Structure of an H1-Bound 6-Nucleosome Array Reveals an Untwisted Two-Start Chromatin Fiber Conformation.

Chromatin adopts a diversity of regular and irregular fiber structures in vitro and in vivo. However, how an array of nucleosomes folds into and switches between different fiber conformations is poorly understood. We report the 9.7 Å resolution crystal structure of a 6-nucleosome array bound to linker histone H1 determined under ionic conditions that favor incomplete chromatin condensation. The structure reveals a flat two-start helix with uniform nucleosomal stacking interfaces and a nucleosome packing density that is only half that of a twisted 30-nm fiber. Hydroxyl radical footprinting indicates that H1 binds the array in an on-dyad configuration resembling that observed for mononucleosomes. Biophysical, cryo-EM, and crosslinking data validate the crystal structure and reveal that a minor change in ionic environment shifts the conformational landscape to a more compact, twisted form. These findings provide insights into the structural plasticity of chromatin and suggest a possible assembly pathway for a 30-nm fiber.

NAP1-Related Protein 1 (NRP1) has multiple interaction modes for chaperoning histones H2A-H2B.

Nucleosome Assembly Protein 1 (NAP1) family proteins are evolutionarily conserved histone chaperones that play important roles in diverse biological processes. In this study, we determined the crystal structure of Arabidopsis NAP1-Related Protein 1 (NRP1) complexed with H2A-H2B and uncovered a previously unknown interaction mechanism in histone chaperoning. Both in vitro binding and in vivo plant rescue assays proved that interaction mediated by the N-terminal α-helix (αN) domain is essential for NRP1 function. In addition, the C-terminal acidic domain (CTAD) of NRP1 binds to H2A-H2B through a conserved mode similar to other histone chaperones. We further extended previous knowledge of the NAP1-conserved earmuff domain by mapping the amino acids of NRP1 involved in association with H2A-H2B. Finally, we showed that H2A-H2B interactions mediated by αN, earmuff, and CTAD domains are all required for the effective chaperone activity of NRP1. Collectively, our results reveal multiple interaction modes of a NAP1 family histone chaperone and shed light on how histone chaperones shield H2A-H2B from nonspecific interaction with DNA.

Structural evidence for Nap1-dependent H2A-H2B deposition and nucleosome assembly.

Nap1 is a histone chaperone involved in the nuclear import of H2A-H2B and nucleosome assembly. Here, we report the crystal structure of Nap1 bound to H2A-H2B together with in vitro and in vivo functional studies that elucidate the principles underlying Nap1-mediated H2A-H2B chaperoning and nucleosome assembly. A Nap1 dimer provides an acidic binding surface and asymmetrically engages a single H2A-H2B heterodimer. Oligomerization of the Nap1-H2A-H2B complex results in burial of surfaces required for deposition of H2A-H2B into nucleosomes. Chromatin immunoprecipitation-exonuclease (ChIP-exo) analysis shows that Nap1 is required for H2A-H2B deposition across the genome. Mutants that interfere with Nap1 oligomerization exhibit severe nucleosome assembly defects showing that oligomerization is essential for the chaperone function. These findings establish the molecular basis for Nap1-mediated H2A-H2B deposition and nucleosome assembly.

Characterization of Caenorhabditis elegans Nucleosome Assembly Protein 1 Uncovers the Role of Acidic Tails in Histone Binding.

Nucleosome assembly proteins (Naps) influence chromatin dynamics by directly binding to histones. Here we provide a comprehensive structural and biochemical analysis of a Nap protein from Caenorhabditis elegans (CeNap1). CeNap1 naturally lacks the acidic N-terminal tail and has a short C-terminal tail compared to many other Nap proteins. Comparison of CeNap1 with full length and tail-less constructs of Saccharomyces cerevisiae Nap1 uncovers the role of these tails in self-association, histone binding, and Nap competition with DNA for H2A-H2B. We find that the presence of tails influences the stoichiometry of H2A-H2B binding and is required to complete the interactions between H2A-H2B and DNA. The absolute stoichiometry of the Nap protein and H2A-H2B complex is 2:1 or 2:2, with only a very small population of higher-order oligomers occurring at 150 mM NaCl. We also show that H3-H4 binds differently than H2A-H2B and that an (H3-H4)2 tetramer can simultaneously bind two Nap2 protein homodimers.

