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.

H2A Ubiquitination Alters H3-tail Dynamics on Linker-DNA to Enhance H3K27 Methylation.

Polycomb repressive complex 1 (PRC1) and PRC2 are responsible for epigenetic gene regulation. PRC1 ubiquitinates histone H2A (H2Aub), which subsequently promotes PRC2 to introduce the H3 lysine 27 tri-methyl (H3K27me3) repressive chromatin mark. Although this mechanism provides a link between the two key transcriptional repressors, PRC1 and PRC2, it is unknown how histone-tail dynamics contribute to this process. Here, we have examined the effect of H2A ubiquitination and linker-DNA on H3-tail dynamics and H3K27 methylation by PRC2. In naïve nucleosomes, the H3-tail dynamically contacts linker DNA in addition to core DNA, and the linker-DNA is as important for H3K27 methylation as H2A ubiquitination. H2A ubiquitination alters contacts between the H3-tail and DNA to improve the methyltransferase activity of the PRC2-AEBP2-JARID2 complex. Collectively, our data support a model in which H2A ubiquitination by PRC1 synergizes with linker-DNA to hold H3 histone tails poised for their methylation by PRC2-AEBP2-JARID2.

Erratum: Characteristic H3 N-tail dynamics in the nucleosome core particle, nucleosome, and chromatosome.

[This corrects the article DOI: 10.1016/j.isci.2022.103937.].

FACT modulates the conformations of histone H2A and H2B N-terminal tails within nucleosomes.

Gene expression is regulated by the modification and accessibility of histone tails within nucleosomes. The histone chaperone FACT (facilitate chromatin transcription), comprising SPT16 and SSRP1, interacts with nucleosomes through partial replacement of DNA with the phosphorylated acidic intrinsically disordered (pAID) segment of SPT16; pAID induces an accessible conformation of the proximal histone H3 N-terminal tail (N-tail) in the unwrapped nucleosome with FACT. Here, we use NMR to probe the histone H2A and H2B tails in the unwrapped nucleosome. Consequently, both the H2A and H2B N-tails on the pAID-proximal side bind to pAID with robust interactions, which are important for nucleosome assembly with FACT. Furthermore, the conformations of these N-tails on the distal DNA-contact site are altered from those in the canonical nucleosome. Our findings highlight that FACT both proximally and distally regulates the conformations of the H2A and H2B N-tails in the asymmetrically unwrapped nucleosome.

Characteristic H3 N-tail dynamics in the nucleosome core particle, nucleosome, and chromatosome.

The nucleosome core particle (NCP) comprises a histone octamer, wrapped around by ∼146-bp DNA, while the nucleosome additionally contains linker DNA. We previously showed that, in the nucleosome, H4 N-tail acetylation enhances H3 N-tail acetylation by altering their mutual dynamics. Here, we have evaluated the roles of linker DNA and/or linker histone on H3 N-tail dynamics and acetylation by using the NCP and the chromatosome (i.e., linker histone H1.4-bound nucleosome). In contrast to the nucleosome, H3 N-tail acetylation and dynamics are greatly suppressed in the NCP regardless of H4 N-tail acetylation because the H3 N-tail is strongly bound between two DNA gyres. In the chromatosome, the asymmetric H3 N-tail adopts two conformations: one contacts two DNA gyres, as in the NCP; and one contacts linker DNA, as in the nucleosome. However, the rate of H3 N-tail acetylation is similar in the chromatosome and nucleosome. Thus, linker DNA and linker histone both regulate H3-tail dynamics and acetylation.

The N-terminal Tails of Histones H2A and H2B Adopt Two Distinct Conformations in the Nucleosome with Contact and Reduced Contact to DNA.

The nucleosome comprises two histone dimers of H2A-H2B and one histone tetramer of (H3-H4)2, wrapped around by ~145 bp of DNA. Detailed core structures of nucleosomes have been established by X-ray and cryo-EM, however, histone tails have not been visualized. Here, we have examined the dynamic structures of the H2A and H2B tails in 145-bp and 193-bp nucleosomes using NMR, and have compared them with those of the H2A and H2B tail peptides unbound and bound to DNA. Whereas the H2A C-tail adopts a single but different conformation in both nucleosomes, the N-tails of H2A and H2B adopt two distinct conformations in each nucleosome. To clarify these conformations, we conducted molecular dynamics (MD) simulations, which suggest that the H2A N-tail can locate stably in either the major or minor grooves of nucleosomal DNA. While the H2B N-tail, which sticks out between two DNA gyres in the nucleosome, was considered to adopt two different orientations, one toward the entry/exit side and one on the opposite side. Then, the H2A N-tail minor groove conformation was obtained in the H2B opposite side and the H2B N-tail interacts with DNA similarly in both sides, though more varied conformations are obtained in the entry/exit side. Collectively, the NMR findings and MD simulations suggest that the minor groove conformer of the H2A N-tail is likely to contact DNA more strongly than the major groove conformer, and the H2A N-tail reduces contact with DNA in the major groove when the H2B N-tail is located in the entry/exit side.

