Micro-C Analysis Workflow Using Pairtools and Juicer.

Three-dimensional (3D) chromosome structures are closely related to various chromosomal functions, and deep analysis of the structures is crucial for the elucidation of the functions. In recent years, chromosome conformation capture (3C) techniques combined with next-generation sequencing analysis have been developed to comprehensively reveal 3D chromosome structures. Micro-C is one such method that can detect the structures at nucleosome resolution. In this chapter, I provide a basic method for Micro-C analysis. I present and discuss a series of data analyses ranging from mapping to basic downstream analyses, including loop detection.

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.

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.

Human 8-oxoguanine glycosylase OGG1 binds nucleosome at the dsDNA ends and the super-helical locations.

The human glycosylase OGG1 extrudes and excises the oxidized DNA base 8-oxoguanine (8-oxoG) to initiate base excision repair and plays important roles in many pathological conditions such as cancer, inflammation, and neurodegenerative diseases. Previous structural studies have used a truncated protein and short linear DNA, so it has been unclear how full-length OGG1 operates on longer DNA or on nucleosomes. Here we report cryo-EM structures of human OGG1 bound to a 35-bp long DNA containing an 8-oxoG within an unmethylated Cp-8-oxoG dinucleotide as well as to a nucleosome with an 8-oxoG at super-helical location (SHL)-5. The 8-oxoG in the linear DNA is flipped out by OGG1, consistent with previous crystallographic findings with a 15-bp DNA. OGG1 preferentially binds near dsDNA ends at the nucleosomal entry/exit sites. Such preference may underlie the enzyme's function in DNA double-strand break repair. Unexpectedly, we find that OGG1 bends the nucleosomal entry DNA, flips an undamaged guanine, and binds to internal nucleosomal DNA sites such as SHL-5 and SHL+6. We suggest that the DNA base search mechanism by OGG1 may be chromatin context-dependent and that OGG1 may partner with chromatin remodelers to excise 8-oxoG at the nucleosomal internal sites.

Creating a bacterium that forms eukaryotic nucleosome core particles.

The nucleosome is one of the hallmarks of eukaryotes, a dynamic platform that supports many critical functions in eukaryotic cells. Here, we engineer the in vivo assembly of the nucleosome core in the model bacterium Escherichia coli. We show that bacterial chromosome DNA and eukaryotic histones can assemble in vivo to form nucleosome complexes with many features resembling those found in eukaryotes. The formation of nucleosomes in E. coli was visualized with atomic force microscopy and using tripartite split green fluorescent protein. Under a condition that moderate histones expression was induced at 1 µM IPTG, the nucleosome-forming bacterium is viable and has sustained growth for at least 110 divisions in longer-term growth experiments. It exhibits stable nucleosome formation, a consistent transcriptome across passages, and reduced growth fitness under stress conditions. In particular, the nucleosome arrays in E. coli genic regions have profiles resembling those in eukaryotic cells. The observed compatibility between the eukaryotic nucleosome and the bacterial chromosome machinery may reflect a prerequisite for bacteria-archaea union, providing insight into eukaryogenesis and the origin of the nucleosome.

Identification of a novel DNA oxidative damage repair pathway, requiring the ubiquitination of the histone variant macroH2A1.1.

BACKGROUND: The histone variant macroH2A (mH2A), the most deviant variant, is about threefold larger than the conventional histone H2A and consists of a histone H2A-like domain fused to a large Non-Histone Region responsible for recruiting PARP-1 to chromatin. The available data suggest that the histone variant mH2A participates in the regulation of transcription, maintenance of heterochromatin, NAD+ metabolism, and double-strand DNA repair. RESULTS: Here, we describe a novel function of mH2A, namely its implication in DNA oxidative damage repair through PARP-1. The depletion of mH2A affected both repair and cell survival after the induction of oxidative lesions in DNA. PARP-1 formed a specific complex with mH2A nucleosomes in vivo. The mH2A nucleosome-associated PARP-1 is inactive. Upon oxidative damage, mH2A is ubiquitinated, PARP-1 is released from the mH2A nucleosomal complex, and is activated. The in vivo-induced ubiquitination of mH2A, in the absence of any oxidative damage, was sufficient for the release of PARP-1. However, no release of PARP-1 was observed upon treatment of the cells with either the DNA alkylating agent MMS or doxorubicin. CONCLUSIONS: Our data identify a novel pathway for the repair of DNA oxidative lesions, requiring the ubiquitination of mH2A for the release of PARP-1 from chromatin and its activation.

