H2A-H2B Dimers Research Articles

Effects of charge-modifying mutations in histone H2A \u03b13-domain on nucleosome stability assessed by single-pair FRET and MD simulations

Nucleosomes are important for chromatin compaction and gene regulation; their integrity depends crucially on the structural properties of the histone tails. Recent all-atom molecular dynamics simulations revealed that removal of the N-terminal tails of histone H3, known to destabilize nucleosomes, causes a rearrangement of two arginines of histone H2A, namely R81 and R88 by altering the electrostatic environment of the H2A α3 domain. Whether this rearrangement is the cause or the effect of decreased stability, is unclear. Here, we emulate the altered electrostatic environment that was found after H3 tail clipping through charge-modifying mutations to decouple its impact on intranucleosomal interactions from that of the histone tails. Förster resonance energy transfer experiments on recombinant nucleosomes and all-atom molecular dynamics simulations reveal a compensatory role of those amino acids in nucleosome stability. The simulations indicate a weakened interface between H2A-H2B dimers and the (H3-H4)2 tetramer, as well as between dimers and DNA. These findings agree with the experimental observations of position and charge dependent decreased nucleosome stability induced by the introduced mutations. This work highlights the importance of the H2A α3 domain and suggests allosteric effects between this domain and the outer DNA gyre as well as the H3 N-terminal tail.

Regulation of the Stability of the Histone H2A-H2B Dimer by H2A Tyr57 Phosphorylation.

Histone H2A and H2B form a H2A-H2B heterodimer, which is a fundamental unit of nucleosome assembly and disassembly. Several posttranslational modifications change the interface between the H2A-H2B dimer and the H3-H4 tetramer and regulate nucleosome stability. However, posttranslational modifications associated with the interface between H2A and H2B have not been discussed. In this paper, it is shown that Tyr57 phosphorylation in H2A strongly influences H2A-H2B dimerization. Tyr57-phosphorylated H2A was chemically synthesized and utilized to reconstitute the H2A-H2B dimer and nucleosome as well as canonical H2A. Thermal shift assays showed that phosphorylation destabilized the dimer and facilitated dissociation of H2A and H2B from the nucleosome structure. The proximity between H2A Tyr57 and the H2B αC helix is assumed to lead the destabilization. The DNA accessibility of the nucleosome was estimated by using micrococcal nuclease. The phosphorylated nucleosome did not change DNA accessibility compared to that of the canonical nucleosome. It is demonstrated that phosphorylation at Tyr57 changes the H2A-H2B dimer interaction but does not interfere with histone-DNA interactions. This work on the destabilization of the H2A-H2B dimer by Tyr57 phosphorylation is a promising step in elucidating control mechanisms of dynamic behavior of H2A and H2B through posttranslational modifications.

Dual function of Swc5 in SWR remodeling ATPase activation and histone H2A eviction.

The chromatin remodeler SWR deposits histone H2A.Z at promoters and other regulatory sites via an ATP-driven histone exchange reaction that replaces nucleosomal H2A with H2A.Z. Simultaneous binding of SWR to both H2A nucleosome and free H2A.Z induces SWR ATPase activity and engages the histone exchange mechanism. Swc5 is a conserved subunit of the 14-polypeptide SWR complex that is required for the histone exchange reaction, but its molecular role is unknown. We found that Swc5, although not required for substrate binding, is required for SWR ATPase stimulation, suggesting that Swc5 is required to couple substrate recognition to ATPase activation. A biochemical complementation assay was developed to show that a unique, conserved domain at the C-terminus of Swc5, called Bucentaur (BCNT), is essential for the histone exchange activity of SWR, whereas an acidic region at the N-terminus is required for optimal SWR function. In vitro studies showed the acidic N-terminus of Swc5 preferentially binds to the H2A–H2B dimer and exhibits histone chaperone activity. We propose that an auxiliary function of Swc5 in SWR is to assist H2A ejection as H2A.Z is inserted into the nucleosome.

Residues in the Nucleosome Acidic Patch Regulate Histone Occupancy and Are Important for FACT Binding in Saccharomyces cerevisiae.

