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Cytosine can undergo modifications, forming 5-methylcytosine (5-mC) and its oxidized products 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Despite their importance as epigenetic markers and as central players in cellular processes, it is not well understood how these modifications influence physical properties of DNA and chromatin. Here we report a comprehensive survey of the effect of cytosine modifications on DNA flexibility. We find that even a single copy of 5-fC increases DNA flexibility markedly. 5-mC reduces and 5-hmC enhances flexibility, and 5-caC does not have a measurable effect. Molecular dynamics simulations show that these modifications promote or dampen structural fluctuations, likely through competing effects of base polarity and steric hindrance, without changing the average structure. The increase in DNA flexibility increases the mechanical stability of the nucleosome and vice versa, suggesting a gene regulation mechanism where cytosine modifications change the accessibility of nucleosomal DNA through their effects on DNA flexibility. Cytosine modifications are important epigenetic markers yet their physical influence on DNA is not well understood. Here, Ngo et al . show that different alterations affect DNA flexibility, suggesting a mechanism where modifications change accessibility of nucleosome bound DNA.

In inflammation, extensive cell death may occur, which results in the release of chromatin components into the extracellular environment. Individually, the purified chromatin components double stranded (ds)DNA and histones have been demonstrated, both in vitro and in vivo , to display various immunostimulatory effects, for example, histones induce cytotoxicity and proinflammatory signaling through toll-like receptor (TLR)2 and 4, while DNA induces signaling through TLR9 and intracellular nucleic acid sensing mechanisms. However, DNA and histones are organized in nucleosomes in the nucleus, and evidence suggests that nucleosomes are released as such in inflammation. The cytotoxicity and proinflammatory signaling induced by nucleosomes have not been studied as extensively as the separate effects brought about by histones and dsDNA, and there appear to be some marked differences. Remarkably, little distinction between the different forms in which histones circulate has been made throughout literature. This is partly due to the limitations of existing techniques to differentiate between histones in their free or DNA-bound form. Here we review the current understanding of immunostimulation induced by extracellular histones, dsDNA and nucleosomes, and discuss the importance of techniques that in their detection differentiate between these different chromatin components.

Biochemical reconstitution of a synthetic human kinetochore with 21 protein subunits and centromeric nucleosomal DNA unveils fundamental principles of kinetochore organization and function. A reconstituted 21-subunit kinetochore Kinetochores are multisubunit complexes that assemble on centromeres and connect chromosomes with microtubules in the mitotic spindle during cell division. Andrea Musacchio and colleagues have reconstituted a synthetic human kinetochore with 21 protein subunits and centromeric nucleosomal DNA. A seven-subunit subcomplex determines the binding selectivity for the centromeric histone CENP-A and binds directly to the microtubule-binding subcomplex of the outer kinetochore — an architecture reminiscent of that in Saccharomyces cerevisiae . Reconstituting this minimal linkage between centromeric chromatin and the mitotic spindle in vitro should allow further insights into kinetochore structure and function. Chromosomes are carriers of genetic material and their accurate transfer from a mother cell to its two daughters during cell division is of paramount importance for life. Kinetochores are crucial for this process, as they connect chromosomes with microtubules in the mitotic spindle 1 . Kinetochores are multi-subunit complexes that assemble on specialized chromatin domains, the centromeres, that are able to enrich nucleosomes containing the histone H3 variant centromeric protein A (CENP-A) 2 . A group of several additional CENPs, collectively known as constitutive centromere associated network (CCAN) 3 , 4 , 5 , 6 , establish the inner kinetochore, whereas a ten-subunit assembly known as the KMN network creates a microtubule-binding site in the outer kinetochore 7 , 8 . Interactions between CENP-A and two CCAN subunits, CENP-C and CENP-N, have been previously described 9 , 10 , 11 , but a comprehensive understanding of CCAN organization and of how it contributes to the selective recognition of CENP-A has been missing. Here we use biochemical reconstitution to unveil fundamental principles of kinetochore organization and function. We show that cooperative interactions of a seven-subunit CCAN subcomplex, the CHIKMLN complex, determine binding selectivity for CENP-A over H3-nucleosomes. The CENP-A:CHIKMLN complex binds directly to the KMN network, resulting in a 21-subunit complex that forms a minimal high-affinity linkage between CENP-A nucleosomes and microtubules in vitro . This structural module is related to fungal point kinetochores, which bind a single microtubule. Its convolution with multiple CENP-A proteins may give rise to the regional kinetochores of higher eukaryotes, which bind multiple microtubules. Biochemical reconstitution paves the way for mechanistic and quantitative analyses of kinetochores.