Single-Molecule Investigations on Histone H2A-H2B Dynamics in the Nucleosome.

Nucleosomes impose physical barriers to DNA-templated processes, playing important roles in eukaryotic gene regulation. DNA is packaged into nucleosomes by histone proteins mainly through strong electrostatic interactions that can be modulated by various post-translational histone modifications. Investigating the dynamics of histone dissociation from the nucleosome and how it is altered upon histone modifications is important for understanding eukaryotic gene regulation mechanisms. In particular, histone H2A-H2B dimer displacement in the nucleosome is one of the most important and earliest steps of histone dissociation. Two conflicting hypotheses on the requirement for dimer displacement are that nucleosomal DNA needs to be unwrapped before a dimer can displace and that a dimer can displace without DNA unwrapping. In order to test the hypotheses, we employed three-color single-molecule FRET and monitored in a time-resolved manner the early kinetics of H2A-H2B dimer dissociation triggered by high salt concentration and by histone chaperone Nap1. The results reveal that dimer displacement requires DNA unwrapping in the vast majority of the nucleosomes in the salt-induced case, while dimer displacement precedes DNA unwrapping in >60% of the nucleosomes in the Nap1-mediated case. We also found that acetylation at histone H4K16 or H3K56 affects the kinetics of Nap1-mediated dimer dissociation and facilitates the process both kinetically and thermodynamically. On the basis of these results, we suggest a mechanism by which histone chaperone facilitates H2A-H2B dimer displacement from the histone core without requiring another factor to unwrap the nucleosomal DNA.

Single-Molecule Studies of the Linker Histone H1 Binding to DNA and the Nucleosome.

Linker histone H1 regulates chromatin structure and gene expression. Investigating the dynamics and stoichiometry of binding of H1 to DNA and the nucleosome is crucial to elucidating its functions. Because of the abundant positive charges and the strong self-affinity of H1, quantitative in vitro studies of its binding to DNA and the nucleosome have generated results that vary widely and, therefore, should be interpreted in a system specific manner. We sought to overcome this limitation by developing a specially passivated microscope slide surface to monitor binding of H1 to DNA and the nucleosome at a single-molecule level. According to our measurements, the stoichiometry of binding of H1 to DNA and the nucleosome is very heterogeneous with a wide distribution whose averages are in reasonable agreement with previously published values. Our study also revealed that H1 does not dissociate from DNA or the nucleosome on a time scale of tens of minutes. We found that histone chaperone Nap1 readily dissociates H1 from DNA and superstoichiometrically bound H1 from the nucleosome, supporting a hypothesis whereby histone chaperones contribute to the regulation of the H1 profile in chromatin.

The histone chaperones Vps75 and Nap1 form ring-like, tetrameric structures in solution.

NAP-1 fold histone chaperones play an important role in escorting histones to and from sites of nucleosome assembly and disassembly. The two NAP-1 fold histone chaperones in budding yeast, Vps75 and Nap1, have previously been crystalized in a characteristic homodimeric conformation. In this study, a combination of small angle X-ray scattering, multi angle light scattering and pulsed electron-electron double resonance approaches were used to show that both Vps75 and Nap1 adopt ring-shaped tetrameric conformations in solution. This suggests that the formation of homotetramers is a common feature of NAP-1 fold histone chaperones. The tetramerisation of NAP-1 fold histone chaperones may act to shield acidic surfaces in the absence of histone cargo thus providing a 'self-chaperoning' type mechanism.

Large multimeric assemblies of nucleosome assembly protein and histones revealed by small-angle X-ray scattering and electron microscopy.