Local volume concentration, packing domains, and scaling properties of chromatin.

We propose the Self Returning Excluded Volume (SR-EV) model for the structure of chromatin based on stochastic rules and physical interactions. The SR-EV rules of return generate conformationally defined domains observed by single-cell imaging techniques. From nucleosome to chromosome scales, the model captures the overall chromatin organization as a corrugated system, with dense and dilute regions alternating in a manner that resembles the mixing of two disordered bi-continuous phases. This particular organizational topology is a consequence of the multiplicity of interactions and processes occurring in the nuclei, and mimicked by the proposed return rules. Single configuration properties and ensemble averages show a robust agreement between theoretical and experimental results including chromatin volume concentration, contact probability, packing domain identification and size characterization, and packing scaling behavior. Model and experimental results suggest that there is an inherent chromatin organization regardless of the cell character and resistant to an external forcing such as RAD21 degradation.

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.

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.

Finish the unfinished: Chd1 resolving hexasome-nucleosome complex with FACT.

In this issue of Molecular Cell, Engeholm et al.1 present cryo-EM structures of the chromatin remodeler Chd1 bound to a hexasome-nucleosome complex, an intermediate state during transcription either with or without FACT to restore the missing H2A-H2B dimer. These two binding modes reveal how Chd1 and FACT cooperate in nucleosome re-establishment during transcription.

Protocol for preparing SUV420H1 in complex with the nucleosome containing H2A.Z and H4K20Ecx for structure determination.

The histone lysine methyltransferase SUV420H1 preferentially targets the H2A.Z-containing nucleosome core particle (H2A.Z-NCP) and catalyzes the H4K20me2 modification at replication origins. Here, we present a protocol for preparing SUV420H1 in complex with the nucleosome containing H2A.Z and H4K20Ecx for structure determination. We describe steps for the installation of S-ethyl-cysteine (Ecx), nucleosome and complex preparation, and performing the cryoelectron microscopy (cryo-EM) sample check. This protocol substitutes lysine 20 in histone H4 with S-ethyl-cysteine (H4K20Ecx), which enhances the stability of the interaction between SUV420H1 and nucleosomes. For complete details on the use and execution of this protocol, please refer to Huang et al.1.

Protocol for evaluating the E3 ligase activity of BRCA1-BARD1 and its variants by nucleosomal histone ubiquitylation.

The tumor suppressor breast cancer 1 (BRCA1) complexed with BRCA1-associated RING domain 1 (BARD1), a RING-type E3 ligase, facilitates the attachment of ubiquitin onto the substrate protein. Here, we present a protocol for evaluating the E3 ligase activity of BRCA1-BARD1 and its variants by nucleosomal histone ubiquitylation. We describe steps for isolating 147 bp Widom 601 DNA and assembling nucleosome core particles (NCPs). We then detail procedures for the in vitro ubiquitylation of nucleosome histone H2A by BRCA1-BARD1 and its variants. For complete details on the use and execution of this protocol, please refer to Wang et al.1.

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy.

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding their biophysical properties and interactions with chromatin-binding proteins. Nucleosome reconstitution on DNA for these studies typically involves a salt dialysis procedure that provides precise control over the placement and number of nucleosomes formed along a DNA tether. However, this protocol is time-consuming and requires a substantial amount of DNA and histone octamers as inputs. To offer an alternative strategy, an in situ nucleosome reconstitution method for single-molecule force and fluorescence microscopy that utilizes the histone chaperone Nap1 is described. This method enables users to assemble nucleosomes on any DNA template without the need for strong nucleosome positioning sequences, adjust nucleosome density on demand, and use fewer reagents. In situ nucleosome formation occurs within seconds, offering a simpler experimental workflow and a convenient transition into single-molecule measurements. Examples of two downstream assays for probing nucleosome mechanics and visualizing the behavior of individual proteins on chromatin are further described.

Nucleosomal DNA unwinding pathway through canonical and non-canonical histone disassembly.

The nucleosome including H2A.B, a mammalian-specific H2A variant, plays pivotal roles in spermatogenesis, embryogenesis, and oncogenesis, indicating unique involvement in transcriptional regulation distinct from canonical H2A nucleosomes. Despite its significance, the exact regulatory mechanism remains elusive. This study utilized solid-state nanopores to investigate DNA unwinding dynamics, applying local force between DNA and histones. Comparative analysis of canonical H2A and H2A.B nucleosomes demonstrated that the H2A.B variant required a lower voltage for complete DNA unwinding. Furthermore, synchronization analysis and molecular dynamics simulations indicate that the H2A.B variant rapidly unwinds DNA, causing the H2A-H2B dimer to dissociate from DNA immediately upon disassembly of the histone octamer. In contrast, canonical H2A nucleosomes unwind DNA at a slower rate, suggesting that the H2A-H2B dimer undergoes a state of stacking at the pore. These findings suggest that nucleosomal DNA in the H2A.B nucleosomes undergoes a DNA unwinding process involving histone octamer disassembly distinct from that of canonical H2A nucleosomes, enabling smoother unwinding. The integrated approach of MD simulations and nanopore measurements is expected to evolve into a versatile tool for studying molecular interactions, not only within nucleosomes but also through the forced dissociation of DNA-protein complexes.

Extracting regulatory active chromatin footprint from cell-free DNA.

Cell-free DNA (cfDNA) has emerged as a pivotal player in precision medicine, revolutionizing the diagnostic and therapeutic landscape. While its clinical applications have significantly increased in recent years, current cfDNA assays have limited ability to identify the active transcriptional programs that govern complex disease phenotypes and capture the heterogeneity of the disease. To address these limitations, we have developed a non-invasive platform to enrich and examine the active chromatin fragments (cfDNAac) in peripheral blood. The deconvolution of the cfDNAac signal from traditional nucleosomal chromatin fragments (cfDNAnuc) yields a catalog of features linking these circulating chromatin signals in blood to specific regulatory elements across the genome, including enhancers, promoters, and highly transcribed genes, mirroring the epigenetic data from the ENCODE project. Notably, these cfDNAac counts correlate strongly with RNA polymerase II activity and exhibit distinct expression patterns for known circadian genes. Additionally, cfDNAac signals across gene bodies and promoters show strong correlations with whole blood gene expression levels defined by GTEx. This study illustrates the utility of cfDNAac analysis for investigating epigenomics and gene expression, underscoring its potential for a wide range of clinical applications in precision medicine.

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.

Histones and histone variant families in prokaryotes.

Histones are important chromatin-organizing proteins in eukaryotes and archaea. They form superhelical structures around which DNA is wrapped. Recent studies have shown that some archaea and bacteria contain alternative histones that exhibit different DNA binding properties, in addition to highly divergent sequences. However, the vast majority of these histones are identified in metagenomes and thus are difficult to study in vivo. The recent revolutionary breakthroughs in computational protein structure prediction by AlphaFold2 and RoseTTAfold allow for unprecedented insights into the potential function and structure of previously uncharacterized proteins. Here, we categorize the prokaryotic histone space into 17 distinct groups based on AlphaFold2 predictions. We identify a superfamily of histones, termed α3 histones, which are common in archaea and present in several bacteria. Importantly, we establish the existence of a large family of histones throughout archaea and in some bacteriophages that, instead of wrapping DNA, bridge DNA, thereby diverging from conventional nucleosomal histones.

