at a salt concentration lower than two molar.Transfer the mix to a six kilodalton cutoff dialysis membrane and dialyze against one liter of refolding buffer overight at four degrees Celsius. On the following day, reduce the salt concentration of the dialysis buffer in a stepwise manner. Keeping the sample in each sodium chloride buffer for at least three hours.Next, transfer the dialysis membrane to a beaker with one liter of assay buffer and continue dialysis overnight at four degrees Celsius. On the following day, collect the dialyzed sample. Remove any precipitate formed by centrifugation in a microfuge for five minutes at four degrees Celsius on maximum speed.Concentrate the sample using a 30 kilodalton cutoff centrifugal filter unit to a final volume of about 300 microliters. Measure the DNA concentration at 260 nanometers. The final concentration should be in the range of 10 to 20 micromolar.Store the sample at four degrees Celsius for up to several weeks. A gel filtration elution profile of the refolded histone mixture shows the histone octamers eluting at about 70 milliliters. An SDS page gel shows the correct histone stoichiometry.The reconstituted octameric pool containing the three types of octamers is subjected to a tandem affinity purification scheme. The isolation of asymmetric species is verified by the presence of the affinity tag. The affinity tags are subsequently removed with TEV protease.A small scale nucleosome reconstitution using different molar ratios of DNA to octamer showed that a ratio of one to one was optimal. A large scale nucleosome reconstitution was performed using the optimized DNA to octamer molar ratio, and the correct stoichiometry of the four core histones was observed. NMR spectroscopy of differentially isotope-labeled nucleosomes confirmed the presence of nitrogen 15 labeled and carbon 13 labeled sister H3 histones.In this application example, nucleosomes that were differentially isotope-labeled and asymmetrically phosphorylated on C ring 10 of histone H3 were reacted with GCN5 acetyltransferase. And acetylation of histone H3 lysine 14 on both H3 copies was followed in real time by NMR spectroscopy. The analysis revealed that C ring 10 of histone H3 phosphorylation stimulates histone H3 lysine 14 acetylation by GCN5 in cis compared to in trans.Once mastered, this technique can be done in five days if it's performed properly. After watching this video you should have a good understanding of how to reconstitute nucleosomes containing sister histones with different features.

Research

JoVE Journal

Biochemistry

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become ...

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts ...

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

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

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

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

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

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

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully ...