Integrative Modeling of 3D Genome Organization by Bayesian Molecular Dynamics Simulations with Hi-C Metainference.

Polymer modeling has been playing an increasingly important role in complementing 3D genome experiments, both to aid their interpretation and to reveal the underlying molecular mechanisms. This chapter illustrates an application of Hi-C metainference, a Bayesian approach to explore the 3D organization of a target genomic region by integrating experimental contact frequencies into a prior model of chromatin. The method reconstructs the conformational ensemble of the target locus by combining molecular dynamics simulation and Monte Carlo sampling from the posterior probability distribution given the data. Using prior chromatin models at both 1 kb and nucleosome resolution, we apply this approach to a 30 kb locus of mouse embryonic stem cells consisting of two well-defined domains linking several gene promoters together. Retaining the advantages of both physics-based and data-driven strategies, Hi-C metainference can provide an experimentally consistent representation of the system while at the same time retaining molecular details necessary to derive physical insights.

Molecular basis of global promoter sensing and nucleosome capture by the SWR1 chromatin remodeler.

The SWR1 chromatin remodeling complex is recruited to +1 nucleosomes downstream of transcription start sites of eukaryotic promoters, where it exchanges histone H2A for the specialized variant H2A.Z. Here, we use cryoelectron microscopy (cryo-EM) to resolve the structural basis of the SWR1 interaction with free DNA, revealing a distinct open conformation of the Swr1 ATPase that enables sliding from accessible DNA to nucleosomes. A complete structural model of the SWR1-nucleosome complex illustrates critical roles for Swc2 and Swc3 subunits in oriented nucleosome engagement by SWR1. Moreover, an extended DNA-binding α helix within the Swc3 subunit enables sensing of nucleosome linker length and is essential for SWR1-promoter-specific recruitment and activity. The previously unresolved N-SWR1 subcomplex forms a flexible extended structure, enabling multivalent recognition of acetylated histone tails by reader domains to further direct SWR1 toward the +1 nucleosome. Altogether, our findings provide a generalizable mechanism for promoter-specific targeting of chromatin and transcription complexes.

Visualizing, quantifying and mapping chromatin remodelers at work with single-molecule and single-cell imaging.

Chromatin remodeling, carried out by four major subfamilies of ATP-dependent remodeler complexes across eukaryotes, alleviates the topological challenge posed by nucleosomes to regulate genome access. Recently, single-molecule and single-cell imaging techniques have been widely employed to probe this crucial process, both in vitro and in cellulo. Herein, we provide an integrated account of key recent efforts that leverage these approaches to visualize, quantify and map chromatin remodelers at work, elucidating diverse aspects of the remodeling process in both space and time, including molecular mechanisms of DNA wrapping/unwrapping, nucleosome translocation and histone exchange, dynamics of chromatin binding/target search and their intranuclear organization into hotspots or phase condensates, as well as functional coupling with transcription. The mechanistic insights and quantitative parameters revealed shed light on a multi-modal yet shared landscape for regulating remodeling across molecular and cellular scales, and pave the way for further interrogating the implications of its misregulation in disease contexts.

Structural mechanism of HP1⍺-dependent transcriptional repression and chromatin compaction.

Heterochromatin protein 1 (HP1) plays a central role in establishing and maintaining constitutive heterochromatin. However, the mechanisms underlying HP1-nucleosome interactions and their contributions to heterochromatin functions remain elusive. Here, we present the cryoelectron microscopy (cryo-EM) structure of an HP1α dimer bound to an H2A.Z-nucleosome, revealing two distinct HP1α-nucleosome interfaces. The primary HP1α binding site is located at the N terminus of histone H3, specifically at the trimethylated lysine 9 (K9me3) region, while a secondary binding site is situated near histone H2B, close to nucleosome superhelical location 4 (SHL4). Our biochemical data further demonstrates that HP1α binding influences the dynamics of DNA on the nucleosome. It promotes DNA unwrapping near the nucleosome entry and exit sites while concurrently restricting DNA accessibility in the vicinity of SHL4. Our study offers a model for HP1α-mediated heterochromatin maintenance and gene silencing. It also sheds light on the H3K9me-independent role of HP1 in responding to DNA damage.

Analysis of the primary factors influencing donor derived cell-free DNA testing in kidney transplantation.