RAD51 is the central eukaryotic recombinase required for meiotic recombination and mitotic repair of double-strand DNA breaks (DSBs) 1 , 2 . However, the mechanism by which RAD51 functions at DSB sites in chromatin has remained elusive. Here we report the cryo-electron microscopy structures of human RAD51–nucleosome complexes, in which RAD51 forms ring and filament conformations. In the ring forms, the N-terminal lobe domains (NLDs) of RAD51 protomers are aligned on the outside of the RAD51 ring, and directly bind to the nucleosomal DNA. The nucleosomal linker DNA that contains the DSB site is recognized by the L1 and L2 loops—active centres that face the central hole of the RAD51 ring. In the filament form, the nucleosomal DNA is peeled by the RAD51 filament extension, and the NLDs of RAD51 protomers proximal to the nucleosome bind to the remaining nucleosomal DNA and histones. Mutations that affect nucleosome-binding residues of the RAD51 NLD decrease nucleosome binding, but barely affect DNA binding in vitro. Consistently, yeast Rad51 mutants with the corresponding mutations are substantially defective in DNA repair in vivo. These results reveal an unexpected function of the RAD51 NLD, and explain the mechanism by which RAD51 associates with nucleosomes, recognizes DSBs and forms the active filament in chromatin. Cryo-electron microscopy structures of human RAD51 in complex with the nucleosome show that RAD51 can adopt two conformations—rings and filaments—and reveal how RAD51 binds to the nucleosome through its N-terminal lobe domain.

The deubiquitinating enzyme BAP1 is an important tumor suppressor that has drawn attention in the clinic since its loss leads to a variety of cancers. BAP1 is activated by ASXL1 to deubiquitinate mono-ubiquitinated H2A at K119 in Polycomb gene repression, but the mechanism of this reaction remains poorly defined. Here we show that the BAP1 C-terminal extension is important for H2A deubiquitination by auto-recruiting BAP1 to nucleosomes in a process that does not require the nucleosome acidic patch. This initial encounter-like complex is unproductive and needs to be activated by the DEUBAD domains of ASXL1, ASXL2 or ASXL3 to increase BAP1’s affinity for ubiquitin on H2A, to drive the deubiquitination reaction. The reaction is specific for Polycomb modifications of H2A as the complex cannot deubiquitinate the DNA damage-dependent ubiquitination at H2A K13/15. Our results contribute to the molecular understanding of this important tumor suppressor. The tumor suppressor BAP1 is activated by ASXL1 to deubiquitinate mono-ubiquitinated H2A at K119 in Polycomb gene repression. Here, the authors show how BAP1’s C-terminal extension auto-recruits it to nucleosomes, where the DEUBAD domain of ASXL1 increases BAP1’s affinity for ubiquitin to drive deubiquitination.

Neutrophils release the serine proteases neutrophil elastase and cathepsin G, which have microbicidal activity and thereby contribute to the innate immune response. Steffen Massberg et al . now show that these neutrophil serine proteases, in association with extracellular nucleosomes, can also promote coagulation and thrombosis within large blood vessels. In a mouse model of systemic bacterial infection, these proteases spurred intravascular coagulation in the microcirculation of the liver, limiting bacterial tissue invasion. These findings point to a role for thrombosis in antimicrobial defense. Blood neutrophils provide the first line of defense against pathogens but have also been implicated in thrombotic processes. This dual function of neutrophils could reflect an evolutionarily conserved association between blood coagulation and antimicrobial defense, although the molecular determinants and in vivo significance of this association remain unclear. Here we show that major microbicidal effectors of neutrophils, the serine proteases neutrophil elastase and cathepsin G, together with externalized nucleosomes, promote coagulation and intravascular thrombus growth in vivo . The serine proteases and extracellular nucleosomes enhance tissue factor– and factor XII–dependent coagulation in a process involving local proteolysis of the coagulation suppressor tissue factor pathway inhibitor. During systemic infection, activation of coagulation fosters compartmentalization of bacteria in liver microvessels and reduces bacterial invasion into tissue. In the absence of a pathogen challenge, neutrophil-derived serine proteases and nucleosomes can contribute to large-vessel thrombosis, the main trigger of myocardial infarction and stroke. The ability of coagulation to suppress pathogen dissemination indicates that microvessel thrombosis represents a physiological tool of host defense.

