Histone H2B Research Articles

MLKL promotes extracellular trap releasing in Vibrio splendidus AJ01-challenged coelomocytes of sea cucumber Apostichopus japonicus

Extracellular traps (ETs) is a novel host-defense mechanism for the immobilization and killing of invading microorganisms, of which pseudokinase mixed lineage kinase-like protein (MLKL) is necessary for ET formation. Here, we aimed to investigate the effects of various stimulations (including classic ET inducers and pathogen) on ET formation in the coelomocytes of invertebrate sea cucumber Apostichopus japonicus. Our results show that stimulation of sea cucumber coelomocytes with phorbol 12-myristate 13-acetate (PMA), zymosan, Vibrio splendidus AJ01, and AJ01 flagella all led to the formation of typical ET fibers, which can be further digested by DNase I. This finding suggests that DNA is an essential component of coelomocyte ETs. Meanwhile, the signals of histones H2A and H2B transferred to extracellular spaces and colocalized with AJ01, which indicates that histones are also crucial components of coelomocyte ETs. Scanning electron microscopy observation and antimicrobial activity analysis successfully revealed that AJ01 was trapped and killed by ET fibers, and this finding implies the antimicrobial role of sea cucumber coelomocyte ETs. More importantly, silencing and inhibition of AjMLKL after AJ01 infection not only considerably depressed ET generation but also increased the survival rate of AJ01. All our results indicate that AjMLKL mediates sea cucumber ET formation and plays critical roles during pathogen infection.

9366 The Rnf20 Histone Modifier Is A Crucial Mediator Of Pancreatic Beta Cell Identity And Function

Abstract Disclosure: T.H. Pierre: None. Y. Liu: None. M.M. Bethea: None. K. Coutinho: None. C. Hunter: None. Diabetes is characterized by the loss of pancreatic β-cell mass. As diabetes incidence continues to elevate, it is critical that we develop a better understanding of the factors that define functional β-cells in order to improve current therapeutics. One approach is through the study of transcription factors (TFs), which are essential for β-cell development and function. Prior work from our lab and others has examined Islet-1 (Isl1) a LIM-homeodomain TF that is required for the maturation of the endocrine pancreas and adult β-cell function. We have also extensively characterized several Isl1-interacting co-regulators (e.g., Ldb1 and SSBP3) that facilitate its roles in maintaining glucose homeostasis. Most recently, we have identified that Isl1 interacts with the ubiquitin ligases Ring Finger 20 (Rnf20) and Rnf40, whose histone H2B monoubiquitation (H2Bub1) activity support the gene regulatory functions of Isl1. With in vitro experiments, we demonstrated that Rnf20 and Rnf40 are important for glucose stimulated insulin secretion (GSIS) and mediate the expression of β-cell identity genes. Based on these findings, we hypothesize that Isl1-Rnf complexes modify histones to regulate β-cell gene expression and function. To address this hypothesis in vivo, we developed an adult inducible β-cell knockout of Rnf20 (Rnf20Δβ-cell). Rnf20Δβ-cell mice display fasting hyperglycemia and severe glucose intolerance that is driven by impaired GSIS, reduced insulin content, and the dysregulation of critical β-cell genes including Ins1, Ucn3, and Slc2a2. Additionally, loss of Rnf20 perturbs insulin processing and overall β-cell maturation as observed by measurement of proinsulin:insulin ratios, C-peptide levels, and whole islet RNA-seq. Phenotypes exhibited by Rnf20Δβ-cell mice are similar to those observed in Isl1 β-cell knockout mice, and comparative transcriptomics revealed an overlap of 570 genes involved in metabolism, zinc homeostasis, and islet proliferation including Slc30a8 and Matn2, both of which we found are occupied by Isl1 and Rnf20. Collectively, our findings demonstrate the importance of the Rnf20 histone modifier in regulating β-cell function and expand our understanding of the Isl1 regulatory network. Presentation: 6/2/2024

Immunohistochemical expression of netosis markers (MPO, H2A, H2B) in placenta of RPL women in Basrah Province

