Since the first high-resolution structure of the nucleosome was reported in 1997, the available information on chromatin structure has increased very rapidly. Here, we review insights derived from cutting-edge biophysical and structural approaches applied to the study of nucleosome dynamics and nucleosome-binding factors, with a focus on the experimental advances driving the research. In addition, we highlight emerging challenges in nucleosome structural biology.

/pdf/nucleosome-structure-and-dynamics-are-coming-of-age-55ix2a8p11.pdf

Nucleosome structure and dynamics are coming of age.

The histone variant H2A.Z is involved in several processes such as transcriptional control, DNA repair, regulation of centromeric heterochromatin and, not surprisingly, is implicated in diseases such as cancer. Here, we review the recent developments on H2A.Z focusing on its role in transcriptional activation and repression. H2A.Z, as a replication-independent histone, has been studied in several model organisms and inducible mammalian model systems. Its loading machinery and several modifying enzymes have been recently identified, and some of the long-standing discrepancies in transcriptional activation and/or repression are about to be resolved. The buffering functions of H2A.Z, as supported by genome-wide localization and analyzed in several dynamic systems, are an excellent example of transcriptional control. Posttranslational modifications such as acetylation and ubiquitination of H2A.Z, as well as its specific binding partners, are in our view central players in the control of gene expression. Understanding the key-mechanisms in either turnover or stabilization of H2A.Z-containing nucleosomes as well as defining the H2A.Z interactome will pave the way for therapeutic applications in the future.

/pdf/the-histone-variant-h2a-z-in-gene-regulation-2p6h677qk5.pdf

The histone variant H2A.Z in gene regulation

Mammalian SWI/SNF family chromatin remodelers, BRG1/BRM-associated factor (BAF) and polybromo-associated BAF (PBAF), regulate chromatin structure and transcription, and their mutations are linked to cancers. The 3.7-angstrom-resolution cryo-electron microscopy structure of human BAF bound to the nucleosome reveals that the nucleosome is sandwiched by the base and the adenosine triphosphatase (ATPase) modules, which are bridged by the actin-related protein (ARP) module. The ATPase motor is positioned proximal to nucleosomal DNA and, upon ATP hydrolysis, engages with and pumps DNA along the nucleosome. The C-terminal α helix of SMARCB1, enriched in positively charged residues frequently mutated in cancers, mediates interactions with an acidic patch of the nucleosome. AT-rich interactive domain-containing protein 1A (ARID1A) and the SWI/SNF complex subunit SMARCC serve as a structural core and scaffold in the base module organization, respectively. Our study provides structural insights into subunit organization and nucleosome recognition of human BAF complex.

Structure of nucleosome-bound human BAF complex.

Genetic Control of Expression and Splicing in Developing Human Brain Informs Disease Mechanisms.

In the eukaryotic nucleus, DNA is packaged in the form of nucleosomes, each of which comprises about 147 base pairs of DNA wrapped around a histone protein octamer. The position and histone composition of nucleosomes is governed by ATP-dependent chromatin remodellers1–3 such as the 15-subunit INO80 complex4. INO80 regulates gene expression, DNA repair and replication by sliding nucleosomes, the exchange of histone H2A.Z with H2A, and the positioning of + 1 and −1 nucleosomes at promoter DNA5–8. The structures and mechanisms of these remodelling reactions are currently unknown. Here we report the cryo-electron microscopy structure of the evolutionarily conserved core of the INO80 complex from the fungus Chaetomium thermophilum bound to a nucleosome, at a global resolution of 4.3 A and with major parts at 3.7 A. The INO80 core cradles one entire gyre of the nucleosome through multivalent DNA and histone contacts. An Rvb1/Rvb2 AAA+ ATPase heterohexamer is an assembly scaffold for the complex and acts as a ‘stator’ for the motor and nucleosome-gripping subunits. The Swi2/Snf2 ATPase motor binds to nucleosomal DNA at superhelical location −6, unwraps approximately 15 base pairs, disrupts the H2A–DNA contacts and is poised to pump entry DNA into the nucleosome. Arp5 and Ies6 bind superhelical locations −2 and −3 to act as a counter grip for the motor, on the other side of the H2A–H2B dimer. The Arp5 insertion domain forms a grappler element that binds the nucleosome dyad, connects the Arp5 actin-fold and entry DNA over a distance of about 90 A and packs against histone H2A–H2B near the ‘acidic patch’. Our structure together with biochemical data8 suggests a unified mechanism for nucleosome sliding and histone editing by INO80. The motor is part of a macromolecular ratchet, persistently pumping entry DNA across the H2A–H2B dimer against the Arp5 grip until a large nucleosome translocation step occurs. The transient exposure of H2A–H2B by motor activity as well as differential recognition of H2A.Z and H2A may regulate histone exchange. Cryo-electron microscopy structures of the evolutionarily conserved core of a fungal INO80 complex bound to the nucleosomal substrate reveal the mechanism underlying nucleosome sliding and histone editing used by this ATP-dependent chromatin remodeller.

