Molecular and structural basis of the chromatin remodeling activity by Arabidopsis DDM1.

The histone H2A variant H2A.W occupies transposons and thus prevents access to them in Arabidopsis thaliana. H2A.W is deposited by the chromatin remodeler DDM1, which also promotes the accessibility of chromatin writers to heterochromatin by an unknown mechanism. To shed light on this question, we solve the cryo-EM structures of nucleosomes containing H2A and H2A.W, and the DDM1-H2A.W nucleosome complex. These structures show that the DNA end flexibility of the H2A nucleosome is higher than that of the H2A.W nucleosome. In the DDM1-H2A.W nucleosome complex, DDM1 binds to the N-terminal tail of H4 and the nucleosomal DNA and increases the DNA end flexibility of H2A.W nucleosomes. Based on these biochemical and structural results, we propose that DDM1 counters the low accessibility caused by nucleosomes containing H2A.W to enable the maintenance of repressive epigenetic marks on transposons and prevent their activity.

Structural basis for the H2AK119ub1-specific DNMT3A-nucleosome interaction.

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 DNMT3A1's 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 UDR's 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.

Studying Structure and Functions of Nucleosomes with Atomic Force Microscopy.

Chromatin is an epigenetic platform for implementation of DNA-dependent processes. Nucleosome, as a basic level of chromatin compaction, largely determines its properties and structure. In the study of nucleosomes structure and functions physicochemical tools are actively used, such as magnetic and optical "tweezers", "DNA curtains", nuclear magnetic resonance, X-ray crystallography, and cryogenic electron microscopy, as well as optical methods based on Förster resonance energy transfer. Despite the fact that these approaches make it possible to determine a wide range of structural and functional characteristics of chromatin and nucleosomes with high spatial and time resolution, atomic force microscopy (AFM) complements the capabilities of these methods. The results of structural studies of nucleosome focusing on the AFM method development are presented in this review. The possibilities of AFM are considered in the context of application of other physicochemical approaches.

Angle between DNA linker and nucleosome core particle regulates array compaction revealed by individual-particle cryo-electron tomography.

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.

To slide or not to slide: key role of the hexasome in chromatin remodeling revealed.

Hexasomes are non-canonical nucleosomes that package DNA with six instead of eight histones. First discovered 40 years ago as a consequence of transcription, two near-atomic-resolution cryo-EM structures of the hexasome in complex with the chromatin remodeler INO80 have now started to unravel its mechanistic impact on the regulatory landscape of chromatin. Loss of one histone H2A-H2B dimer converts inactive nucleosomes into distinct and favorable substrates for ATP-dependent chromatin remodeling.

Cryo-EM structure and functional analysis of the chromatin remodeler RSF.

The RSF complex belongs to the ISWI chromatin-remodeling family and is composed of two subunits: RSF1 (remodeling and spacing factor 1) and SNF2h (sucrose nonfermenting protein 2 homolog). The RSF complex participates in nucleosome spacing and assembly, and subsequently promotes nucleosome maturation. Although SNF2h has been extensively studied in the last few years, the structural and functional properties of the remodeler RSF1 still remain vague. Here, a cryo-EM structure of the RSF-nucleosome complex is reported. The 3D model shows a two-lobe architecture of RSF, and the structure of the RSF-nucleosome (flanked with linker DNA) complex shows that the RSF complex moves the DNA away from the histone octamer surface at the DNA-entry point. Additionally, a nucleosome-sliding assay and a restriction-enzyme accessibility assay show that the RSF1 subunit may cause changes in the chromatin-remodeling properties of SNF2h. As a `nucleosome ruler', the results of an RSF-dinucleosome binding affinity test led to the proposal that the critical distance that RSF `measures' between two nucleosomes is about 24 base pairs.

High-Speed Atomic Force Microscopy Reveals the Nucleosome Sliding and DNA Unwrapping/Wrapping Dynamics of Tail-less Nucleosomes.