Background. Recurrent Pregnancy Loss (RPL) is a multifactorial disorder significantly impacting women’s health globally. Emerging research has implicated Neutrophil Extracellular Traps (NETs) in the pathophysi­ology of RPL, suggesting an immunological underpinning to miscarriage occurrences. Aim. The study aim to evaluate the role and expression of NETosis markers (MPO, histones H2A, H2B) in women with RPL in comparison to pregnant women. Methods. This prospective cohort study, conducted from January 2022 to July 2023, included 30 participants (20 RPL patients and 10 controls) from Al Basrah Maternity and Child Hospital. Immunohistochemical technique were employed to detect Myeloperoxidase and histones in placental samples. Statistical analysis was performed using SPSS, with significance set at p<90.05. Results. Immunohistochemical analysis of placental tissues indicated significant overexpression of Myelo­peroxidase (MPO), and histones H2A and H2B, in RPL patients compared to control, emphasizing the potential involvement of NETs in placental pathology associated with miscarriage. The expression levels of MPO were positive in 80% of RPL patients but undetected in control (p = 0.0001). Histone expressions also differed significantly, with H2A detected in 55% of RPL patients compared to none in control (p = 0.001) and H2B in 70% versus 30% (both p <0.001).

RNF20-mediated transcriptional pausing and VEGFA splicing orchestrate vessel growth

Signal-responsive gene expression is essential for vascular development, yet the mechanisms integrating signaling inputs with transcriptional activities are largely unknown. Here we show that RNF20, the primary E3 ubiquitin ligase for histone H2B, plays a multifaceted role in sprouting angiogenesis. RNF20 mediates RNA polymerase (Pol II) promoter-proximal pausing at genes highly paused in endothelial cells, involved in VEGFA signaling, stress response, cell cycle control and mRNA splicing. It also orchestrates large-scale mRNA processing events that alter the bioavailability and function of critical pro-angiogenic factors, such as VEGFA. Mechanistically, RNF20 restricts ERG-dependent Pol II pause release at highly paused genes while binding to Notch1 to promote H2B monoubiquitination at Notch target genes and Notch-dependent gene expression. This balance is crucial, as loss of Rnf20 leads to uncontrolled tip cell specification. Our findings highlight the pivotal role of RNF20 in regulating VEGF–Notch signaling circuits during vessel growth, underscoring its potential for therapeutic modulation of angiogenesis.

Roles of Histone H2B, H3 and H4 Variants in Cancer Development and Prognosis.

Histone variants are the paralogs of core histones (H2A, H2B, H3 and H4). They are stably expressed throughout the cell cycle in a replication-independent fashion and are capable of replacing canonical counterparts under different fundamental biological processes. Variants have been shown to take part in multiple processes, including DNA damage repair, transcriptional regulation and X chromosome inactivation, with some of them even specializing in lineage-specific roles like spermatogenesis. Several reports have recently identified some unprecedented variants from different histone families and exploited their prognostic value in distinct types of cancer. Among the four classes of canonical histones, the H2A family has the greatest number of variants known to date, followed by H2B, H3 and H4. In our prior review, we focused on summarizing all 19 mammalian histone H2A variants. Here in this review, we aim to complete the full summary of the roles of mammalian histone variants from the remaining histone H2B, H3, and H4 families, along with an overview of their roles in cancer biology and their prognostic value in a clinical context.

Molecular characterization of histone gene in golden pompano (Trachinotus ovatus) and antimicrobial activity of its derived peptides

