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.

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.

The Expanding Constellation of Histone Post-Translational Modifications in the Epigenetic Landscape.

The emergence of a nucleosome-based chromatin structure accompanied the evolutionary transition from prokaryotes to eukaryotes. In this scenario, histones became the heart of the complex and precisely timed coordination between chromatin architecture and functions during adaptive responses to environmental influence by means of epigenetic mechanisms. Notably, such an epigenetic machinery involves an overwhelming number of post-translational modifications at multiple residues of core and linker histones. This review aims to comprehensively describe old and recent evidence in this exciting field of research. In particular, histone post-translational modification establishing/removal mechanisms, their genomic locations and implication in nucleosome dynamics and chromatin-based processes, as well as their harmonious combination and interdependence will be discussed.

Cryo-EM structure of the nucleosome core particle containing Giardia lamblia histones.

Giardia lamblia is a pathogenic unicellular eukaryotic parasite that causes giardiasis. Its genome encodes the canonical histones H2A, H2B, H3, and H4, which share low amino acid sequence identity with their human orthologues. We determined the structure of the G. lamblia nucleosome core particle (NCP) at 3.6 Å resolution by cryo-electron microscopy. G. lamblia histones form a characteristic NCP, in which the visible 125 base-pair region of the DNA is wrapped in a left-handed supercoil. The acidic patch on the G. lamblia octamer is deeper, due to an insertion extending the H2B α1 helix and L1 loop, and thus cannot bind the LANA acidic patch binding peptide. The DNA and histone regions near the DNA entry-exit sites could not be assigned, suggesting that these regions are asymmetrically flexible in the G. lamblia NCP. Characterization by thermal unfolding in solution revealed that both the H2A-H2B and DNA association with the G. lamblia H3-H4 were weaker than those for human H3-H4. These results demonstrate the uniformity of the histone octamer as the organizing platform for eukaryotic chromatin, but also illustrate the unrecognized capability for large scale sequence variations that enable the adaptability of histone octamer surfaces and confer internal stability.

BRCA1/BARD1 site-specific ubiquitylation of nucleosomal H2A is directed by BARD1.

Mutations in the E3 ubiquitin ligase RING domains of BRCA1/BARD1 predispose carriers to breast and ovarian cancers. We present the structure of the BRCA1/BARD1 RING heterodimer with the E2 enzyme UbcH5c bound to its cellular target, the nucleosome, along with biochemical data that explain how the complex selectively ubiquitylates lysines 125, 127 and 129 in the flexible C-terminal tail of H2A in a fully human system. The structure reveals that a novel BARD1-histone interface couples to a repositioning of UbcH5c compared to the structurally similar PRC1 E3 ligase Ring1b/Bmi1 that ubiquitylates H2A Lys119 in nucleosomes. This interface is sensitive to both H3 Lys79 methylation status and mutations found in individuals with cancer. Furthermore, NMR reveals an unexpected mode of E3-mediated substrate regulation through modulation of dynamics in the C-terminal tail of H2A. Our findings provide insight into how E3 ligases preferentially target nearby lysine residues in nucleosomes by a steric occlusion and distancing mechanism.

Structures of Native-like Nucleosomes: One Step Closer toward Understanding the Structure and Function of Chromatin.

Genomic DNA in eukaryotes is organized into chromatin through association with core histone proteins to form nucleosomes. To understand the structure and function of chromatin, we must determine the structures of nucleosomes containing native DNA sequences. However, to date, our knowledge of nucleosome structures is mainly based on the crystallographic studies of the nucleosomes containing non-native DNA sequences. Here, we discuss the technical issues related to the determination of the nucleosome structures and review the few structural studies on native-like nucleosomes. We show how an antibody fragment-aided single-particle cryo-EM can be a useful method to determine the structures of nucleosomes containing genomic DNA. Finally, we provide a perspective for future structural studies of some native-like nucleosomes that play critical roles in chromatin functions.

Molecular basis of nucleosomal H3K36 methylation by NSD methyltransferases.