The donor-derived cell-free DNA (ddcfDNA) is found in the plasma and urine of kidney transplant recipients and displays notable potential in diagnosing rejection, specifically antibody-mediated rejection (ABMR). Nonetheless, the quantitative methods of ddcfDNA lacking standardization and diverse detection techniques can impact the test outcomes. Besides, both the fraction and absolute values of ddcfDNA have been reported as valuable markers for rejection diagnosis, but they carry distinct meanings and are special in various pathological conditions. Additionally, ddcfDNA is highly sensitive to kidney transplant injury. The various sampling times and combination with other diseases can indeed impact ddcfDNA detection values. This review comprehensively analyses the various factors affecting ddcfDNA detection in kidney transplantation, including the number of SNPs and sequencing depths. Furthermore, different pathological conditions, distinct sampling time points, and the presence of complex heterologous signals can influence ddcfDNA testing results in kidney transplantation. The review also provides insights into ddcfDNA testing on different platforms along with key considerations.

Targeting NETosis in Acute Brain Injury: A Systematic Review of Preclinical and Clinical Evidence.

Acute brain injury (ABI) remains one of the leading causes of death and disability world-wide. Its treatment is challenging due to the heterogeneity of the mechanisms involved and the variability among individuals. This systematic review aims at evaluating the impact of anti-histone treatments on outcomes in ABI patients and experimental animals and defining the trend of nucleosome levels in biological samples post injury. We performed a search in Pubmed/Medline and Embase databases for randomized controlled trials and cohort studies involving humans or experimental settings with various causes of ABI. We formulated the search using the PICO method, considering ABI patients or animal models as population (P), comparing pharmacological and non-pharmacological therapy targeting the nucleosome as Intervention (I) to standard of care or no treatment as Control (C). The outcome (O) was mortality or functional outcome in experimental animals and patients affected by ABI undergoing anti-NET treatments. We identified 28 studies from 1246 articles, of which 7 were experimental studies and 21 were human clinical studies. Among these studies, only four assessed the effect of anti-NET therapy on circulating markers. Three of them were preclinical and reported better outcome in the interventional arm compared to the control arm. All the studies observed a significant reduction in circulating NET-derived products. NETosis could be a target for new treatments. Monitoring NET markers in blood and cerebrospinal fluid might predict mortality and long-term outcomes. However, longitudinal studies and randomized controlled trials are warranted to fully evaluate their potential, as current evidence is limited.

PICKLE-mediated nucleosome condensing drives H3K27me3 spreading for the inheritance of Polycomb memory during differentiation.

Spreading of H3K27me3 is crucial for the maintenance of mitotically inheritable Polycomb-mediated chromatin silencing in animals and plants. However, how Polycomb repressive complex 2 (PRC2) accesses unmodified nucleosomes in spreading regions for spreading H3K27me3 remains unclear. Here, we show in Arabidopsis thaliana that the chromatin remodeler PICKLE (PKL) plays a specialized role in H3K27me3 spreading to safeguard cell identity during differentiation. PKL specifically localizes to H3K27me3 spreading regions but not to nucleation sites and physically associates with PRC2. Loss of PKL disrupts the occupancy of the PRC2 catalytic subunit CLF in spreading regions and leads to aberrant dedifferentiation. Nucleosome density increase endowed by the ATPase function of PKL ensures that unmodified nucleosomes are accessible to PRC2 catalytic activity for H3K27me3 spreading. Our findings demonstrate that PKL-dependent nucleosome compaction is critical for PRC2-mediated H3K27me3 read-and-write function in H3K27me3 spreading, thus revealing a mechanism by which repressive chromatin domains are established and propagated.

NuRD chromatin remodeling is required to repair exogenous DSBs in the Caenorhabditis elegans germline.

Organisms rely on coordinated networks of DNA repair pathways to protect genomes against toxic double-strand breaks (DSBs), particularly in germ cells. All repair mechanisms must successfully negotiate the local chromatin environment in order to access DNA. For example, nucleosomes can be repositioned by the highly conserved Nucleosome Remodeling and Deacetylase (NuRD) complex. In Caenorhabditis elegans, NuRD functions in the germline to repair DSBs - the loss of NuRD's ATPase subunit, LET-418/CHD4, prevents DSB resolution and therefore reduces fertility. In this study, we challenge germlines with exogenous DNA damage to better understand NuRD's role in repairing DSBs. We find that let-418 mutants are hypersensitive to cisplatin and hydroxyurea: exposure to either mutagen impedes DSB repair, generates aneuploid oocytes, and severely reduces fertility and embryonic survival. These defects resemble those seen when the Fanconi anemia (FA) DNA repair pathway is compromised, and we find that LET-418's activity is epistatic to that of the FA component FCD-2/FANCD2. We propose a model in which NuRD is recruited to the site of DNA lesions to remodel chromatin and allow access for FA pathway components. Together, these results implicate NuRD in the repair of both endogenous DSBs and exogenous DNA lesions to preserve genome integrity in developing germ cells.

