Native Histone Research Articles

Glycoxidation of mammalian whole histone generates highly immunogenic aggregates: Sera of SLE patients contain autoantibodies against aggregates.

Non-enzymatic glycation and oxidation of self-proteins, causing formation and accumulation of advanced glycation end products (AGEs), have been reported in an array of pathologies, including systemic lupus erythematosus (SLE). Such modifications may generate neo-epitopes, break immunological tolerance, and induce antibody response. In this study, we have first analysed the structural modifications of whole histone in the presence of deoxyribose followed by oxidation with hydroxyl radicals. Changes in the secondary and tertiary structure of the whole histone were determined by spectroscopic techniques and biochemical assays. Fluorescence spectroscopy and UPLC-MS showed the generation of AGEs such as carboxymethyl lysine and pentosidine, while DLS and TEM indicated the presence of amorphous AGE-aggregates. Moreover, rabbits immunized with these histone-AGEs exhibited enhanced immunogenicity and ELISA and western immunoblot of IgG antibodies from SLE patients' sera showed a significantly higher specificity towards modified histone-AGEs than the native histone.

MacroH2A1 drives nucleosome dephasing and genome instability in histone humanized yeast

In addition to replicative histones, eukaryotic genomes encode a repertoire of non-replicative variant histones, providing additional layers of structural and epigenetic regulation. Here, we systematically replace individual replicative human histones with non-replicative human variant histones using a histone replacement system in yeast. We show that variants H2A.J, TsH2B, and H3.5 complement their respective replicative counterparts. However, macroH2A1 fails to complement, and its overexpression is toxic in yeast, negatively interacting with yeast's native histones and kinetochore genes. To isolate yeast with macroH2A1 chromatin, we uncouple the effects of its macro and histone fold domains, revealing that both domains suffice to override native nucleosome positioning. Furthermore, both uncoupled constructs of macroH2A1 exhibit lower nucleosome occupancy, decreased short-range chromatin interactions (<20 kb), disrupted centromeric clustering, and increased chromosome instability. Our observations demonstrate that lack of a canonical histone H2A dramatically alters chromatin organization in yeast, leading to genome instability and substantial fitness defects.

Native and tagged CENP-A histones are functionally inequivalent

BackgroundOver the past several decades, the use of biochemical and fluorescent tags has elucidated mechanistic and cytological processes that would otherwise be impossible. The challenging nature of certain nuclear proteins includes low abundancy, poor antibody recognition, and transient dynamics. One approach to get around those issues is the addition of a peptide or larger protein tag to the target protein to improve enrichment, purification, and visualization. However, many of these studies were done under the assumption that tagged proteins can fully recapitulate native protein function.ResultsWe report that when C-terminally TAP-tagged CENP-A histone variant is introduced, it undergoes altered kinetochore protein binding, differs in post-translational modifications (PTMs), utilizes histone chaperones that differ from that of native CENP-A, and can partially displace native CENP-A in human cells. Additionally, these tagged CENP-A-containing nucleosomes have reduced centromeric incorporation at early G1 phase and poorly associates with linker histone H1.5 compared to native CENP-A nucleosomes.ConclusionsThese data suggest expressing tagged versions of histone variant CENP-A may result in unexpected utilization of non-native pathways, thereby altering the biological function of the histone variant.

Micromechanical Study of Hyperacetylated Nucleosomes Using Single Molecule Transverse Magnetic Tweezers.

Nucleosomes are stable complexes of DNA and histone proteins that are essential for the proper functioning of the genome. These structures must be unwrapped and disassembled for processes such as gene expression, replication, and repair. Histone post-translational modifications (PTMs) are known to play a significant role in regulating the structural changes of nucleosomes. However, the underlying mechanisms by which these modifications function remain unclear. In this study, we report the results of single molecule micromanipulation experiments on DNA-protein complexes composed of hyperacetylated histone proteins using transverse magnetic tweezers. The experiments were conducted by pre-extending λ-DNA with a force less than 4 pN before introducing hyperacetylated histones into the sample chamber. The DNA shortened as the histones formed complexes with it and the nucleosome arrays were then exposed to increasing tension, resulting in quantized changes in the DNA's extension with step sizes of (integral multiples of) ~50 nm. We also compared results of experiments using PTM histones and native histones with data collected for both types of histones for the same force ranges (2-80 pN) and loading rates. Our data show that hyperacetylated nucleosomes require an unbinding force of around ~2.5 pN, which is similar to that required for native histones. Moreover, we identified clear differences between the step-size distributions of native and hyperacetylated histones and found that in contrast to tethers reconstituted with native histones, the majority of nucleosomes in tethers compacted with hyperacetylated histones underwent disassembly at forces significantly lower than 6 pN.