The nucleosome assembly protein (NAP) family represents a key group of histone chaperones that are essential for cell viability. Several x-ray structures of NAP1 dimers are available; however, there are currently no structures of this ubiquitous chaperone in complex with histones. We have characterized NAP1 from Xenopus laevis and reveal that it forms discrete multimers with histones H2A/H2B and H3/H4 at a stoichiometry of one NAP dimer to one histone fold dimer. These complexes have been characterized by size exclusion chromatography, analytical ultracentrifugation, multiangle laser light scattering, and small-angle x-ray scattering to reveal their oligomeric assembly states in solution. By employing single-particle cryo-electron microscopy, we visualized these complexes for the first time and show that they form heterogeneous ring-like structures, potentially acting as large scaffolds for histone assembly and exchange.

Non-rigid image registration to reduce beam-induced blurring of cryo-electron microscopy images.

The typical dose used to record cryo-electron microscopy images from vitrified biological specimens is so high that radiation-induced structural alterations are bound to occur during data acquisition. Integration of all scattered electrons into one image can lead to significant blurring, particularly if the data are collected from an unsupported thin layer of ice suspended over the holes of a support film. Here, the dose has been fractioned and exposure series have been acquired in order to study beam-induced specimen movements under low dose conditions, prior to bubbling. Gold particles were added to the protein sample as fiducial markers. These were automatically localized and tracked throughout the exposure series and showed correlated motions within small patches, with larger amplitudes of motion vectors at the start of a series compared with the end of each series. A non-rigid scheme was used to register all images within each exposure series, using natural neighbor interpolation with the gold particles as anchor points. The procedure increases the contrast and resolution of the examined macromolecules.

Dynamic Solution Structures of Whole Human NAP1 Dimer Bound to One and Two Histone H2A-H2B Heterodimers Obtained by Integrative Methods.

Nucleosome assembly protein 1 (NAP1) binds to histone H2A-H2B heterodimers, mediating their deposition on and eviction from the nucleosome. Human NAP1 (hNAP1) consists of a dimerization core domain and intrinsically disordered C-terminal acidic domain (CTAD), both of which are essential for H2A-H2B binding. Several structures of NAP1 proteins bound to H2A-H2B exhibit binding polymorphisms of the core domain, but the distinct structural roles of the core and CTAD domains remain elusive. Here, we have examined dynamic structures of the full-length hNAP1 dimer bound to one and two H2A-H2B heterodimers by integrative methods. Nuclear magnetic resonance (NMR) spectroscopy of full-length hNAP1 showed CTAD binding to H2A-H2B. Atomic force microscopy revealed that hNAP1 forms oligomers of tandem repeated dimers; therefore, we generated a stable dimeric hNAP1 mutant exhibiting the same H2A-H2B binding affinity as wild-type hNAP1. Size exclusion chromatography (SEC), multi-angle light scattering (MALS) and small angle X-ray scattering (SAXS), followed by modelling and molecular dynamics simulations, have been used to reveal the stepwise dynamic complex structures of hNAP1 binding to one and two H2A-H2B heterodimers. The first H2A-H2B dimer binds mainly to the core domain of hNAP1, while the second H2A-H2B binds dynamically to both CTADs. Based on our findings, we present a model of the eviction of H2A-H2B from nucleosomes by NAP1.

Assembly states of the nucleosome assembly protein 1 (NAP-1) revealed by sedimentation velocity and non-denaturing MS.

Proteins often exist as ensembles of interconverting states in solution which are often difficult to quantify. In the present manuscript we show that the combination of MS under nondenaturing conditions and AUC-SV (analytical ultracentrifugation sedimentation velocity) unambiguously clarifies a distribution of states and hydrodynamic shapes of assembled oligomers for the NAP-1 (nucleosome assembly protein 1). MS established the number of associated units, which was utilized as input for the numerical analysis of AUC-SV profiles. The AUC-SV analysis revealed that less than 1% of NAP-1 monomer exists at the micromolar concentration range and that the basic assembly unit consists of dimers of yeast or human NAP-1. These dimers interact non-covalently to form even-numbered higher-assembly states, such as tetramers, hexamers, octamers and decamers. MS and AUC-SV consistently showed that the formation of the higher oligomers was suppressed with increasing ionic strength, implicating electrostatic interactions in the formation of higher oligomers. The hydrodynamic shapes of the NAP-1 tetramer estimated from AUC-SV agreed with the previously proposed assembly models built using the known three-dimensional structure of yeast NAP-1. Those of the hexamer and octamer could be represented by new models shown in the present study. Additionally, MS was used to measure the stoichiometry of the interaction between the human NAP-1 dimer and the histone H2A-H2B dimer or H3-H4 tetramer. The present study illustrates a rigorous procedure for the analysis of protein assembly and protein-protein interactions in solution.