The essential histone chaperone FACT plays a critical role in DNA replication, repair, and transcription, primarily by binding to histone H2A-H2B dimers and regulating their assembly into nucleosomes. While FACT histone chaperone activity has been extensively studied, the exact nature of the H2A and H2B residues important for FACT binding remains controversial. In this study, we characterized the functions of residues in the histone H2A and H2B acidic patch, which is important for binding many chromatin-associated factors. We found that mutations in essential acidic patch residues cause a defect in histone occupancy in yeast, even though most of these histone mutants are expressed normally in yeast and form stable nucleosomes in vitro Instead, we show that two acidic patch residues, H2B L109 and H2A E57, are important for histone binding to FACT in vivo We systematically screened mutants in other H2A and H2B residues previously suspected to be important for FACT binding and confirmed the importance of H2B M62 using an in-vivo FACT-binding assay. Furthermore, we show that, like deletion mutants in FACT subunits, an H2A E57 and H2B M62 double mutant is lethal in yeast. In summary, we show that residues in the nucleosome acidic patch promote histone occupancy and are important for FACT binding to H2A-H2B dimers in yeast.

INO80 exchanges H2A.Z for H2A by translocating on DNA proximal to histone dimers

ATP-dependent chromatin remodellers modulate nucleosome dynamics by mobilizing or disassembling nucleosomes, as well as altering nucleosome composition. These chromatin remodellers generally function by translocating along nucleosomal DNA at the H3–H4 interface of nucleosomes. Here we show that, unlike other remodellers, INO80 translocates along DNA at the H2A–H2B interface of nucleosomes and persistently displaces DNA from the surface of H2A–H2B. DNA translocation and DNA torsional strain created near the entry site of nucleosomes by INO80 promotes both the mobilization of nucleosomes and the selective exchange of H2A.Z–H2B dimers out of nucleosomes and replacement by H2A–H2B dimers without any additional histone chaperones. We find that INO80 translocates and mobilizes H2A.Z-containing nucleosomes more efficiently than those containing H2A, partially accounting for the preference of INO80 to replace H2A.Z with H2A. Our data suggest that INO80 has a mechanism for dimer exchange that is distinct from other chromatin remodellers including its paralogue SWR1.

Single-Molecule Investigations on Histone H2A-H2B Dynamics in the Nucleosome.

Nucleosomes impose physical barriers to DNA-templated processes, playing important roles in eukaryotic gene regulation. DNA is packaged into nucleosomes by histone proteins mainly through strong electrostatic interactions that can be modulated by various post-translational histone modifications. Investigating the dynamics of histone dissociation from the nucleosome and how it is altered upon histone modifications is important for understanding eukaryotic gene regulation mechanisms. In particular, histone H2A-H2B dimer displacement in the nucleosome is one of the most important and earliest steps of histone dissociation. Two conflicting hypotheses on the requirement for dimer displacement are that nucleosomal DNA needs to be unwrapped before a dimer can displace and that a dimer can displace without DNA unwrapping. In order to test the hypotheses, we employed three-color single-molecule FRET and monitored in a time-resolved manner the early kinetics of H2A-H2B dimer dissociation triggered by high salt concentration and by histone chaperone Nap1. The results reveal that dimer displacement requires DNA unwrapping in the vast majority of the nucleosomes in the salt-induced case, while dimer displacement precedes DNA unwrapping in >60% of the nucleosomes in the Nap1-mediated case. We also found that acetylation at histone H4K16 or H3K56 affects the kinetics of Nap1-mediated dimer dissociation and facilitates the process both kinetically and thermodynamically. On the basis of these results, we suggest a mechanism by which histone chaperone facilitates H2A-H2B dimer displacement from the histone core without requiring another factor to unwrap the nucleosomal DNA.

Nucleosome Opening Kinetics and the Influence of Histone Modifications Studied by Single Molecule FRET

Nucleosomes compact the genome and regulate access by wrapping and unwrapping DNA. Here we characterized NaCl-induced opening of mononucleosomes by single-molecule FRET, using donor/acceptor labels on the DNA and various histones. By species-selective fluorescence lifetime and photon distribution analysis we identified new nucleosome opening intermediates and developed a kinetic model [1]. Opening proceeds through a weakening of the H2A-H2B dimer/(H3-H4)2 tetramer interface on a 0.1 ms time scale, then by a slower two-step release of the dimers coupled to DNA unwrapping, extending from several ms to minutes. Nucleosome opening and detachment of histone dimers proceed asymmetrically depending on the DNA sequence.Mutations at the H2A/H3 interface (H2A R81A, R88A, R81A/R88A, R81E/R88E) facilitate the initial opening, confirming the importance of the dimer:tetramer interface for nucleosome stability. This is also supported by molecular dynamics (MD) simulations that show enhanced DNA fluctuations for the mutants. Partially opened states such as described here might be a convenient nucleation point for DNA-recognizing proteins.For characterizing the role of histone tails in nucleosome interaction and stability, we performed MD simulations in explicit and implicit solvent on the H4 tail interacting with the acidic patch of a neighboring nucleosome. The simulations show that H4K16 acetylation decreases the interactions between H4 tail and the acidic patch of the neighboring nucleosome, a process that is central to the regulation of chromatin compaction [2,3].