Synthetic maize centromeres transmit chromosomes across generations.

Centromeres are long, often repetitive regions of genomes that bind kinetochore proteins and ensure normal chromosome segregation. Engineering centromeres that function in vivo has proven to be difficult. Here we describe a tethering approach that activates functional maize centromeres at synthetic sequence arrays. A LexA-CENH3 fusion protein was used to recruit native Centromeric Histone H3 (CENH3) to long arrays of LexO repeats on a chromosome arm. Newly recruited CENH3 was sufficient to organize functional kinetochores that caused chromosome breakage, releasing chromosome fragments that were passed through meiosis and into progeny. Several fragments formed independent neochromosomes with centromeres localized over the LexO repeat arrays. The new centromeres were self-sustaining and transmitted neochromosomes to subsequent generations in the absence of the LexA-CENH3 activator. Our results demonstrate the feasibility of using synthetic centromeres for karyotype engineering applications.

Autoantibodies against citrullinated and native proteins and prediction of rheumatoid arthritis-associated interstitial lung disease: A nested case-control study.

To identify fine specificity anti-citrullinated protein antibodies (ACPA) associated with incident rheumatoid arthritis-associated interstitial lung disease (RA-ILD). This nested case-control study within the Brigham RA Sequential Study matched incident RA-ILD cases to RA-noILD controls on time of blood collection, age, sex, RA duration, and rheumatoid factor status. A multiplex assay measured ACPA and anti-native protein antibodies from stored serum prior to RA-ILD onset. Logistic regression models calculated odds ratios (OR) with 95% confidence intervals (CI) for RA-ILD, adjusting for prospectively-collected covariates. We estimated optimism-corrected area under the curves (AUC) using internal validation. Model coefficients generated a risk score for RA-ILD. We analyzed 84 incident RA-ILD cases (mean age 67 years, 77% female, 90% White) and 233 RA-noILD controls (mean age 66 years, 80% female, 94% White). We identified six fine specificity antibodies that were associated with RA-ILD. The antibody isotypes and targeted proteins were: IgA2 to citrullinated histone 4 (OR 0.08 per log-transformed unit, 95% CI 0.03-0.22), IgA2 to citrullinated histone 2A (OR 4.03, 95% CI 2.03-8.00), IgG to cyclic citrullinated filaggrin (OR 3.47, 95% CI 1.71-7.01), IgA2 to native cyclic histone 2A (OR 5.52, 95% CI 2.38-12.78), IgA2 to native histone 2A (OR 4.60, 95% CI 2.18-9.74), and IgG to native cyclic filaggrin (OR 2.53, 95% CI 1.47-4.34). These six antibodies predicted RA-ILD risk better than all clinical factors combined (optimism-corrected AUC=0·84 versus 0·73). We developed a risk score for RA-ILD combining these antibodies with the clinical factors (smoking, disease activity, glucocorticoid use, obesity). At 50% predicted RA-ILD probability, the risk scores both without (score=2·6) and with (score=5·9) biomarkers achieved specificity ≥93% for RA-ILD. Specific ACPA and anti-native protein antibodies improve RA-ILD prediction. These findings implicate synovial protein antibodies in the pathogenesis of RA-ILD and suggest clinical utility in predicting RA-ILD once validated in external studies. National Institutes of Health.

Histone chaperone-mediated co-expression assembly of tetrasomes and nucleosomes.

The nucleosome, a basic unit of chromatin found in all eukaryotes, is thought to be assembled through the orchestrated activity of several histone chaperones and chromatin assembly factors in a stepwise manner, proceeding from tetrasome assembly, to H2A/H2B deposition, and finally to formation of the mature nucleosome. In this study, we demonstrate chaperone‐mediated assembly of both tetrasomes and nucleosomes on the well‐defined Widom 601 positioning sequence using a co‐expression/reconstitution wheat germ cell‐free system. The purified tetrasomes and nucleosomes were positioned around the center of a given sequence. The heights and diameters were measured by atomic force microscopy. Together with the reported unmodified native histones produced by the wheat germ cell‐free platform, our method is expected to be useful for downstream applications in the field of chromatin research.

