Core Histone Proteins Research Articles

Characterization of Medusavirus encoded histones reveals nucleosome-like structures and a unique linker histone

The organization of DNA into nucleosomes is a ubiquitous and ancestral feature that was once thought to be exclusive to the eukaryotic domain of life. Intriguingly, several representatives of the Nucleocytoplasmic Large DNA Viruses (NCLDV) encode histone-like proteins that in Melbournevirus were shown to form nucleosome-like particles. Medusavirus medusae (MM), a distantly related giant virus, encodes all four core histone proteins and, unique amongst most giant viruses, a putative acidic protein with two domains resembling eukaryotic linker histone H1. Here, we report the structure of nucleosomes assembled with MM histones and highlight similarities and differences with eukaryotic and Melbournevirus nucleosomes. Our structure provides insight into how variations in histone tail and loop lengths are accommodated within the context of the nucleosome. We show that MM-histones assemble into tri-nucleosome arrays, and that the putative linker histone H1 does not function in chromatin compaction. These findings expand our limited understanding of chromatin organization by virus-encoded histones.

The Structures, Functions, and Roles of Class III HDACs (Sirtuins) in Neuropsychiatric Diseases.

Over the past two decades, epigenetic regulation has become a rapidly growing and influential field in biology and medicine. One key mechanism involves the acetylation and deacetylation of lysine residues on histone core proteins and other critical proteins that regulate gene expression and cellular signaling. Although histone deacetylases (HDACs) have received significant attention, the roles of individual HDAC isoforms in the pathogenesis of psychiatric diseases still require further research. This is particularly true with regard to the sirtuins, class III HDACs. Sirtuins have unique functional activity and significant roles in normal neurophysiology, as well as in the mechanisms of addiction, mood disorders, and other neuropsychiatric abnormalities. This review aims to elucidate the differences in catalytic structure and function of the seven sirtuins as they relate to psychiatry.

Gibberellin Signaling through RGA Suppresses GCN5 Effects on Arabidopsis Developmental Stages.

Histone acetyltransferases (HATs) modify the amino-terminal tails of the core histone proteins via acetylation, regulating chromatin structure and transcription. GENERAL CONTROL NON-DEREPRESSIBLE 5 (GCN5) is a HAT that specifically acetylates H3K14 residues. GCN5 has been associated with cell division and differentiation, meristem function, root, stem, foliar, and floral development, and plant environmental response. The flowers of gcn5 plants display a reduced stamen length and exhibit male sterility relative to the wild-type plants. We show that these effects may arise from gibberellin (GA)-signaling defects. The signaling pathway of bioactive GAs depends on the proteolysis of their repressors, DELLA proteins. The repressor GA (RGA) DELLA protein represses plant growth, inflorescence, and flower and seed development. Our molecular data indicate that GCN5 is required for the activation and H3K14 acetylation of genes involved in the late stages of GA biosynthesis and catabolism. We studied the genetic interaction of the RGA and GCN5; the RGA can partially suppress GCN5 action during the whole plant life cycle. The reduced elongation of the stamen filament of gcn5-6 mutants is reversed in the rga-t2;gcn5-6 double mutants. RGAs suppress the GCN5 effect on the gene expression and histone acetylation of GA catabolism and GA signaling. Interestingly, the RGA and RGL2 do not suppress ADA2b function, suggesting that ADA2b acts downstream of GA signaling and is distinct from GCN5 activity. In conclusion, we propose that the action of GCN5 on stamen elongation is partially mediated by RGA and GA signaling.

Histone Variants and Their Chaperones in Hematological Malignancies.

Epigenetic regulation occurs on the level of compacting DNA into chromatin. The functional unit of chromatin is the nucleosome, which consists of DNA wrapped around a core of histone proteins. While canonical histone proteins are incorporated into chromatin through a replication-coupled process, structural variants of histones, commonly named histone variants, are deposited into chromatin in a replication-independent manner. Specific chaperones and chromatin remodelers mediate the locus-specific deposition of histone variants. Although histone variants comprise one of the least understood layers of epigenetic regulation, it has been proposed that they play an essential role in directly regulating gene expression in health and disease. Here, we review the emerging evidence suggesting that histone variants have a role at different stages of hematopoiesis, with a particular focus on the histone variants H2A, H3, and H1. Moreover, we discuss the current knowledge on how the dysregulation of histone variants can contribute to hematopoietic malignancies.