Each nucleosome contains four types of histone proteins, each with a histone tail. These tails are essential for the epigenetic regulation of gene expression through post-translational modifications (PTMs). However, their influence on nucleosome dynamics at the single-molecule level remains undetermined. Here, we employed high-speed atomic force microscopy to visualize nucleosome dynamics in the absence of the N-terminal tail of each histone or all of the N-terminal tails. Loss of all tails stripped 6.7 base pairs of the nucleosome from the histone core, and the DNA entry-exit angle expanded by 18° from that of wild-type nucleosomes. Tail-less nucleosomes, particularly those without H2B and H3 tails, showed a 10-fold increase in dynamics, such as nucleosome sliding and DNA unwrapping/wrapping, within 0.3 s, emphasizing their role in histone-DNA interactions. Our findings illustrate that N-terminal histone tails stabilize the nucleosome structure, suggesting that histone tail PTMs modulate nucleosome dynamics.

Cryo-EM structures of RAD51 assembled on nucleosomes containing a DSB site.

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.

Parental histone transfer caught at the replication fork.

In eukaryotes, DNA compacts into chromatin through nucleosomes1,2. Replication of the eukaryotic genome must be coupled to the transmission of the epigenome encoded in the chromatin3,4. Here we report cryo-electron microscopy structures of yeast (Saccharomyces cerevisiae) replisomes associated with the FACT (facilitates chromatin transactions) complex (comprising Spt16 and Pob3) and an evicted histone hexamer. In these structures, FACT is positioned at the front end of the replisome by engaging with the parental DNA duplex to capture the histones through the middle domain and the acidic carboxyl-terminal domain of Spt16. The H2A-H2B dimer chaperoned by the carboxyl-terminal domain of Spt16 is stably tethered to the H3-H4 tetramer, while the vacant H2A-H2B site is occupied by the histone-binding domain of Mcm2. The Mcm2 histone-binding domain wraps around the DNA-binding surface of one H3-H4 dimer and extends across the tetramerization interface of the H3-H4 tetramer to the binding site of Spt16 middle domain before becoming disordered. This arrangement leaves the remaining DNA-binding surface of the other H3-H4 dimer exposed to additional interactions for further processing. The Mcm2 histone-binding domain and its downstream linker region are nested on top of Tof1, relocating the parental histones to the replisome front for transfer to the newly synthesized lagging-strand DNA. Our findings offer crucial structural insights into the mechanism of replication-coupled histone recycling for maintaining epigenetic inheritance.

Nucleosome-bound NR5A2 structure reveals pioneer factor mechanism by DNA minor groove anchor competition.

Gene expression during natural and induced reprogramming is controlled by pioneer transcription factors that initiate transcription from closed chromatin. Nr5a2 is a key pioneer factor that regulates zygotic genome activation in totipotent embryos, pluripotency in embryonic stem cells and metabolism in adult tissues, but the mechanism of its pioneer activity remains poorly understood. Here, we present a cryo-electron microscopy structure of human NR5A2 bound to a nucleosome. The structure shows that the conserved carboxy-terminal extension (CTE) loop of the NR5A2 DNA-binding domain competes with a DNA minor groove anchor of the nucleosome and releases entry-exit site DNA. Mutational analysis showed that NR5A2 D159 of the CTE is dispensable for DNA binding but required for stable nucleosome association and persistent DNA 'unwrapping'. These findings suggest that NR5A2 belongs to an emerging class of pioneer factors that can use DNA minor groove anchor competition to destabilize nucleosomes and facilitate gene expression during reprogramming.

From Nucleosomes to Compartments: Physicochemical Interactions Underlying Chromatin Organization.