Genetically Encoded Benzoyllysines Serve as Versatile Probes for Interrogating Histone Benzoylation and Interactions in Living Cells.

Histone posttranslational modifications (PTMs) are vital epigenetic regulators in many fundamental cell signaling pathways and diverse biological processes. Histone lysine benzoylation is a recently identified epigenetic mark associated with active transcription; however, it remains to be explored. Herein, we first report the genetic encoding of benzoyllysine and fluorinated benzoyllysines into full-length histone proteins in a site-specific manner in live cells, based on our rationally designed synthetase and fine-integrated fluorine element into benzoyllysines. The incorporated unnatural amino acids integrating unique features were demonstrated as versatile probes for investigating histone benzoylation under biological environments, conferring multiplex signals such as 19F NMR spectra with chemical clarity and fluorescence signals for benzoylation. Moreover, the site specifically incorporated lysine benzoylation within native full-length histone proteins revealed distinct dynamics of debenzoylation in the presence of debenzoylase sirtuin 2 (SIRT2). Our developed strategy for genetic encoding of benzoyllysines offers a general and novel approach to gain insights into interactions of site-specific histone benzoylation modifications with interactomes and molecular mechanisms in physiological settings, which could not be accessible with fragment histone peptides. This versatile chemical tool enables a direct and new avenue to explore benzoylation, interactions, and histone epigenetics, which will provide broad utilities in chemical biology, protein science, and basic biology research.

Neutrophil stimulation with citrullinated histone H4 slows down calcium influx and reduces NET formation compared with native histone H4.

Peptidylarginine deiminase 4 (PAD4) catalyzes posttranslational modification of many target proteins through converting protein arginine or mono-methylarginine to citrulline. Neutrophil extracellular trap (NET) formation is the most dramatic manifestation of PAD4-mediated hypercitrullination reaction in neutrophils, which is characterized by the release of nuclear chromatin to form a chromatin network in the extracellular space. Histones H4, one of the major protein components of chromatin, is released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury and can also be released during the process of NET formation, along with its citrullinated form. The present study showed that histone H4 can induce NET formation in a calcium and PAD4 dependent manner. Histone H4 caused permeabilization of the neutrophil membrane and sustained rise in intracellular calcium that is necessary for activation of PAD4. In comparison, citrullinated histone H4 induced less calcium influx compared with its native form, leading to reduced NET formation. These studies suggest that citrullinated histone H4 could serve as a brake in the pathology of NETs, slowing down the vicious circle between histone H4 and NETs.

NMR Analyses of Acetylated H2A.Z Isoforms Identify Differential Binding Interactions with the Bromodomain of the NURF Nucleosome Remodeling Complex

Gene specific recruitment of bromodomain-containing proteins to chromatin is affected by post-translational acetylation of lysine on histones. Whereas interactions of the bromodomain with acetylation patterns of native histones (H2A, H2B, H3, and H4) have been well characterized, the motif for recognition for histone variants H2A.Z I and H2A.Z II by bromodomains has yet to be fully investigated. Elucidating these molecular mechanisms is crucial for understanding transcriptional regulation in cellular processes involved in both development and disease. Here, we have used protein-observed fluorine NMR to fully characterize the affinities of H2A.Z I and II acetylation patterns for BPTF's bromodomain and found the diacetylated mark of lysine 7 and 13 on H2A.Z II to have the strongest interaction with K7ac preferentially engaging the binding site. We further examined the selectivity of H2A.Z histones against a variety of bromodomains, revealing that the bromodomain of CECR2 binds with the highest affinity and specificity for acetylated H2A.Z I over isoform II. These results support a possible role for different H2A.Z transcriptional activation mechanisms that involve recruitment of chromatin remodeling complexes.