Spontaneous histone exchange between nucleosomes

The nucleosome is the fundamental gene-packing unit in eukaryotes. Nucleosomes comprise ∼147bp DNA wrapped around an octameric histone protein core composed of two H2A-H2B dimers and one (H3-H4)2 tetramer. The strong yet flexible DNA-histone interactions are the physical basis of the dynamic regulation of genes packaged in chromatin. The dynamic nature of DNA-histone interactions also implies thatnucleosomes dissociate DNA-histone contacts both transiently and repeatedly. This kinetic instability may lead to spontaneous nucleosome disassembly or histone exchange between nucleosomes. At high nucleosome concentrations, nucleosome-nucleosome collisions and subsequent histone exchange would be a more likely event, where nucleosomes could act as their own histone chaperone. This spontaneous histone exchange could serve as a mechanism for maintaining overall chromatin stability, although it has never been reported. Here we employed three-color single-molecule FRET (smFRET) to demonstrate that histone H2A-H2B dimers are exchanged spontaneously between nucleosomes on a time scale of a few tens of seconds at a physiological nucleosome concentration. We show that the rate of histone exchange increases at a higher monovalent salt concentration, with histone-acetylated nucleosomes, and in the presence of histone chaperone Nap1, while it remains unchanged at a higher temperature, and decreases upon DNA methylation. These results support the notion of histone exchange via transient and repetitive partial disassembly of the nucleosome and corroborate spontaneous histone diffusion in a compact chromatin context, modulating the local concentrations of histone modifications and variants.

How Do Nucleosome Dynamics Regulate Protein Search on DNA?

Nearly three-fourths of all eukaryotic DNA is occupied by nucleosomes, protein-DNA complexes comprising octameric histone core proteins and ∼150 base pairs of DNA. In addition to acting as a DNA compaction vehicle, the dynamics of nucleosomes regulate the DNA site accessibility for the nonhistone proteins, thereby controlling regulatory processes involved in determining the cell identity and cell fate. Here, we propose an analytical framework to analyze the role of nucleosome dynamics on the target search process of transcription factors through a simple discrete-state stochastic description of the search process. By considering the experimentally determined kinetic rates associated with protein and nucleosome dynamics as the only inputs, we estimate the target search time of a protein via first-passage probability calculations separately during nucleosome breathing and sliding dynamics. Although both the nucleosome dynamics permit transient access to the DNA sites that are otherwise occluded by the histone proteins, our result suggests substantial differences between the protein search mechanism on a nucleosome performing breathing and sliding dynamics. Furthermore, we identify the molecular factors that influence the search efficiency and demonstrate how these factors together portray a highly dynamic landscape of gene regulation. Our analytical results are validated using extensive Monte Carlo simulations.

Histone acetyltransferases and histone deacetyl transferases play crucial role during oogenesis and early embryo development.

Dynamic epigenetic regulation is critical for proper oogenesis and early embryo development. During oogenesis, fully grown germinal vesicle oocytes develop to mature Metaphase II oocytes which are ready for fertilization. Fertilized oocyte proliferates mitotically until blastocyst formation and the process is called early embryo development. Throughout oogenesis and early embryo development, spatio-temporal gene expression takes place, and this dynamic gene expression is controlled with the aid of epigenetics. Epigenetic means that gene expression can be altered without changing DNA itself. Epigenome is regulated through DNA methylation and histone modifications. While DNA methylation generally ends up with repression of gene expression, histone modifications can result in expression or repression depending on type of modification, type of histone protein and its specific residue. One of the modifications is histone acetylation which generally ends up with gene expression. Histone acetylation occurs through the addition of acetyl group onto amino terminal of the core histone proteins by histone acetyltransferases (HATs). Contrarily, histone deacetylation is associated with repression of gene expression, and it is catalyzed by histone deacetylases (HDACs). This review article focuses on what is known about alterations in the expression of HATs and HDACs and emphasizes importance of HATs and HDACs during oogenesis and early embryo development.