Chromatin organization plays a critical role in cellular function by regulating access to genetic information. However, understanding chromatin folding is challenging due to its complex, multiscale nature. Significant progress has been made in studying in vitro systems, uncovering the structure of individual nucleosomes and their arrays, and elucidating the role of physicochemical forces in stabilizing these structures. Additionally, remarkable advancements have been achieved in characterizing chromatin organization in vivo, particularly at the whole-chromosome level, revealing important features such as chromatin loops, topologically associating domains, and nuclear compartments. However, bridging the gap between in vitro and in vivo studies remains challenging. The resemblance between in vitro and in vivo chromatin conformations and the relevance of internucleosomal interactions for chromatin folding in vivo are subjects of debate. This article reviews experimental and computational studies conducted at various length scales, highlighting the significance of intrinsic interactions between nucleosomes and their roles in chromatin folding in vivo.

The CRL5-SPSB3 ubiquitin ligase targets nuclear cGAS for degradation.

Cyclic GMP-AMP synthase (cGAS) senses aberrant DNA during infection, cancer and inflammatory disease, and initiates potent innate immune responses through the synthesis of 2'3'-cyclic GMP-AMP (cGAMP)1-7. The indiscriminate activity of cGAS towards DNA demands tight regulatory mechanisms that are necessary to maintain cell and tissue homeostasis under normal conditions. Inside the cell nucleus, anchoring to nucleosomes and competition with chromatin architectural proteins jointly prohibit cGAS activation by genomic DNA8-15. However, the fate of nuclear cGAS and its role in cell physiology remains unclear. Here we show that the ubiquitin proteasomal system (UPS) degrades nuclear cGAS in cycling cells. We identify SPSB3 as the cGAS-targeting substrate receptor that associates with the cullin-RING ubiquitin ligase 5 (CRL5) complex to ligate ubiquitin onto nuclear cGAS. A cryo-electron microscopy structure of nucleosome-bound cGAS in a complex with SPSB3 reveals a highly conserved Asn-Asn (NN) minimal degron motif at the C terminus of cGAS that directs SPSB3 recruitment, ubiquitylation and cGAS protein stability. Interference with SPSB3-regulated nuclear cGAS degradation primes cells for type I interferon signalling, conferring heightened protection against infection by DNA viruses. Our research defines protein degradation as a determinant of cGAS regulation in the nucleus and provides structural insights into an element of cGAS that is amenable to therapeutic exploitation.

Histone modifications regulate pioneer transcription factor cooperativity.

Pioneer transcription factors have the ability to access DNA in compacted chromatin1. Multiple transcription factors can bind together to a regulatory element in a cooperative way, and cooperation between the pioneer transcription factors OCT4 (also known as POU5F1) and SOX2 is important for pluripotency and reprogramming2-4. However, the molecular mechanisms by which pioneer transcription factors function and cooperate on chromatin remain unclear. Here we present cryo-electron microscopy structures of human OCT4 bound to a nucleosome containing human LIN28B or nMATN1 DNA sequences, both of which bear multiple binding sites for OCT4. Our structural and biochemistry data reveal that binding of OCT4 induces changes to the nucleosome structure, repositions the nucleosomal DNA and facilitates cooperative binding of additional OCT4 and of SOX2 to their internal binding sites. The flexible activation domain of OCT4 contacts the N-terminal tail of histone H4, altering its conformation and thus promoting chromatin decompaction. Moreover, the DNA-binding domain of OCT4 engages with the N-terminal tail of histone H3, and post-translational modifications at H3K27 modulate DNA positioning and affect transcription factor cooperativity. Thus, our findings suggest that the epigenetic landscape could regulate OCT4 activity to ensure proper cell programming.

Structure of the NuA4 acetyltransferase complex bound to the nucleosome.

