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Recent studies showed an interphase chromosome architecture-a specific coiled nucleosome structure-derived from cryopreserved EM tomograms, and dispersed throughout the nucleus. The images were computationally processed to fill in the missing wedges of data caused by incomplete tomographic tilts. The resulting structures increased z-resolution enabling an extension of the proposed architecture to that of mitotic chromosomes. Here, we provide additional insights into the chromosome architecture that was recently published [M. Elbaum et al., Proc. Natl. Acad. Sci. U.S.A. 119, e2119101119 (2022)]. We build on the defined chromosomes time-dependent structures in an effort to probe their dynamics. Variants of the coiled chromosome structures, possibly further defining specific regions, are discussed. We propose, based on generalized specific uncoiling of mitotic chromosomes in telophase, large-scale reorganization of interphase chromosomes. Chromosome territories, organized as micron-sized small patches, are constructed, satisfying complex volume considerations. Finally, we unveiled the structures of replicated coiled chromosomes, still attached to centromeres, as part of chromosome architecture.

Isoform 1 of DNA methyltransferase DNMT3A (DNMT3A1) specifically recognizes nucleosome monoubiquitylated at histone H2A lysine-119 (H2AK119ub1) for establishment of DNA methylation. Mis-regulation of this process may cause aberrant DNA methylation and pathogenesis. However, the molecular basis underlying DNMT3A1-nucleosome interaction remains elusive. Here we report the cryo-EM structure of DNMT3A1s ubiquitin-dependent recruitment (UDR) fragment complexed with H2AK119ub1-modified nucleosome. DNMT3A1 UDR occupies an extensive nucleosome surface, involving the H2A-H2B acidic patch, a surface groove formed by H2A and H3, nucleosomal DNA, and H2AK119ub1. The DNMT3A1 UDRs interaction with H2AK119ub1 affects the functionality of DNMT3A1 in cells in a context-dependent manner. Our structural and biochemical analysis also reveals competition between DNMT3A1 and JARID2, a cofactor of polycomb repression complex 2 (PRC2), for nucleosome binding, suggesting the interplay between different epigenetic pathways. Together, this study reports a molecular basis for H2AK119ub1-dependent DNMT3A1-nucleosome association, with important implications in DNMT3A1-mediated DNA methylation in development.

Transcriptional silencing in Saccharomyces cerevisiae involves the generation of a chromatin state that stably represses transcription. Using multiple reporter assays, a diverse set of upstream activating sequence enhancers and core promoters were investigated for their susceptibility to silencing. We show that heterochromatin stably silences only weak and stress-induced regulatory elements but is unable to stably repress housekeeping gene regulatory elements, and the partial repression of these elements did not result in bistable expression states. Permutation analysis of enhancers and promoters indicates that both elements are targets of repression. Chromatin remodelers help specific regulatory elements to resist repression, most probably by altering nucleosome mobility and changing transcription burst duration. The strong enhancers/promoters can be repressed if silencer-bound Sir1 is increased. Together, our data suggest that the heterochromatic locus has been optimized to stably silence the weak mating-type gene regulatory elements but not strong housekeeping gene regulatory sequences.

Single-particle cryo-EM is widely used to determine enzyme-nucleosome complex structures. However, cryo-EM sample preparation remains challenging and inconsistent due to complex denaturation at the air-water interface (AWI). Here, to address this issue, we develop graphene-oxide-coated EM grids functionalized with either single-stranded DNA (ssDNA) or thiol-poly(acrylic acid-co-styrene) (TAASTY) co-polymer. These grids protect complexes between the chromatin remodeler SNF2h and nucleosomes from the AWI and facilitate collection of high-quality micrographs of intact SNF2h-nucleosome complexes in the absence of crosslinking. The data yields maps ranging from 2.3 to 3 Å in resolution. 3D variability analysis reveals nucleotide-state linked conformational changes in SNF2h bound to a nucleosome. In addition, the analysis provides structural evidence for asymmetric coordination between two SNF2h protomers acting on the same nucleosome. We envision these grids will enable similar detailed structural analyses for other enzyme-nucleosome complexes and possibly other protein-nucleic acid complexes in general.

The meiotic chromosome axis coordinates chromosome organization and interhomolog recombination in meiotic prophase and is essential for fertility. In S. cerevisiae, the HORMAD protein Hop1 mediates the enrichment of axis proteins at nucleosome-rich islands through a central chromatin-binding region (CBR). Here, we use cryoelectron microscopy to show that the Hop1 CBR directly recognizes bent nucleosomal DNA through a composite interface in its PHD and winged helix-turn-helix domains. Targeted disruption of the Hop1 CBR-nucleosome interface causes a localized reduction of axis protein binding and meiotic DNA double-strand breaks (DSBs) in axis islands and leads to defects in chromosome synapsis. Synthetic effects with mutants of the Hop1 regulator Pch2 suggest that nucleosome binding delays a conformational switch in Hop1 from a DSB-promoting, Pch2-inaccessible state to a DSB-inactive, Pch2-accessible state to regulate the extent of meiotic DSB formation. Phylogenetic analyses of meiotic HORMADs reveal an ancient origin of the CBR, suggesting that the mechanisms we uncover are broadly conserved.

Tau is a microtubule-associated protein often found in neurofibrillary tangles (NFTs) in the brains of patients with Alzheimers disease. Beyond this context, mounting evidence suggests that tau localizes into the nucleus, where it may play a role in DNA protection and heterochromatin regulation. The molecular mechanisms behind these observations are currently unclear. Using in vitro biophysical experiments, here we demonstrate that tau can undergo liquid-liquid phase separation (LLPS) with DNA, mononucleosomes, and reconstituted nucleosome arrays under low salt conditions. Low concentrations of tau promote chromatin compaction and protect DNA from digestion. While the material state of samples at physiological salt is dominated by chromatin oligomerization, tau can still associate strongly and reversibly with nucleosome arrays. These properties are driven by taus strong interactions with linker and nucleosomal DNA. In addition, tau co-localizes into droplets formed by nucleosome arrays and phosphorylated HP1α, a key heterochromatin constituent thought to function through an LLPS mechanism. Importantly, LLPS and chromatin interactions are disrupted by aberrant tau hyperphosphorylation. These biophysical properties suggest that tau may directly impact DNA and chromatin accessibility and that loss of these interactions could contribute to the aberrant nuclear effects seen in tau pathology.

