Core Histones Research Articles

Cryo-EM Structures of Chromatosomes Containing Human Linker Histone Variants

Linker and core histones together condense genomic DNA by forming the chromatosome, the structural unit of metazoan chromatin. There are 11 linker histone variants in human but no high-resolution structures of chromatosomes are available. Aided by a single-chain antibody fragment, we determined the cryo-EM structures of three chromatosomes containing different linker histone variants at near atomic resolution. In these structures, the globular domains of the linker histones bind to the nucleosome dyad, but the linker DNAs displayed different conformations. We observed no well-defined densities for linker histones tails but NMR studies showed that many residues in their C-terminal tails were not observable, suggesting that the C-terminal tails likely interact with the linker DNA dynamically. Our study provides insights into the structures and dynamics of chromatosomes, which have implications for understanding the roles of different linker histone variants in chromatin structure and function.

DNA Sequence and Histone Core Composition Determine the Unwrapping Pathways in Nucleosomes

Interactions with histone core proteins in the cell efficiently store DNA and regulate access by partner molecules. In this process multiple histone core variants interact with a myriad of diverse sequences of DNA. Understanding how these interactions are influenced by histone composition and DNA sequence is crucial for unraveling their regulatory and packaging mechanisms. We use small angle X-ray scattering with contrast variation (CV-SAXS) to measure DNA unwrapping from nucleosomes as they are electrostatically destabilized by salt. This process allows many different unwrapping states to become accessible. The unwrapping states that are sampled in any given condition are determined from the scattering profiles using an ensemble optimization method. With this method we are able to map the unfolding landscape from fully wrapped to fully unwrapped, allowing the protein-nucleic acid interactions to be probed. We report on the results of multiple sequences and histone variants, including both natural and synthetic DNA sequences.

Histone-DNA Interactions in the Archaeon Methanocaldococcus Jannaschii

The requirement for DNA compaction is universal across all domains of life. Although the histone proteins of Archaea and Eukaryotes have low sequence homology, the core histone fold is well-conserved and structures are virtually identical. However, small structural differences mean that Archaeal nucleosomes might assemble into a continuous solenoid-like fiber: the ‘hypernucleosome’. Given that M. jannaschii thrives at 80oC and its genomic DNA is positively supercoiled (the opposite to mesophiles like us), the mechanism of DNA packaging may show interesting adaptations and diversity that will inform us about the biophysics of chromatin in general. Here, we have used magnetic tweezers to control the supercoiling of DNA while quantifying the kinetics and energetics of DNA-histone binding, wrapping and compaction. Using this technique, we have investigated the properties of M. jannaschii chromatin with its most abundant histone protein “A3”. We propose a model for the force-extension properties of Archaeal chromatin, analogous to that for Eukaryotic chromatin, and demonstrate that the force-extension properties are dependent upon protein concentration. We find that, under moderate load, histone binding confers stability to DNA by preventing base-pair unstacking when negative supercoiling is applied, and the chromatin fiber shows a sharp buckling transition between both positive and negative supercoiling regimes. At high load (>5pN), the chromatin fiber is disrupted and the force-extension diagram returns to the worm-like chain, typical of naked DNA. Our model suggests a mechanism by which DNA processing enzymes may shunt or displace nucleosomes, obviating the need for active chromatin remodeling enzymes.

Unraveling the Nucleosome through Microscopic Simulations

The DNA of higher organisms associates with histone proteins to organize into an array of nucleosomes. The nucleosome, however, is not one monolithic structure, but rather, refers to an ensemble of assemblies that vary in the chemical modifications present, histone composition, and surrounding DNA sequence. Here, we connect computation with experimental efforts to reveal the effects of acetylation on the H4 histone tail, the impact of a centromere-specific variant on histone assembly structure and dynamics, and the influence of nucleotide sequence on nucleosome breathing. We discover increasing levels of acetylation introduce greater transient order to the H4 histone tail with the mono-acetylation of lysine 16 causing specific structural effects; replacing histone H3 with centromere-specific CENP-A confers enhanced flexibility to dimers and entire nucleosomes, but, interestingly, more rigidity to tetramers; and lastly, that DNA sequences with greater CG content form more stable nucleosomes, undergoing fewer events in which the outer stretches of DNA spontaneously detach and reattach to the histone core.