Deoxyribonucleic acid in eukaryotes wraps around the histone octamer to form nucleosomes1, the fundamental unit of chromatin. The N termini of histone H4 interact with nearby nucleosomes and play an important role in the formation of high-order chromatin structure and heterochromatin silencing2-4. NuA4 in yeast and its homologue Tip60 complex in mammalian cells are the key enzymes that catalyse H4 acetylation, which in turn regulates chromatin packaging and function in transcription activation and DNA repair5-10. Here we report the cryo-electron microscopy structure of NuA4 from Saccharomyces cerevisiae bound to the nucleosome. NuA4 comprises two major modules: the catalytic histone acetyltransferase (HAT) module and the transcription activator-binding (TRA) module. The nucleosome is mainly bound by the HAT module and is positioned close to a polybasic surface of the TRA module, which is important for the optimal activity of NuA4. The nucleosomal linker DNA carrying the upstream activation sequence is oriented towards the conserved, transcription activator-binding surface of the Tra1 subunit, which suggests a potential mechanism of NuA4 to act as a transcription co-activator. The HAT module recognizes the disk face of the nucleosome through the H2A-H2B acidic patch and nucleosomal DNA, projecting the catalytic pocket of Esa1 to the N-terminal tail of H4 and supporting its function in selective acetylation of H4. Together, our findings illustrate how NuA4 is assembled and provide mechanistic insights into nucleosome recognition and transcription co-activation by a HAT.

Columnar structure of human telomeric chromatin.

Telomeres, the ends of eukaryotic chromosomes, play pivotal parts in ageing and cancer and are targets of DNA damage and the DNA damage response1-5. Little is known about the structure of telomeric chromatin at the molecular level. Here we used negative stain electron microscopy and single-molecule magnetic tweezers to characterize 3-kbp-long telomeric chromatin fibres. We also obtained the cryogenic electron microscopy structure of the condensed telomeric tetranucleosome and its dinucleosome unit. The structure displayed close stacking of nucleosomes with a columnar arrangement, and an unusually short nucleosome repeat  length that comprised about 132 bp DNA wound in a continuous superhelix around histone octamers. This columnar structure is primarily stabilized by the H2A carboxy-terminal and histone amino-terminal tails in a synergistic manner. The columnar conformation results in exposure of the DNA helix, which may make it susceptible to both DNA damage and the DNA damage response. The conformation also exists in an alternative open state, in which one nucleosome is unstacked and flipped out, which exposes the acidic patch of the histone surface. The structural features revealed in this work suggest mechanisms by which protein factors involved in telomere maintenance can access telomeric chromatin in its compact form.

Transcription destabilizes centromere function.

Bi-oriented attachment of microtubules to the centromere is a pre-requisite for faithful chromosome segregation during mitosis. Budding yeast have point centromeres containing the cis-element proteins CDE-I, -II, and -III, which interact with trans-acting factors such as Cbf1, Cse4, and Ndc10. Our previous genetic screens, using a comprehensive library of histone point mutants, revealed that the TBS-I, -II, and -III regions of nucleosomes are required for faithful chromosome segregation. In TBS-III deficient cells, peri-centromeric nucleosomes containing the H2A.Z homolog Htz1 are lacking, however, it is unclear why chromosome segregation is defective in these cells. Here, we show that, in cells lacking TBS-III, both chromatin binding at the centromere and the total amount of some of the centromere proteins are reduced, and transcription through the centromere is up-regulated during M-phase. Moreover, the chromatin binding of Cse4, Mif2, Cbf1, Ndc10, and Scm3 was reduced upon ectopic transcription through the centromere in wild-type cells. These results suggest that transcription through the centromere displaces key centromere proteins and, consequently, destabilizes the interaction between centromeres and microtubules, leading to defective chromosome segregation. The identification of new roles for histone binding residues in TBS-III will shed new light on nucleosome function during chromosome segregation.

Unique protein interaction networks define the chromatin remodelling module of the NuRD complex.