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140-kb unique coding region flanked by 30-40 copies of a 0.8-kb terminal repeat (TR) sequence. A gene enhancer recruits transcription-related enzymes by having arrays of transcription factor binding sites. Here, we show that KSHV TR possesses transcription regulatory function with latency-associated nuclear antigen (LANA). Cleavage under targets and release using nuclease demonstrated that TR fragments were occupied by LANA-interacting histone-modifying enzymes in naturally infected cells. The TR was enriched with histone H3K27 acetylation (H3K27Ac) and H3K4 tri-methylation (H3K4me3) modifications and also expressed nascent RNAs. The sites of H3K27Ac and H3K4me3 modifications were also conserved in the KSHV unique region among naturally infected primary effusion lymphoma cells. KSHV origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies with that of TR. In the Ori-Lyt region, the LANA and LANA-interacting proteins colocalized with an H3K27Ac-modified nucleosome along with paused RNA polymerase II. The KSHV transactivator KSHV replication and transcription activator (K-Rta) recruitment sites franked the LANA-bound nucleosome, and reactivation evicted the LANA-bound nucleosome. Including TR fragments in reporter plasmid enhanced inducible viral gene promoter activities independent of the orientations. In the presence of TR in reporter plasmids, K-Rta transactivation was drastically increased, while LANA acquired the promoter repression function. KSHV TR, therefore, functions as an enhancer for KSHV inducible genes. However, in contrast to cellular enhancers bound by multiple transcription factors, perhaps the KSHV enhancer is predominantly regulated by the LANA nuclear body.IMPORTANCEEnhancers are a crucial regulator of differential gene expression programs. Enhancers are the cis-regulatory sequences determining target genes' spatiotemporal and quantitative expression. Here, we show that Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeats fulfill the enhancer definition for KSHV inducible gene promoters. The KSHV enhancer is occupied by latency-associated nuclear antigen (LANA) and its interacting proteins, such as CHD4. Neighboring terminal repeat (TR) fragments to lytic gene promoters drastically enhanced KSHV replication and transcription activator and LANA transcription regulatory functions. This study, thus, proposes a new latency-lytic switch model in which TR accessibility to the KSHV gene promoters regulates viral inducible gene expression.

The conformational dynamics of nucleosome arrays generate a diverse spectrum of microscopic states, posing challenges to their structural determination. Leveraging cryogenic electron tomography (cryo-ET), we determine the three-dimensional (3D) structures of individual mononucleosomes and arrays comprising di-, tri-, and tetranucleosomes. By slowing the rate of condensation through a reduction in ionic strength, we probe the intra-array structural transitions that precede inter-array interactions and liquid droplet formation. Under these conditions, the arrays exhibite irregular zig-zag conformations with loose packing. Increasing the ionic strength promoted intra-array compaction, yet we do not observe the previously reported regular 30-nanometer fibers. Interestingly, the presence of H1 do not induce array compaction; instead, one-third of the arrays display nucleosomes invaded by foreign DNA, suggesting an alternative role for H1 in chromatin network construction. We also find that the crucial parameter determining the structure adopted by chromatin arrays is the angle between the entry and exit of the DNA and the corresponding tangents to the nucleosomal disc. Our results provide insights into the initial stages of intra-array compaction, a critical precursor to condensation in the regulation of chromatin organization.

Xudong Cui,1,&ast; Tiewei Li,2,&ast; Jingping Yang,3 Xiaojuan Li,2 Pengfei Xuan,3 Hongyan Wang3 1Respiratory and Critical Care Medicine Department, Inner Mongolia Baogang Hospital, Inner Mongolia Medical University, Hohhot, People’s Republic of China; 2Department of Clinical Laboratory, Children’s Hospital Affiliated to Zhengzhou University, Zhengzhou Key Laboratory of Children’s Infection and Immunity, Zhengzhou, People’s Republic of China; 3Respiratory and Critical Care Medicine Department, Inner Mongolia Baogang Hospital, The Third Affiliated Hospital of Inner Mongolia Medical University, Baotou, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Tiewei Li; Jingping Yang, Email litieweind@163.com; yangron@sina.comBackground: Previous studies have demonstrated that neutrophil extracellular traps (NETs) are crucial in infectious diseases. This study aims to evaluate the clinical value of NET-related biomarkers in identifying the risk of COVID-19 and diagnosing the disease.Methods: This study involved 32 patients who tested positive for COVID-19 via polymerase chain reaction (PCR) between April and August 2023. During the same period, 30 healthy volunteers were enrolled as a control group. The principal biomarkers related to NETs are citrullinated histone H3 (CitH3), double-stranded DNA (dsDNA), myeloperoxidase-DNA complex (MPO-DNA), and Nucleosome. Elevated levels in two or more of these biomarkers indicate raised NET concentrations. Multivariable logistic regression analysis was employed to assess whether NET-related biomarkers were the independent risk factor of COVID-19. The diagnostic value of NET-related biomarkers in COVID-19 was further evaluated using receiver operating characteristic (ROC) curve analysis. Statistical procedures were executed in SPSS software (version 24.0, USA).Results: Compared with the control group, patients infected with COVID-19 had higher levels of dsDNA and nucleosomes (P < 0.001). Correlation analysis revealed a positive correlation between dsDNA levels and neutrophil count (r = 0.309, P = 0.015) as well as between nucleosome levels and neutrophil count (r = 0.446, P < 0.001). Further analysis showed that dsDNA and nucleosomes were independent risk factors for COVID-19 infection. ROC curve analysis showed that dsDNA area under the curve (AUC) = 0.777, 95% confidence interval (CI), 0.661– 0.893, P < 0.001, and nucleosomes (AUC = 0.884, 95% CI, 0.778– 0.991, P < 0.001) had well diagnostic value in the diagnosing COVID-19 infection.Conclusion: NET-related biomarkers, dsDNA and nucleosomes, were independent risk factors of COVID-19 infection and potentially useful biomarkers in diagnosing COVID-19 infection in older patients.Keywords: neutrophil extracellular traps, COVID-19, pneumonia, dsDNA, nucleosomes, older patient

In the nucleus, transcriptionally silent genes are sequestered into heterochromatin compartments comprising nucleosomes decorated with histone H3 Lys9 trimethylation and a protein called HP1α. This protein can form liquid-liquid droplets in vitro and potentially organize heterochromatin through a phase separation mechanism that is promoted by phosphorylation. Elucidating the molecular interactions that drive HP1α phase separation and its consequences on nucleosome structure and dynamics has been challenging due to the viscous and heterogeneous nature of such assemblies. Here, we tackle this problem by a combination of solution and solid-state NMR spectroscopy, which allows us to dissect the interactions of phosphorylated HP1α with nucleosomes in the context of phase separation. Our experiments indicate that phosphorylated human HP1α does not cause any major rearrangements to the nucleosome core, in contrast to the yeast homologue Swi6. Instead, HP1α interacts specifically with the methylated H3 tails and slows the dynamics of the H4 tails. Our results shed light on how phosphorylated HP1α proteins may regulate the heterochromatin landscape, while our approach provides an atomic resolution view of a heterogeneous and dynamic biological system regulated by a complex network of interactions and post-translational modifications.

Nearly all essential nuclear processes act on DNA packaged into arrays of nucleosomes. However, our understanding of how these processes (for example, DNA replication, RNA transcription, chromatin extrusion and nucleosome remodeling) occur on individual chromatin arrays remains unresolved. Here, to address this deficit, we present SAMOSA-ChAAT: a massively multiplex single-molecule footprinting approach to map the primary structure of individual, reconstituted chromatin templates subject to virtually any chromatin-associated reaction. We apply this method to distinguish between competing models for chromatin remodeling by the essential imitation switch (ISWI) ATPase SNF2h: nucleosome-density-dependent spacing versus fixed-linker-length nucleosome clamping. First, we perform in vivo single-molecule nucleosome footprinting in murine embryonic stem cells, to discover that ISWI-catalyzed nucleosome spacing correlates with the underlying nucleosome density of specific epigenomic domains. To establish causality, we apply SAMOSA-ChAAT to quantify the activities of ISWI ATPase SNF2h and its parent complex ACF on reconstituted nucleosomal arrays of varying nucleosome density, at single-molecule resolution. We demonstrate that ISWI remodelers operate as density-dependent, length-sensing nucleosome sliders, whose ability to program DNA accessibility is dictated by single-molecule nucleosome density. We propose that the long-observed, context-specific regulatory effects of ISWI complexes can be explained in part by the sensing of nucleosome density within epigenomic domains. More generally, our approach promises molecule-precise views of the essential processes that shape nuclear physiology.