In addition to controlling gene expression, mediating DNA folding into chromatin, and responding to immunological stimuli, histones are also thought to have antimicrobial effects. This study identified the molecular characteristics of core Histone MacroH2A2 (TOMacroH2A2) and Histone H2B 1/2 (TOH2B) from Trachinotus ovatus, and the antimicrobial potential of their derived peptides (To.mh2a and To. h2b). The open reading frames (ORFs) of TOMacroH2A2 and TOH2B from T. ovatus were 1010 bp and 375 bp, encoding polypeptides of 369 and 124 amino acids, respectively. The TOMacroH2A2 included an H2A domain and an A1pp domain, while TOH2B included an H2B domain. The amino acid sequences of TOMacroH2A2 and TOH2B demonstrated high homology with other teleost's sequences of histone macroh2a2 and histone h2b, with homologies exceeding 90 %. Expression analysis showed high expression of TOMacroH2A2 in brain, stomach, heart, and skin tissues and TOH2B in gill, brain, and skin tissues. In addition, the histone-derived peptides To. mh2a and To. h2b, synthesized based on two histone sequences from T. ovatus, exhibited typical physical characteristics of antimicrobial peptides, including positive charges, amphipathicity, hydrophobicity, and rich α-helix structure. Crucially, the vitro antibacterial results demonstrated that To. mh2a and To. h2b can inhibit the growth of various aquatic pathogens including Streptococcus agalactiae, Staphylococcus aureus, Bacillus subtilis, Acinetobacter baumannii, Aeromonas hydrophila, and Escherichia coli to varying degrees. Specifically, To. mh2a and To. h2b were capable of disrupting the cell surface structures of S. aureus and penetrating the cell membrane, leading to the leakage of cellular contents, thereby exerting their antibacterial effects. Furthermore, gel electrophoresis migration assays showed that To. mh2a and To. h2b participated in antimicrobial activity by binding to bacterial genomic DNA and reducing the migration rate of gDNA in a dose-dependent manner. The minimum effective concentration for binding to DNA was approximately 50 μM. In conclusion, our study suggested that To. mh2a and To. h2b can act as antimicrobial peptides, providing a potential strategy for controlling bacterial diseases in T. ovatus.

Genome-wide analysis of the biophysical properties of chromatin and nuclear proteins in living cells with Hi-D.

To understand the dynamic nature of the genome, the localization and rearrangement of DNA and DNA-binding proteins must be analyzed across the entire nucleus of single living cells. Recently, we developed a computational light microscopy technique, called high-resolution diffusion (Hi-D) mapping, which can accurately detect, classify and map diffusion dynamics and biophysical parameters such as the diffusion constant, the anomalous exponent, drift velocity and model physical diffusion from the data at a high spatial resolution across the genome in living cells. Hi-D combines dense optical flow to detect and track local chromatin and nuclear protein motion genome-wide and Bayesian inference to characterize this local movement at nanoscale resolution. Here we present the Python implementation of Hi-D, with an option for parallelizing the calculations to run on multicore central processing units (CPUs). The functionality of Hi-D is presented to the users via user-friendly documented Python notebooks. Hi-D reduces the analysis time to less than 1 h using a multicore CPU with a single compute node. We also present different applications of Hi-D for live-imaging of DNA, histone H2B and RNA polymerase II sequences acquired with spinning disk confocal and super-resolution structured illumination microscopy.

Revealing cis- and trans-regulatory elements underlying nuclear distribution and function of the Arabidopsis histone H2B.8 variant

The H2B.8 variant has been diverged from other variants by its extended N-terminal region that possesses a conserved domain. We generated transgenic Arabidopsis plants expressing H2B.9 (class I), H2B.5 (class II) and H2B.8 (class III) fused to GFP under the 35 S promoter and studied their nuclear distribution and function. H2B.8-GFP showed peculiar nuclear localization at chromocenters in all cell types examined, while H2B.5-GFP and H2B.9-GFP displayed various patterns often dependent on cell types. H2B variants faithfully assembled onto nucleosomes showing no effect on nuclear organization; H2B.8-GFP appeared as three distinct isoforms in which one isoform appeared to be SUMOylated. Interestingly, transient expression in protoplasts revealed H2B.8 nuclear localization distinct from transgenic plants as it was restricted to the nuclear periphery generating a distinctive ring-like appearance accompanied by nuclear size reduction. This unique appearance was abolished by deletion of the N-terminal conserved domain or when H2B.8-GFP is transiently expressed in ddm1 protoplasts. GFP-TRAP-coupled proteome analysis uncovered H2B.8-partner proteins including H2A.W.12, which characterizes heterochromatin. Thus, our data highlight H2B.8 as a unique variant evolved in angiosperms to control chromatin compaction/aggregation and uncover cis- and trans-regulatory elements underlying its nuclear distribution and function.