Functional characterization of human nucleosome assembly protein 1-like proteins as histone chaperones.

Nucleosome Assembly Protein 1 (NAP1) is a highly conserved histone chaperone protein suspected to be involved in the dynamical regulation of the histone H2A-H2B hetero-dimer. However, the exact mechanism by which NAP1-like proteins act is currently unknown. In this work, we characterized the biochemical properties of two human NAP1-like proteins, hNAP1L1 and hNAP1L4, including a previously uncharacterized subtype, with the aim of determining their exact mechanistic role. Both hNAP1L1 and hNAP1L4 were found to be localized mainly to the cytoplasm and a minor population of them was suggested to be in the nucleus. Biochemical analyses demonstrated that both hNAP1L1 and hNAP1L4 mediated nucleosome formation. In addition, hNAP1L1 was shown to possess a significantly greater nucleosome disassembly activity than hNAP1L4, suggesting that hNAP1L1 and hNAP1L4 may play distinct roles in the regulation of histone dynamics. Building upon this initial discovery we also found that histone H2A-H2B and various histone H2A variants-H2B dimers were found to associate with both hNAP1L1 and hNAP1L4 in cell extracts. These results suggest that human NAP1-like proteins play overlapping roles in transport and deposition of histone H2A-H2B or H2A variants-H2B dimers on chromatin and nonoverlapping roles in nucleosome disassembly.

New nuclear partners for nucleosome assembly protein 1: unexpected associations.

Histone chaperones are important players in chromatin dynamics. They are instrumental in nucleosome assembly and disassembly and in histone variant exchange reactions that occur during DNA transactions. The molecular mechanisms of their action are not well understood and may involve interactions with various protein partners in the context of the nucleus. In an attempt to further elucidate nuclear roles of histone chaperones, we performed a proteomic search for nuclear partners of a particular histone chaperone, nucleosome assembly protein 1 (Nap1). Proteins recognized as Nap1 partners by immuno-affinity capture and Far Western blots were identified by mass spectrometry. The identified partners are known to participate in a number of nuclear processes, including DNA replication, recombination, and repair as well as RNA transcription and splicing. Finding nuclear actin among the Nap1 partners may be of particular significance, in view of actin's role in transcription, transcription regulation, and RNA splicing. We are proposing a model of how actin-Nap1 interaction may be involved in transcription elongation through chromatin. In addition, awareness of the interactions between Nap1 and Hsp70, another identified partner, may help to understand nucleosome dynamics around sites of single-strand DNA break repair. These studies represent a starting point for further investigation of Nap1 associations in human cells.

The intrinsic stability of H2B-ubiquitylated nucleosomes and their in vitro assembly/disassembly by histone chaperone NAP1.

BACKGROUND: Apart the gene-regulatory functions as docking sites for histone 'readers', some histone modifications could directly affect nucleosome structure. The H2BK34-ubiquitylation deposited by MOF-MSL complex, increases nucleosome dynamics in vitro and promotes donation of one H2A/H2B dimer to histone acceptors. METHODS: We evaluated temperature-depended stability of H2BK34-ubiquitylated nucleosomes under 'physiological' ionic conditions in the presence or absence of histone acceptor, and examined assembly and disassembly of ubiquitylated nucleosomes in vitro by recombinant mouse NAP1. RESULTS: H2BK34ub modification is sufficient to promote selective eviction of only one H2A/H2B dimer independently of histone-binding agents. Despite the robust H2A/H2B dimer-displacement effect of mNAP1 with the H2BK34ub (but not unmodified) nucleosomes, NAP1 could assemble symmetrically- or asymmetrically ubiquitylated nucleosomes under 'physiological' conditions in vitro. CONCLUSIONS AND GENERAL SIGNIFICANCE: The increased mobility of one nucleosomal H2A/H2B dimer is an intrinsic nucleosome destabilizing property of H2BK34 ubiquitylation that has the intranucleosome bases. The ability of NAP to reasonably efficiently assemble H2BK34-ubiquitylated nucleosomes supposes a potential mechanism for deposition/distribution of H2BK34ub mark in the MOF-MSL independent manner (for example, during histone dimer exchange upon transcription elongation).