A repressive H3K36me2 reader mediates Polycomb silencing.

In animals, evolutionarily conserved Polycomb repressive complex 2 (PRC2) catalyzes histone H3 lysine 27 trimethylation (H3K27me3) and PRC1 functions in recruitment and transcriptional repression. However, the mechanisms underlying H3K27me3-mediated stable transcriptional silencing are largely unknown, as PRC1 subunits are poorly characterized in fungi. Here, we report that in the filamentous fungus Magnaporthe oryzae, the N-terminal chromodomain and C-terminal MRG domain of Eaf3 play key roles in facultative heterochromatin formation and transcriptional silencing. Eaf3 physically interacts with Ash1, Eed, and Sin3, encoding an H3K36 methyltransferase, the core subunit of PRC2, and a histone deacetylation co-suppressor, respectively. Eaf3 co-localizes with a set of repressive Ash1-H3K36me2 and H3K27me3 loci and mediates their transcriptional silencing. Furthermore, Eaf3 acts as a histone reader for the repressive H3K36me2 and H3K27me3 marks. Eaf3-occupied regions are associated with increased nucleosome occupancy, contributing to transcriptional silencing in M. oryzae. Together, these findings reveal that Eaf3 is a repressive H3K36me2 reader and plays a vital role in Polycomb gene silencing and the formation of facultative heterochromatin in fungi.

Ubiquitinated histone H2B as gatekeeper of the nucleosome acidic patch.

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

The role of hexokinases in epigenetic regulation: altered hexokinase expression and chromatin stability in yeast.

BACKGROUND: Human hexokinase 2 (HK2) plays an important role in regulating Warburg effect, which metabolizes glucose to lactate acid even in the presence of ample oxygen and provides intermediate metabolites to support cancer cell proliferation and tumor growth. HK2 overexpression has been observed in various types of cancers and targeting HK2-driven Warburg effect has been suggested as a potential cancer therapeutic strategy. Given that epigenetic enzymes utilize metabolic intermediates as substrates or co-factors to carry out post-translational modification of histones and nucleic acids modifications in cells, we hypothesized that altering HK2 expression could impact the epigenome and, consequently, chromatin stability in yeast. To test this hypothesis, we established genetic models with different yeast hexokinase 2 (HXK2) expression in Saccharomyces cerevisiae yeast cells and investigated the effect of HXK2-dependent metabolism on parental nucleosome transfer, a key DNA replication-coupled epigenetic inheritance process, and chromatin stability. RESULTS: By comparing the growth of mutant yeast cells carrying single deletion of hxk1Δ, hxk2Δ, or double-loss of hxk1Δ hxk2Δ to wild-type cells, we firstly confirmed that HXK2 is the dominant HXK in yeast cell growth. Surprisingly, manipulating HXK2 expression in yeast, whether through overexpression or deletion, had only a marginal impact on parental nucleosome assembly, but a noticeable trend with decrease chromatin instability. However, targeting yeast cells with 2-deoxy-D-glucose (2-DG), a clinical glycolysis inhibitor that has been proposed as an anti-cancer treatment, significantly increased chromatin instability. CONCLUSION: Our findings suggest that in yeast cells lacking HXK2, alternative HXKs such as HXK1 or glucokinase 1 (GLK1) play a role in supporting glycolysis at a level that adequately maintains epigenomic stability. While our study demonstrated an increase in epigenetic instability with 2-DG treatment, the observed effect seemed to occur dependent on non-glycolytic function of Hxk2. Thus, additional research is needed to identify the molecular mechanism through which 2-DG influences chromatin stability.