Structural Insights into the Mammalian Late-Stage Initiation Complexes

SummaryIn higher eukaryotes, the mRNA sequence in the direct vicinity of the start codon, called the Kozak sequence (CRCCaugG, where R is a purine), is known to influence the rate of the initiation process. However, the molecular basis underlying its role remains poorly understood. Here, we present the cryoelectron microscopy (cryo-EM) structures of mammalian late-stage 48S initiation complexes (LS48S ICs) in the presence of two different native mRNA sequences, β-globin and histone 4, at overall resolution of 3 and 3.5 Å, respectively. Our high-resolution structures unravel key interactions from the mRNA to eukaryotic initiation factors (eIFs): 1A, 2, 3, 18S rRNA, and several 40S ribosomal proteins. In addition, we are able to study the structural role of ABCE1 in the formation of native 48S ICs. Our results reveal a comprehensive map of ribosome/eIF-mRNA and ribosome/eIF-tRNA interactions and suggest the impact of mRNA sequence on the structure of the LS48S IC.

In Vivo Histone Labeling Using Ultrafast trans-Splicing Inteins.

The development of expressed protein ligation(EPL) widened the scope of questions that could be addressed by mechanistic biochemistry. Protein trans-splicing (PTS) relies on the same basic chemical principles, but utilizes split inteins to tracelessly ligate distinct peptide or polypeptide fragments together with native peptide bonds. Here we present a method to adapt PTS methodologies for their use in live cells, in order to deliver synthetic or native histone modifications. As an example, we provide a protocol to incorporate a small molecule fluorophore into chromatinized histones. The protocol should be easily adaptable to incorporate other modifications to chromatin in vivo.

Differential ACPA Binding to Nuclear Antigens Reveals a PAD-Independent Pathway and a Distinct Subset of Acetylation Cross-Reactive Autoantibodies in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) associated anti-citrullinated protein autoantibodies (ACPA) target a wide range of modified proteins. Citrullination occurs during physiological processes such as apoptosis, yet little is known about the interaction of ACPA with nuclear antigens or apoptotic cells. Since uncleared apoptotic cells and neutrophil extracellular trap (NET) products have been postulated to be central sources of autoantigen and immunostimulation in autoimmune disease, we sought to characterize the anti-nuclear and anti-neutrophil reactivities of ACPA. Serology showed that a subset of anti-CCP2 seropositive RA patients had high reactivity to full-length citrullinated histones. In contrast, seronegative RA patients displayed elevated IgG reactivity to native histone compared to controls, but no citrulline-specific reactivity. Screening of 10 single B-cell derived monoclonal ACPA from RA patients revealed that four ACPA exhibited strong binding to apoptotic cells and three of these had anti-nuclear (ANA) autoantibody reactivity. Modified histones were confirmed to be the primary targets of this anti-nuclear ACPA subset following immunoprecipitation from apoptotic cell lysates. Monoclonal ACPA were also screened for reactivities against stimulated murine and human neutrophils, and all the nuclear-reactive monoclonal ACPA bound to NETs. Intriguingly, one ACPA mAb displayed a contrasting cytoplasmic perinuclear neutrophil binding and may represent a different NET-reactive ACPA subset. Notably, studies of CRISPR-Cas9 PAD4 KO cells and cells from PAD KO mice showed that the cytoplasmic NET-binding was fully dependent on PAD4, whilst nuclear- and histone-mediated NET reactivity was largely PAD-independent. Our further analysis revealed that the nuclear binding could be explained by consensus-motif driven ACPA cross-reactivity to acetylated histones. Specific acetylated histone peptides targeted by the monoclonal antibodies were identified and the anti-modified protein autoantibody (AMPA) profile of the ACPA was found to correlate with the functional activity of the antibodies. In conclusion, when investigating monoclonal ACPA, we could group ACPA into distinct subsets based on their nuclear binding-patterns and acetylation-mediated binding to apoptotic cells, neutrophils, and NETs. Differential anti-modified protein reactivities of RA-autoantibody subsets could have an important functional impact and provide insights in RA pathogenesis.

Nucleotide Excision Repair and Impact of Site-Specific 5',8-Cyclopurine and Bulky DNA Lesions on the Physical Properties of Nucleosomes.