Structural basis for ATP-dependent chromatin remodelling by the INO80 complex.

Access to DNA within nucleosomes is required for a variety of processes in cells including transcription, replication and repair. Consequently, cells encode multiple systems that remodel nucleosomes. These complexes can be simple, involving one or a few protein subunits, or more complicated multi-subunit machines
 1
 . Biochemical studies2–4 have placed the motor domains of several chromatin remodellers in the superhelical location 2 region of the nucleosome. Structural studies of yeast Chd1 and Snf2—a subunit in the complex with the capacity to remodel the structure of chromatin (RSC)—in complex with nucleosomes5–7 have provided insights into the basic mechanism of nucleosome sliding performed by these complexes. However, how larger, multi-subunit remodelling complexes such as INO80 interact with nucleosomes and how remodellers carry out functions such as nucleosome sliding
 8
 , histone exchange
 9
 and nucleosome spacing10–12 remain poorly understood. Although some remodellers work as monomers
 13
 , others work as highly cooperative dimers11, 14, 15. Here we present the structure of the human INO80 chromatin remodeller with a bound nucleosome, which reveals that INO80 interacts with nucleosomes in a previously undescribed manner: the motor domains are located on the DNA at the entry point to the nucleosome, rather than at superhelical location 2. The ARP5–IES6 module of INO80 makes additional contacts on the opposite side of the nucleosome. This arrangement enables the histone H3 tails of the nucleosome to have a role in the regulation of the activities of the INO80 motor domain—unlike in other characterized remodellers, for which H4 tails have been shown to regulate the motor domains.

