Core Histones Research Articles

Regulation of telomere silencing by the core histones-autophagy-Sir2 axis.

Telomeres contain compacted heterochromatin, and genes adjacent to telomeres are subjected to transcription silencing. Maintaining telomere structure integrity and transcription silencing is important to prevent the occurrence of premature aging and aging-related diseases. How telomere silencing is regulated during aging is not well understood. Here, we find that the four core histones are reduced during yeast chronological aging, leading to compromised telomere silencing. Mechanistically, histone loss promotes the nuclear export of Sir2 and its degradation by autophagy. Meanwhile, reducing core histones enhances the autophagy pathway, which further accelerates autophagy-mediated Sir2 degradation. By screening the histone mutant library, we identify eight histone mutants and one histone modification (histone methyltransferase Set1-catalyzed H3K4 trimethylation) that regulate telomere silencing by modulating the core histones-autophagy-Sir2 axis. Overall, our findings reveal core histones and autophagy as causes of aging-coupled loss of telomere silencing and shed light on dynamic regulation of telomere structure during aging.

HMGA1 Regulates the Expression of Replication-Dependent Histone Genes and Cell-Cycle in Breast Cancer Cells.

Breast cancer (BC) is the primary cause of cancer mortality in women and the triple-negative breast cancer (TNBC) is the most aggressive subtype characterized by poor differentiation and high proliferative properties. High mobility group A1 (HMGA1) is an oncogenic factor involved in the onset and progression of the neoplastic transformation in BC. Here, we unraveled that the replication-dependent-histone (RD-HIST) gene expression is enriched in BC tissues and correlates with HMGA1 expression. We explored the role of HMGA1 in modulating the RD-HIST genes expression in TNBC cells and show that MDA-MB-231 cells, depleted of HMGA1, express low levels of core histones. We show that HMGA1 participates in the activation of the HIST1H4H promoter and that it interacts with the nuclear protein of the ataxia-telangiectasia mutated locus (NPAT), the coordinator of the transcription of the RD-HIST genes. Moreover, we demonstrate that HMGA1 silencing increases the percentage of cells in G0/G1 phase both in TNBC and epirubicin resistant TNBC cells. Moreover, HMGA1 silencing causes an increase in epirubicin IC50 both in parental and epirubicin resistant cells thus suggesting that targeting HMGA1 could affect the efficacy of epirubicin treatment.

The role of histone deacetylases in embryonic development.

The basic units of chromatin are nucleosomes, that are made up of DNA wrapped around histone cores. Histone lysine residue is a common location for posttranslational modifications, with acetylation being the second most prevalent. Histone acetyltransferases (HATs/KATs) and histone deacetylases (HDACs/KDACs) regulate histone acetylation, which is important in gene expression control. HDACs/KDACsregulate gene expressions through the repression of the transcription machinery. HDAC/KDAC isoforms play a major role during various stages of embryo development and neurogenesis. In specific, class I and II HDACs/KDACs are involved in cardiac muscle differentiation and development. An insight into different pathways and genes associated with embryonic development, the effect of HDAC/KDAC activity during the embryonic stem cell differentiation, preimplantation, embryo development, gastrulation, and the role of different HDAC/KDAC inhibitors during the process of embryogenesis is summarized in the present review article.

Identification of ocular regulatory functions of core histone variant H3.2

The posttranscriptional modifications (PTM) of the Histone H3 family play an important role in ocular system differentiation. However, there has been no study on the nature of specific Histone H3 subtype carrying these modifications. Fortuitously, we had previously identified a dominant small-eye mutant Aey69 mouse with a mutation in the H3.2 encoding Hist2h3c1 gene (Vetrivel et al., 2019). In continuation, in the present study, the role of Histone H3.2 with relation to the microphtalmic Aey69 has been elaborated. Foremost, a transgenic mouse line expressing the fusion protein H3.2-GFP was generated using Crispr/Cas9. The approach was intended to confer a unique tag to the Hist2h3c1 gene which is similar in sequence and encoded protein structure to other histones. The GFP tag was then used for ChIP Seq analysis of the genes regulated by H3.2. The approach revealed ocular specific H3.2 targets including Ephrin family genes. Altered enrichment of H3.2 was found in the mutant Aey69 mouse, specifically around the ligand Efna5 and the receptor Ephb2. The effect of this altered enrichment on Ephrin signaling was further analysed by QPCR and immunohistochemistry. This study identifies Hist2h3c1 encoded H3.2 as an important epigenetic player in ocular development. By binding to specific regions of ocular developmental factors Histone H3.2 facilitates the function of these genes for successful early ocular development.