Abstract Background Histone ubiquitination modification is emerging as a critical epigenetic mechanism involved in a range of biological processes. In vitro reconstitution of ubiquitinated nucleosomes is pivotal for elucidating the influence of histone ubiquitination on chromatin dynamics. Results In this study, we introduce a Non-Denatured Histone Octamer Ubiquitylation (NDHOU) approach for generating ubiquitin or ubiquitin-like modified histone octamers. The method entails the co-expression and purification of histone octamers, followed by their chemical cross-linking to ubiquitin using 1,3-dibromoacetone. We demonstrate that nucleosomes reconstituted with these octamers display a high degree of homogeneity, rendering them highly compatible with in vitro biochemical assays. These ubiquitinated nucleosomes mimic physiological substrates in function and structure. Additionally, we have extended this method to cross-linking various histone octamers and three types of ubiquitin-like proteins. Conclusions Overall, our findings offer an efficient strategy for producing ubiquitinated nucleosomes, advancing biochemical and biophysical studies in the field of chromatin biology.

Transcription has a mechanical component, as the translocation of the transcription machinery or RNA polymerase (RNAP) on DNA or chromatin is dynamically coupled to the chromatin torsion. This posits chromatin mechanics as a possible regulator of eukaryotic transcription, however, the modes and mechanisms of this regulation are elusive. Here, we first take a statistical mechanics approach to model the torsional response of topology-constrained chromatin. Our model recapitulates the experimentally observed weaker torsional stiffness of chromatin compared to bare DNA and proposes structural transitions of nucleosomes into chirally distinct states as the driver of the contrasting torsional mechanics. Coupling chromatin mechanics with RNAP translocation in stochastic simulations, we reveal a complex interplay of DNA supercoiling and nucleosome dynamics in governing RNAP velocity. Nucleosomes play a dual role in controlling the transcription dynamics. The steric barrier aspect of nucleosomes in the gene body counteracts transcription via hindering RNAP motion, whereas the chiral transitions facilitate RNAP motion via driving a low restoring torque upon twisting the DNA. While nucleosomes with low dissociation rates are typically transcriptionally repressive, highly dynamic nucleosomes offer less of a steric barrier and enhance the transcription elongation dynamics of weakly transcribed genes via buffering DNA twist. We use the model to predict transcription-dependent levels of DNA supercoiling in segments of the budding yeast genome that are in accord with available experimental data. The model unveils a paradigm of DNA supercoilingmediated interaction between genes and makes testable predictions that will guide experimental design. [ABSTRACT FROM AUTHOR]

Understanding the function of eukaryotic genomes, including the human genome, is undoubtedly one of the major scientific challenges of the 21st century. The cornerstone of eukaryotic genome organization is nucleosomes—elementary building blocks of chromatin about 10 nm in size that wrap DNA around an octamer of histone proteins. Nucleosomes are integral players in all genomic processes, including transcription, DNA replication and repair. They mediate genome regulation at the epigenetic level, bridging the discrete nature of the genetic information encoded in DNA with the analog physical nature of the intermolecular interactions required to access that information. Due to their relatively large size and dynamic nature, nucleosomes are difficult objects for experimental characterization. Molecular dynamics (MD) simulations have emerged over the years as a useful tool to complement experimental studies. Particularly in recent years, advances in computing power, refinement of MD force fields and codes have opened up new frontiers in terms of simulation timescales and quality for nucleosomes and related systems. It has become possible to elucidate in atomistic detail their functional dynamics modes such as DNA unwrapping and sliding, to characterize the effects of epigenetic modifications, DNA and protein sequence variation on nucleosome structure and stability, to describe the mechanisms governing nucleosome interactions with chromatin‐associated proteins and the formation of supranucleosome structures. In this review, we systematically analyzed all‐atom MD simulation studies of nucleosomes and related structures published since 2018 and discussed their relevance in the context of older studies, experimental data, and related coarse‐grained and multiscale studies. This article is categorized under:Software > Molecular ModelingMolecular and Statistical Mechanics > Molecular Dynamics and Monte‐Carlo MethodsStructure and Mechanism > Computational Biochemistry and Biophysics [ABSTRACT FROM AUTHOR]

Background: Histone ubiquitination modification is emerging as a critical epigenetic mechanism involved in a range of biological processes. In vitro reconstitution of ubiquitinated nucleosomes is pivotal for elucidating the influence of histone ubiquitination on chromatin dynamics. Results: In this study, we introduce a Non-Denatured Histone Octamer Ubiquitylation (NDHOU) approach for generating ubiquitin or ubiquitin-like modified histone octamers. The method entails the co-expression and purification of histone octamers, followed by their chemical cross-linking to ubiquitin using 1,3-dibromoacetone. We demonstrate that nucleosomes reconstituted with these octamers display a high degree of homogeneity, rendering them highly compatible with in vitro biochemical assays. These ubiquitinated nucleosomes mimic physiological substrates in function and structure. Additionally, we have extended this method to cross-linking various histone octamers and three types of ubiquitin-like proteins. Conclusions: Overall, our findings offer an efficient strategy for producing ubiquitinated nucleosomes, advancing biochemical and biophysical studies in the field of chromatin biology. [ABSTRACT FROM AUTHOR]

Chromatin remodelers are molecular motors that act on nucleosomes: they move them along DNA or (dis-)assemble them. Despite the fact that they perform essential regulatory functions in cells—their deregulation can contribute to the development of cancers and lead to cell death—chromatin remodelers have only received meager attention in the biophysics community so far. In this short text, we attempt to present the key features of this interesting class of enzymes obtained with different experimental and theoretical methods, thereby providing a concise introduction for biophysicists to further stimulate interest in their properties. [ABSTRACT FROM AUTHOR]

Unlike other chromatin remodelers, INO80 preferentially mobilizes hexasomes, which can form during transcription. Why INO80 prefers hexasomes over nucleosomes remains unclear. Here, we report structures of Saccharomyces cerevisiae INO80 bound to a hexasome or a nucleosome. INO80 binds the two substrates in substantially different orientations. On a hexasome, INO80 places its ATPase subunit, Ino80, at superhelical location -2 (SHL -2), in contrast to SHL -6 and SHL -7, as previously seen on nucleosomes. Our results suggest that INO80 action on hexasomes resembles action by other remodelers on nucleosomes such that Ino80 is maximally active near SHL -2. The SHL -2 position also plays a critical role for nucleosome remodeling by INO80. Overall, the mechanistic adaptations used by INO80 for preferential hexasome sliding imply that subnucleosomal particles play considerable regulatory roles.