Partial Replacement of Nucleosomal DNA with Human FACT Induces Dynamic Exposure and Acetylation of Histone H3 N-Terminal Tails.

The FACT (facilitates chromatin transcription) complex, comprising SPT16 and SSRP1, conducts structural alterations during nucleosome unwrapping. Our previous cryoelectron microscopic (cryo-EM) analysis revealed the first intermediate structure of an unwrapped nucleosome with human FACT, in which 112-bp DNA and the phosphorylated intrinsically disordered (pAID) segment of SPT16 jointly wrapped around the histone core instead of 145-bp DNA. Using NMR, here we clarified that the histone H3 N-terminal tails, unobserved in the cryo-EM structure, adopt two different conformations reflecting their asymmetric locations at entry/exit sites: one corresponds to the original nucleosome site buried in two DNA gyres (DNA side), whereas the other, comprising pAID and DNA, is more exposed to the solvent (pAID side). NMR real-time monitoring showed that H3 acetylation is faster on the pAID side than on the DNA side. Our findings highlight that accessible conformations of H3 tails are created by the replacement of nucleosomal DNA with pAID.

Acetylated histone H4 tail enhances histone H3 tail acetylation by altering their mutual dynamics in the nucleosome.

The structural unit of eukaryotic chromatin is a nucleosome, comprising two histone H2A-H2B heterodimers and one histone (H3-H4)2 tetramer, wrapped around by ∼146 bp of DNA. The N-terminal flexible histone tails stick out from the histone core and have extensive posttranslational modifications, causing epigenetic changes of chromatin. Although crystal and cryogenic electron microscopy structures of nucleosomes are available, the flexible tail structures remain elusive. Using NMR, we have examined the dynamics of histone H3 tails in nucleosomes containing unmodified and tetra-acetylated H4 tails. In unmodified nucleosome, the H3 tail adopts a dynamic equilibrium structure between DNA-contact and reduced-contact states. In acetylated H4 nucleosome, however, the H3 tail equilibrium shifts to a mainly DNA-contact state with a minor reduced-contact state. The acetylated H4 tail is dynamically released from its own DNA-contact state to a reduced-contact state, while the H3 tail DNA-contact state becomes major. Notably, H3 K14 in the acetylated H4 nucleosome is much more accessible to acetyltransferase Gcn5 relative to unmodified nucleosome, possibly due to the formation of a favorable H3 tail conformation for Gcn5. In summary, each histone tail adopts a characteristic dynamic state but regulates one other, probably creating a histone tail network even on a nucleosome.

Structural Basis of Homology-Directed DNA Repair Mediated by RAD52.

RAD52 mediates homologous recombination by annealing cDNA strands. However, the detailed mechanism of DNA annealing promoted by RAD52 has remained elusive. Here we report two crystal structures of human RAD52 single-stranded DNA (ssDNA) complexes that probably represent key reaction intermediates of RAD52-mediated DNA annealing. The first structure revealed a "wrapped" conformation of ssDNA around the homo-oligomeric RAD52 ring, in which the edges of the bases involved in base pairing are exposed to the solvent. The ssDNA conformation is close to B-form and appears capable of engaging in Watson-Crick base pairing with the cDNA strand. The second structure revealed a "trapped" conformation of ssDNA between two RAD52 rings. This conformation is stabilized by a different RAD52 DNA binding site, which promotes the accumulation of multiple RAD52 rings on ssDNA and the aggregation of ssDNA. These structures provide a structural framework for understanding the mechanism of RAD52-mediated DNA annealing.

Solution structure of the isolated histone H2A-H2B heterodimer.