Abstract 3484: Oncohistone H2B-E76K alters gene expression & cellular phenotype in a bronchial epithelial cell line

Abstract The nucleosome is the basic unit of chromatin, comprising 147 bp of DNA wrapped around an octamer of core histone proteins H2A, H2B, H3 and H4. Compact chromatin acts as a barrier to gene expression and is a key property governing DNA replication and gene expression. Analyzing over 46,000 in cancer patient samples in cBioPortal, we characterized mutations located in canonical histone genes and found several recurrent missense mutations. Foremost among the identified mutations was a glutamate to lysine missense mutation at amino acid 76 of histone H2B (H2B-E76K) that disrupts histone H2B-H4 interaction. Previously, we demonstrated that exogenous expression of H2B-E76K disrupted nucleosome stability, altered chromatin accessibility, and gene expression. We developed a new model utilizing CRISPR “knock-in” technology to integrate the E76K mutation into the endogenous H2BC4 gene of the lung epithelial cell line BEAS-2B to create a homozygous and heterozygous H2B-E76K cell line. Although the mutant histone only accounts for about 2-5% of total histone H2B, expression of H2B-E76K catalyzed distinct changes in cell migration, proliferation, colony formation and gene expression. CUT&RUN technology revealed mutant histones broadly incorporate throughout the genome. Gene set enrichment analysis (GSEA) revealed that H2B-E76K homozygous cells displayed expression changes similar to polycomb repressive complex 1 (PRC1) disruption or oncogenic KRAS expression. Using available datasets, we discovered that expression of H2B-E76K caused increased expression of PTN, PKIB, and CDH2, genes important in migration, epithelial-mesenchymal transition (EMT), and adhesion pathways. Furthermore, analysis of chromatin accessibility changes indicated that increased accessibility was significant in and around genes governing cell migration. Notably, H2B-E76K significantly increased heterogeneity of gene expression as measured using scRNA-seq. Together these results indicate that mutations disrupting histone structure may create millions of dysfunctional nucleosomes and new sites of “open” chromatin, resulting in loss of nucleosome-mediated gene repression and/or altered interactions with regulators of chromatin, fundamentally changing cell growth and migration properties to favor tumorigenesis. Citation Format: Kimberly Espinoza Pereira, Richard L. Bennett, Jixiu Shan, Jay Sarthy, Jonathan D. Licht. Oncohistone H2B-E76K alters gene expression & cellular phenotype in a bronchial epithelial cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3484.

Ionic strength modulates excision of uracil by SMUG1 from nucleosome core particles

Ionic strength affects many cellular processes including the packaging of genetic material in eukaryotes. For example, chromatin fibers are compacted in high ionic strength environments as are the minimal unit of packaging in chromatin, nucleosome core particles (NCPs). Furthermore, ionic strength is known to modulate several aspects of NCP dynamics including transient unwrapping of DNA from the histone protein core, nucleosome gaping, and intra- and internucleosomal interactions of the N-terminal histone tails. Changes in NCP structure may also impact interactions of transcriptional, repair, and other cellular machinery with nucleosomal DNA. One repair process, base excision repair (BER), is impacted by NCP structure and may be further influenced by changes in ionic strength. Here we examine the effects of ionic strength on the initiation of BER using biochemical assays. Using a population of NCPs containing uracil (U) at dozens of geometric locations, excision of U by single-strand selective monofunctional uracil DNA glycosylase (SMUG1) is assessed at higher and lower ionic strengths. SMUG1 has increased excision activity in the lower ionic strength conditions. On duplex DNA, however, SMUG1 activity is largely unaffected by ionic strength except at short incubation times, suggesting that changes in SMUG1 activity are likely due to alterations in NCP structure and dynamics. These results allow us to further understand the cellular role of SMUG1 in a changing ionic environment and broadly contribute to the understanding of BER on chromatin and genomic stability.