How Protein Binding Sensitizes the Nucleosome to Histone H3K56 Acetylation.

The nucleosome, the fundamental gene-packing unit comprising an octameric histone protein core wrapped with DNA, has a flexible structure that enables dynamic gene regulation mechanisms. Histone lysine acetylation at H3K56 removes a positive charge from the histone core where it interacts with the termini of the nucleosomal DNA and acts as a critical gene regulatory signal that is implicated in transcription initiation and elongation. The predominant proposal for the biophysical role of H3K56 acetylation (H3K56ac) is that weakened electrostatic interaction between DNA termini and the histone core results in facilitated opening and subsequent disassembly of the nucleosome. However, this effect alone is too weak to account for the strong coupling between H3K56ac and its regulatory outcomes. Here we utilized a semisynthetically modified nucleosome with H3K56ac in order to address this discrepancy. Based on the results, we propose an innovative mechanism by which the charge neutralization effect of H3K56ac is significantly amplified via protein binding. We employed three-color single-molecule fluorescence resonance energy transfer (smFRET) to monitor the opening rate of nucleosomal DNA termini induced by binding of histone chaperone Nap1. We observed an elevated opening rate upon H3K56ac by 5.9-fold, which is far larger than the 1.5-fold previously reported for the spontaneous opening dynamics in the absence of Nap1. Our proposed mechanism successfully reconciles this discrepancy because DNA opening for Nap1 binding must be larger than the average spontaneous opening. This is a novel mechanism that can explain how a small biophysical effect of histone acetylation results in a significant change in protein binding rate.

Structural Insights into ceNAP1 Chaperoning Activity toward ceH2A-H2B.

In eukaryotes, nucleosome assembly is crucial for genome integrity. The histone chaperone NAP1 plays an important role in histone folding, storage, and transport, as well as histone exchange and nucleosome assembly. At present, the molecular basis of these activities is not fully understood. We have solved high-resolution crystal structures of Caenorhabditis elegans NAP1 (ceNAP1) in complex with its cognate substrates: the C. elegans H2A-H2B dimer (ceH2A-H2B) and the H2A.Z-H2B dimer (ceH2A.Z-H2B). Our structural and biochemical data reveals the acidic concave surface is relevant to tetramerization, and uncovers how a ceNAP1 homodimer uses its concave surface to asymmetrically recognize a ceH2A-H2B or ceH2A.Z-H2B heterodimer. Intriguingly, an "acidic strip" within the concave surface of ceNAP1 is crucial for binding histones, including H2A-H2B, H3-H4, and histone variants. Thus, our results provide insight into the molecular mechanisms of NAP1 histone chaperone activity.

Biochemical and Biophysical Characterisation of Higher Oligomeric Structure of Rat Nucleosome Assembly Protein 1.