The basic helix-loop-helix (bHLH) family of transcription factors recognizes DNA motifs known as E-boxes (CANNTG) and includes 108 members1. Here we investigate how chromatinized E-boxes are engaged by two structurally diverse bHLH proteins: the proto-oncogene MYC-MAX and the circadian transcription factor CLOCK-BMAL1 (refs. 2,3). Both transcription factors bind to E-boxes preferentially near the nucleosomal entry-exit sites. Structural studies with engineered or native nucleosome sequences show that MYC-MAX or CLOCK-BMAL1 triggers the release of DNA from histones to gain access. Atop the H2A-H2B acidic patch4, the CLOCK-BMAL1 Per-Arnt-Sim (PAS) dimerization domains engage the histone octamer disc. Binding of tandem E-boxes5-7 at endogenous DNA sequences occurs through direct interactions between two CLOCK-BMAL1 protomers and histones and is important for circadian cycling. At internal E-boxes, the MYC-MAX leucine zipper can also interact with histones H2B and H3, and its binding is indirectly enhanced by OCT4 elsewhere on the nucleosome. The nucleosomal E-box position and the type of bHLH dimerization domain jointly determine the histone contact, the affinity and the degree of competition and cooperativity with other nucleosome-bound factors.

CD8+ T cells provide host protection against pathogens by differentiating into distinct effector and memory cell subsets, but how chromatin is site-specifically remodeled during their differentiation is unclear. Due to its critical role in regulating chromatin and enhancer accessibility through its nucleosome remodeling activities, we investigated the role of the canonical BAF (cBAF) chromatin remodeling complex in antiviral CD8+ T cells during infection. ARID1A, a subunit of cBAF, was recruited early after activation and established de novo open chromatin regions (OCRs) at enhancers. Arid1a deficiency impaired the opening of thousands of activation-induced enhancers, leading to loss of TF binding, dysregulated proliferation and gene expression, and failure to undergo terminal effector differentiation. Although Arid1a was dispensable for circulating memory cell formation, tissue-resident memory (Trm) formation was strongly impaired. Thus, cBAF governs the enhancer landscape of activated CD8+ T cells that orchestrates TF recruitment and activity and the acquisition of specific effector and memory differentiation states.

ABSTRACT Isw2 proteins, ubiquitous across eukaryotes, exhibit a propensity for DNA binding and exert dynamic influences on local chromosome condensation in an ATP-dependent fashion, thereby modulating the accessibility of neighboring genes to transcriptional machinery. Here, we report the deletion of a putative MoISW2 gene, yielding substantial ramifications on plant pathogenicity. Subsequent gene complementation and chromatin immunoprecipitation sequencing (ChIP-seq) analyses were conducted to delineate binding sites. RNA sequencing (RNA-seq) assays revealed discernible impacts on global gene regulation along chromosomes in both mutant and wild-type strains, with comparative analyses against 55 external RNA-seq data sets corroborating these findings. Notably, MoIsw2-mediated binding and activities delineate genomic loci characterized by pronounced gene expression variability proximal to MoIsw2 binding sites, juxtaposed with comparatively stable expression in surrounding regions. The contingent genes influenced by MoIsw2 activity predominantly encompass niche-determinant genes, including those encoding secreted proteins, secondary metabolites, and stress-responsive elements, alongside avirulence genes. Furthermore, our investigations unveil a spatial correlation between MoIsw2 binding motifs and known transposable elements (TEs), suggesting a potential interplay wherein TE transposition at these loci could modulate the transcriptional landscape of Magnaporthe oryzae in a strain-specific manner. Collectively, these findings position MoIsw2 as a plausible master regulator orchestrating the delicate equilibrium between genes vital for biomass proliferation, akin to housekeeping genes, and niche-specific determinants crucial for ecological adaptability. Stress-induced TE transposition, in conjunction with MoIsw2 activity, emerges as a putative mechanism fostering enhanced mutagenesis and accelerated evolution of niche-determinant genes relative to housekeeping counterparts.IMPORTANCEIsw2 proteins are conserved in plants, fungi, animals, and other eukaryotes. We show that a fungal Isw2 protein in the rice pathogen Magnaporthe oryzae binds to retrotransposon (RT) DNA motifs and affects the epigenetic gene expression landscape of the fungal genome. Mainly ecological niche determinant genes close to the binding motifs are affected. RT elements occur frequently in DNA between genes in most organisms. They move place and multiply in the genome, especially under physiological stress. We further discuss the Isw2 and RT combined activities as a possible sought-after mechanism that can cause biased mutation rates and faster evolution of genes necessary for reacting to abiotic and biotic challenges. The most important biotic challenges for plant pathogens are the ones from the host plants’ innate immunity. The overall result of these combined activities will be an adaptation-directed evolution of niche-determinant genes.

Sirtuin 6 (SIRT6) is a multifaceted protein deacetylase/deacylase and a major target for small-molecule modulators of longevity and cancer. In the context of chromatin, SIRT6 removes acetyl groups from histone H3 in nucleosomes, but the molecular basis for its nucleosomal substrate preference is unknown. Our cryo-electron microscopy structure of human SIRT6 in complex with the nucleosome shows that the catalytic domain of SIRT6 pries DNA from the nucleosomal entry-exit site and exposes the histone H3 N-terminal helix, while the SIRT6 zinc-binding domain binds to the histone acidic patch using an arginine anchor. In addition, SIRT6 forms an inhibitory interaction with the C-terminal tail of histone H2A. The structure provides insights into how SIRT6 can deacetylate both H3 K9 and H3 K56.

Macromolecular machines acting on genes are at the core of life's fundamental processes, including DNA replication and repair, gene transcription and regulation, chromatin packaging, RNA splicing, and genome editing. Here, we report the increasing role of computational biophysics in characterizing the mechanisms of "machines on genes", focusing on innovative applications of computational methods and their integration with structural and biophysical experiments. We showcase how state-of-the-art computational methods, including classical and ab initio molecular dynamics to enhanced sampling techniques, and coarse-grained approaches are used for understanding and exploring gene machines for real-world applications. As this review unfolds, advanced computational methods describe the biophysical function that is unseen through experimental techniques, accomplishing the power of the "computational microscope", an expression coined by Klaus Schulten to highlight the extraordinary capability of computer simulations. Pushing the frontiers of computational biophysics toward a pragmatic representation of large multimegadalton biomolecular complexes is instrumental in bridging the gap between experimentally obtained macroscopic observables and the molecular principles playing at the microscopic level. This understanding will help harness molecular machines for medical, pharmaceutical, and biotechnological purposes.

Eukaryotes have evolved multiple ATP-dependent chromatin remodelers to shape the nucleosome landscape. We recently uncovered an evolutionarily conserved SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeler complex in plants reminiscent of the mammalian BAF subclass, which specifically incorporates the MINUSCULE (MINU) catalytic subunits and the TRIPLE PHD FINGERS (TPF) signature subunits. Here we report experimental evidence that establishes the functional relevance of TPF proteins for the complex activity. Our results show that depletion of TPF triggers similar pleiotropic phenotypes and molecular defects to those found in minu mutants. Moreover, we report the genomic location of MINU2 and TPF proteins as representative members of this SWI/SNF complex and their impact on nucleosome positioning and transcription. These analyses unravel the binding of the complex to thousands of genes where it modulates the position of the +1 nucleosome. These targets tend to produce 5-shifted transcripts in the tpf and minu mutants pointing to the participation of the complex in alternative transcription start site usage. Interestingly, there is a remarkable correlation between +1 nucleosome shift and 5 transcript length change suggesting their functional connection. In summary, this study unravels the function of a plant SWI/SNF complex involved in +1 nucleosome positioning and transcription start site determination.