Stabilization of Nucleosomes by Histone Tails and by FACT Revealed by spFRET Microscopy.

A correct chromatin structure is important for cell viability and is tightly regulated by numerous factors. Human protein complex FACT (facilitates chromatin transcription) is an essential factor involved in chromatin transcription and cancer development. Here FACT-dependent changes in the structure of single nucleosomes were studied with single-particle Förster resonance energy transfer (spFRET) microscopy using nucleosomes labeled with a donor-acceptor pair of fluorophores, which were attached to the adjacent gyres of DNA near the contact between H2A-H2B dimers. Human FACT and its version without the C-terminal domain (CTD) and the high mobility group (HMG) domain of the structure-specific recognition protein 1 (SSRP1) subunit did not change the structure of the nucleosomes, while FACT without the acidic C-terminal domains of the suppressor of Ty 16 (Spt16) and the SSRP1 subunits caused nucleosome aggregation. Proteolytic removal of histone tails significantly disturbed the nucleosome structure, inducing partial unwrapping of nucleosomal DNA. Human FACT reduced DNA unwrapping and stabilized the structure of tailless nucleosomes. CTD and/or HMG domains of SSRP1 are required for this FACT activity. In contrast, previously it has been shown that yeast FACT unfolds (reorganizes) nucleosomes using the CTD domain of SSRP1-like Pol I-binding protein 3 subunit (Pob3). Thus, yeast and human FACT complexes likely utilize the same domains for nucleosome reorganization and stabilization, respectively, and these processes are mechanistically similar.

An Assay for Measuring Histone Variant Exchange within Nucleosomes In Vitro.

The incorporation of histone variants into specific chromatin regions is a mechanism by which cells can regulate many important biological processes. One such example is H2A.Z, a highly conserved variant of H2A that is incorporated in genomic regulatory regions and contributes to control gene expression. H2A.Z variant exchange involves the removal of H2A-H2B dimers from a preassembled nucleosome and their replacement with H2A.Z-H2B dimers. A specific family of chromatin remodeling complexes, homologous to the yeast Swr1 complex, have been shown to be capable of this histone exchange activity both in vivo and in vitro. Here, we describe an assay to measure the histone H2A.Z exchange activity of recombinant human p400 on immobilized mononucleosomes in vitro. The assay can be adapted to other histone exchange complexes/catalytic subunits purified from any species.

Structural Insights into the Association of Hif1 with Histones H2A-H2B Dimer and H3-H4 Tetramer

Histone chaperones are critical for guiding specific post-transcriptional modifications of histones, safeguarding the histone deposition (or disassociation) of nucleosome (dis)assembly, and regulating chromatin structures to change gene activities. HAT1-interacting factor 1 (Hif1) has been reported to be an H3-H4 chaperone and to be involved in telomeric silencing and nucleosome (dis)assembly. However, the structural basis for the interaction of Hif1 with histones remains unknown. Here, we report the complex structure of Hif1 binding to H2A-H2B for uncovering the chaperone specificities of Hif1 on binding to both the H2A-H2B dimer and the H3-H4 tetramer. Our findings reveal that Hif1 interacts with the H2A-H2B dimer and the H3-H4 tetramer via distinct mechanisms, suggesting that Hif1 is a pivotal scaffold on alternate binding of H2A-H2B and H3-H4. These specificities are conserved features of the Sim3-Hif1-NASP interrupted tetratricopeptide repeat proteins, which provide clues for investigating their potential roles in nucleosome (dis)assembly.

Structural and histone binding ability characterization of the ARB2 domain of a histone deacetylase Hda1 from Saccharomyces cerevisiae.