Circulating nucleosomes as a potential cancer biomarker in dogs with splenic nodular lesions.

Splenic nodular lesions in dogs can be either benign or malignant. They might be discovered incidentally or, in case of rupture, they may lead to hemoabdomen. Nevertheless, splenectomy followed by histopathology is essential for diagnosis and to prevent rupture. Yet, this invasive procedure might be postponed for dogs with benign splenic nodular lesions. Conversely, owners may opt for euthanasia over surgery for malignancies with poor prognosis like hemangiosarcoma. Thus, anticipating diagnosis with non-invasive biomarkers is crucial for proper patient management. In this prospective study, plasma samples were collected from 66 dogs with histologically confirmed splenic nodular lesions. A canine-specific ELISA kit was applied to assess nucleosome concentration, with histopathology of the spleen serving as the gold standard. Nucleosome concentration was found to be significantly higher in dogs with malignant splenic nodular lesions, particularly in those with hemangiosarcoma and other malignancies. The presence of hemoabdomen, more prevalent in dogs with splenic malignancy, also resulted in increased plasmatic nucleosome concentrations. Plasma nucleosomes could serve as a biomarker for detecting malignant splenic nodular lesions in dogs. More research is needed to understand how nucleosome concentration relate to disease stage and prognosis in dogs with hemangiosarcoma.

Impacts of Nucleosome Positioning Elements and Pre-Assembled Chromatin States on Expression and Retention of Transgenes.

BACKGROUND/OBJECTIVES: Transgene applications, ranging from gene therapy to the development of stable cell lines and organisms, rely on maintaining the expression of transgenes. To date, the use of plasmid-based transgenes has been limited by the loss of their expression shortly after their delivery into the target cells. The short-lived expression of plasmid-based transgenes has been largely attributed to host-cell-mediated degradation and/or silencing of transgenes. The development of chromatin-based strategies for gene delivery has the potential to facilitate defining the requirements for establishing epigenetic states and to enhance transgene expression for numerous applications. METHODS: To assess the impact of "priming" plasmid-based transgenes to adopt accessible chromatin states to promote gene expression, nucleosome positioning elements were introduced at promoters of transgenes, and vectors were pre-assembled into nucleosomes containing unmodified histones or mutants mimicking constitutively acetylated states at residues 9 and 14 of histone H3 or residue 16 of histone H4 prior to their introduction into cells, then the transgene expression was monitored over time. RESULTS: DNA sequences capable of positioning nucleosomes could positively impact the expression of adjacent transgenes in a distance-dependent manner in the absence of their pre-assembly into chromatin. Intriguingly, the pre-assembly of plasmids into chromatin facilitated the prolonged expression of transgenes relative to plasmids that were not pre-packaged into chromatin. Interactions between pre-assembled chromatin states and nucleosome positioning-derived effects on expression were also assessed and, generally, nucleosome positioning played the predominant role in influencing gene expression relative to priming with hyperacetylated chromatin states. CONCLUSIONS: Strategies incorporating nucleosome positioning elements and the pre-assembly of plasmids into chromatin prior to nuclear delivery can modulate the expression of plasmid-based transgenes.

Resolution of transcription-induced hexasome-nucleosome complexes by Chd1 and FACT.

To maintain the nucleosome organization of transcribed genes, ATP-dependent chromatin remodelers collaborate with histone chaperones. Here, we show that at the 5' ends of yeast genes, RNA polymerase II (RNAPII) generates hexasomes that occur directly adjacent to nucleosomes. The resulting hexasome-nucleosome complexes are then resolved by Chd1. We present two cryoelectron microscopy (cryo-EM) structures of Chd1 bound to a hexasome-nucleosome complex before and after restoration of the missing inner H2A/H2B dimer by FACT. Chd1 uniquely interacts with the complex, positioning its ATPase domain to shift the hexasome away from the nucleosome. In the absence of the inner H2A/H2B dimer, its DNA-binding domain (DBD) packs against the ATPase domain, suggesting an inhibited state. Restoration of the dimer by FACT triggers a rearrangement that displaces the DBD and stimulates Chd1 remodeling. Our results demonstrate how chromatin remodelers interact with a complex nucleosome assembly and suggest how Chd1 and FACT jointly support transcription by RNAPII.

Structural and dynamic studies of chromatin by solid-state NMR spectroscopy.