Ubiquitinated histone H2B as gatekeeper of the nucleosome acidic patch.

Monoubiquitination of histones H2B-K120 (H2BK120ub) and H2A-K119 (H2AK119ub) play opposing roles in regulating transcription and chromatin compaction. H2BK120ub is a hallmark of actively transcribed euchromatin, while H2AK119ub is highly enriched in transcriptionally repressed heterochromatin. Whereas H2BK120ub is known to stimulate the binding or activity of various chromatin-modifying enzymes, this post-translational modification (PTM) also interferes with the binding of several proteins to the nucleosome H2A/H2B acidic patch via an unknown mechanism. Here, we report cryoEM structures of an H2BK120ub nucleosome showing that ubiquitin adopts discrete positions that occlude the acidic patch. Molecular dynamics simulations show that ubiquitin remains stably positioned over this nucleosome region. By contrast, our cryoEM structures of H2AK119ub nucleosomes show ubiquitin adopting discrete positions that minimally occlude the acidic patch. Consistent with these observations, H2BK120ub, but not H2AK119ub, abrogates nucleosome interactions with acidic patch-binding proteins RCC1 and LANA, and single-domain antibodies specific to this region. Our results suggest a mechanism by which H2BK120ub serves as a gatekeeper to the acidic patch and point to distinct roles for histone H2AK119 and H2BK120 ubiquitination in regulating protein binding to nucleosomes.

Daxx mediated histone H3.3 deposition on HSV-1 DNA restricts genome decompaction and the progression of immediate-early transcription.

Herpesviruses are ubiquitous pathogens that cause a wide range of disease. Upon nuclear entry, their genomes associate with histones and chromatin modifying enzymes that regulate the progression of viral transcription and outcome of infection. While the composition and modification of viral chromatin has been extensively studied on bulk populations of infected cells by chromatin immunoprecipitation, this key regulatory process remains poorly defined at single-genome resolution. Here we use high-resolution quantitative imaging to investigate the spatial proximity of canonical and variant histones at individual Herpes Simplex Virus 1 (HSV-1) genomes within the first 90 minutes of infection. We identify significant population heterogeneity in the stable enrichment and spatial proximity of canonical histones (H2A, H2B, H3.1) at viral DNA (vDNA) relative to established promyelocytic leukaemia nuclear body (PML-NB) host factors that are actively recruited to viral genomes upon nuclear entry. We show the replication-independent histone H3.3/H4 chaperone Daxx to cooperate with PML to mediate the enrichment and spatial localization of variant histone H3.3 at vDNA that limits the rate of HSV-1 genome decompaction to restrict the progress of immediate-early (IE) transcription. This host response is counteracted by the viral ubiquitin ligase ICP0, which degrades PML to disperse Daxx and variant histone H3.3 from vDNA to stimulate the progression of viral genome expansion, IE transcription, and onset of HSV-1 replication. Our data support a model of intermediate and sequential histone assembly initiated by Daxx that limits the rate of HSV-1 genome decompaction independently of the stable enrichment of histones H2A and H2B at vDNA required to facilitate canonical nucleosome assembly. We identify HSV-1 genome decompaction upon nuclear infection to play a key role in the initiation and functional outcome of HSV-1 lytic infection, findings pertinent to the transcriptional regulation of many nuclear replicating herpesvirus pathogens.

Microsporidian EnP1 alters host cell H2B monoubiquitination and prevents ferroptosis facilitating microsporidia survival

Microsporidia are intracellular eukaryotic pathogens that pose a substantial threat to immunocompromised hosts. The way these pathogens manipulate host cells during infection remains poorly understood. Using a proximity biotinylation strategy we established that microsporidian EnP1 is a nucleus-targeted effector that modifies the host cell environment. EnP1's translocation to the host nucleus is meditated by nuclear localization signals (NLSs). In the nucleus, EnP1 interacts with host histone H2B. This interaction disrupts H2B monoubiquitination (H2Bub), subsequently impacting p53 expression. Crucially, this inhibition of p53 weakens its control over the downstream target gene SLC7A11, enhancing the host cell's resilience against ferroptosis during microsporidian infection. This favorable condition promotes the proliferation of microsporidia within the host cell. These findings shed light on the molecular mechanisms by which microsporidia modify their host cells to facilitate their survival.