Loss of the Ash2l subunit of histone H3K4 methyltransferase complexes reduces chromatin accessibility at promoters

Changes in gene expression programs are intimately linked to cell fate decisions. Post-translational modifications of core histones contribute to control gene expression. Methylation of lysine 4 of histone H3 (H3K4) correlates with active promoters and gene transcription. This modification is catalyzed by KMT2 methyltransferases, which require interaction with 4 core subunits, WDR5, RBBP5, ASH2L and DPY30, for catalytic activity. Ash2l is necessary for organismal development and for tissue homeostasis. In mouse embryo fibroblasts (MEFs), Ash2l loss results in gene repression, provoking a senescence phenotype. We now find that upon knockout of Ash2l both H3K4 mono- and tri-methylation (H3K4me1 and me3, respectively) were deregulated. In particular, loss of H3K4me3 at promoters correlated with gene repression, especially at CpG island promoters. Ash2l loss resulted in increased loading of histone H3 and reduced chromatin accessibility at promoters, accompanied by an increase of repressing and a decrease of activating histone marks. Moreover, we observed altered binding of CTCF upon Ash2l loss. Lost and gained binding was noticed at promoter-associated and intergenic sites, respectively. Thus, Ash2l loss and reduction of H3K4me3 correlate with altered chromatin accessibility and transcription factor binding. These findings contribute to a more detailed understanding of mechanistic consequences of H3K4me3 loss and associated repression of gene transcription and thus of the observed cellular consequences.

Genome-wide survey of D/E repeats in human proteins uncovers their instability and aids in identifying their role in the chromatin regulator ATAD2.

D/E repeats are stretches of aspartic and/or glutamic acid residues found in over 150 human proteins. We examined genomic stability of D/E repeats and functional characteristics of D/E repeat-containing proteins vis-à-vis the proteins with poly-Q or poly-A repeats, which are known to undergo pathologic expansions. Mining of tumor sequencing data revealed that D/E repeat-coding regions are similar to those coding poly-Qs and poly-As in increased incidence of trinucleotide insertions/deletions but differ in types and incidence of substitutions. D/E repeat-containing proteins preferentially function in chromatin metabolism and are the more likely to be nuclear and interact with core histones, the longer their repeats are. One of the longest D/E repeats of unknown function is in ATAD2, a bromodomain family ATPase frequently overexpressed in tumors. We demonstrate that D/E repeat deletion in ATAD2 suppresses its binding to nascent and mature chromatin and to the constitutive pericentromeric heterochromatin, where ATAD2 represses satellite transcription.

Profiling Accessible Chromatin and Nucleosomes in the Mammalian Genome.

Genomic DNA wraps around core histones to form nucleosomes, which provides steric constraints on how transcription factors (TFs) can interact with gene regulatory sequences. It is increasingly apparent that well-positioned, accessible nucleosomes are an inherent feature of active enhancers and can facilitate cooperative TF binding, referred to as nucleosome-mediated cooperativity. Thus, profiling chromatin and nucleosome properties (accessibility, positioning, and occupancy) on the genome is crucial to understand cell-type-specific gene regulation. Here we describe a simplified protocol to profile accessible nucleosomes in the mammalian genome using low-level and high-level micrococcal nuclease (MNase) digestion followed by genome-wide sequencing.