Dynamics of the nucleosomal histone H3 N-terminal tail revealed by high precision single-molecule FRET.

Chromatin compaction and gene accessibility are orchestrated by assembly and disassembly of nucleosomes. Although the disassembly process was widely studied, little is known about the structure and dynamics of the disordered histone tails, which play a pivotal role for nucleosome integrity. This is a gap filling experimental FRET study from the perspective of the histone H3 N-terminal tail (H3NtT) of reconstituted mononucleosomes. By systematic variation of the labeling positions we monitored the motions of the H3NtT relative to the dyad axis and linker DNA. Single-molecule FRET unveiled that H3NtTs do not diffuse freely but follow the DNA motions with multiple interaction modes with certain permitted dynamic transitions in the μs to ms time range. We also demonstrate that the H3NtT can allosterically sense charge-modifying mutations within the histone core (helix α3 of histone H2A (R81E/R88E)) resulting in increased dynamic transitions and lower rate constants. Those results complement our earlier model on the NaCl induced nucleosome disassembly as changes in H3NtT configurations coincide with two major steps: unwrapping of one linker DNA and weakening of the internal DNA - histone interactions on the other side. This emphasizes the contribution of the H3NtT to the fine-tuned equilibrium between overall nucleosome stability and DNA accessibility.

Nucleus-specific linker histones Hho1 and Mlh1 form distinct protein interactions during growth, starvation and development in Tetrahymena thermophila

Chromatin organization influences most aspects of gene expression regulation. The linker histone H1, along with the core histones, is a key component of eukaryotic chromatin. Despite its critical roles in chromatin structure and function and gene regulation, studies regarding the H1 protein-protein interaction networks, particularly outside of Opisthokonts, are limited. The nuclear dimorphic ciliate protozoan Tetrahymena thermophila encodes two distinct nucleus-specific linker histones, macronuclear Hho1 and micronuclear Mlh1. We used a comparative proteomics approach to identify the Hho1 and Mlh1 protein-protein interaction networks in Tetrahymena during growth, starvation, and sexual development. Affinity purification followed by mass spectrometry analysis of the Hho1 and Mlh1 proteins revealed a non-overlapping set of co-purifying proteins suggesting that Tetrahymena nucleus-specific linker histones are subject to distinct regulatory pathways. Furthermore, we found that linker histones interact with distinct proteins under the different stages of the Tetrahymena life cycle. Hho1 and Mlh1 co-purified with several Tetrahymena-specific as well as conserved interacting partners involved in chromatin structure and function and other important cellular pathways. Our results suggest that nucleus-specific linker histones might be subject to nucleus-specific regulatory pathways and are dynamically regulated under different stages of the Tetrahymena life cycle.

A chromatin scaffold for DNA damage recognition: how histone methyltransferases prime nucleosomes for repair of ultraviolet light-induced lesions.

The excision of mutagenic DNA adducts by the nucleotide excision repair (NER) pathway is essential for genome stability, which is key to avoiding genetic diseases, premature aging, cancer and neurologic disorders. Due to the need to process an extraordinarily high damage density embedded in the nucleosome landscape of chromatin, NER activity provides a unique functional caliper to understand how histone modifiers modulate DNA damage responses. At least three distinct lysine methyltransferases (KMTs) targeting histones have been shown to facilitate the detection of ultraviolet (UV) light-induced DNA lesions in the difficult to access DNA wrapped around histones in nucleosomes. By methylating core histones, these KMTs generate docking sites for DNA damage recognition factors before the chromatin structure is ultimately relaxed and the offending lesions are effectively excised. In view of their function in priming nucleosomes for DNA repair, mutations of genes coding for these KMTs are expected to cause the accumulation of DNA damage promoting cancer and other chronic diseases. Research on the question of how KMTs modulate DNA repair might pave the way to the development of pharmacologic agents for novel therapeutic strategies.