Light-Dependent Circadian Rhythm Governs O-GlcNAc Cycling to Influence Cognitive Function in Adult Zebrafish.

This study explores the 24-h rhythmic cycle of protein O-GlcNAcylation within the brain and highlights its crucial role in regulating the circadian cycle and neuronal function based on zebrafish as an animal model. In our experiments, disruption of the circadian rhythm, achieved through inversion of the light-dark cycle or daytime melatonin treatment, not only impaired the rhythmic changes of O-GlcNAcylation along with altering expression patterns of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in zebrafish brain but also significantly impeded learning and memory function. In particular, circadian disruption affected rhythmic expression of protein O-GlcNAcylation and OGT in the nuclear fraction. Notably, the circadian cycle induces rhythmic alterations in O-GlcNAcylation of H2B histone protein that correspond to changes in H3 trimethylation. Disruption of the cycle interfered with these periodic histone code alterations. Pharmacological inhibition of OGT with OSMI-1 disrupted the wake-sleep patterns of zebrafish without affecting expression of circadian rhythm-regulating genes. OSMI-1 inhibited the expression of c-fos, bdnf, and calm1, key genes associated with brain function and synaptic plasticity, and decreased the binding of O-GlcNAcylated H2B and OGT to promoter regions of these genes. The collective findings support the potential involvement of circadian cycling of the O-GlcNAc histone code in regulating synaptic plasticity and brain function. Overall, data from this study provide evidence that protein O-GlcNAcylation serves as a pivotal posttranslational mechanism integrating circadian signals and neuronal function to regulate rhythmic physiology.

Adverse Effects of Nicotine on Human Sperm Nuclear Proteins.

The effects of smoking on human health have long been documented. However, only a few studies have highlighted the direct effects of nicotine on sperm function. Nicotine, as a chemical compound found in tobacco, has been shown to modulate different aspects of spermatogenesis and sperm functions. Nicotine can lead to a reduction in the number of sperm, their motility and functionality. It can change the molecular expressions involved in sperm function, including genes encoding sperm nuclear proteins. The most important nuclear proteins that play a critical role in sperm function are known as H2B histone family, member W, testis-specific (H2BFWT), transition protein 1 (TNP1), transition protein 2 (TNP2), protamine-1 (PRM1), and protamine-2 (PRM2). These proteins are involved in sperm chromatin condensation, which in turn affects fertilization and embryonic development. Any alteration in the expression of these genes due to nicotine exposure/usage may lead to adverse implications in couples' fertility and the health of future generations. Since research in this area is still relatively new, it underscores the importance of understanding the potential side effects of environmental factors such as nicotine on reproductive health.

Nanoscale Structure, Interactions, and Dynamics of Centromere Nucleosomes.

Centromeres are specific segments of chromosomes comprising two types of nucleosomes: canonical nucleosomes containing an octamer of H2A, H2B, H3, and H4 histones and CENP-A nucleosomes in which H3 is replaced with its analogue CENP-A. This modification leads to a difference in DNA wrapping (∼121 bp), considerably less than 147 bp in canonical nucleosomes. We used atomic force microscopy (AFM) and high-speed AFM (HS-AFM) to characterize nanoscale features and dynamics for both types of nucleosomes. For both nucleosomes, spontaneous asymmetric unwrapping of DNA was observed, and this process occurs via a transient state with ∼100 bp DNA wrapped around the core, followed by a rapid dissociation of DNA. Additionally, HS-AFM revealed higher stability of CENP-A nucleosomes compared with H3 nucleosomes in which dissociation of the histone core occurs prior to the nucleosome dissociation. These results help elucidate the differences between these nucleosomes and the potential biological necessity for CENP-A nucleosomes.