Nucleosome assembly protein 1 (NAP1) is a histone chaperone that exchanges histone H2A-H2B dimer from chromatin templates. Studies with yeast NAP1 (yNAP1) have revealed its existence as multiple oligomeric species in solution. Here, rat NAP1 (rNAP1), which is 98% identical to the human NAP1 (hNAP1) was used as a model to characterize the oligomeric structures of this protein in higher eukaryotes. Gel filtration chromatography and Dynamic light scattering of recombinant rNAP1 indicated that the protein exists as a complex mixture of multimeric species even at 500 mM ionic strength. The solution-state complexity remains unchanged even at higher ionic strengths. Equilibrium unfolding (ΔG 14.6 kcal mol- 1) shows that rNAP1, both dimeric and oligomeric, follow the two-state model of unfolding with no detectable intermediates. Homology modelling suggests that rat and yeast NAP1 share an overall similar structure with conserved domains. However, dissimilar substitutions like threonine and lysine with glycine in the ß-hairpin involved in oligomerization, possibly leads to the observed differences in the oligomerization propensity of the two proteins. Molecular dynamic simulation (MDS) of the two structures also revealed that rNAP1 dimer is more stable owing to the extensive hydrogen bonding in comparison to yNAP1. Further, in vitro kinase assay showed that the phosphorylation of rNAP1 favors oligomerization with no effect on its histone binding capacity. Our results clearly suggest that there are differences in the in-solution behavior of rNAP1 compared to yNAP1 which may have in vivo functional implications for the regulation of these complexes during chromatin assembly and rearrangement.

Histone octamer rearranges to adapt to DNA unwrapping.

Nucleosomes, the basic units of chromatin, package and regulate expression of eukaryotic genomes. Although the structure of the intact nucleosome is well characterized, little is known about structures of partially unwrapped, transient intermediates. In this study, we present nine cryo-EM structures of distinct conformations of nucleosome and subnucleosome particles. These structures show that initial DNA breathing induces conformational changes in the histone octamer, particularly in histone H3, that propagate through the nucleosome and prevent symmetrical DNA opening. Rearrangements in the H2A-H2B dimer strengthen interaction with the unwrapping DNA and promote nucleosome stability. In agreement with this, cross-linked H2A-H2B that cannot accommodate unwrapping of the DNA is not stably maintained in the nucleosome. H2A-H2B release and DNA unwrapping occur simultaneously, indicating that DNA is essential in stabilizing the dimer in the nucleosome. Our structures reveal intrinsic nucleosomal plasticity that is required for nucleosome stability and might be exploited by extrinsic protein factors.

Redundant Functions for Nap1 and Chz1 in H2A.Z Deposition.

H2A.Z is a histone H2A variant that contributes to transcriptional regulation, DNA damage response and limits heterochromatin spreading. In Saccharomyces cerevisiae, H2A.Z is deposited by the SWR-C complex, which relies on several histone chaperones including Nap1 and Chz1 to deliver H2A.Z-H2B dimers to SWR-C. However, the mechanisms by which Nap1 and Chz1 cooperate to bind H2A.Z and their contribution to H2A.Z deposition in chromatin is not well understood. Using structural modeling and molecular dynamics simulations, we identify a series of H2A.Z residues that form a chaperone-specific binding surface. Mutation of these residues revealed different surface requirements for Nap1 and Chz1 interaction with H2A.Z. Consistent with this result, we found that loss of Nap1 or Chz1 individually resulted in mild defects in H2A.Z deposition, but that deletion of both Nap1 and Chz1 resulted in a significant reduction of H2A.Z deposition at promoters and led to heterochromatin spreading. Together, our findings reveal unique H2A.Z surface dependences for Nap1 and Chz1 and a redundant role for these chaperones in H2A.Z deposition.

Coordinated Action of Nap1 and RSC in Disassembly of Tandem Nucleosomes.

The SWI/SNF and RSC family of ATP-dependent chromatin remodelers disassembles nucleosomes by moving nucleosomes into the vicinity of adjoining nucleosomes. We found that the histone chaperone Nap1 efficiently promotes disassembly of adjacent nucleosomes with which RSC collides and not the disassembly of nucleosomes mobilized by RSC. Nap1 is specific to RSC, as it does not target SWI/SNF, its paralog in Saccharomyces cerevisiae Extensive mutational analysis of Nap1 has revealed that Nap1 affinity for histones H2A-H2B and H3-H4 and its ability to displace histones from DNA are required for Nap1 to enhance RSC-mediated disassembly. Other histone chaperones, such as Vps75, that also bind histones are not able to enhance RSC-mediated disassembly. Our study suggests a mechanism by which Nap1 is recruited to actively transcribed regions and assists in the passage of the transcription complex through chromatin, and it provides a novel mechanism for the coordinated action of RSC and Nap1.