The protein deacetylase SIRT6 maintains cellular homeostasis through multiple pathways that include the deacetylation of histone H3 and repression of transcription. Prior work suggests that SIRT6 is associated with chromatin and can substantially reduce global levels of H3 acetylation, but how SIRT6 is able to accomplish this feat is unknown. Here, we describe an exquisitely tight interaction between SIRT6 and nucleosome core particles, in which a 2:1 enzyme:nucleosome complex assembles via asymmetric binding with distinct affinities. While both SIRT6 molecules associate with the acidic patch on the nucleosome, we find that the intrinsically disordered SIRT6 C-terminus promotes binding at the higher affinity site through recognition of nucleosomal DNA. Together, multivalent interactions couple productive binding to efficient deacetylation of histones on endogenous chromatin. Unique among histone deacetylases, SIRT6 possesses the intrinsic capacity to tightly interact with nucleosomes for efficient activity. SIRT6 plays essential roles in metabolism, tumor suppression, and DNA repair through the deacetylation of histone substrates. Here the authors use biophysical methods to investigate the molecular basis for SIRT6 interaction with the nucleosome core particle.

Many stem cells undergo asymmetric division to produce a self-renewing stem cell and a differentiating daughter cell. Here we show that, similarly to H3, histone H4 is inherited asymmetrically in Drosophila melanogaster male germline stem cells undergoing asymmetric division. In contrast, both H2A and H2B are inherited symmetrically. By combining super-resolution microscopy and chromatin fiber analyses with proximity ligation assays on intact nuclei, we find that old H3 is preferentially incorporated by the leading strand, whereas newly synthesized H3 is enriched on the lagging strand. Using a sequential nucleoside analog incorporation assay, we detect a high incidence of unidirectional replication fork movement in testes-derived chromatin and DNA fibers. Biased fork movement coupled with a strand preference in histone incorporation would explain how asymmetric old and new H3 and H4 are established during replication. These results suggest a role for DNA replication in patterning epigenetic information in asymmetrically dividing cells in multicellular organisms.

Genetic and genomic techniques have proven incredibly powerful for identifying and studying molecular players implicated in the epigenetic regulation of DNA-templated processes such as transcription. However, achieving a mechanistic understanding of how these molecules interact with chromatin to elicit a functional output is non-trivial, owing to the tremendous complexity of the biochemical networks involved. Advances in protein engineering have enabled the reconstitution of ‘designer’ chromatin containing customized post-translational modification patterns, which, when used in conjunction with sophisticated biochemical and biophysical methods, allow many mechanistic questions to be addressed. In this Review, we discuss how such tools complement established ‘omics’ techniques to answer fundamental questions on chromatin regulation, focusing on chromatin mark establishment and protein–chromatin interactions.In this Review, Hananya et al. discuss how designer chromatin containing specific patterns of post-translational modifications is being used to study the mechanisms underlying the epigenetic regulation of gene expression.

Interaction proteomics studies have provided fundamental insights into multimeric biomolecular assemblies and cell-scale molecular networks. Significant recent developments in mass spectrometry-based interaction proteomics have been fueled by rapid advances in label-free, isotopic, and isobaric quantitation workflows. Here, we report a quantitative protein–DNA and protein–nucleosome binding assay that uses affinity purifications from nuclear extracts coupled with isobaric chemical labeling and mass spectrometry to quantify apparent binding affinities proteome-wide. We use this assay with a variety of DNA and nucleosome baits to quantify apparent binding affinities of monomeric and multimeric transcription factors and chromatin remodeling complexes. Quantitative mass spectrometry enables the proteome-wide assessment of biomolecular binding affinities. While previous approaches mainly focused on protein–small molecule interactions, the authors here present a method to probe protein–DNA and protein–nucleosome binding affinities at proteome scale.