Structure and regulation of the human INO80–nucleosome complex

INTRODUCTION Canonical nucleosomes contain two copies of each of four histone proteins: H2A, H2B, H3, and H4. However, variants of these histones can be inserted by adenosine triphosphate (ATP)–dependent chromatin-remodeling machines. The yeast SWR1 chromatin-remodeling complex, a member of the INO80 remodeler family, catalyzes the exchange of H2A-H2B dimers for dimers containing Htz1 (H2A.Z in human) in an ATP-dependent manner. However, the mechanism by which SWR1 exchanges histones is poorly understood. Despite having a DNA translocase subunit similar to that in the INO80 complex that slides nucleosomes, no net translocation of nucleosomes has been reported for SWR1. Consequently, the function of the ATPase activity, which is required for histone exchange in SWR1, has remained enigmatic. RATIONALE To obtain sufficient quantities for structural analysis, we generated the complete 14-subunit yeast SWR1 complex in insect cells. Binding of nucleosomes to SWR1 is stabilized in the presence of an ATP analog (ADP•BeF 3 ), which we used to prepare a complex with a canonical yeast H2A-containing nucleosome. Structural analysis was undertaken by cryo–electron microscopy (cryo-EM). We also used single-molecule FRET (smFRET) techniques to probe the dynamics of nucleosomes bound to SWR1. Fluorescent probes were positioned on the H2A histones and the end of the DNA to monitor changes in nucleosome dynamics upon binding of SWR1 and ATP (or ATP analogs). RESULTS We determined the cryo-EM structure of the SWR1-nucleosome complex at 3.6-A resolution. The architecture of the complex shows how the SWR1 complex is assembled around a heterohexameric core of the RuvBL1 and RuvBL2 subunits. The Swr1 motor subunit binds at superhelical location 2 (SHL2), a position it shares in common with other remodelers but not with its most closely related complex, INO80, which binds at SHL6-SHL7. Binding of ATP or ADP•BeF 3 to the SWR1-nucleosome complex induces substantial unwrapping of the DNA wrap. Conformational changes in the motor domains of the Swr1 subunit drive a single–base pair translocation of the DNA wrap from the DNA entry site. The single–base pair DNA translocation accompanies conformational changes in the histone core that begin to destabilize the histone dimer interface. Using smFRET methods, we further probed these conformational changes to show how an increase in the dynamics of the SWR1-bound nucleosomes is dependent on binding of ATP but not hydrolysis. CONCLUSION The cryo-EM structure of the SWR1 complex bound to a nucleosome reveals details of the intricate interactions between components of the SWR1 complex and its nucleosome substrate. Interactions between the Swr1 motor domains and the DNA wrap at SHL2 distort the DNA, causing a bulge with concomitant translocation of the DNA by one base pair, coupled to conformational changes of the histone core that likely destabilize the dimer interface. Furthermore, partial unwrapping of the DNA from the histone core takes place upon binding of nucleosomes to the SWR1 complex. Single-molecule data monitor this unwrapping and show how the dynamics are altered by ATP binding prior to hydrolysis.

Structure and dynamics of the yeast SWR1-nucleosome complex

Several chromatin remodellers have the ability to space nucleosomes on DNA. For ISWI remodellers, this involves an interplay between H4 histone tails, the AutoN and NegC motifs of the motor domains that together regulate ATPase activity and sense the length of DNA flanking the nucleosome. By contrast, the INO80 complex also spaces nucleosomes but is not regulated by H4 tails and lacks the AutoN and NegC motifs. Instead nucleosome sliding requires cooperativity between two INO80 complexes that monitor DNA length simultaneously on either side of the nucleosome during sliding. The C-terminal domain of the human Ino80 subunit (Ino80CTD) binds cooperatively to DNA and dimerisation of these domains provides crosstalk between complexes. ATPase activity, rather than being regulated, instead gradually becomes uncoupled as nucleosome sliding reaches an end point and this is controlled by the Ino80CTD. A single active ATPase motor within the dimer is sufficient for sliding.

/pdf/crosstalk-within-a-functional-ino80-complex-dimer-regulates-38go6frvd3.pdf

Crosstalk within a functional INO80 complex dimer regulates nucleosome sliding.

We have prepared recombinant fourteen subunit yeast SWR1 complex from insect cells using a modified MultiBac system. The 1.07 MDa recombinant protein complex has histone-exchange activity. Full exchange activity is realized with a single SWR1 complex bound to a nucleosome. We also prepared mutant complexes that lack a variety of subunits or combinations of subunits and these start to reveal roles for some of these subunits as well as indicating interactions between them in the full complex. Complexes containing a series of N-terminally and C-terminally truncated Swr1 subunits reveal further details about interactions between subunits as well as their binding sites on the Swr1 subunit. Finally, we present electron microscopy studies revealing the dynamic nature of the complex and a 21 A resolution reconstruction of the intact complex provides details not apparent in previously reported structures, including a large central cavity of sufficient size to accommodate a nucleosome.

/pdf/functional-characterization-and-architecture-of-recombinant-1c60es1gq8.pdf

Functional characterization and architecture of recombinant yeast SWR1 histone exchange complex.

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Structure and regulation of the human INO80–nucleosome complex

Structure and dynamics of the yeast SWR1-nucleosome complex

Crosstalk within a functional INO80 complex dimer regulates nucleosome sliding.

Functional characterization and architecture of recombinant yeast SWR1 histone exchange complex.