Histone methyltransferases of the nuclear receptor-binding SET domain protein (NSD) family, including NSD1, NSD2 and NSD3, have crucial roles in chromatin regulation and are implicated in oncogenesis1,2. NSD enzymes exhibit an autoinhibitory state that is relieved by binding to nucleosomes, enabling dimethylation of histone H3 at Lys36 (H3K36)3-7. However, the molecular basis that underlies this mechanism is largely unknown. Here we solve the cryo-electron microscopy structures of NSD2 and NSD3 bound to mononucleosomes. We find that binding of NSD2 and NSD3 to mononucleosomes causes DNA near the linker region to unwrap, which facilitates insertion of the catalytic core between the histone octamer and the unwrapped segment of DNA. A network of DNA- and histone-specific contacts between NSD2 or NSD3 and the nucleosome precisely defines the position of the enzyme on the nucleosome, explaining the specificity of methylation to H3K36. Intermolecular contacts between NSD proteins and nucleosomes are altered by several recurrent cancer-associated mutations in NSD2 and NSD3. NSDs that contain these mutations are catalytically hyperactive in vitro and in cells, and their ectopic expression promotes the proliferation of cancer cells and the growth of xenograft tumours. Together, our research provides molecular insights into the nucleosome-based recognition and histone-modification mechanisms of NSD2 and NSD3, which could lead to strategies for therapeutic targeting of proteins of the NSD family.

Structural basis for sequestration and autoinhibition of cGAS by chromatin.

Cyclic GMP-AMP synthase (cGAS) is an innate immune sensor for cytosolic microbial DNA1. After binding DNA, cGAS synthesizes the messenger 2'3'-cyclic GMP-AMP (cGAMP)2-4, which triggers cell-autonomous defence and the production of type I interferons and pro-inflammatory cytokines via the activation of STING5. In addition to responding to cytosolic microbial DNA, cGAS also recognizes mislocalized cytosolic self-DNA and has been implicated in autoimmunity and sterile inflammation6,7. Specificity towards pathogen- or damage-associated DNA was thought to be caused by cytosolic confinement. However, recent findings place cGAS robustly in the nucleus8-10, where tight tethering of chromatin is important to prevent autoreactivity to self-DNA8. Here we show how cGAS is sequestered and inhibited by chromatin. We provide a cryo-electron microscopy structure of the cGAS catalytic domain bound to a nucleosome, which shows that cGAS does not interact with the nucleosomal DNA, but instead interacts with histone 2A-histone 2B, and is tightly anchored to the 'acidic patch'. The interaction buries the cGAS DNA-binding site B, and blocks the formation of active cGAS dimers. The acidic patch robustly outcompetes agonistic DNA for binding to cGAS, which suggests that nucleosome sequestration can efficiently inhibit cGAS, even when accessible DNA is nearby, such as in actively transcribed genomic regions. Our results show how nuclear cGAS is sequestered by chromatin and provides a mechanism for preventing autoreactivity to nuclear self-DNA.

Mechanical properties of nucleoprotein complexes determined by nanoindentation spectroscopy.

The interplay between transcription factors, chromatin remodelers, 3-D organization, and mechanical properties of the chromatin fiber controls genome function in eukaryotes. Besides the canonical histones which fold the bulk of the chromatin into nucleosomes, histone variants create distinctive chromatin domains that are thought to regulate transcription, replication, DNA damage repair, and faithful chromosome segregation. Whether histone variants translate distinctive biochemical or biophysical properties to their associated chromatin structures, and whether these properties impact chromatin dynamics as the genome undergoes a multitude of transactions, is an important question in biology. Here, we describe single-molecule nanoindentation tools that we developed specifically to determine the mechanical properties of histone variant nucleosomes and their complexes. These methods join an array of cutting-edge new methods that further our quantitative understanding of the response of chromatin to intrinsic and extrinsic forces which act upon it during biological transactions in the nucleus.

A dynamic view of DNA structure within the nucleosome: Biological implications.