The nonbulky 5',8-cyclopurine DNA lesions (cP) and the bulky, benzo[ a]pyrene diol epoxide-derived stereoisomeric cis- and trans- N2-guanine adducts (BPDE-dG) are good substrates of the human nucleotide excision repair (NER) mechanism. These DNA lesions were embedded at the In or Out rotational settings near the dyad axis in nucleosome core particles reconstituted either with native histones extracted from HeLa cells (HeLa-NCP) or with recombinant histones (Rec-NCP). The cP lesions are completely resistant to NER in human HeLa cell extracts. The BPDE-dG adducts are also NER-resistant in Rec-NCPs but are good substrates of NER in HeLa-NCPs. The four BPDE-dG adduct samples are excised with different efficiencies in free DNA, but in HeLa-NCPs, the efficiencies are reduced by a common factor of 2.2 ± 0.2 relative to the NER efficiencies in free DNA. The NER response of the BPDE-dG adducts in HeLa-NCPs is not directly correlated with the observed differences in the thermodynamic destabilization of HeLa-NCPs, the Förster resonance energy transfer values, or hydroxyl radical footprint patterns and is weakly dependent on the rotational settings. These and other observations suggest that NER is initiated by the binding of the DNA damage-sensing NER factor XPC-RAD23B to a transiently opened BPDE-modified DNA sequence that corresponds to the known footprint of XPC-DNA-RAD23B complexes (≥30 bp). These observations are consistent with the hypothesis that post-translational modifications and the dimensions and properties of the DNA lesions are the major factors that have an impact on the dynamics and initiation of NER in nucleosomes.

Histone-Mimetic Gold Nanoparticles as Versatile Scaffolds for Gene Transfer and Chromatin Analysis.

Histone-inspired polymer assemblies (polyplexes) can regulate gene expression and subcellular transport in plasmids by harnessing the cellular machinery normally used for histone proteins. When grafted to polyplexes, histone tails promote nuclear accumulation, trigger plasmid DNA (pDNA) release, and enhance transcription. Herein, we developed multifunctional gold nanoparticles (AuNPs) decorated by histone motifs as histone-inspired scaffolds with improved pDNA binding, easy bioimaging, and increased potential for gene delivery and chromatin analysis applications. We hypothesized that polycationic AuNPs coupled to histone motifs would mimic the native presentation of these sequences on the histone octamer and thereby create structures with the capacity to both engage native histone effectors and condense pDNA into nucleosome-inspired nanostructures. AuNPs bearing ∼2 nm cores were prepared based on the well-established Brust-Schiffrin two-phase method involving tetrachloroaurate reduction in the presence of 1-pentanethiol. Solid phase peptide synthesis was employed to generate thiolated polycationic ligands and histone tail motifs, and the AuNPs and peptide ligands were combined in a two-step Murray place exchange reaction at various ratios to produce a collection of polycationic AuNPs modified with varying amounts of histone tails. Electron microscopy and thermal analyses demonstrated that these modified AuNPs exhibited tunable biochemical and biophysical properties that closely mimicked the properties of native histones. The histone-mimetic nanoscaffolds efficiently and sequence-specifically engaged histone effectors responsible for activating transcription. In addition, the nanoscaffolds condensed pDNA into complexes with high stability in the presence of physiological concentrations of heparin, a common extracellular polyanion. These combined properties of histone engagement and high stability led to a ∼6-fold enhancement in transfection efficiency as compared with typical polymeric transfection reagents, with the increased transfection efficiency correlated to the presence and amount of histone tails displayed on the surface of the nanoscaffolds. These findings demonstrate the utility of employing a biomimetic materials design approach to develop more effective and stable delivery vehicles for gene transfer and chromatin analysis applications.

Relevance of arginine residues in Cu(II)-induced DNA breakage and Proteinase K resistance of H1 histones

This work analyzes the involvement of arginines in copper/H2O2-induced DNA breakage. Copper is a highly redox active metal which has been demonstrated to form compounds with arginines. For this aim we used mixtures of pGEM3 DNA plasmid and two types of H1 histones which differ only in their arginine content. The sperm H1 histone from the annelid worm Chaetopterus variopedatus (arginine content 12.6 mol% K/R ratio 2) and the somatic H1 histone from calf thymus (arginine content 1.8 mol% and K/R ratio 15). Copper/H2O2-induced DNA breakage was observed only in presence of sperm H1 histones, but it was more relevant for the native molecule than for the deguanidinated derivative (K/R ratio 14), in which 80% of arginine residues were converted to ornithine. Further, copper induced proteinase K resistance and increase of DNA binding affinity on native sperm H1 histones. These observations are consistent with a copper induced reorganization of the side-chains of arginine residues. Copper, instead, did not affect DNA binding affinity of somatic and deguanidinated H1 histones, which show similar K/R ratio and DNA binding mode. These results indicate that arginine residues could affect these H1 histones properties and provide new insights into copper toxicity mechanisms.