Photobuforin II, a fluorescent photoswitchable peptide

Antimicrobial peptide buforin II translocates across the cell membrane and binds to DNA. Its sequence is identical to a portion of core histone protein H2A making it a highly charged peptide. Buforin II has a proline residue in the middle of its sequence that creates a helix-hinge-helix motif which has been found to play a key role in its ability to translocate across the cell membrane. To explore the structure-function relationship of this proline residue this study has replaced P11 with a meta-substituted azobenzene amino acid (Z). The resultant peptide, photobuforin II, retained the secondary structure and membrane activity of the naturally occurring peptide while gaining new spectroscopic properties. Photobuforin II can be isomerized from its trans to cis isomer upon irradiation with ultra-violet (UV) light and from its cis to trans isomer upon irradiation with visible (VL). Photobuforin II is also fluorescent with an emission peak at 390 nm. The intrinsic fluorescence of the peptide was used to determine binding to the membrane and to DNA. VL-treated photobuforin II has a 2-fold lower binding constant compared to UV-treated photobuforin and causes 11-fold more membrane leakage in 3:1 POPC:POPG vesicles. Photobuforin II provides insights into the importance of structure function relationships in membrane active peptides while also demonstrating that azobenzene can be used in certain peptide sequences to produce intrinsic fluorescence.

Abnormal histone replacement following BPA exposure affects spermatogenesis and fertility sequentially

Bisphenol A (BPA) is an endocrine-disrupting chemical widely distributed in the environment. Its exposure has been linked to male infertility in animals and humans due to its ability to induce epigenetic modification. Despite extensive research confirming the impact of BPA on epigenetic regulation, fundamental concerns about how BPA causes epigenetic changes and the underlying mechanism of BPA on the male reproductive system remain unresolved. Therefore, we sought to investigate the effects of BPA on epigenetic regulation and the histone-to-protamine (PRM) transition, which is fundamental process for male fertility in testes and spermatozoa by exposing male mice to BPA for 6 weeks while giving the mice in the control group corn oil by oral gavage. Our results demonstrated that the mRNA levels of the histone family and PRMs were significantly altered by BPA exposure in testes and spermatozoa. Subsequently, core histone proteins, the PRM1/PRM2 ratio, directly linked to male fertility, and transition proteins were significantly reduced. Furthermore, we discovered that BPA significantly caused abnormal histone-to-protamine replacement during spermiogenesis by increased histone variants-related to histone-to-PRM transition. The levels of histone H3 modification in the testes and DNA methylation in spermatozoa were significantly increased. Consequently, sperm concentration/motility/hyperactivation, fertilization, and early embryonic development were adversely affected as a consequence of altered signaling proteins following BPA exposure. To our knowledge, this is the first study to indicate that BPA exposure influences the histone-to-PRM transition via altering epigenetic modification and eventually causing reduced male fertility.

Lysine Crotonylation: An Emerging Player in DNA Damage Response.

The DNA damage response (DDR) system plays an important role in maintaining genome stability and preventing related diseases. The DDR network comprises many proteins and posttranslational modifications (PTMs) to proteins, which work in a coordinated manner to counteract various genotoxic stresses. Lysine crotonylation (Kcr) is a newly identified PTM occurring in both core histone and non-histone proteins in various organisms. This novel PTM is classified as a reversible acylation modification, which is regulated by a variety of acylases and deacylases and the intracellular crotonyl-CoA substrate concentration. Recent studies suggest that Kcr links cellular metabolism with gene regulation and is involved in numerous cellular processes. In this review, we summarize the regulatory mechanisms of Kcr and its functions in DDR, including its involvement in double-strand break (DSB)-induced transcriptional repression, DSB repair, and the DNA replication stress response.