During chromatin-regulated processes, the histone H2A-H2B heterodimer functions dynamically in and out of the nucleosome. Although detailed crystal structures of nucleosomes have been established, that of the isolated full-length H2A-H2B heterodimer has remained elusive. Here, we have determined the solution structure of human H2A-H2B by NMR coupled with CS-Rosetta. H2A and H2B each contain a histone fold, comprising four α-helices and two ß-strands (α1-ß1-α2-ß2-α3-αC), together with the long disordered N- and C-terminal H2A tails and the long N-terminal H2B tail. The N-terminal αN helix, C-terminal ß3 strand, and 310 helix of H2A observed in the H2A-H2B nucleosome structure are disordered in isolated H2A-H2B. In addition, the H2A α1 and H2B αC helices are not well fixed in the heterodimer, and the H2A and H2B tails are not completely random coils. Comparison of hydrogen-deuterium exchange, fast hydrogen exchange, and {(1)H}-(15)N hetero-nuclear NOE data with the CS-Rosetta structure indicates that there is some conformation in the H2A 310 helical and H2B Lys11 regions, while the repression domain of H2B (residues 27-34) exhibits an extended string-like structure. This first structure of the isolated H2A-H2B heterodimer provides insight into its dynamic functions in chromatin.

C-terminal acidic domain of histone chaperone human NAP1 is an efficient binding assistant for histone H2A-H2B, but not H3-H4.

Nucleosome assembly protein 1 (NAP1) binds both the (H3-H4)2 tetramer and two H2A-H2B dimers, mediating their sequential deposition on DNA. NAP1 contains a C-terminal acidic domain (CTAD) and a core domain that promotes dimer formation. Here, we have investigated the roles of the core domain and CTAD of human NAP1 in binding to H2A-H2B and H3-H4 by isothermal calorimetry and native mass spectrometry and compared them with the roles of yeast NAP1. We show that the hNAP1 and yNAP1 dimers bind H2A-H2B by two different modes: a strong endothermic interaction and a weak exothermic interaction. A mutant hNAP1, but not yNAP1, dimer lacking CTAD loses the exothermic interaction and shows greatly reduced H2A-H2B binding activity. The isolated CTAD of hNAP1 binds H2A-H2B only exothermically with relatively stronger binding as compared with the exothermic interaction observed for the full-length hNAP1 dimer. Thus, the two CTADs in the hNAP1 dimer seem to provide binding assistance for the strong endothermic interaction of the core domain with H2A-H2B. By contrast, in the relatively weaker binding of hNAP1 to H3-H4 as compared with yNAP1, CTAD of hNAP1 has no significant role. To our knowledge, this is the first distinct role identified for the hNAP1 CTAD.

Electrostatic Repulsion during Ferritin Assembly and Its Screening by Ions.

Escherichia coli non-heme-binding ferritin A (EcFtnA) is a spherical cagelike protein that is composed of 24 identical subunits. EcFtnA dissociates into 2-mers under acidic conditions and can reassemble into the native structure when the pH is increased. To understand how electrostatic interactions influence the assembly reaction, the dependence of the process on ionic strength and pH was investigated. The assembly reaction was initiated by stopped-flow mixing of the acid-dissociated EcFtnA solution and high-pH buffer solutions and monitored by time-resolved small-angle X-ray scattering. The rate of assembly increased with increasing ionic strength and decreased with increasing pH from 6 to 8. These dependences were thought to originate from repulsion between assembly units (2-mer in the case of EcFtnA) with the same net charge sign; therefore, to test this assumption, mutants with different net charges (net-charge mutants) were prepared. In buffers with a low ionic strength, the rate of assembly increased with a decreasing net charge. Thus, repulsion between the assembly unit net charges was demonstrated to be an important factor determining the rate of assembly. However, the difference in the assembly rate among net-charge mutants was not significant in buffers with an ionic strength of >0.1. Notably, under such high-ionic strength conditions, the assembly rate increased with an increasing ionic strength, suggesting that local electrostatic interactions are also responsible for the ionic strength dependence of the rate of assembly and are repulsive on average.

Ferritin Assembly Revisited: A Time-Resolved Small-Angle X-ray Scattering Study.