Most of eukaryotic cellular DNA is packed in nucleosome core particles (NCPs), in which the DNA (DNANCP) is wrapped around histones. The influence of this organization on the intrinsic local dynamics of DNA is largely unknown, in particular because capturing such information from experiments remains notoriously challenging. Given the importance of dynamical properties in DNA functions, we addressed this issue using CHARMM36 MD simulations of a nucleosome containing the NCP positioning 601 sequence and four related free dodecamers. Comparison between DNANCP and free DNA reveals a limited impact of the dense DNA-histone interface on correlated motions of dinucleotide constituents and on fluctuations of inter base pair parameters. A characteristic feature intimately associated with the DNANCP super-helical path is a set of structural periodicities that includes a marked alternation of regions enriched in backbone BI and BII conformers. This observation led to uncover a convincing correspondence between the sequence effect on BI/BII propensities in both DNANCP and free DNA, strengthening the idea that the histone preference for particular DNA sequences relies on those intrinsic structural properties. These results offer for the first time a detailed view of the DNA dynamical behavior within NCP. They show in particular that the DNANCP dynamics is substantial enough to preserve the ability to structurally adjust to external proteins, for instance remodelers. Also, fresh structural arguments highlight the relevance of relationships between DNA sequence and structural properties for NCP formation. Overall, our work offers a more rational framework to approach the functional, biological roles of NCP.

Role of nucleosome positioning in 3D chromatin organization and loop formation.

We present a physics-based polymer model that can investigate 3D organization of chromatin accounting for DNA elasticity, DNA-bending due to nucleosomes, and 1D organization of nucleosomes along DNA. We find that the packing density of chromatin oscillates between densities corresponding to highly folded and extended configurations as we change the nucleosome organization (length of linker DNA). We compute the looping probability of chromatin and show that the presence of nucleosomes increases the looping probability of the chain compared to that of a bare DNA. We also show that looping probability has a large variability depending on the nature of nucleosome organization and density of linker histones.

Computational approaches for inferring 3D conformations of chromatin from chromosome conformation capture data.

Chromosome conformation capture (3C) and its variants are powerful experimental techniques for probing intra- and inter-chromosomal interactions within cell nuclei at high resolution and in a high-throughput, quantitative manner. The contact maps derived from such experiments provide an avenue for inferring the 3D spatial organization of the genome. This review provides an overview of the various computational methods developed in the past decade for addressing the very important but challenging problem of deducing the detailed 3D structure or structure population of chromosomal domains, chromosomes, and even entire genomes from 3C contact maps.

PARP1 exhibits enhanced association and catalytic efficiency with γH2A.X-nucleosome.

The poly(ADP-ribose) polymerase, PARP1, plays a key role in maintaining genomic integrity by detecting DNA damage and mediating repair. γH2A.X is the primary histone marker for DNA double-strand breaks and PARP1 localizes to H2A.X-enriched chromatin damage sites, but the basis for this association is not clear. We characterize the kinetics of PARP1 binding to a variety of nucleosomes harbouring DNA double-strand breaks, which reveal that PARP1 associates faster with (γ)H2A.X- versus H2A-nucleosomes, resulting in a higher affinity for the former, which is maximal for γH2A.X-nucleosome that is also the activator eliciting the greatest poly-ADP-ribosylation catalytic efficiency. The enhanced activities with γH2A.X-nucleosome coincide with increased accessibility of the DNA termini resulting from the H2A.X-Ser139 phosphorylation. Indeed, H2A- and (γ)H2A.X-nucleosomes have distinct stability characteristics, which are rationalized by mutational analysis and (γ)H2A.X-nucleosome core crystal structures. This suggests that the γH2A.X epigenetic marker directly facilitates DNA repair by stabilizing PARP1 association and promoting catalysis.

Structural Basis of H2B Ubiquitination-Dependent H3K4 Methylation by COMPASS.

The COMPASS (complex of proteins associated with Set1) complex represents the prototype of the SET1/MLL family of methyltransferases that controls gene transcription by H3K4 methylation (H3K4me). Although H2B monoubiquitination (H2Bub) is well known as a prerequisite histone mark for COMPASS activity, how H2Bub activates COMPASS remains unclear. Here, we report the cryoelectron microscopy (cryo-EM) structures of an extended COMPASS catalytic module (CM) bound to the H2Bub and free nucleosome. The COMPASS CM clamps onto the nucleosome disk-face via an extensive interface to capture the flexible H3 N-terminal tail. The interface also sandwiches a critical Set1 arginine-rich motif (ARM) that autoinhibits COMPASS. Unexpectedly, without enhancing COMPASS-nucleosome interaction, H2Bub activates the enzymatic assembly by packing against Swd1 and alleviating the inhibitory effect of the Set1 ARM upon fastening it to the acidic patch. By delineating the spatial configuration of the COMPASS-H2Bub-nucleosome assembly, our studies establish the structural framework for understanding the long-studied H2Bub-H3K4me histone modification crosstalk.