Hda1 is the catalytic core component of the H2B- and H3- specific histone deacetylase (HDAC) complex from Saccharomyces cerevisiae, which is involved in the epigenetic repression and plays a crucial role in transcriptional regulation and developmental events. Though the N-terminal catalytic HDAC domain of Hda1 is well characterized, the function of the C-terminal ARB2 domain remains unknown. In this study, we determine the crystal structure of the ARB2 domain from S. cerevisiae Hda1 at a resolution of 2.7 Å. The ARB2 domain displays an α/β sandwich architecture with an arm protruding outside. Two ARB2 domain molecules form a compact homo-dimer via the arm elements, and assemble as an inverse “V” shape. The pull-down and ITC results reveal that the ARB2 domain possesses the histone binding ability, recognizing both the H2A-H2B dimer and H3-H4 tetramer. Perturbation of the dimer interface abolishes the histone binding ability of the ARB2 domain, indicating that the unique dimer architecture of the ARB2 domain coincides with the function for anchoring to histone. Collectively, our data report the first structure of the ARB2 domain and disclose its histone binding ability, which is of benefit for understanding the deacetylation reaction catalyzed by the class II Hda1 HDAC complex.

Nucleosome assembly and disassembly activity of GRWD1, a novel Cdt1-binding protein that promotes pre-replication complex formation

GRWD1 was previously identified as a novel Cdt1-binding protein that possesses histone-binding and nucleosome assembly activities and promotes MCM loading, probably by maintaining chromatin openness at replication origins. However, the molecular mechanisms underlying these activities remain unknown. We prepared reconstituted mononucleosomes from recombinant histones and a DNA fragment containing a nucleosome positioning sequence, and investigated the effects of GRWD1 on them. GRWD1 could disassemble these preformed mononucleosomes in vitro in an ATP-independent manner. Thus, our data suggest that GRWD1 facilitates removal of H2A-H2B dimers from nucleosomes, resulting in formation of hexasomes. The activity was compromised by deletion of the acidic domain, which is required for efficient histone binding. In contrast, nucleosome assembly activity of GRWD1 was not affected by deletion of the acidic domain. In HeLa cells, the acidic domain of GRWD1 was necessary to maintain chromatin openness and promote MCM loading at replication origins. Taken together, our results suggest that GRWD1 promotes chromatin fluidity by influencing nucleosome structures, e.g., by transient eviction of H2A-H2B, and thereby promotes efficient MCM loading at replication origins.

Single-Molecule Observation Reveals Spontaneous Protein Dynamics in the Nucleosome.

Structural dynamics of a protein molecule is often critical to its function. Single-molecule methods provide efficient ways to investigate protein dynamics, although it is very challenging to achieve a millisecond or higher temporal resolution. Here we report spontaneous structural dynamics of the histone protein core in the nucleosome based on a single-molecule method that can reveal submillisecond dynamics by combining maximum likelihood estimation and fluorescence correlation spectroscopy. The nucleosome, comprising ∼147 bp DNA and an octameric histone protein core consisting of H2A, H2B, H3, and H4, is the fundamental packing unit of the eukaryotic genome. The nucleosome imposes a physical barrier that should be overcome during various DNA-templated processes. Structural fluctuation of the nucleosome in the histone core has been hypothesized to be required for nucleosome disassembly but has yet to be directly probed. Our results indicate that at 100 mM NaCl the histone H2A-H2B dimer dissociates from the histone core transiently once every 3.6 ± 0.6 ms and returns to its position within 2.0 ± 0.3 ms. We also found that the motion is facilitated upon H3K56 acetylation and inhibited upon replacing H2A with H2A.Z. These results provide the first direct examples of how a localized post-translational modification or an epigenetic variation affects the kinetic and thermodynamic stabilities of a macromolecular protein complex, which may directly contribute to its functions.