The combination of four proteins and their paralogues including MBD2/3, GATAD2A/B, CDK2AP1 and CHD3/4/5, which we refer to as the MGCC module, form the chromatin remodelling module of the nucleosome remodelling and deacetylase (NuRD) complex. To date, mechanisms by which the MGCC module acquires paralogue-specific function and specificity have not been addressed. Understanding the protein-protein interaction (PPI) network of the MGCC subunits is essential for defining underlying mechanisms of gene regulation. Therefore, using pulldown followed by mass spectrometry analysis (PD-MS), we report a proteome-wide interaction network of the MGCC module in a paralogue-specific manner. Our data also demonstrate that the disordered C-terminal region of CHD3/4/5 is a gateway to incorporate remodelling activity into both ChAHP (CHD4, ADNP, HP1γ) and NuRD complexes in a mutually exclusive manner. We define a short aggregation-prone region (APR) within the C-terminal segment of GATAD2B that is essential for the interaction of CHD4 and CDK2AP1 with the NuRD complex. Finally, we also report an association of CDK2AP1 with the nuclear receptor co-repressor (NCOR) complex. Overall, this study provides insight into the possible mechanisms through which the MGCC module can achieve specificity and diverse biological functions.

Super resolution microscopy reveals how elongating RNA polymerase II and nascent RNA interact with nucleosome clutches.

Transcription and genome architecture are interdependent, but it is still unclear how nucleosomes in the chromatin fiber interact with nascent RNA, and which is the relative nuclear distribution of these RNAs and elongating RNA polymerase II (RNAP II). Using super-resolution (SR) microscopy, we visualized the nascent transcriptome, in both nucleoplasm and nucleolus, with nanoscale resolution. We found that nascent RNAs organize in structures we termed RNA nanodomains, whose characteristics are independent of the number of transcripts produced over time. Dual-color SR imaging of nascent RNAs, together with elongating RNAP II and H2B, shows the physical relation between nucleosome clutches, RNAP II, and RNA nanodomains. The distance between nucleosome clutches and RNA nanodomains is larger than the distance measured between elongating RNAP II and RNA nanodomains. Elongating RNAP II stands between nascent RNAs and the small, transcriptionally active, nucleosome clutches. Moreover, RNA factories are small and largely formed by few RNAP II. Finally, we describe a novel approach to quantify the transcriptional activity at an individual gene locus. By measuring local nascent RNA accumulation upon transcriptional activation at single alleles, we confirm the measurements made at the global nuclear level.

Unusual nucleosome formation and transcriptome influence by the histone H3mm18 variant.

Histone H3mm18 is a non-allelic H3 variant expressed in skeletal muscle and brain in mice. However, its function has remained enigmatic. We found that H3mm18 is incorporated into chromatin in cells with low efficiency, as compared to H3.3. We determined the structures of the nucleosome core particle (NCP) containing H3mm18 by cryo-electron microscopy, which revealed that the entry/exit DNA regions are drastically disordered in the H3mm18 NCP. Consistently, the H3mm18 NCP is substantially unstable in vitro. The forced expression of H3mm18 in mouse myoblast C2C12 cells markedly suppressed muscle differentiation. A transcriptome analysis revealed that the forced expression of H3mm18 affected the expression of multiple genes, and suppressed a group of genes involved in muscle development. These results suggest a novel gene expression regulation system in which the chromatin landscape is altered by the formation of unusual nucleosomes with a histone variant, H3mm18, and provide important insight into understanding transcription regulation by chromatin.

Effect of histone H4 tail on nucleosome stability and internucleosomal interactions.

Chromatin structure is dictated by nucleosome assembly and internucleosomal interactions. The tight wrapping of nucleosomes inhibits gene expression, but modifications to histone tails modulate chromatin structure, allowing for proper genetic function. The histone H4 tail is thought to play a large role in regulating chromatin structure. Here we investigated the structure of nucleosomes assembled with a tail-truncated H4 histone using Atomic Force Microscopy. We assembled tail-truncated H4 nucleosomes on DNA templates allowing for the assembly of mononucleosomes or dinucleosomes. Mononucleosomes assembled on nonspecific DNA led to decreased DNA wrapping efficiency. This effect is less pronounced for nucleosomes assembled on positioning motifs. Dinucleosome studies resulted in the discovery of two effects- truncation of the H4 tail does not diminish the preferential positioning observed in full-length nucleosomes, and internucleosomal interaction eliminates the DNA unwrapping effect. These findings provide insight on the role of histone H4 in chromatin structure and stability.