Many large protein-nucleic acid complexes exhibit allosteric regulation. In these systems, the propagation of the allosteric signaling is strongly coupled to conformational dynamics and catalytic function, challenging state-of-the-art analytical methods. Here, we review established and innovative approaches used to elucidate allosteric mechanisms in these complexes. Specifically, we report network models derived from graph theory and centrality analyses in combination with molecular dynamics (MD) simulations, introducing novel schemes that implement the synergistic use of graph theory with enhanced simulations methods and ab-initio MD. Accelerated MD simulations are used to construct "enhanced network models", describing the allosteric response over long timescales and capturing the relation between allostery and conformational changes. "Ab-initio network models" combine graph theory with ab-initio MD and quantum mechanics/molecular mechanics (QM/MM) simulations to describe the allosteric regulation of catalysis by following the step-by-step dynamics of biochemical reactions. This approach characterizes how the allosteric regulation changes from reactants to products and how it affects the transition state, revealing a tense-to-relaxed allosteric regulation along the chemical step. Allosteric models and applications are showcased for three paradigmatic examples of allostery in protein-nucleic acid complexes: (i) the nucleosome core particle, (ii) the CRISPR-Cas9 genome editing system and (iii) the spliceosome. These methods and applications create innovative protocols to determine allosteric mechanisms in protein-nucleic acid complexes that show tremendous promise for medicine and bioengineering.

Nucleosome DNA unwrapping and its disassembly into hexasomes and tetrasomes is necessary for genomic access and plays an important role in transcription regulation. Previous single-molecule mechanical nucleosome unwrapping revealed a low- and a high-force transitions, and force-FRET pulling experiments showed that DNA unwrapping is asymmetric, occurring always first from one side before the other. However, the assignment of DNA segments involved in these transitions remains controversial. Here, using high-resolution optical tweezers with simultaneous single-molecule FRET detection, we show that the low-force transition corresponds to the undoing of the outer wrap of one side of the nucleosome (∼27 bp), a process that can occur either cooperatively or noncooperatively, whereas the high-force transition corresponds to the simultaneous unwrapping of ∼76 bp from both sides. This process may give rise stochastically to the disassembly of nucleosomes into hexasomes and tetrasomes whose unwrapping/rewrapping trajectories we establish. In contrast, nucleosome rewrapping does not exhibit asymmetry. To rationalize all previous nucleosome unwrapping experiments, it is necessary to invoke that mechanical unwrapping involves two nucleosome reorientations: one that contributes to the change in extension at the low-force transition and another that coincides but does not contribute to the high-force transition.

It has been proposed that the intrinsic property of nucleosome arrays to undergo liquid-liquid phase separation (LLPS) in vitro is responsible for chromatin domain organization in vivo. However, understanding nucleosomal LLPS has been hindered by the challenge to characterize the structure of the resulting heterogeneous condensates. We used cryo-electron tomography and deep-learning-based 3D reconstruction/segmentation to determine the molecular organization of condensates at various stages of LLPS. We show that nucleosomal LLPS involves a two-step process: a spinodal decomposition process yielding irregular condensates, followed by their unfavorable conversion into more compact, spherical nuclei that grow into larger spherical aggregates through accretion of spinodal materials or by fusion with other spherical condensates. Histone H1 catalyzes more than 10-fold the spinodal-to-spherical conversion. We propose that this transition involves exposure of nucleosome hydrophobic surfaces causing modified inter-nucleosome interactions. These results suggest a physical mechanism by which chromatin may transition from interphase to metaphase structures.

The genomic positions of nucleosomes are a defining feature of the cell's epigenomic state, but signal-dependent transcription factors (SDTFs), upon activation, bind to specific genomic locations and modify nucleosome positioning. Here we leverage SDTFs as perturbation probes to learn about nucleosome dynamics in living cells. We develop Markov models of nucleosome dynamics and fit them to time course sequencing data of DNA accessibility. We find that (1) the dynamics of DNA unwrapping are significantly slower in cells than reported from cell-free experiments, (2) only models with cooperativity in wrapping and unwrapping fit the available data, (3) SDTF activity produces the highest eviction probability when its binding site is adjacent to but not on the nucleosome dyad, and (4) oscillatory SDTF activity results in high location variability. Our work uncovers the regulatory rules governing SDTF-induced nucleosome dynamics in live cells, which can predict chromatin accessibility alterations during inflammation at single-nucleosome resolution.

Cryoelectron tomography of the cell nucleus using scanning transmission electron microscopy and deconvolution processing technology has highlighted a large-scale, 100- to 300-nm interphase chromosome structure, which is present throughout the nucleus. This study further documents and analyzes these chromosome structures. The paper is divided into four parts: 1) evidence (preliminary) for a unified interphase chromosome structure; 2) a proposed unified interphase chromosome architecture; 3) organization as chromosome territories (e.g., fitting the 46 human chromosomes into a 10-μm-diameter nucleus); and 4) structure unification into a polytene chromosome architecture and lampbrush chromosomes. Finally, the paper concludes with a living light microscopy cell study showing that the G1 nucleus contains very similar structures throughout. The main finding is that this chromosome structure appears to coil the 11-nm nucleosome fiber into a defined hollow structure, analogous to a Slinky helical spring [https://en.wikipedia.org/wiki/Slinky; motif used in Bowerman et al., eLife 10, e65587 (2021)]. This Slinky architecture can be used to build chromosome territories, extended to the polytene chromosome structure, as well as to the structure of lampbrush chromosomes.

The critical role of the INO80 chromatin remodeling complex in transcription is commonly attributed to its nucleosome sliding activity. Here, we have found that INO80 prefers to mobilize hexasomes over nucleosomes. INO80's preference for hexasomes reaches up to ∼60 fold when flanking DNA overhangs approach ∼18-bp linkers in yeast gene bodies. Correspondingly, deletion of INO80 significantly affects the positions of hexasome-sized particles within yeast genes in vivo. Our results raise the possibility that INO80 promotes nucleosome sliding by dislodging an H2A-H2B dimer, thereby making a nucleosome transiently resemble a hexasome. We propose that this mechanism allows INO80 to rapidly mobilize nucleosomes at promoters and hexasomes within gene bodies. Rapid repositioning of hexasomes that are generated in the wake of transcription may mitigate spurious transcription. More generally, such versatility may explain how INO80 regulates chromatin architecture during the diverse processes of transcription, replication, and repair.