Abstract 1972: Changes in the architecture, interactions and chromatin remodeling of SWI/SNF due to loss of Snf5

Abstract The mammalian SWI/SNF complex is a family of an estimated ∼100 complexes, each with different combinations of related subunits. Efforts in sequencing many cancer genomes has revealed that mammalian SWI/SNF complexes is one of the most frequently mutated epigenetic factors found in a broad range of cancers. In order to understand more about the subunit organization of SWI/SNF and because yeast and mammalian SWI/SNF complexes share many conserved subunits and domains; we have studied the role of the Snf5 subunit in the yeast SWI/SNF to better understand the role of the mammalian homolog SNF5 that is a known tumor suppressor gene. The basis for the loss of mammalian SNF5 being a driver mutation in pediatric rhabdoid tumors is currently not well understood or how the loss of SNF5 effects the composition, recruitment and nucleosome remodeling activity of the SWI/SNF complex. We have assessed the effects on deletion of Snf5 on the compositional integrity of SWI/SNF and have evidence for Snf5 forming a sub-complex with two other highly conserved subunits of SWI/SNF that are lost from the complex when Snf5 is deleted. In addition we have mapped the inter-subunit and intra-subunit interactions of the yeast SWI/SNF complex with lysine specific homo bi-functional crosslinkers and mass spectrometry. These data confirm the formation of a tri-subunit Snf5 sub-complex and show that this module uniquely interacts with the ATPase domain of the catalytic subunit of SWI/SNF (Snf2). These observations lead to a further investigation into the regulation of the remodeling activity of SWI/SNF by the Snf5 subunit. We have found that Snf5 is required for the ATPase domain to make stable interactions with nucleosomal DNA. And the loss of these interactions in turn also causes a reduction in the remodeling efficiency of the SWI/SNF complex. We have done additional mapping of the interactions of Snf5 as part of the SWI/SNF complex and find that the highly conserved region of yeast Snf5 corresponding to the human SNF5 protein associates with the surface of the nucleosome near the histone H2A-H2B dimer. We will discuss the relevance of these findings in highlighting how an aberrant form of SWI/SNF could be created by loss of Snf5 and provide an example of how it alters the normal function and structure of SWI/SNF. Citation Format: Blaine Bartholomew, Jim Persinger, Payel Sen, Mekonnen Dechassa Lemma, Jie Luo, Jeff Ranish. Changes in the architecture, interactions and chromatin remodeling of SWI/SNF due to loss of Snf5. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1972.

A basic domain in the histone H2B N-terminal tail is important for nucleosome assembly by FACT.

Nucleosome assembly in vivo requires assembly factors, such as histone chaperones, to bind to histones and mediate their deposition onto DNA. In yeast, the essential histone chaperone FACT (FAcilitates Chromatin Transcription) functions in nucleosome assembly and H2A–H2B deposition during transcription elongation and DNA replication. Recent studies have identified candidate histone residues that mediate FACT binding to histones, but it is not known which histone residues are important for FACT to deposit histones onto DNA during nucleosome assembly. In this study, we report that the histone H2B repression (HBR) domain within the H2B N-terminal tail is important for histone deposition by FACT. Deletion of the HBR domain causes significant defects in histone occupancy in the yeast genome, particularly at HBR-repressed genes, and a pronounced increase in H2A–H2B dimers that remain bound to FACT in vivo. Moreover, the HBR domain is required for purified FACT to efficiently assemble recombinant nucleosomes in vitro. We propose that the interaction between the highly basic HBR domain and DNA plays an important role in stabilizing the nascent nucleosome during the process of histone H2A–H2B deposition by FACT.

Exploring the Free Energy Landscape of Nucleosomes.

The nucleosome is the fundamental unit for packaging the genome. A detailed molecular picture for its conformational dynamics is crucial for understanding transcription and gene regulation. We investigate the disassembly of single nucleosomes using a predictive coarse-grained protein DNA model with transferable force fields. This model quantitatively describes the thermodynamic stability of both the histone core complex and the nucleosome and predicts rates of transient nucleosome opening that match experimental measurements. Quantitative characterization of the free-energy landscapes reveals the mechanism of nucleosome unfolding in which DNA unwinding and histone protein disassembly are coupled. The interfaces between H2A-H2B dimers and the (H3-H4)2 tetramer are first lost when the nucleosome opens releasing a large fraction but not all of its bound DNA. For the short strands studied in single molecule experiments, the DNA unwinds asymmetrically from the histone proteins, with only one of its two ends preferentially exposed. The detailed molecular mechanism revealed in this work provides a structural basis for interpreting experimental studies of nucleosome unfolding.

Regulation of MicroRNA-Mediated Developmental Changes by the SWR1 Chromatin Remodeling Complex.