Imaging of native transcription factors and histone phosphorylation at high resolution in live cells.

Fluorescent labeling of endogenous proteins for live-cell imaging without exogenous expression of tagged proteins or genetic manipulations has not been routinely possible. We describe a simple versatile antibody-based imaging approach (VANIMA) for the precise localization and tracking of endogenous nuclear factors. Our protocol can be implemented in every laboratory allowing the efficient and nonharmful delivery of organic dye-conjugated antibodies, or antibody fragments, into different metazoan cell types. Live-cell imaging permits following the labeled probes bound to their endogenous targets. By using conventional and super-resolution imaging we show dynamic changes in the distribution of several nuclear transcription factors (i.e., RNA polymerase II or TAF10), and specific phosphorylated histones (γH2AX), upon distinct biological stimuli at the nanometer scale. Hence, considering the large panel of available antibodies and the simplicity of their implementation, VANIMA can be used to uncover novel biological information based on the dynamic behavior of transcription factors or posttranslational modifications in the nucleus of single live cells.

Purification of Histone Variant-Interacting Chaperone Complexes.

Identification of the interaction partners of a protein is one of useful straightforward methods to gain insight into the molecular functions of the protein in cells. The pre-deposited forms of histones are associated with the specific histone chaperones to assemble into chromatin. Here, I describe an affinity purification method using the FLAG/HA double epitope-tagging technique and its application to purify particular histone variant-interacting chaperone complexes from soluble fraction to study dynamic chromatin functions. The purification is performed under low salt condition to obtain native histone variant complexes, and it would be useful to identify the specific chaperone proteins involved in the specific chromatin functions via histone variants.

A simple and versatile system for the ATP-dependent assembly of chromatin

Chromatin is the natural form of DNA in the eukaryotic nucleus and is the substrate for diverse biological phenomena. The functional analysis of these processes ideally would be carried out with nucleosomal templates that are assembled with customized core histones, DNA sequences, and chromosomal proteins. Here we report a simple, reliable, and versatile method for the ATP-dependent assembly of evenly spaced nucleosome arrays. This minimal chromatin assembly system comprises the Drosophila nucleoplasmin-like protein (dNLP) histone chaperone, the imitation switch (ISWI) ATP-driven motor protein, core histones, template DNA, and ATP. The dNLP and ISWI components were synthesized in bacteria, and each protein could be purified in a single step by affinity chromatography. We show that the dNLP-ISWI system can be used with different DNA sequences, linear or circular DNA, bulk genomic DNA, recombinant or native Drosophila core histones, native human histones, the linker histone H1, the non-histone chromosomal protein HMGN2, and the core histone variants H3.3 and H2A.V. The dNLP-ISWI system should be accessible to a wide range of researchers and enable the assembly of customized chromatin with specifically desired DNA sequences, core histones, and other chromosomal proteins.

Mass Spectrometry-Based Methodology for Identification of Native Histone Variant Modifications From Mammalian Tissues and Solid Tumors.

Histone posttranslational modifications (PTMs) are key epigenetic marks involved in gene silencing or activation. Histone modifications impact chromatin organization and transcriptional processes through the changes in charge density between histones and DNA. They also serve as recognition and binding sites for specific binding proteins. Histone tails and globular cores contain many basic amino acid residues, which are subject to various dynamic modifications, making the modification repertoire extremely diverse. Consequently, determination of histone PTM identity and quantity has been a challenging task. In recent years, mass spectrometry-based methods have proven useful in histone PTM characterization. This chapter provides a brief overview of these methods and describes the approach to analyze the PTMs of the histone variant CENP-A, essential for the cell cycle progression, when present in minute amounts from tumor and mammalian tissues. Because this method does not rely on antibody-based immunopurification, we anticipate that these tools could be readily adaptable to the investigation to other histone variants in a range of mammalian tissues and solid tumors.