Chromatin is a complex of DNA with histone proteins organized into nucleosomes that regulates genome accessibility and controls transcription, replication and repair by dynamically switching between open and compact states as a function of different parameters including histone post-translational modifications and interactions with chromatin modulators. Continuing advances in structural biology techniques including X-ray crystallography, cryo-electron microscopy and nuclear magnetic resonance (NMR) spectroscopy have facilitated studies of chromatin systems, in spite of challenges posed by their large size and dynamic nature, yielding important functional and mechanistic insights. In this review we highlight recent applications of magic angle spinning solid-state NMR - an emerging technique that is uniquely-suited toward providing atomistic information for rigid and flexible regions within biomacromolecular assemblies - to detailed characterization of structure, conformational dynamics and interactions for histone core and tail domains in condensed nucleosomes and oligonucleosome arrays mimicking chromatin at high densities characteristic of the cellular environment.

Studying large biomolecules as sedimented solutes with solid-state NMR.

Sedimentation solid-state NMR is a novel method for sample preparation in solid-state NMR (ssNMR) studies. It involves the sedimentation of soluble macromolecules such as large protein complexes. By utilizing ultra-high centrifugal forces, the molecules in solution are driven into a high-concentrated hydrogel, resulting in a sample suitable for solid-state NMR. This technique has the advantage of avoiding the need for chemical treatment, thus minimizing the loss of sample biological activity. Sediment ssNMR has been successfully applied to a variety of non-crystalline protein solids, significantly expanding the scope of solid-state NMR research. In theory, using this method, any biological macromolecule in solution can be transferred into a sedimented solute appropriate for solid-state NMR analysis. However, specialized equipment and careful handling are essential for effectively collecting and loading the sedimented solids to solid-state NMR rotors. To improve efficiency, we have designed a series of loading tools to achieve the loading process from the solution to the rotor in one step. In this paper, we illustrate the sample preparation process of sediment NMR using the H1.4-NCP167 complex, which consists of linker histone H1.4 and nucleosome core particle, as an example.

Anticancer Plant Secondary Metabolites Induce Linker Histone Depletion from Chromatin.

BACKGROUND: Many plant secondary metabolites (PSMs) were shown to intercalate into DNA helix or interact with DNA grooves. This may influence histone-DNA interactions changeing chromatin structure and genome functioning. METHODS: Nucleosome stability and linker histone H1.2, H1.4 and H1.5 localizations were studied in HeLa cells after the treatment with 15 PSMs, which are DNA-binders and possess anticancer activity according to published data. Chromatin remodeler CBL0137 was used as a control. Effects of PSMs were studied using fluorescent microscopy, flowcytometry, quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), western-blotting. RESULTS: We showed that 1-hour treatment with CBL0137 strongly inhibited DNA synthesis and caused intensive linker histone depletion consistent with nucleosome destabilization. None of PSMs caused nucleosome destabilization, while most of them demonstrated significant influence on linker histone localizations. In particular, cell treatment with 11 PSMs at non-toxic concentrations induced significant translocation of the histone H1.5 to nucleoli and most of PSMs caused depletion of the histones H1.2 and H1.4 from chromatin fraction. Curcumin, resveratrol, berberine, naringenin, and quercetin caused significant redistribution of all three variants of the studied linker histones showing some overlap of PSM effects on linker histone DNA-binding. We demonstrated that PSMs, which induced the most significant redistribution of the histone H1.5 (berberine, curcumin and naringenin), influence the proportion of cells synthesizing DNA, expressing or non-expressing cyclin B and influence cell cycle distribution. Berberine induction of H1.5 translocations to nucleoli was shown to occur independently on the phases of cell cycle (metaphase was not analyzed). CONCLUSIONS: For the first time we revealed PSM influence on linker histone location in cell nuclei that opens a new direction of PSM research as anticancer agents.

RNA polymerases reshape chromatin architecture and couple transcription on individual fibers.

RNA polymerases must initiate and pause within a complex chromatin environment, surrounded by nucleosomes and other transcriptional machinery. This environment creates a spatial arrangement along individual chromatin fibers ripe for both competition and coordination, yet these relationships remain largely unknown owing to the inherent limitations of traditional structural and sequencing methodologies. To address this, we employed long-read chromatin fiber sequencing (Fiber-seq) in Drosophila to visualize RNA polymerase (Pol) within its native chromatin context with single-molecule precision along up to 30 kb fibers. We demonstrate that Fiber-seq enables the identification of individual Pol II, nucleosome, and transcription factor footprints, revealing Pol II pausing-driven destabilization of downstream nucleosomes. Furthermore, we demonstrate pervasive direct distance-dependent transcriptional coupling between nearby Pol II genes, Pol III genes, and transcribed enhancers, modulated by local chromatin architecture. Overall, transcription initiation reshapes surrounding nucleosome architecture and couples nearby transcriptional machinery along individual chromatin fibers.