Histone variants H3.3 and H2A.Z/H3.3 facilitate excision of uracil from nucleosome core particles

At the most fundamental level of chromatin organization, DNA is packaged as nucleosome core particles (NCPs) where DNA is wound around a core of histone proteins. This ubiquitous sequestration of DNA within NCPs presents a significant barrier to many biological processes, including DNA repair. We previously demonstrated that histone variants from the H2A family facilitate excision of uracil (U) lesions by DNA base excision repair (BER) glycosylases. Here, we consider how the histone variant H3.3 and double-variant H2A.Z/H3.3 modulate the BER enzymes uracil DNA glycosylase (UDG) and single-strand selective monofunctional uracil DNA glycosylase (SMUG1). Using an NCP model system with U:G base pairs at a wide variety of geometric positions we generate the global repair profile for both glycosylases. Enhanced excision of U by UDG and SMUG1 is observed with the H3.3 variant. We demonstrate that these H3.3-containing NCPs form two species: (1) octasomes, which contain the full complement of eight histone proteins and (2) hexasomes which are sub-nucleosomal particles that contain six histones. Both the octasome and hexasome species facilitate excision activity of UDG and SMUG1, with the largest impacts observed at sterically-occluded lesion sites and in terminal regions of DNA of the hexasome that do not closely interact with histones. For the double-variant H2A.Z/H3.3 NCPs, which exist as octasomes, the global repair profile reveals that UDG but not SMUG1 has increased U excision activity. The enhanced glycosylase activity reveals potential functions for these histone variants to facilitate BER in packaged DNA and contributes to our understanding of DNA repair in chromatin and its significance regarding mutagenesis and genomic integrity.

Reading the message: Connections between the epigenetic landscape bromodomain activity

Bromodomains are conserved epigenetic reader domains known to recognize acetylated lysine residues on the N‐terminal histone tails protruding from the nucleosome. The 61 human bromodomains are found in 41 different proteins, and have been classified into eight different subfamilies based on sequence conservation. These subfamilies often share unique sequence variations that drive differences in histone ligand preference for specific patterns of post‐translational modifications on core and variant histone proteins. The BET bromodomains, which are an attractive drug target in multiple disease states, have been the focus of many studies to elucidate the molecular mechanism of histone recognition. However, much less is known about non‐BET bromodomain‐containing proteins. In this study, we examined the ligand specificity the family IV ATAD2B bromodomain and compared it to its closely related paralog in ATAD2. We show that the ATAD2B bromodomain recognizes mono‐ and di‐acetyllysine modifications on histones H4 and H2A. A structure‐function approach using X‐ray crystallography, isothermal titration calorimetry experiments, and site‐directed mutagenesis coupled to ligand binding assays identified key residues in the acetyllysine binding pocket that dictate the molecular recognition process. Furthermore, our results demonstrate how cross‐talk between multiple modifications alters the binding activity of the ATAD2B bromodomain. The structure of the ATAD2B bromodomain in complex with a small molecule ATAD2 bromodomain inhibitor revealed that critical contacts required for coordination are conserved between the ATAD2/B bromodomains, and many of these residues play a dual role in acetyllysine recognition. We further characterized an alternative splice variant of ATAD2B that results in a loss of bromodomain function. Our results outline the structural and functional features of the ATAD2B bromodomain, and identify novel mechanisms regulating the interaction of the ATAD2B protein with chromatin.

In Vivo Crosslinking and Mass Spectral Analysis of the Yeast Nucleosomal Protein Interactome