Functional Role of Histone Variant macroH2A1 (H2AFY) in Acute Myeloid Leukemia (AML)

Histone variants can replace replication-coupled histones in the nucleosome. Among core histones, the H2A family exhibits highest sequence divergence resulting in the largest number of variants. Dysregulated expression of macrohistone (macroH2A) variants has been reported in different cancers. Their role in normal hematopoiesis and hematopoietic cancers remained so far poorly understood. We found high expression of macrohistone H2AFY (macroH2A1) in hematopoietic stem and progenitor cells (HSPCs) and primary AML as compared to differentiated hematopoietic cells. To assess for the functional dependency of human AML cells on macroH2A1, we performed in vitro CRISPR-Cas9 negative selection competition assays. Genetic inactivation of H2AFY in 4/9 AML cell lines (Molm-13, MV4;11, THP-1, OCI-AML3) resulted in outcompetition of H2AFY-deleted cells against non-deleted competitors. To confirm these findings in vivo, we performed xenotransplantation experiments. Genetic inactivation of H2AFY in human AML cells resulted in delayed leukemia development in recipient mice (median survival for NT-control: 35.5 days vs. H2AFY-KO: 44.0 days; p=0.002). To validate the functional impact of macroH2A1 on leukemic stem cells (LSCs), we generated a novel conditional knockout mouse model for H2AFY [B6(Cg)-H2afytm1Flh/J (H2afy flox/flox)]. Here, isolated lineage-depleted H2afy+/+, Mx1-Cre+ and H2afyfl/fl, Mx1-Cre+ bone marrow cells (Lin-BMCs) were retrovirally infected with MLL-AF9-GFP (MA9) and transplanted into sub-lethally irradiated recipient mice. Following establishment of leukemia, we transplanted 1x105 leukemic cells and inactivated H2afy in the leukemic cells by pIpC injection on day 7, 9 and 11 post-transplantation. Genetic deletion of H2afy resulted in delayed disease progression (median survival +/+: 83.5 days vs. -/-: 162.0 days, p=0.0013) and reduction of disease penetrance to 61%. Organ infiltration by leukemic cells led to disease development in a limited number of H2afy-/- recipient mice. Isolated H2afy-/- leukemic cells showed a decreased in proliferation and an increased in differentiation. Importantly, 37% of H2afy-/- recipient mice that developed AML showed only partial excision of H2afy, indicating selection of partially excised clones. Long-term survivors without leukemia development showed no molecular evidence for counterselection. Irrespective of the time of disease development and survival, immunophenotypic analysis of (Lin-cKit+Sca-1-FcγR+CD34+GFP+) LGMPs revealed decreased numbers of LSCs following H2afy-deletion (+/+: 2631/106 BMC; -/-: 727/106 BMC; p=0.0002). To assess for the functional consequences of H2afy-deletion on the LSC pool, we performed limiting dilution assays. When transplanting 1x105, 1x104, 1x103 and 1x102 H2afy+/+ or -/- leukemic cells into sub-lethally irradiated recipients, LGMP frequency in H2afy-deficient cells was significantly reduced (1/1 vs. 1/29928; p=1.25e-19). The reduction of LGMP frequency correlated with reduced plating efficiency and increased differentiation. Consistently, primary human AML cells showed impaired colony forming capacity following H2afy deletion (mean colony number NT-control: 60.0±17.1 vs. H2afy-KD: 30.1±8.9; p=0.0023). To assess for the impact of H2afy deletion on normal HSPCs, we performed competitive repopulation assays. Immunophenotypic abundance of HSPC in H2afy+/+ vs. -/- animals revealed comparable numbers in all relevant subpopulations. Serial competitive transplantation of H2afy+/+ and -/- BMC into primary and secondary recipient animals showed no competitive disadvantage or lack of self-renewal capacity of H2afy-/- cells. Likewise, colony formation of healthy human donor CD34+ cells was not impaired following depletion of H2AFY by RNAi. Transcriptome analysis of LGMP populations indicated increased of myeloid cell development and loss of LSC signatures in H2afy-deficient LGMPs. Likewise, ATAC-seq of H2afy+/+ and -/- LGMP population revealed profound changes in the chromatin structure and a strong correlation between chromatin accessibility and mRNA expression. Together, our results provide first evidence for a critical function of H2afy (macroH2A1) in models of AML while being dispensable for normal HSPC function. Experiments to assess for the mechanistic consequences of H2afy deletion on chromatin are currently under way.