The role of histone tails in nucleosome stability: An electrostatic perspective

We propose a methodology for the study of protein-DNA electrostatic interactions and apply it to clarify the effect of histone tails in nucleosomes. This method can be used to correlate electrostatic interactions to structural and functional features of protein-DNA systems, and can be combined with coarse-grained representations. In particular, we focus on the electrostatic field and resulting forces acting on the DNA. We investigate the electrostatic origins of effects such as different stages in DNA unwrapping, nucleosome destabilization upon histone tail truncation, and the role of specific arginines and lysines undergoing Post-Translational Modifications. We find that the positioning of the histone tails can oppose the attractive pull of the histone core, locally deform the DNA, and tune DNA unwrapping. Small conformational variations in the often overlooked H2A C-terminal tails had significant electrostatic repercussions near the DNA entry and exit sites. The H2A N-terminal tail exerts attractive electrostatic forces towards the histone core in positions where Polymerase II halts its progress. We validate our results with comparisons to previous experimental and computational observations.

Linker histone H1.5 is an underestimated factor in differentiation and carcinogenesis.

Human histone H1.5, in mice called H1b, belongs to the family of linker histones (H1), which are key players in chromatin organization. These proteins sit on top of nucleosomes, in part to stabilize them, and recruit core histone modifying enzymes. Through subtype-specific deposition patterns and numerous post-translational modifications, they fine-tune gene expression and chromatin architecture, and help to control cell fate and homeostasis. However, even though it is increasingly implicated in mammalian development, H1.5 has not received as much research attention as its relatives. Recent studies have focused on its prognostic value in cancer patients and its contribution to tumorigenesis through specific molecular mechanisms. However, many functions of H1.5 are still poorly understood. In this review, we will summarize what is currently known about H1.5 and its function in cell differentiation and carcinogenesis. We will suggest key experiments that are required to understand the molecular network, in which H1.5 is embedded. These experiments will advance our understanding of the epigenetic reprogramming occurring in developmental and carcinogenic processes.

Remodeling of the H3 nucleosomal landscape during mouse aging

In multi-cellular organisms, the control of gene expression is key not only for development, but also for adult cellular homeostasis, and deregulation of gene expression correlates with aging. A key layer in the study of gene regulation mechanisms lies at the level of chromatin: cellular chromatin states (i.e. the ‘epigenome’) can tune transcriptional profiles, and, in line with the prevalence of transcriptional alterations with aging, accumulating evidence suggests that the chromatin landscape is altered with aging across cell types and species. However, although alterations in the chromatin make-up of cells are considered to be a hallmark of aging, little is known of the genomic loci that are specifically affected by age-related chromatin state remodeling and of their biological significance. Here, we report the analysis of genome-wide profiles of core histone H3 occupancy in aging male mouse tissues (i.e. heart, liver, cerebellum and olfactory bulb) and primary cultures of neural stem cells. We find that, although no drastic changes in H3 levels are observed, local changes in H3 occupancy occur with aging across tissues and cells with both regions of increased or decreased occupancy. These changes are compatible with a general increase in chromatin accessibility at pro-inflammatory genes and may thus mechanistically underlie known shift in gene expression programs during aging.

Reprogramming of H3K9bhb at Regulatory Elements is an Epigenetic Feature of Fasting in the Small Intestine

β-hydroxybutyrate (β-OHB) provides an essential metabolic energy source during fasting and functions as an epigenetic regulator by lysine β-hydroxybutyrylation (Kbhb) modification of core histones H3 and H4. We report that H3K9bhb, but not H4K12bhb, becomes highly enriched at proximal promoters of genes regulating lipolytic and ketogenic metabolic programs in small intestinal (SI) crypts during fasting and these pathways are transcriptionally activated in Lgr5 + SI stem cells. Similar Kbhb enrichment is observed in Lgr5 + stem cell-enriched epithelial spheroids treated with β-OHB in vitro. Combinatorial chromatin state analysis reveals that H3K9bhb enrichment is associated with active chromatin states and that fasting enriches for an H3K9bhb–H3K27ac signature at active metabolic gene promoters and distal DNA enhancer elements. Intestinal knockout of Hmgcs2 results in a marked loss of H3K9bhb associated fasting-induced gene programs suggesting that local production of β-OHB is responsible for epigenetic reprogramming. We conclude that specific modulation of H3K9bhb in SI crypts is a key epigenetic event in response to fasting.