Chromatin is essential for proper DNA storage and gene regulation. The repeating unit of chromatin, the nucleosome, is composed of DNA wrapped around an octameric core of histone proteins. Histones interact with DNA, but they also make contacts to chromosomal regulating proteins. Histones undergo an array of dynamic posttranslational modifications (PTMs), each having an essential role in chromosomal structuring, function, and maintenance. To understand how nucleosomes regulate chromatin processes and how the dynamic PTM landscape influences histone‐protein interactions, it is important to identify interacting partners. We use UV crosslinking unnatural amino acids that allow for site‐selective photo‐crosslinking from histones, in vivo. This allows for the study of chromatin protein‐protein interactive dynamics in the physiological environment of the nucleus. We pair crosslinking techniques with mass spectral (MS) analysis to identify histone interacting proteins from cell lysates providing an unbiased overview of the site‐specific interactome. Our assays reveal many well‐established chromatin partners; however, they also provided insight to nucleosomal contacts that have been less reported in the literature. Of interest, we identify several histone contacts to ribosomal subunits, both large and small. We are interested in the extra ribosomal function of these proteins and their roles in chromatin regulation. To verify MS hits, we crosslink in yeast strains harboring a C‐terminal fusion tag (Myc) to the gene of interest, while expressing the crosslinking histone with a separate tag (HA). Products are precipitated and analyzed via western blot by decorating with antibodies to the opposing tag. We have verified several histone‐ribosomal protein interactions, including Rpl7A, Rpl3, and Rpl10. Using this technique, we investigate PTM mutations and their effects on the ability to form crosslinked products, providing insight on sequestering and regulatory mechanisms. These initial investigations suggest that UV crosslinking paired with MS analysis is an effective tool for studying yeast nucleosomal protein contacts.

Some assembly required: structural and mechanistic studies of histone chaperones, nucleosome assembly factors and ATP dependent chromatin remodelers

All eukaryotes organize their DNA into nucleosomes, consisting of an octamer of the four core histone proteins H2A, H2B, H3, and H4, around which 147 base pairs of DNA are wrapped in two tight superhelical turns. Nucleosomes pack into higher order structures and require histone chaperones and ATP‐dependent remodelers to make the DNA accessible. I will discuss our recent progress in understanding the higher order structure of chromatin at the centromere, and the role of centromere‐binding proteins in locally folding the chromatin fiber. I will also discuss mechanisms by which nucleosomes are remodeled and assembled in an ATP dependent and ATP‐independent manner. The histone chaperone FACT binds and stabilizes partially disassembled nucleosomes by employing an acidic C‐terminal domain as a DNA mimic to clamp down a histone dimer. The chromatin remodeler SMARCAD1 requires ATP hydrolysis to remove histones from a nucleosome, which it then can deposit onto a new DNA segment in an ATP‐independent mechanism, and I will present biochemical and structural evidence for this mode of action.

Intracellular Formation of a DNA Damage-Induced, Histone Post-Translational Modification Following Bleomycin Treatment.

Evaluating the significance of various forms of DNA damage is complicated by discoveries that some lesions inactivate repair enzymes or produce more deleterious forms of damage. Histone lysines within nucleosomes react with the commonly produced C4'-oxidized abasic site (C4-AP) to concomitantly yield an electrophilic modification (KMP) on lysine and DNA strand scission. We developed a chemoproteomic approach to identify KMP in HeLa cells. More than 60 000 KMP-modified histones are produced per cell. Using LC-MS/MS, we detected KMP at 17 of the 57 lysine residues distributed throughout the four core histone proteins. Therefore, KMP constitutes a DNA damage-induced, nonenzymatic histone post-translational modification. KMP formation suggests that downstream processes resulting from DNA damage could have ramifications on cells.

Participation of Histones in DNA Damage and Repair within Nucleosome Core Particles: Mechanism and Applications.