A giant virus genome is densely packaged by stable nucleosomes within virions

The two doublet histones of Marseillevirus are distantly related to the four eukaryotic core histones and wrap 121 base pairs of DNA to form remarkably similar nucleosomes. By permeabilizing Marseillevirus virions and performing genome-wide nuclease digestion, chemical cleavage, and mass spectrometry assays, we find that the higher-order organization of Marseillevirus chromatin fundamentally differs from that of eukaryotes. Marseillevirus nucleosomes fully protect DNA within virions as closely abutted 121-bp DNA-wrapped cores without linker DNA or phasing along genes. Likewise, we observed that nucleosomes reconstituted onto multi-copy tandem repeats of a nucleosome-positioning sequence are tightly packed. Dense promiscuous packing of fully wrapped nucleosomes rather than "beads on a string" with genic punctuation represents a distinct mode of DNA packaging by histones. We suggest that doublet histones have evolved for viral genome protection and may resemble an early stage of histone differentiation leading to the eukaryotic octameric nucleosome.

Extracellular histones as damage-associated molecular patterns in neuroinflammatory responses.

The four core histones H2A, H2B, H3, H4, and the linker histone H1 primarily bind DNA and regulate gene expression within the nucleus. Evidence collected mainly from the peripheral tissues illustrates that histones can be released into the extracellular space by activated or damaged cells. In this article, we first summarize the innate immune-modulatory properties of extracellular histones and histone-containing complexes, such as nucleosomes, and neutrophil extracellular traps (NETs), described inperipheral tissues. There, histones act as damage-associated molecular patterns (DAMPs), which are a class of endogenous molecules that trigger immune responses by interacting directly with the cellular membranes andactivating pattern recognition receptors (PRRs), suchas toll-like receptors (TLR) 2, 4, 9 and the receptor foradvanced glycation end-products (RAGE). We then focuson the available evidence implicating extracellular histones as DAMPs of the central nervous system (CNS). It is becoming evident that histones are present in the brain parenchyma after crossing the blood-brain barrier (BBB) or being released by several types of brain cells, including neurons, microglia, and astrocytes. However, studies on the DAMP-like effects of histones on CNS cells are limited. For example, TLR4 is the only known molecular target of CNS extracellular histones and their interactions with other PRRs expressed by brain cells have not been observed. Nevertheless, extracellular histones are implicated in thepathogenesis of a variety of neurological disorders characterized by sterile neuroinflammation; therefore, detailed studies on the role these proteins and their complexes play in these pathologies could identify novel therapeutic targets.

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.

Accurate Prediction of Epigenetic Multi-Targets with Graph Neural Network-Based Feature Extraction.

Epigenetics, referring to genetic modifications that change gene expression, but which are not encoded in DNA, has been shown to be related to oncology, with the potential to influence associated treatments. As such, epigenetic drugs comprise an important new field in cancer therapy; however, drug development is a high-cost and time-consuming procedure. Different epigenetic modifications, such as mutations in DNA methyltransferase and somatic mutations in core histone genes that lead to a global loss of the histone modifications, have innumerable relationships. In this article, we propose a graph neural network-based model for the extraction of molecular features, thus reducing the computational requirements. Through integration with a popular and efficient supervised learner, our model achieves higher prediction accuracy in both single- and multi-target tasks and can determine the pleiotropy associated with drugs, providing theoretical support for drug combination and discovery research.

Structural insights into acetylated histone ligand recognition by the BDP1 bromodomain of Plasmodium falciparum

Plasmodium falciparum requires a two-host system, moving between Anopheles mosquito and humans, to complete its life cycle. To overcome such dynamic growth conditions its histones undergo various post-translational modifications to regulate gene expression. The P. falciparum Bromodomain Protein 1 (PfBDP1) has been shown to interact with acetylated lysine modifications on histone H3 to regulate the expression of invasion-related genes. Here, we investigated the ability of the PfBDP1 bromodomain to interact with acetyllsyine modifications on additional core and variant histones. A crystal structure of the PfBDP1 bromodomain (PfBDP1-BRD) reveals it contains the conserved bromodomain fold, but our comparative analysis between the PfBDP1-BRD and human bromodomain families indicates it has a unique binding mechanism. Solution NMR spectroscopy and ITC binding assays carried out with acetylated histone ligands demonstrate that it preferentially recognizes tetra-acetylated histone H4, and we detected weaker interactions with multi-acetylated H2A.Z in addition to the previously reported interactions with acetylated histone H3. Our findings indicate PfBDP1 may play additional roles in the P. falciparum life cycle, and the distinctive features of its bromodomain binding pocket could be leveraged for the development of new therapeutic agents to help overcome the continuously evolving resistance of P. falciparum against currently available drugs.