Liquid-Liquid Phase Separation of Histone Proteins in Cells: Role in Chromatin Organization

Liquid-liquid phase separation (LLPS) of proteins and nucleic acids has emerged as an important phenomenon in membraneless intracellular organization. We demonstrate that the linker histone H1 condenses into liquid-like droplets in the nuclei of HeLa cells. The droplets, observed during the interphase of the cell cycle, are colocalized with DNA-dense regions indicative of heterochromatin. In vitro, H1 readily undergoes LLPS with both DNA and nucleosomes of varying lengths but does not phase separate in the absence of DNA. The nucleosome core particle maintains its structural integrity inside the droplets, as demonstrated by FRET. Unexpectedly, H2A also forms droplets in the presence of DNA and nucleosomes in vitro, whereas the other core histones precipitate. The phase diagram of H1 with nucleosomes is invariant to the nucleosome length at physiological salt concentration, indicating that H1 is capable of partitioning large segments of DNA into liquid-like droplets. Of the proteins tested (H1, core histones, and the heterochromatin protein HP1α), this property is unique to H1. In addition, free nucleotides promote droplet formation of H1 nucleosome in a nucleotide-dependent manner, with droplet formation being most favorable with ATP. Although LLPS of HP1α is known to contribute to the organization of heterochromatin, our results indicate that H1 also plays a role. Based on our study, we propose that H1 and DNA act as scaffolds for phase-separated heterochromatin domains.

Hdac3, Setdb1, and Kap1 mark H3K9me3/H3K14ac bivalent regions in young and aged liver.

Post‐translational modifications of histone tails play a crucial role in gene regulation. Here, we performed chromatin profiling by quantitative targeted mass spectrometry to assess all possible modifications of the core histones. We identified a bivalent combination, a dually marked H3K9me3/H3K14ac modification in the liver, that is significantly decreased in old hepatocytes. Subsequent sequential ChIP‐Seq identified dually marked single nucleosome regions, with reduced number of sites and decreased signal in old livers, confirming mass spectrometry results. We detected H3K9me3 and H3K14ac bulk ChIP‐Seq signal in reChIP nucleosome regions, suggesting a correlation between H3K9me3/H3K14ac bulk bivalent genomic regions and dually marked single nucleosomes. Histone H3K9 deacetylase Hdac3, as well as H3K9 methyltransferase Setdb1, found in complex Kap1, occupied both bulk and single nucleosome bivalent regions in both young and old livers, correlating to presence of H3K9me3. Expression of genes associated with bivalent regions in young liver, including those regulating cholesterol secretion and triglyceride synthesis, is upregulated in old liver once the bivalency is lost. Hence, H3K9me3/H3K14ac dually marked regions define a poised inactive state that is resolved with loss of one or both of the chromatin marks, which subsequently leads to change in gene expression.

Combination of a novel microtubule inhibitor MBRI-001 and gemcitabine synergistically induces cell apoptosis by increasing DNA damage in pancreatic cancer cell lines.

Pancreatic cancer (PC) is a highly malignant cancer with poor prognosis. Although gemcitabine (GEM; 2',2'-difluoro-deoxycytidine) has been used as the first-line chemotherapeutic agent in PC treatment for decades, its limited efficacy remains a significant clinical issue, which may be resolved by GEM combination therapy. In this study, we aimed to investigate the anti-tumor effects of MBRI-001 in combination with GEM in BxPC-3 and MIA PaCa-2 human PC cell lines. In vitro and in vivo results indicate that MBRI-001 showed synergistic activity with GEM. GEM induced apoptosis by increasing DNA damage (phosphorylated core histone protein H2AX (γ-H2AX)), MBRI-001 activated mitochondrial-apoptotic pathway (cleaved poly-ADP ribose polymerase (PARP)). Thus, the combination of the two intensified both apoptosis and DNA damage and showed significantly superior anti-tumor activity compared to each agent alone. The adoption of combination of MBRI-001 with GEM may be beneficial as they act synergistically and thus, can be a potential therapeutic choice for improving the prognosis of PC patients in the future.