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

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

Composition and function of plant chromatin remodeling complexes.

ATP-dependent chromatin remodelers play a crucial role in modifying chromatin configuration by utilizing the energy of ATP hydrolysis. They are involved in various processes, including transcription, DNA replication, and maintaining genome stability. These remodeling remodelers usually form multi-subunit chromatin remodeling complexes in eukaryotes. In plants, chromatin remodeling complexes have diverse functions in regulating plant development and stress response. Recent studies have conducted extensive research on plant chromatin remodeling complexes. This review focuses on recent advances in the classification and composition of plant chromatin remodeling complexes, the protein-protein interactions within the complexes, their impact on chromatin configuration, and their interactions with chromatin modifications and transcription factors.

FACT mediates the depletion of macroH2A1.2 to expedite gene transcription.

The histone variant macroH2A is generally linked to transcriptionally inactive chromatin, but how macroH2A regulates chromatin structure and functions in the transcriptional process remains elusive. This study reveals that while the integration of human macroH2A1.2 into nucleosomes does not affect their stability or folding dynamics, it notably hinders the maintenance of facilitates chromatin transcription's (FACT's) function. We show that FACT effectively diminishes the stability of macroH2A1.2-nucleosomes and expedites their depletion subsequent to the initial unfolding process. Furthermore, we identify the residue S139 in macroH2A1.2 as a critical switch to modulate FACT's function in nucleosome maintenance. Genome-wide analyses demonstrate that FACT-mediated depletion of macroH2A-nucleosomes allows the correct localization of macroH2A, while the S139 mutation reshapes macroH2A distribution and influences stimulation-induced transcription and cellular response in macrophages. Our findings provide mechanistic insights into the intricate interplay between macroH2A and FACT at the nucleosome level and elucidate their collective role in transcriptional regulation and immune response of macrophages.

Structural insights into the cooperative nucleosome recognition and chromatin opening by FOXA1 and GATA4.

Mouse FOXA1 and GATA4 are prototypes of pioneer factors, initiating liver cell development by binding to the N1 nucleosome in the enhancer of the ALB1 gene. Using cryoelectron microscopy (cryo-EM), we determined the structures of the free N1 nucleosome and its complexes with FOXA1 and GATA4, both individually and in combination. We found that the DNA-binding domains of FOXA1 and GATA4 mainly recognize the linker DNA and an internal site in the nucleosome, respectively, whereas their intrinsically disordered regions interact with the acidic patch on histone H2A-H2B. FOXA1 efficiently enhances GATA4 binding by repositioning the N1 nucleosome. In vivo DNA editing and bioinformatics analyses suggest that the co-binding mode of FOXA1 and GATA4 plays important roles in regulating genes involved in liver cell functions. Our results reveal the mechanism whereby FOXA1 and GATA4 cooperatively bind to the nucleosome through nucleosome repositioning, opening chromatin by bending linker DNA and obstructing nucleosome packing.

Assessing the COX-2/PGE2 Ratio and Anti-Nucleosome Autoantibodies as Biomarkers of Autism Spectrum Disorders: Using Combined ROC Curves to Improve Diagnostic Values.

Autism spectrum disorder (ASD) is a neurodevelopmental condition marked by restricted and repetitive behaviors as well as difficulties with social interaction. Numerous studies have revealed aberrant lipid mediators and autoimmunity as a recognized etiological cause of ASD that is amenable to therapeutic intervention. In this study, the relationship between the relative cyclooxygenase-2/prostaglandin E2 ratio (COX-2/PGE2) as a lipid mediator marker and anti-nucleosome autoantibodies as an autoimmunity marker of ASD was investigated using multiple regression and combined receiver operating characteristic (ROC) curve analyses. The study also sought to identify the linear combination of these variables that optimizes the partial area under the ROC curves. There were forty ASD children and forty-two age- and gender-matched controls included in the current study. Using combined ROC curve analysis, a notable increase in the area under the curve was seen in the patient group, using the control group as a reference group. Additionally, it was reported that the combined markers had improved specificity and sensitivity. This study demonstrates how the predictive value of particular biomarkers associated with lipid metabolism and autoimmunity in children with ASD can be measured using a ROC curve analysis. This technique should help us better understand the etiological mechanism of ASD and how it may adversely affect cellular homeostasis, which is essential to maintaining healthy metabolic pathways. Early diagnosis and intervention may be facilitated by this knowledge.