H3K27me3 in Diffuse Midline Glioma and Epithelial Ovarian Cancer: Opposing Epigenetic Changes Leading to the Same Poor Outcomes.

Histone post-translational modifications modulate gene expression through epigenetic gene regulation. The core histone H3 family members, H3.1, H3.2, and H3.3, play a central role in epigenetics. H3 histones can acquire many post-translational modifications, including the trimethylation of H3K27 (H3K27me3), which represses transcription. Triple methylation of H3K27 is performed by the histone methyltransferase Enhancer of Zeste Homologue 2 (EZH2), a component of the Polycomb Repressive Complex 2. Both global increases and decreases in H3K27me3 have been implicated in a wide range of cancer types. Here, we explore how opposing changes in H3K27me3 contribute to cancer by highlighting its role in two vastly different cancer types; (1) a form of glioma known as diffuse midline glioma H3K27-altered and (2) epithelial ovarian cancer. These two cancers vary widely in the age of onset, sex, associated mutations, and cell and organ type. However, both diffuse midline glioma and ovarian cancer have dysregulation of H3K27 methylation, triggering changes to the cancer cell transcriptome. In diffuse midline glioma, the loss of H3K27 methylation is a primary driving factor in tumorigenesis that promotes glial cell stemness and silences tumor suppressor genes. Conversely, hypermethylation of H3K27 occurs in late-stage epithelial ovarian cancer, which promotes tumor vascularization and tumor cell migration. By using each cancer type as a case study, this review emphasizes the importance of H3K27me3 in cancer while demonstrating that the mechanisms of histone H3 modification and subsequent gene expression changes are not a one-size-fits-all across cancer types.

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.

Research Status of Death Domain-associated Protein

Overexpression of death domain-associated protein (DAXX) is a common feature of many cancers, and it structurally has binding sites for many interactors (eg, ARTX, HDAC3). According to functional studies, DAXX interacts with a number of DNA-binding transcription factors (TFs), epigenetic regulators, core histones, and proteins involved in chromatin to induce apoptosis via the extrinsic death receptor pathway. Functions as a co-activator or transcriptional inhibitor of cobalt diplus to regulate gene expression. Current studies have found that DAXX is expressed upregulated in ovarian cancer. Additionally, research has demonstrated that DAXX increases the tumorigenicity of prostate cancer by preventing autophagy pathways. In contrast, patients with pancreatic neuroendocrine tumors have lower survival rates when their DAXX is lost. A powerful breast tumor-initiating cell (TIC) inhibitor, DAXX also inhibits the production of pluripotent and EMT genes via promoters that may bind to pluripotent TIC-related genes. As a result, DAXX has powerful carcinogenic properties and potential new therapeutic targets. This article will introduce the protein from the aspects of DAXX's structure, function, and relationship with cancer.

Mapping the modification of histones by the myeloperoxidase-derived oxidant hypochlorous acid (HOCl)

Histones are critical for the packaging of nuclear DNA and chromatin assembly, which is facilitated by the high abundance of Lys and Arg residues within these proteins. These residues are also the site of a range of post-translational modifications, which influence the regulatory function of histones. Histones are also present in the extracellular environment, following release by various pathways, particularly neutrophil extracellular traps (NETs). NETs contain myeloperoxidase, which retains its enzymatic activity and produces hypochlorous acid (HOCl). This suggests that histones could be targets for HOCl under conditions where aberrant NET release is prevalent, such as chronic inflammation. In this study, we examine the reactivity of HOCl with a mixture of linker (H1) and core (H2A, H2B, H3 and H4) histones. HOCl modified the histones in a dose- and time-dependent manner, resulting in structural changes to the proteins and the formation of a range of post-translational modification products. N-Chloramines are major products following exposure of the histones to HOCl and decompose over 24 h forming Lys nitriles and carbonyls (aminoadipic semialdehydes). Chlorination and dichlorination of Tyr, but not Trp residues, is also observed. Met sulfoxide and Met sulfones are formed, though these oxidation products are also detected albeit at a lower extent, in the non-treated histones. Evidence for histone fragmentation and aggregation was also obtained. These results could have implications for the development of chronic inflammatory diseases, given the key role of Lys residues in regulating histone function.