Usp7 Regulates Glial Lineage Cell-Specific Transcription Factors by Modulating Histone H2B Monoubiquitination.

Histone H2B monoubiquitination (H2Bub1) is a dynamic posttranslational modification which are linked to DNA damage and plays a key role in a wide variety of regulatory transcriptional programs. Cancer cells exhibit a variety of epigenetic changes, particularly any aberrant H2Bub1 has frequently been associated with the development of tumors. Nevertheless, our understanding of the mechanisms governing the histone H2B deubiquitination and their associated functions during stem cell differentiation remain only partially understood. In this study, we wished to investigate the role of deubiquitinating enzymes (DUBs) on H2Bub1 regulation during stem cell differentiation. In a search for potential DUBs for H2B monoubiquitination, we identified Usp7, a ubiquitin-specific protease that acts as a negative regulator of H2B ubiquitination during the neuronal differentiation of mouse embryonic carcinoma cells. Loss of function of the Usp7 gene by a CRISPR/Cas9 system during retinoic acid-mediated cell differentiation contributes to the increase in H2Bub1. Furthermore, knockout of the Usp7 gene particularly elevated the expression of neuronal differentiation related genes including astryocyte-specific markers and oligodendrocyte-specific markers. In particular, glial lineage cell-specific transcription factors including oligodendrocyte transcription factor 2, glial fibrillary acidic protein, and SRY-box transcription factor 10 was significantly upregulated during neuronal differentiation. Thus, our findings suggest a novel role of Usp7 in gliogenesis in mouse embryonic carcinoma cells.

Bacterial endometritis-induced changes in the endometrial proteome in mares: Potential uterine biomarker for bacterial endometritis

Equine endometritis is one of the main causes of subfertility in the mare. Unraveling the molecular mechanisms involved in this condition and pinpointing proteins with biomarker potential could be crucial in both diagnosing and treating this condition. This study aimed to identify the endometritis-induced changes in the endometrial proteome in mares and to elucidate potential biological processes in which these proteins may be involved. Secondly, biomarkers related to bacterial endometritis (BE) in mares were identified. Uterine lavage fluid samples were collected from 28 mares (14 healthy: negative cytology and culture, and no clinical signs and 14 mares with endometritis: positive cytology and culture, in addition to clinical signs). Proteomic analysis was performed with a UHPLC-MS/MS system and bioinformatic analysis was carried out using Qlucore Omics Explorer. Gene Ontology enrichment and pathway analysis (PANTHER and KEGG) of the uterine proteome were performed to identify active biological pathways in enriched proteins from each group. Quantitative analysis revealed 38 proteins differentially abundant in endometritis mares when compared to healthy mares (fold changes >4.25, and q-value = 0.002). The proteins upregulated in the secretome of mares with BE were involved in biological processes related to the generation of energy and REDOX regulation and to the defense response to bacterium. A total of 24 biomarkers for BE were identified using the biomarker workbench algorithm. Some of the proteins identified were related to the innate immune system such as isoforms of histones H2A and H2B involvement in neutrophil extracellular trap (NET) formation, complement C3a, or gelsolin and profilin, two actin-binding proteins which are essential for dynamic remodeling of the actin cytoskeleton during cell migration. The other group of biomarkers were three known antimicrobial peptides (lysosome, equine cathelicidin 2 and myeloperoxidase (MPO)) and two uncharacterized proteins with a high homology with cathelicidin families.Findings in this study provide the first evidence that innate immune cells in the equine endometrium undergo reprogramming of metabolic pathways similar to the Warburg effect during activation. In addition, biomarkers of BE in uterine fluid of mares including the new proteins identified, as well as other antimicrobial peptides already known, offer future lines of research for alternative treatments to antibiotics.

Conformational Dynamics of the Nucleosomal Histone H2B Tails Revealed by Molecular Dynamics Simulations.