HECTD1 promotes base excision repair in nucleosomes through chromatin remodelling.

Base excision repair (BER) is the major cellular DNA repair pathway that recognises and excises damaged DNA bases to help maintain genome stability. Whilst the major enzymes and mechanisms co-ordinating BER are well known, the process of BER in chromatin where DNA is compacted with histones, remains unclear. Using reconstituted mononucleosomes containing a site-specific synthetic abasic site (tetrahydrofuran, THF), we demonstrate that the DNA damage is less efficiently incised by recombinant AP endonuclease 1 (APE1) when the DNA backbone is facing the histone core (THF-in) compared to that orientated away (THF-out). However, when utilizing HeLa whole cell extracts, the difference in incision of THF-in versus THF-out is less pronounced suggesting the presence of chromatin remodelling factors that stimulate THF accessibility to APE1. We subsequently purified an activity from HeLa cell extracts and identify this as the E3 ubiquitin ligase, HECTD1. We demonstrate that a recombinant truncated form of HECTD1 can stimulate incision of THF-in by APE1 in vitro by histone ubiquitylation, and that siRNA-mediated depletion of HECTD1 leads to deficiencies in DNA damage repair and decreased cell survival following x-ray irradiation, particularly in normal fibroblasts. Thus, we have now identified HECTD1 as an important factor in promoting BER in chromatin.

Validation of the Genetically-Defined DLBCL Subtypes and Generation of a Parsimonious Probabilistic Classifier

Diffuse large B-cell lymphoma (DLBCL) is a clinically and molecularly heterogeneous disease with recognized transcriptional subtypes associated with normal cells of origin, activated B-cell (ABC) and germinal center B-cell (GCB) tumors. Emerging data suggested that additional heterogeneity existed, prompting us to comprehensively characterize genomic signatures of 304 newly diagnosed DLBCLs from patients treated with state-of-the-art therapy. We integrated recurrent mutations, somatic copy number alterations (SCNAs) and structural variants (SVs) and identified 5 genetically distinct DLBCL clusters (C1- C5 DLBCLs; Chapuy, Stewart, Dunford, et al. Nat Med 2018). Specifically, we identified two genetically distinct ABC subtypes, including favorable-risk C1 DLBCLs with features of extrafollicular origin and alterations also seen in transformed marginal zone lymphomas (NOTCH2 and NF-κB pathway member mutations and BCL6 SVs). Unfavorable-risk C5 ABC DLBCLs harbored frequent 18q/BCL2 copy gain and co-occurring CD79B and MYD88L265P mutations. We also identified two genetically distinct GCB subtypes, including unfavorable-risk C3 DLBCLs with frequent BCL2 SVs, mutations in chromatin-modifying enzymes (CREBBP, MLL2, EZH2) and BCR/PI3K signaling pathway members (including inactivating PTEN mutations and copy loss). Favorable-risk C4 GCB DLBCLs had frequent mutations in core and linker histones and signaling intermediates (SGK1, BRAF and STAT3). Additionally, we identified an ABC/GCB-independent subtype, C2 DLBCLs, characterized by frequent bi-allelic TP53 inactivation, 9p21.23/CDKN2A copy loss and associated genomic instability reflected in recurrent SCNAs, increased genome doublings and a distinct outcome following induction therapy. A next step in utilizing the characterized genetic substructure was to confirm it in an independent series and develop a molecular classifier that allows prospective identification of C1-C5 DLBCLs. To this end, we accessed whole exome sequencing, copy number and SV data from a recent cohort of newly diagnosed DLBCLs (39 tumor-normal pairs, 462 tumor-only samples; Schmitz et al. NEJM 2018). All samples were re-analyzed using our mutational and SCNA pipelines and our newly generated tumor-only algorithm (Chapuy, Stewart, Dunford, et al. Nat Med 2018) to avoid batch effects and harmonize the datasets. SVs were used as reported. Purity and ploidy were inferred using ABSOLUTE and samples with missing data or low purity were removed. For the combined cohort (579 samples), we assessed our previously characterized 158 genetic drivers (Chapuy, Stewart, Dunford, et al. Nat Med 2018) and confirmed equal distribution of their marginal frequencies (R=0.88, p=1.5e-51), excluding batch effects. Next, we applied non-negative matrix factorization (NNF) consensus clustering to the combined dataset (158 genetic drivers vs. 579 tumors) and confirmed the C1-C5 DLBCL genetic clusters. Notably, tumors from both series contributed at comparable frequencies to the respective C1-C5 DLBCLs. We also noted an enrichment of the alternative genetic labels from Schmitz et al. in 3 of our C1-C5 DLBCL subtypes (B2N in C1 DLBCLs, p<0.0001; EZB in C3 DLBCLs, p<0.0001; MCD in C5 DLBCLs, p<0.0001). These data confirmed the identity of the C1-C5 DLBCL clusters in an independent cohort. Next, we developed a molecular classifier that prospectively identified C1-C5 DLBCLs using a minimum number of easy-to-measure features. The NMF-defined classes of the combined cohort were used as gold-standard training and validation datasets. We tested different models for classification and selected an artificial neural network approach which provides accurate classification of individual samples and well-calibrated confidence metrics. To minimize potential overtraining, we developed a reduced input feature set of the 22 most discriminating features, constructed confidence metrics for each sample and trained an ensemble of Feed-Forward Neural Networks via 10-fold cross validation. With this approach, our classifier had 84% accuracy for the total set and 94% accuracy for the high-confidence samples (70% of all samples). The newly developed parsimonious classifier will allow prospective identification of the independently confirmed C1-C5 DLBCL subtypes in newly diagnosed patients, a necessity for clinical application. Disclosures Getz: Pharmacyclics: Research Funding; IBM: Research Funding; MuTect, ABSOLTUE, MutSig and POLYSOLVER: Patents & Royalties: MuTect, ABSOLTUE, MutSig and POLYSOLVER. Shipp:BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck & Co.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees.