The Effects of Histone H2B Ubiquitylations on the Nucleosome Structure and Internucleosomal Interactions.

Eukaryotic gene compaction takes place at multiple levels to package DNA to chromatin and chromosomes. Two of the most fundamental levels of DNA packaging are at the nucleosome and dinucleosome stacks. The nucleosome is the basic gene-packing unit and is composed of DNA wrapped around a histone core. Nucleosomes stack with one another for further compaction of DNA. The first stacking step leads to dinucleosome formation, which is driven by internucleosomal interactions between various parts of two nucleosomes. Histone proteins are rich targets for post-translational modifications, some of which affect the structure of the nucleosome and the interactions between nucleosomes. These effects are often implicated in the regulation of various genomic transactions. In particular, histone H2B ubiquitylation has been associated with facilitated transcription and hexasome formation. Here, we employed semi-synthetically ubiquitylated histone H2B and single-molecule FRET to investigate the effects of H2B ubiquitylations at lysine 34 (H2BK34) and lysine 120 (H2BK120) on the structure of the nucleosome and the interactions between two nucleosomes. Our results suggest that H2BK34 ubiquitylation widens the DNA gyre gap in the nucleosome and stabilizes long- and short-range internucleosomal interactions while H2BK120 ubiquitylation does not affect the nucleosome structure or internucleosomal interactions. These results suggest potential roles for H2B ubiquitylations in facilitated transcription and hexasome formation while maintaining the structural integrity of chromatin.

Global analysis of lysine 2-hydroxyisobutyrylation during Fusarium graminearum infection in maize.

Proteins post-translational modification (PTMs) is necessary in the whole life process of organisms. Among them, lysine 2-hydroxyisobutyrylation (Khib) plays an important role in protein synthesis, transcriptional regulation, and cell metabolism. Khib is a newly identified PTM in several plant species. However, the function of Khib in maize was unclear. In this study, western blotting results showed that Khib modification level increased significantly after Fusarium graminearum infection, and 2,066 Khib modified sites on 728 proteins were identified in maize, among which 24 Khib sites occurred on core histones. Subcellular localization results showed that these Khib modified proteins were localized in cytoplasm, chloroplast, and nucleus. Then, comparative proteomic analysis of the defense response to F. graminearum infection showed that Khib modification participated in plant resistance to pathogen infection by regulating glycolysis, TCA cycle, protein synthesis, peroxisome, and secondary metabolic processes, such as benzoxazinoid biosynthesis, phenylpropanoid biosynthesis, jasmonic acid synthesis, and tyrosine and tryptophan biosynthesis. In addition, we also demonstrated that lysine 2-hydroxyisobutyrylation sites on histones were involved in the gene expression of pathogenesis-related proteins. Our results provide a new perspective for the study of plant disease resistance, and had directive significance of maize disease resistance for molecular breeding.

Structure of IMPORTIN-4 bound to the H3–H4–ASF1 histone–histone chaperone complex

IMPORTIN-4, the primary nuclear import receptor of core histones H3 and H4, binds the H3-H4 dimer and histone chaperone ASF1 prior to nuclear import. However, how H3-H3-ASF1 is recognized for transport cannot be explained by available crystal structures of IMPORTIN-4-histone tail peptide complexes. Our 3.5-Å IMPORTIN-4-H3-H4-ASF1 cryoelectron microscopy structure reveals the full nuclear import complex and shows a binding mode different from suggested by previous structures. The N-terminal half of IMPORTIN-4 clamps the globular H3-H4 domain and H3 αN helix, while its C-terminal half binds the H3 N-terminal tail weakly; tail contribution to binding energy is negligible. ASF1 binds H3-H4 without contacting IMPORTIN-4. Together, ASF1 and IMPORTIN-4 shield nucleosomal H3-H4 surfaces to chaperone and import it into the nucleus where RanGTP binds IMPORTIN-4, causing large conformational changes to release H3-H4-ASF1. This work explains how full-length H3-H4 binds IMPORTIN-4 in the cytoplasm and how it is released in the nucleus.