The ATP-dependent SWR1 chromatin remodeling complex (SWR1-C) exchanges the histone H2A-H2B dimer with the H2A.Z-H2B dimer, producing variant nucleosomes. Arabidopsis thaliana SWR1-C contributes to the active transcription of many genes, but also to the repression of genes that respond to environmental and developmental stimuli. Unlike other higher eukaryotic H2A.Z deposition mutants (which are embryonically lethal), Arabidopsis SWR1-C component mutants, including arp6, survive and display a pleiotropic developmental phenotype. However, the molecular mechanisms of early flowering, leaf serration, and the production of extra petals in arp6 have not been completely elucidated. We report here that SWR1-C is required for miRNA-mediated developmental control via transcriptional regulation. In the mutants of the components of SWR1-C such as arp6, sef, and pie1, miR156 and miR164 levels are reduced at the transcriptional level, which results in the accumulation of target mRNAs and associated morphological changes. Sequencing of small RNA libraries confirmed that many miRNAs including miR156 decreased in arp6, though some miRNAs increased. The arp6 mutation suppresses the accumulation of not only unprocessed primary miRNAs, but also miRNA-regulated mRNAs in miRNA processing mutants, hyl1 and serrate, which suggests that arp6 has a transcriptional effect on both miRNAs and their targets. We consistently detected that the arp6 mutant exhibits increased nucleosome occupancy at the tested MIR gene promoters, indicating that SWR1-C contributes to transcriptional activation via nucleosome dynamics. Our findings suggest that SWR1-C contributes to the fine control of plant development by generating a balance between miRNAs and target mRNAs at the transcriptional level.

Expression and purification of the full murine NPM2 and study of its interaction with protamines and histones

Mouse nucleoplasmin M.NPM2 was recombinantly expressed and the protein consisting of the complete sequence was purified and characterized. Similar to its Xenopus laevis X.NPM2 counterpart, the protein forms stable pentameric complexes and exhibits an almost undistinguishable hydrodynamic ionic strength-dependent unfolding behavior. The interaction of N.PM2 with histones and mouse P1/P2 protamines revealed that these chromosomal proteins bind preferentially to the distal part of the nucleoplasmin pentamer. Moreover, the present work highlights the critical role played by histones H2B and H4 in the association of the histone H2A-H2B dimers and histone octamer with nucleoplasmin.

Stepwise nucleosome translocation by RSC remodeling complexes.

The SWI/SNF-family remodelers regulate chromatin structure by coupling the free energy from ATP hydrolysis to the repositioning and restructuring of nucleosomes, but how the ATPase activity of these enzymes drives the motion of DNA across the nucleosome remains unclear. Here, we used single-molecule FRET to monitor the remodeling of mononucleosomes by the yeast SWI/SNF remodeler, RSC. We observed that RSC primarily translocates DNA around the nucleosome without substantial displacement of the H2A-H2B dimer. At the sites where DNA enters and exits the nucleosome, the DNA moves largely along or near its canonical wrapping path. The translocation of DNA occurs in a stepwise manner, and at both sites where DNA enters and exits the nucleosome, the step size distributions exhibit a peak at approximately 1-2 bp. These results suggest that the movement of DNA across the nucleosome is likely coupled directly to DNA translocation by the ATPase at its binding site inside the nucleosome.

Bioorthogonal Chemistry for the Isolation and Study of Newly Synthesized Histones and Their Modifications

The nucleosome is an octamer containing DNA wrapped around one histone H3-H4 tetramer and two histone H2A-H2B dimers. Within the nucleosome, histones are decorated with post-translational modifications. Previous studies indicate that the H3-H4 tetramer is conserved during DNA replication, suggesting that old tetramers serve as a template for the modification of newly synthesized tetramers. Here, we present a method that merges bioorthogonal chemistry with mass spectrometry for the study of modifications on newly synthesized histones in mammalian cells. HeLa S3 cells are dually labeled with the methionine analog azidohomoalanine and heavy (13)C6,(15)N4 isotope labeled arginine. Heavy amino acid labeling marks newly synthesized histones while azidohomoalanine incorporation allows for their isolation using bioorthogonal ligation. Labeled mononucleosomes were covalently linked via a copper catalyzed reaction to a FLAG-GGR-alkyne peptide, immunoprecipitated, and subjected to mass spectrometry for quantitative modification analysis. Mononucleosomes containing new histones were successfully isolated using this approach. Additionally, the development of this method highlights the potential deleterious effects of azidohomoalanine labeling on protein PTMs and cell cycle progression, which should be considered for future studies utilizing bioorthogonal labeling strategies in mammalian cells.