A Combination Strategy Targeting Enhancer Plasticity Exerts Synergistic Lethality Against Beti-Resistant Leukemia Cells

Primary and acquired drug resistance is a major challenge to achieving optimized clinical outcomes during cancer treatment which can arise from transcription reactivation, bypass and alteration during anticancer treatment [1-3]. Epigenetic dysregulation is emerging as a crucial component involved in drug resistance. Transcriptional adaptation during drug treatment is often mediated by inducible histone modifications, especially histone H3 lysine 27 acetylation (H3K27ac) at the distal enhancer elements, thus activating the transcription of drug resistance-associated genes [4-6]. BRD4 (bromodomain-containing protein 4), a member of the bromodomain and extra-terminal domain (BET) family, acts as a chromatin reader to regulate transcription by linking histone acetylation and core components of the transcriptional apparatus [7]. BET inhibitors (BETi), as exemplified by JQ1 and I-BET151, have been shown to suppress the growth of multiple types of tumor both in vitro and in vivo [8]. However, drug resistance associated with BETi becomes one of the major hurdles hampering the clinical applications of these promising drug candidates [8, 9]. Using BET inhibitor (BETi) resistant leukemia cells as a model system, we demonstrated herein that genome-wide enhancer remodeling played a pivotal role in driving therapeutic resistance via compensational re-expression of pro-survival genes. Capitalizing on CRISPR interference, we identified the second intron of IncRNA, PVT1, as a unique bona fidegained enhancer that restored MYCtranscription independent of BRD4 recruitment. A combined BETi and CDK7 inhibitor treatment abolished MYC transcription by impeding RNAPII loading without affecting PVT1-mediated chromatin looping at the MYClocus in BETi-resistant leukemia cells. Furthermore, recipient mice transferred with BETi-resistant murine MLL-AF9 AML cells receiving the combination treatment showed the most effective therapeutic outcomes, as characterized by prolonged overall survival and reduced tumor burdens in the spleen and bone marrow. Together, our findings have established the feasibility of targeting enhancer plasticity to overcome drug resistance associated with epigenetic therapies.