Simple Summary: This study focused on identifying and evaluating the use of selected potential biomarkers in predicting the treatment response of mast cell tumours (MCT) in dogs using a combination of electrochemotherapy (ECT) and interleukin 12 gene electrotransfer (IL-12 GET). In the study of 48 patients with 86 MCTs, blood samples were taken before, one month and six months after treatment to identify reliable biomarkers that predict response to treatment. An increased plasma nucleosome concentration and increased serum lactate dehydrogenase (LDH) activity one month after treatment correlated with a more effective local response, such as necrosis and swelling. These biomarkers proved to be potential early indicators of treatment success. However, the serum ferritin concentration did not prove to be meaningful. The results of this study provide crucial insights into the use of the plasma nucleosome concentration and serum LDH activity as potential indicators of treatment efficacy in veterinary oncology. Ultimately, this study emphasises the need to identify and validate predictive biomarkers to improve treatment outcomes in dogs with MCT and other malignancies treated similarly. Electrochemotherapy (ECT) in combination with the gene electrotransfer of interleukin 12 (IL-12 GET) has been successfully used in veterinary medicine for the treatment of mast cell tumours (MCT), but the biomarkers that could predict response to this treatment have not yet been investigated. The aim of this study was to determine the plasma nucleosome and serum ferritin concentrations, as well as the lactate dehydrogenase (LDH) activity, in the serum of treated patients before and one and six months after treatment to evaluate their utility as potential biomarkers that could predict response to the combined treatment. The study was conducted in 48 patients with a total of 86 MCTs that we treated with the combined treatment. The blood samples used for analysing the potential predictive biomarkers were taken before treatment and one and six months after treatment, when the response to treatment was also assessed. The Nu. Q® Vet Cancer Test, the Canine Ferritin ELISA Kit, and the RX Daytona+ automated biochemical analyser were used to analyse the blood samples. The results showed that the plasma nucleosome concentration (before treatment (BT): 32.84 ng/mL (median); one month after treatment (1 M AT): 58.89 ng/mL (median); p = 0.010) and serum LDH activity (BT: 59.75 U/L (median); 1 M AT: 107.5 U/L (median); p = 0.012) increased significantly one month after treatment and that the increase correlated significantly with the presence of a more pronounced local reaction (necrosis, swelling, etc.) at that time point for both markers (nucleosome: BT (necrosis): 21.61 ng/mL (median); 1 M AT (necrosis): 69.92 ng/mL (median), p = 0.030; LDH: BT (necrosis): 54.75 U/L (median); 1 M AT (necrosis): 100.3 U/L (median), p = 0.048). Therefore, both the plasma nucleosome concentration and serum LDH activity could serve as early indicators of the effect of the treatment. In this context, the serum ferritin concentration showed no significant predictive potential for treatment response (p > 0.999 for all comparisons). In conclusion, this study provides some new and important observations on the use of predictive biomarkers in veterinary oncology. Furthermore, it emphasises the need for the continued identification and validation of potential predictive biomarkers in dogs with MCT and other malignancies undergoing ECT treatment in combination with IL-12 GET to ultimately improve treatment outcomes. [ABSTRACT FROM AUTHOR]

N6-methyladenine (6mA) of DNA is an emerging epigenetic mark in the genomes of Chlamydomonas, Caenorhabditis elegans, and mammals recently. Levels of 6mA undergo drastic fluctuation and thus affect fertility during meiosis and early embryogenesis. Here, we showed three complex structures of 6mA demethylase C. elegans NMAD-1A, a canonical isoform of NMAD-1 (F09F7.7). Biochemical results revealed that NMAD-1A prefers 6mA Bubble or Bulge DNAs. Structural studies of NMAD-1A revealed an unexpected "stretch-out" conformation of its Flip2 region, a conserved element that is usually bent over the catalytic center to facilitate substrate base flipping in other DNA demethylases. Moreover, the wide channel between the Flip1 and Flip2 of the NMAD-1A explained the observed preference of NMAD-1A for unpairing substrates, of which the flipped 6mA was primed for catalysis. Structural analysis and mutagenesis studies confirmed that key elements such as carboxy-terminal domain (CTD) and hypothetical zinc finger domain (ZFD) critically contributed to structural integrity, catalytic activity, and nucleosome binding. Collectively, our biochemical and structural studies suggest that NMAD-1A prefers to regulate 6mA in the unpairing regions and is thus possibly associated with dynamic chromosome regulation and meiosis regulation. [ABSTRACT FROM AUTHOR]

Histone H3 N-terminal tail (H3NT) clipping has been known for the last four decades, but its functional significance is still poorly understood. H3NT clipping has never received proper attention because it was thought that clipping is the result of the non-specific action of cellular proteases. However, in the last few years, several histone-specific proteases have been identified, and their functions have started to unravel. This paper discusses the recently identified H3-specific proteases, the consequences of histone clipping, and future perspectives. [ABSTRACT FROM AUTHOR]

A molecular architecture is proposed for a representative mitotic chromosome, human chromosome 10. This architecture is built on an interphase chromosome structure based on cryo-electron microscopy (cryo-EM) cellular tomography [J. Sedat et al., Proc. Natl. Acad. Sci. U.S.A., in press], thus unifying chromosome structure throughout the complete mitotic cycle. The basic organizational principle for mitotic chromosomes is specific coiling of the 11-nm nucleosome fiber into large scale, ∼200-nm interphase structures, a Slinky [https://en.wikipedia.org/wiki/Slinky; motif cited in S. Bowerman et al., eLife 10, e65587 (2021)], then further modified with subsequent additional coiling for the final mitotic chromosome structure. The final mitotic chromosome architecture accounts for the dimensional values as well as the well-known cytological configurations. In addition, proof is experimentally provided by digital PCR technology that G1 T cell nuclei are diploid with one DNA molecule per chromosome. Many nucleosome linker DNA sequences, the promotors and enhancers, are suggestive of optimal exposure on the surfaces of the large-scale coils.

The role of histone modifications in transcription remains incompletely understood. Here, we examine the relationship between histone modifications and transcription using experimental perturbations combined with sensitive machine-learning tools. Transcription predicted the variation in active histone marks and complex chromatin states, like bivalent promoters, down to single-nucleosome resolution and at an accuracy that rivaled the correspondence between independent ChIP-seq experiments. Blocking transcription rapidly removed two punctate marks, H3K4me3 and H3K27ac, from chromatin indicating that transcription is required for active histone modifications. Transcription was also required for maintenance of H3K27me3, consistent with a role for RNA in recruiting PRC2. A subset of DNase-I-hypersensitive sites were refractory to prediction, precluding models where transcription initiates pervasively at any open chromatin. Our results, in combination with past literature, support a model in which active histone modifications serve a supportive, rather than an essential regulatory, role in transcription.

RNA polymerase II (Pol II) elongation is a critical step in gene expression. Here we found that NDF, which was identified as a bilaterian nucleosome-destabilizing factor, is also a Pol II transcription factor that stimulates elongation with plain DNA templates in the absence of nucleosomes. NDF binds directly to Pol II and enhances elongation by a different mechanism than that used by transcription factor TFIIS. Moreover, yeast Pdp3, which is related to NDF, binds to Pol II and stimulates elongation. Thus, NDF is a Pol II binding transcription elongation factor that is localized over gene bodies and is conserved from yeast to humans.