DNA is damaged by various endogenous and exogenous sources, leading to a diverse group of reactive intermediates that yield a complex mixture of products. The initially formed products are often metastable and can react to yield lesions that are more biologically deleterious. Mechanistic studies are frequently carried out on free DNA as the substrate. The observations do not necessarily reflect the reaction environment inside human cells where genomic DNA is condensed as chromatin in the nucleus. Chromatin is made up of monomeric structural units called nucleosomes, which are comprised of DNA wrapped around an octameric core of histone proteins (two copies each of histones H2A, H2B, H3, and H4).This account presents a summary of our work in the past decade on the mechanistic studies of DNA damage and repair in reconstituted nucleosome core particles (NCPs). A series of metastable lesions and reactive intermediates, such as abasic sites (AP), N7-methyl-2'-deoxyguanosine (MdG), and 2'-deoxyadenosin-N6-yl radical (dA•), have been independently generated in a site-specific manner in bottom-up-synthesized NCPs. Detailed mechanistic studies on these NCPs revealed that histones actively participate in DNA damage and repair processes in diverse ways. For instance, nucleophilic residues in the flexible histone N-terminal tails, such as Lys and N-terminal α-amine, react with electrophilic DNA damage and reactive intermediates. In some cases, transient intermediates are produced, leading to the promotion or suppression of damage and repair processes. In other examples, reactions with histones yield reversible or stable DNA-protein cross-links (DPCs). Histones also utilize acidic and basic residues, such as histidine and aspartic acid, to catalyze DNA strand cleavage through general acid/base catalysis. Alternatively, a Tyr in histone plays a vital role in nucleosomal DNA damage and repair via radical transfer. Finally, the reactivity discovered during the mechanistic studies has facilitated the development of new reagents and methods with applications in biotechnology.This research has enriched our knowledge of the roles of histone proteins in DNA damage and repair and their contributions to epigenetics and may have significant biological implications. The residues in histone N-terminal tails that react with DNA lesions also play pivotal roles in regulating the structure and function of chromatin, indicating that there may be cross-talk between DNA damage and repair in eukaryotic cells and epigenetic regulation. Also, in view of the biased amino acid composition of histones, these results provide hints about how the proteins have evolved to minimize their deleterious effects but maximize beneficial ones for maintaining genome integrity. Finally, previously unreported DPCs and histone post-translational modifications have been discovered through this research. The effects of these newly identified lesions on the structure and function of chromatin and their fates inside cells remain to be elucidated.

Distinguishing functional exosomes and other extracellular vesicles as a nucleic acid cargo by the anion-exchange method.

The development of a new large‐scale purification protocol is required for research on the reliable bioactivity and drug discovery of extracellular vesicles (EVs). To address this issue, herein, we propose an effective method for preparing high‐performance exosomes (EXOs) by using an anion‐exchange method. Cytotoxic T‐lymphocyte (CTL) EVs from 4 L of culture supernatant through a 220 nm cut‐off filter are divided into two populations at a deproteinization rate of over 99.97%, which are eluted at low (0.15 M–0.3 M) and high (0.3 M–0.5 M) NaCl concentrations (approximately 2 × 1012 and 1.5 × 1012 particles, respectively) through the anion‐exchange column chromatography. The former are abundant in EXO proteins, including late endosome‐associated proteins and rab‐family and integrin‐family proteins, and functional micro (mi) RNAs, and have bioactivity for preventing tumour metastasis by depleting mesenchymal cell populations in the primary tumour lesions. By contrast, the latter is microvesicle (MV)‐like particles including DNA, core histone and ribosomal proteins, and GC‐rich miRNAs with unknown function, and are easily phagocytosed by mannose receptor+ Kupffer cells. Thus, the anion‐exchange method is suitable for the large‐scale separation of bioactive EXOs and MV‐like EVs as a cargo for dangerous nucleic acids at high‐purity.

Histone Variants in the Specialization of Plant Chromatin.

The basic unit of chromatin, the nucleosome, is an octamer of four core histone proteins (H2A, H2B, H3, and H4) and serves as a fundamental regulatory unit in all DNA-templated processes. The majority of nucleosome assembly occurs during DNA replication when these core histones are produced en masse to accommodate the nascent genome. In addition, there are a number of nonallelic sequence variants of H2A and H3 in particular, known as histone variants, that can be incorporated into nucleosomes in a targeted and replication-independent manner. By virtue of their sequence divergence from the replication-coupled histones, these histone variants can impart unique properties onto the nucleosomes they occupy and thereby influence transcription and epigenetic states, DNA repair, chromosome segregation, and other nuclear processes in ways that profoundly affect plant biology. In this review, we discuss the evolutionary origins of these variants in plants, their known roles in chromatin, and their impacts on plant development and stress responses. We focus on the individual and combined roles of histone variants in transcriptional regulation within euchromatic and heterochromatic genome regions. Finally, we highlight gaps in our understanding of plant variants at the molecular, cellular, and organismal levels, and we propose new directions for study in the field of plant histone variants.