Unintegrated HIV-1 DNAs are loaded with core and linker histones and transcriptionally silenced

Upon delivery into the nucleus of the host cell, linear double-stranded retroviral DNAs are either integrated into the host genome to form the provirus or act as a target of the DNA damage response and become circularized. Little is known about the chromatinization status of the unintegrated retroviral DNAs of the human immunodeficiency virus type 1 (HIV-1). In this study, we used chromatin immunoprecipitation to investigate the nature of unintegrated HIV-1 DNAs and discovered that core histones, the histone variant H3.3, and H1 linker histones are all deposited onto extrachromosomal HIV-1 DNA. We performed a time-course analysis and determined that the loading of core and linker histones occurred early after virus application. H3.3 and H1 linker histones were also found to be loaded onto unintegrated DNAs of the Moloney murine leukemia virus. The unintegrated retroviral DNAs are potently silenced, and we provide evidence that the suppression of extrachromosomal HIV-1 DNA is histone-related. Unintegrated DNAs were marked by posttranslational histone modifications characteristic of transcriptionally inactive genes: high levels of H3K9 trimethylation and low levels of H3 acetylation. These findings reveal insights into the nature of unintegrated retroviral DNAs.

Quantitative Modeling of Nucleosome Unwrapping from Both Ends

In eukaryotic cells, DNA is packaged into chromatin where nucleosomes are the basic packaging unit. Important cellular processes including gene expression, DNA replication, and DNA repair require nucleosomal DNA to be unwrapped so that functional proteins can access their target sites, which otherwise are sterically occluded. A key question in this process is what the unwrapped conformations individual nucleosomes adopt within chromatin are. Here, we develop a concurrent nucleosome unwrapping model to address this question. We hypothesize that for a given end-to-end distance of the nucleosomal DNA, the nucleosomal DNA stochastically unwraps from the histone core from both ends independently and that this combination of unwrapping from both sides results in a significant increase in the average distance between the DNA extending from both sides of the nucleosomes. We test our model on recently published experiments using a DNA origami nanocaliper that quantifies nucleosome unwrapping and achieve good agreement between experiment and model prediction. We then investigate the DNA origami caliper distribution when attached to a hexasome (a nucleosome lacking an H2A/H2B dimer). A significant shift in the caliper angle distribution caused by the asymmetric structural features of the hexasome seen experimentally is consistent with the model. Our modeling approach may be more broadly useful to the interpretation of other studies of nucleosome dynamics, chromatin dynamics, and regulatory processes involving nucleosome unwrapping, as well as more generally to optimization of future DNA origami designs to probe mechanical properties of biomolecules.

Metabolic regulation of gene expression by histone lactylation.

The Warburg effect, originally describing augmented lactogenesis in cancer, is associated with diverse cellular processes such as angiogenesis, hypoxia, macrophage polarization, and T-cell activation. This phenomenon is intimately linked with multiple diseases including neoplasia, sepsis, and autoimmune diseases1,2. Lactate, a compound generated during Warburg effect, is widely known as an energy source and metabolic byproduct. However, its non-metabolic functions in physiology and disease remain unknown. Here we report lactate-derived histone lysine lactylation as a new epigenetic modification and demonstrate that histone lactylation directly stimulates gene transcription from chromatin. In total, we identify 28 lactylation sites on core histones in human and mouse cells. Hypoxia and bacterial challenges induce production of lactate through glycolysis that in turn serves as precursor for stimulating histone lactylation. Using bacterially exposed M1 macrophages as a model system, we demonstrate that histone lactylation has different temporal dynamics from acetylation. In the late phase of M1 macrophage polarization, elevated histone lactylation induces homeostatic genes involved in wound healing including arginase 1. Collectively, our results suggest the presence of an endogenous “lactate clock” in bacterially challenged M1 macrophages that turns on gene expression to promote homeostasis. Histone lactylation thus represents a new avenue for understanding the functions of lactate and its role in diverse pathophysiological conditions, including infection and cancer.