The NuA4 protein complex acetylates histones H4 and H2A to activate both transcription and DNA repair. We report the 3.1-Å resolution cryo-electron microscopy structure of the central hub of NuA4, which flexibly tethers the histone acetyltransferase (HAT) and Trimer Independent of NuA4 involved in Transcription Interactions with Nucleosomes (TINTIN) modules. The hub contains the large Tra1 subunit and a core that includes Swc4, Arp4, Act1, Eaf1, and the C-terminal region of Epl1. Eaf1 stands out as the primary scaffolding factor that interacts with the Tra1, Swc4, and Epl1 subunits and contributes the conserved HSA helix to the Arp module. Using nucleosome-binding assays, we find that the HAT module, which is anchored to the core through Epl1, recognizes H3K4me3 nucleosomes with hyperacetylated H3 tails, while the TINTIN module, anchored to the core via Eaf1, recognizes nucleosomes that have hyperacetylated H2A and H4 tails. Together with the known interaction of Tra1 with site-specific transcription factors, our data suggest a model in which Tra1 recruits NuA4 to specific genomic sites then allowing the flexible HAT and TINTIN modules to select nearby nucleosomes for acetylation.

The chromatin architecture in promoters is thought to regulate gene expression, but it remains uncertain how most transcription factors (TFs) impact nucleosome position. The MuvB TF complex regulates cell-cycle dependent gene-expression and is critical for differentiation and proliferation during development and cancer. MuvB can both positively and negatively regulate expression, but the structure of MuvB and its biochemical function are poorly understood. Here we determine the overall architecture of MuvB assembly and the crystal structure of a subcomplex critical for MuvB function in gene repression. We find that the MuvB subunits LIN9 and LIN37 function as scaffolding proteins that arrange the other subunits LIN52, LIN54 and RBAP48 for TF, DNA, and histone binding, respectively. Biochemical and structural data demonstrate that MuvB binds nucleosomes through an interface that is distinct from LIN54-DNA consensus site recognition and that MuvB increases nucleosome occupancy in a reconstituted promoter. We find in arrested cells that MuvB primarily associates with a tightly positioned +1 nucleosome near the transcription start site (TSS) of MuvB-regulated genes. These results support a model that MuvB binds and stabilizes nucleosomes just downstream of the TSS on its target promoters to repress gene expression.

Gene silencing in budding yeast is mediated by Sir protein binding to unacetylated nucleosomes to form a chromatin structure that inhibits transcription. Transcriptional silencing is characterized by the high-fidelity transmission of the silent state. Despite its relative stability, the constituent parts of the silent state are in constant flux, giving rise to a model that silent loci can tolerate such fluctuations without functional consequences. However, the level of tolerance is unknown, and we developed methods to measure the threshold of histone acetylation that causes the silent chromatin state to switch to the active state as well as to measure the levels of the enzymes and structural proteins necessary for silencing. We show that loss of silencing required 50 to 75% acetyl-mimic histones, though the precise levels were influenced by silencer strength and upstream activating sequence (UAS) enhancer/promoter strength. Measurements of repressor protein levels necessary for silencing showed that reducing SIR4 gene dosage two- to threefold significantly weakened silencing, though reducing the gene copy numbers for Sir2 or Sir3 to the same extent did not significantly affect silencing suggesting that Sir4 was a limiting component in gene silencing. Calculations suggest that a mere twofold reduction in the ability of acetyltransferases to acetylate nucleosomes across a large array of nucleosomes may be sufficient to generate a transcriptionally silent domain.

NF-κB is a transcription factor responsible for activating hundreds of genes in mammalian organisms. To accomplish its function, NF-κB must interact with DNA occupied by nucleosomes, but how this interaction occurs is unclear. Here we used Atomic Force Microscopy to characterize complexes of NF-κB with nucleosomes assembled on different DNA templates. The assembly of NF-κB-nucleosome complexes leads to a substantial decrease of DNA wrapping efficiency from 149 ± 2 bp (SEM) for the control nucleosome sample to 135 ± 3 bp for complexes of nucleosomes with NF-κB. Mapping of the nucleosomes did not reveal displacement of under-wrapped nucleosomes from their original position, suggesting that unravelling involves dissociation of one or both flanks of the nucleosomes. Binding of NF-κB to the core was identified by nucleosome core volume measurements. We discovered two binding modes of NF-κB associated with nucleosome unravelling - NF-κB bound to the nucleosome core and to the DNA flanks. From these findings we propose two models explaining the interaction of NF-κB with the nucleosome complex. The partial unravelling of nucleosomes by NF-κB makes the DNA segment at the edge of the nucleosome core accessible, facilitating the transcription process. We speculate that NF-κB can function as a pioneer factor, enhancing its ability to facilitate rapid transcriptional response to cell stress.

In eukaryotic organisms, genomic DNA associates with histone proteins to form nucleosomes. Nucleosomes provide a basis for genome compaction, epigenetic markup, and mediate interactions of nuclear proteins with their target DNA loci. A negatively charged (acidic) patch located on the H2A-H2B histone dimer is a characteristic feature of the nucleosomal surface. The acidic patch is a common site in the attachment of various chromatin proteins, including viral ones. Acidic patch-binding peptides present perspective compounds that can be used to modulate chromatin functioning by disrupting interactions of nucleosomes with natural proteins or alternatively targeting artificial moieties to the nucleosomes, which may be beneficial for the development of new therapeutics. In this work, we used several computational and experimental techniques to improve our understanding of how peptides may bind to the acidic patch and what are the consequences of their binding. Through extensive analysis of the PDB database, histone sequence analysis, and molecular dynamic simulations, we elucidated common binding patterns and key interactions that stabilize peptide–nucleosome complexes. Through MD simulations and FRET measurements, we characterized changes in nucleosome dynamics conferred by peptide binding. Using fluorescence polarization and gel electrophoresis, we evaluated the affinity and specificity of the LANA1-22 peptide to DNA and nucleosomes. Taken together, our study provides new insights into the different patterns of intermolecular interactions that can be employed by natural and designed peptides to bind to nucleosomes, and the effects of peptide binding on nucleosome dynamics and stability. [ABSTRACT FROM AUTHOR]

Histones are an essential part of nucleosomes that regulate chromatin structure and function. Histone exchanges and modifications represent a scaffold for DNA transcription, repair, and replication. Studying histones and histone code is an important and fast-developing branch of epigenetic science. Here we propose a fast, efficient, and versatile assay for nucleosomal histone isolation from mammalian cells, without the use of acids or high salt solutions which are common for other histone isolation techniques. All components used in the protocol are common and inexpensive laboratory chemicals. The protocol has been evaluated on six commonly used cell lines and two animal tissue samples. The mild extraction conditions preserve delicate histone epigenetic changes, allowing its downstream analyses. We have demonstrated the assays' successful application during changes in the transcriptional activity of histone genes, cell cycle transitions, and DNA-damaging conditions. Histone fractions, obtained by the protocol, can be used for further applications, such as electrophoresis, immunoblot, and mass spectrometry. Therefore, the new proposed nucleosomal histone isolation method is sensitive, specific, and suitable for downstream applications of various kinds. [ABSTRACT FROM AUTHOR]

Packaging of the eukaryotic genome into chromatin places fundamental physical constraints on transcription. Clarifying how transcription operates within these constraints is essential to understand how eukaryotic gene expression programs are established and maintained. Here we review what is known about the mechanisms of transcription on chromatin templates. Current models indicate that transcription through chromatin is accomplished by the combination of an inherent nucleosome disrupting activity of RNA polymerase and the action of ATP-dependent chromatin remodeling motors. Collaboration between these two types of molecular motors is proposed to occur at all stages of transcription through diverse mechanisms. Further investigation of how these two motors combine their basic activities is essential to clarify the interdependent relationship between genome structure and transcription.