Epigenetic modifications of histone N-terminal tails play a critical role in regulating the chromatin structure and biological processes such as transcription and DNA repair. One of the key post-translational modifications (PTMs) is the acetylation of lysine residues on histone tails. Epigenetic modifications are ubiquitous in the development of diseases, such as cancer and neurological disorders. Histone H2B tails are critical regulators of nucleosome dynamics, biological processes, and certain diseases. Here, we report all-atomistic molecular dynamics (MD) simulations of the nucleosome to demonstrate that acetylation of the histone tails changes their conformational space and interaction with DNA. We perform simulations of H2B tails, critical regulators of gene regulation, in both the lysine-acetylated (ACK) and unacetylated wild type (WT) states. To explore the effects of salt concentration, we use two different NaCl concentrations to perform simulations at microsecond time scales. Salt can modulate the effects of electrostatic interactions between the DNA phosphate backbone and histone tails. Upon acetylation, H2B tails shift their secondary structure helical propensity. The number of contacts between the DNA and the H2B tail decreases. We characterize the conformational dynamics of the H2B tails by principal component analysis (PCA). The ACK tails become more compact at increased salt concentrations, but conformations from the WT tails display the most contacts with DNA at both salt concentrations. Mainly, H2B acetylation may increase the DNA accessibility for regulatory proteins to bind, which can aid in gene regulation and NCP stability.

Experimental autoimmune encephalomyelitis of mice: Enzymatic cross site-specific hydrolysis of H3 histone by IgGs against H3, H1, H2A, H2B, and H4 histones, and myelin basic protein

Experimental autoimmune encephalomyelitis of mice: Enzymatic cross site-specific hydrolysis of H3 histone by IgGs against H3, H1, H2A, H2B, and H4 histones, and myelin basic protein

Proteomics-based identification of biomarkers reflecting endogenous and exogenous exposure to the advanced glycation end product precursor methylglyoxal in SH-SY5Y human neuroblastoma cells

Methylglyoxal (MGO), a highly reactive precursor of advanced glycation end products, is endogenously produced and prevalent in various food products. This study aimed to characterize protein modifications in SH-SY5Y human neuroblastoma cells induced by MGO and identify potential biomarkers for its exposure and toxicity. A shot-gun proteomic analysis was applied to characterize protein modifications in cells incubated with and without exogenous MGO. Seventy-seven proteins were identified as highly susceptible to MGO modification, among which eight, including vimentin and histone H2B type 2-F, showing concentration-dependent modifications by externally added MGO, were defined as biomarkers for exogenous MGO exposure. Remarkably, up to 10 modification sites were identified on vimentin. Myosin light polypeptide 6 emerged as a biomarker for MGO toxicity, with modifications exclusively observed under cytotoxic MGO levels. Additionally, proteins like serine/threonine-protein kinase SIK2 and calcyphosin, exhibiting comparable or even higher modification levels in control compared to exogenous MGO-treated cells, were defined as biomarkers for endogenous exposure. Bioinformatics analysis revealed that motor proteins, cytoskeleton components, and glycolysis proteins were overrepresented among those highly susceptible to MGO modification. These results identify biomarkers for both endogenous and exogenous MGO exposure and provide insights into the cellular effects of endogenously formed versus externally added MGO.

Dual engagement of the nucleosomal acidic patches is essential for deposition of histone H2A.Z by SWR1C

The yeast SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated key roles for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the acidic patch in the H2A.Z exchange reaction. Utilizing asymmetrically assembled nucleosomes, we demonstrate that the acidic patches on each face of the nucleosome are required for SWR1C-mediated dimer exchange, suggesting SWR1C engages the nucleosome in a ‘pincer-like’ conformation, engaging both patches simultaneously. Loss of a single acidic patch results in loss of high affinity nucleosome binding and nucleosomal stimulation of ATPase activity. We identify a conserved arginine-rich motif within the Swc5 subunit that binds the acidic patch and is key for dimer exchange activity. In addition, our cryoEM structure of a Swc5–nucleosome complex suggests that promoter proximal, histone H2B ubiquitylation may regulate H2A.Z deposition. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.