The assembly reaction of Escherichia coli ferritin A (EcFtnA) was studied using time-resolved small-angle X-ray scattering (TR-SAXS). EcFtnA forms a cagelike structure that consists of 24 identical subunits and dissociates into dimers at acidic pH. The dimer maintains nativelike secondary and tertiary structures and is able to reassemble into a 24-mer when the pH is increased. The reassembly reaction was induced by pH jump, and reassembly was followed by TR-SAXS. Time-dependent changes in the forward scattering intensity and in the gyration radius suggested the existence of a significant population of intermediate oligomers during the assembly reaction. The initial reaction was a mixture of second- and third-order reactions (formation of tetramers and hexamers) from the protein concentration dependence of the initial velocity. The time-dependent change in the SAXS profile was roughly explained by a simple model in which only tetramers, hexamers, and dodecamers were considered as intermediates.

A Physicochemical and Mutational Analysis of Intersubunit Interactions of Escherichia coli Ferritin A.

Ferritin A from Escherichia coli (EcFtnA) is 24-meric protein, which forms spherical cagelike structures called nanocages. The nanocage structure is stabilized by the interface around 4-, 3-, and 2-fold symmetric axes. The subunit structure of EcFtnA comprises a four-helix bundle (helices A-D) and an additional helix E, which forms a 4-fold axis. In this study, we examined the contribution of the interface around three symmetric axes. pH-induced dissociation experiments monitored by analytical ultracentrifugation and small-angle X-ray scattering showed that the dimer related by 2-fold symmetry is the most stable unit. Mutations located near the 3-fold axis revealed that the contribution of each interaction was small. A mutant lacking helix E at the 4-fold axis formed a nanocage, suggesting that helix E is not essential for nanocage formation. Further truncation of the C-terminus of helix D abrogated the formation of the nanocage, suggesting that a few residues located at the C-terminus of helix D are critical for this process. These properties are similar to those known for mammalian ferritins and seem to be common principles for nanocage formation. The difference between EcFtnA and mammalian ferritins was that helix E-truncated EcFtnA maintained an iron-incorporating ability, whereas mammalian mutants lost it.

Relationship between chain collapse and secondary structure formation in a partially folded protein.

Chain collapse and secondary structure formation are frequently observed during the early stages of protein folding. Is the chain collapse brought about by interactions between secondary structure units or is it due to polymer behavior in a poor solvent (coil-globule transition)? To answer this question, we measured small-angle X-ray scattering for a series of ß-lactoglobulin mutants under conditions in which they assume a partially folded state analogous to the folding intermediates. Mutants that were designed to disrupt the secondary structure units showed the gyration radii similar to that of the wild type protein, indicating that chain collapse is due to coil-globule transitions.

Important role of methionine 145 in dimerization of bovine ß-lactoglobulin.

ß-Lactoglobulin (LG) contains nine ß-strands (strands A-I) and one α-helix. Strands A-H form a ß-barrel. At neutral pH, bovine LG (BLG) forms a dimer and the dimer interface consists of AB-loops and the I-strands of two subunits. On the other hand, equine LG (ELG) is monomeric. The residues 145-153 of BLG, which compose a dimer interface, are entirely different from those of ELG. The difference in the association states between BLG and ELG can be attributed to the residues 145-153. To confirm this, we constructed a chimeric LG, ImBLG (I-strand mutated BLG), in which the residues 145-153 were replaced with those of ELG. Gel-filtration chromatography and analytical ultracentrifugation revealed that ImBLG existed as a monomer. To identify the residues important for dimerization, we constructed several revertants and investigated their association. This experiment revealed that, in addition to the interface residues (Ile147, Leu149 and Phe151), Met145 is critical for dimerization. Although Met145 does not contact with the other protomer, it seems to be important in determining the backbone conformation of the I-strand. This was supported by the fact that all Met145-containing mutants showed circular dichroism spectra similar to BLG but different from ImBLG.

Structure and stability of Gyuba, a ß-lactoglobulin chimera.

ß-lactoglobulin (LG) contains nine ß-strands (strands A-I) and one α-helix. Strands A-H form a ß-barrel. At neutral pH, equine LG (ELG) is monomeric, whereas bovine LG (BLG) is dimeric, and the I-strands of its two subunits form an intermolecular ß-sheet. We previously constructed a chimeric ELG in which the sequence of the I-strand was replaced with that of BLG. This chimera did not dimerize. For this study, we constructed the new chimera we call Gyuba (which means cow and horse in Japanese). The amino acid sequence of Gyuba includes the sequences of the BLG secondary structures and those of the ELG loops. The crystal structure of Gyuba is very similar to that of BLG and indicates that Gyuba dimerizes via the intermolecular ß-sheet formed by the two I-strands. Thus, the entire arrangement of the secondary structural elements is important for LG dimer formation.