The epigenome of macrophages can be reprogrammed by extracellular cues, but the extent to which different stimuli achieve this is unclear. Nuclear factor κB (NF-κB) is a transcription factor that is activated by all pathogen-associated stimuli and can reprogram the epigenome by activating latent enhancers. However, we show that NF-κB does so only in response to a subset of stimuli. This stimulus specificity depends on the temporal dynamics of NF-κB activity, in particular whether it is oscillatory or non-oscillatory. Non-oscillatory NF-κB opens chromatin by sustained disruption of nucleosomal histone-DNA interactions, enabling activation of latent enhancers that modulate expression of immune response genes. Thus, temporal dynamics can determine a transcription factor's capacity to reprogram the epigenome in a stimulus-specific manner.

Centromeres play an essential function in cell division by specifying the site of kinetochore formation on each chromosome for mitotic spindle attachment. Centromeres are defined epigenetically by the histone H3 variant Centromere Protein A (Cenpa). Cenpa nucleosomes maintain the centromere by designating the site for new Cenpa assembly after dilution by replication. Vertebrate centromeres assemble on tandem arrays of repetitive sequences, but the function of repeat DNA in centromere formation has been challenging to dissect due to the difficulty in manipulating centromeres in cells. Xenopus laevis egg extracts assemble centromeres in vitro, providing a system for studying centromeric DNA functions. However, centromeric sequences in Xenopus laevis have not been extensively characterized. In this study, we combine Cenpa ChIP-seq with a k-mer based analysis approach to identify the Xenopus laevis centromere repeat sequences. By in situ hybridization, we show that Xenopus laevis centromeres contain diverse repeat sequences, and we map the centromere position on each Xenopus laevis chromosome using the distribution of centromere-enriched k-mers. Our identification of Xenopus laevis centromere sequences enables previously unapproachable centromere genomic studies. Our approach should be broadly applicable for the analysis of centromere and other repetitive sequences in any organism.

Abstract Background Nucleosome remodeling factors regulate the occupancy and positioning of nucleosomes genome-wide through ATP-driven DNA translocation. While many nucleosomes are consistently well-positioned, some nucleosomes and alternative nucleosome structures are more sensitive to nuclease digestion or are transitory. Fragile nucleosomes are nucleosome structures that are sensitive to nuclease digestion and may be composed of either six or eight histone proteins, making these either hexasomes or octasomes. Overlapping dinucleosomes are composed of two merged nucleosomes, lacking one H2A:H2B dimer, creating a 14-mer wrapped by ~ 250 bp of DNA. In vitro studies of nucleosome remodeling suggest that the collision of adjacent nucleosomes by sliding stimulates formation of overlapping dinucleosomes. Results To better understand how nucleosome remodeling factors regulate alternative nucleosome structures, we depleted murine embryonic stem cells of the transcripts encoding remodeler ATPases BRG1 or SNF2H, then performed MNase-seq. We used high- and low-MNase digestion to assess the effects of nucleosome remodeling factors on nuclease-sensitive or “fragile” nucleosome occupancy. In parallel we gel-extracted MNase-digested fragments to enrich for overlapping dinucleosomes. We recapitulate prior identification of fragile nucleosomes and overlapping dinucleosomes near transcription start sites, and identify enrichment of these features around gene-distal DNaseI hypersensitive sites, CTCF binding sites, and pluripotency factor binding sites. We find that BRG1 stimulates occupancy of fragile nucleosomes but restricts occupancy of overlapping dinucleosomes. Conclusions Overlapping dinucleosomes and fragile nucleosomes are prevalent within the ES cell genome, occurring at hotspots of gene regulation beyond their characterized existence at promoters. Although neither structure is fully dependent on either nucleosome remodeling factor, both fragile nucleosomes and overlapping dinucleosomes are affected by knockdown of BRG1, suggesting a role for the complex in creating or removing these structures.

Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) cooperate to determine cell identity by epigenetic gene expression regulation. However, the mechanism of PRC2 recruitment by means of recognition of PRC1-mediated H2AK119ub1 remains poorly understood. Our PRC2 cryo-electron microscopy structure with cofactors JARID2 and AEBP2 bound to a H2AK119ub1-containing nucleosome reveals a bridge helix in EZH2 that connects the SET domain, H3 tail, and nucleosomal DNA. JARID2 and AEBP2 each interact with one ubiquitin and the H2A-H2B surface. JARID2 stimulates PRC2 through interactions with both the polycomb protein EED and the H2AK119-ubiquitin, whereas AEBP2 has an additional scaffolding role. The presence of these cofactors partially overcomes the inhibitory effect that H3K4me3 and H3K36me3 exert on core PRC2 (in the absence of cofactors). Our results support a key role for JARID2 and AEBP2 in the cross-talk between histone modifications and PRC2 activity.

Evolutionary innovations in chromatin biology have been recently discovered through the study of fungi. In Saccharomyces cerevisiae, a prion form of a deacetylase complex assembles over subtelomeric domains that produces a heritable gene expression state that enables resistance to stress. In Candida albicans, stress triggers adaptive chromosome destabilization via erasure a centromeric histone H3, CENP-A; a process that cooperates with a newly evolved H2A variant lacking a mitotic phosphorylation site. Finally, in Cryptococcus neoformans, the loss of a cytosine DNA methyltransferase at least 50 million years ago has enabled the Darwinian evolution of methylation patterns over geological timescales. These studies reveal a remarkable genetic and epigenetic evolutionary plasticity of the chromatin fiber, despite the highly conserved structure of the nucleosome.

DNA methylation and histone tail modifications are interrelated mechanisms involved in a wide range of biological processes, and disruption of this crosstalk is linked to diseases such as acute myeloid leukemia. In addition, DNA methyltransferase 3A (DNMT3A) activity is modulated by several regulatory proteins, including p53 and thymine DNA glycosylase (TDG). However, the relative role of histone tails and regulatory proteins in the simultaneous coordination of DNMT3A activity remains obscure. We observed that DNMT3A binds H3 tails and p53 or TDG at distinct allosteric sites to form DNMT3A-H3 tail-p53 or -TDG multiprotein complexes. Functional characterization of DNMT3A-H3 tail-p53 or -TDG complexes on human-derived synthetic histone H3 tails, mononucleosomes, or polynucleosomes shows p53 and TDG play dominant roles in the modulation of DNMT3A activity. Intriguingly, this dominance occurs even when DNMT3A is actively methylating nucleosome substrates. The activity of histone modifiers is influenced by their ability to sense modifications on histone tails within the same nucleosome or histone tails on neighboring nucleosomes. In contrast, we show here that DNMT3A acts on DNA within a single nucleosome, on nucleosomal DNA within adjacent nucleosomes, and DNA not associated with the DNMT3A-nucleosome complex. Our findings have direct bearing on how the histone code drives changes in DNA methylation and highlight the complex interplay between histone tails, epigenetic enzymes, and modulators of enzymatic activity.