Structural basis of nucleosome dynamics modulation by histone variants H2A.B and H2A.Z.2.2.
ABSTRACT: Nucleosomes are dynamic entities with wide-ranging compositional variations. Human histone variants H2A.B and H2A.Z.2.2 play critical roles in multiple biological processes by forming unstable nucleosomes and open chromatin structures, but how H2A.B and H2A.Z.2.2 confer these dynamic features to nucleosomes remains unclear. Here, we report cryo-EM structures of nucleosome core particles containing human H2A.B (H2A.B-NCP) at atomic resolution, identifying large-scale structural rearrangements in the histone octamer in H2A.B-NCP. H2A.B-NCP compacts approximately 103 bp of DNA wrapping around the core histones in approximately 1.2 left-handed superhelical turns, in sharp contrast to canonical nucleosome encompassing approximately 1.7 turns of DNA. Micrococcal nuclease digestion assay reveals that nineteen H2A.B-specific residues, including a ROF ("regulating-octamer-folding") sequence of six consecutive residues, are responsible for loosening of H2A.B-NCPs. Unlike H2A.B-NCP, the H2A.Z.2.2-containing nucleosome (Z.2.2-NCP) adopts a less-extended structure and compacts around 125 bp of DNA. Further investigation uncovers a crucial role for the H2A.Z.2.2-specific ROF in both H2A.Z.2.2-NCP opening and SWR1-dependent histone replacement. Taken together, these first high-resolution structure of unstable nucleosomes induced by histone H2A variants elucidate specific functions of H2A.B and H2A.Z.2.2 in enhancing chromatin dynamics.
Project description:BACKGROUND:H2A.B, the most divergent histone variant of H2A, can significantly modulate nucleosome and chromatin structures. However, the related structural details and the underlying mechanism remain elusive to date. In this work, we built atomic models of the H2A.B-containing nucleosome core particle (NCP), chromatosome, and chromatin fiber. Multiscale modeling including all-atom molecular dynamics and coarse-grained simulations were then carried out for these systems. RESULTS:It is found that sequence differences at the C-terminal tail, the docking domain, and the L2 loop, between H2A.B and H2A are directly responsible for the DNA unwrapping in the H2A.B NCP, whereas the N-terminus of H2A.B may somewhat compensate for the aforementioned unwrapping effect. The assembly of the H2A.B NCP is more difficult than that of the H2A NCP. H2A.B may also modulate the interactions of H1 with both the NCP and the linker DNA and could further affect the higher-order structure of the chromatin fiber. CONCLUSIONS:The results agree with the experimental results and may shed new light on the biological function of H2A.B. Multiscale modeling may be a valuable tool for investigating structure and dynamics of the nucleosome and the chromatin induced by various histone variants.
Project description:Solution structures of nucleosomes containing a human histone variant, H2A.Z.1, were measured by small-angle X-ray and neutron scatterings (SAXS and SANS). SAXS revealed that the outer shape, reflecting the DNA shape, of the H2A.Z.1 nucleosome is almost the same as that of the canonical H2A nucleosome. In contrast, SANS employing a contrast variation technique revealed that the histone octamer of the H2A.Z.1 nucleosome is smaller than that of the canonical nucleosome. The DNA within the H2A.Z.1 nucleosome was more susceptible to micrococcal nuclease than that within the canonical nucleosome. These results suggested that the DNA is loosely wrapped around the histone core in the H2A.Z.1 nucleosome.
Project description:Nucleosomes, the basic unit of chromatin, are repetitively spaced along DNA and regulate genome expression and maintenance. The long linear chromatin molecule is extensively condensed to fit DNA inside the nucleus. How distant nucleosomes interact to build tertiary chromatin structure remains elusive. In this study, we used cryo-EM to structurally characterize different states of long range nucleosome core particle (NCP) interactions. Our structures show that NCP pairs can adopt multiple conformations, but, commonly, two NCPs are oriented with the histone octamers facing each other. In this conformation, the dyad of both nucleosome core particles is facing the same direction, however, the NCPs are laterally shifted and tilted. The histone octamer surface and histone tails in trans NCP pairs remain accessible to regulatory proteins. The overall conformational flexibility of the NCP pair suggests that chromatin tertiary structure is dynamic and allows access of various chromatin modifying machineries to nucleosomes.
Project description:ATP-dependent chromatin remodeling complexes rearrange nucleosomes by altering the position of DNA around the histone octamer. Although chromatin remodelers and the histone variant H2A.Z colocalize on transcriptional control regions, whether H2A.Z directly affects remodeler association or activity is unclear. We determined the relative association of remodelers with H2A.Z chromatin and tested whether replacement of H2A.Z in a nucleosome altered the activity of remodeling enzymes. Many families of remodelers showed increased association with H2A.Z chromatin, but only the ISWI family of chromatin remodelers showed stimulated activity in vitro. An acidic patch on the nucleosome surface, extended by inclusion of H2A.Z in nucleosomes and essential for viability, is required for ISWI stimulation. We conclude that H2A.Z incorporation increases nucleosome remodeling activity of the largest class of mammalian remodelers (ISWI) and that it correlates with increased association of other remodelers to chromatin. This reveals two possible modes for regulation of a remodeler by a histone variant.
Project description:Nucleosomes containing a human histone variant, H2A.B, in an aqueous solution were analyzed by small-angle neutron scattering utilizing a contrast variation technique. Comparisons with the canonical H2A nucleosome structure revealed that the DNA termini of the H2A.B nucleosome are detached from the histone core surface, and flexibly expanded toward the solvent. In contrast, the histone tails are compacted in H2A.B nucleosomes compared to those in canonical H2A nucleosomes, suggesting that they bind to the surface of the histone core and/or DNA. Therefore, the histone tail dynamics may function to regulate the flexibility of the DNA termini in the nucleosomes.
Project description:Post-translational modifications (PTMs) of histones, such as lysine acetylation of the N-terminal tails, play crucial roles in controlling gene expression. Due to the difficulty in reconstituting site-specifically acetylated nucleosomes with crystallization quality, structural analyses of histone acetylation are currently performed using synthesized tail peptides. Through engineering of the genetic code, translation termination, and cell-free protein synthesis, we reconstituted human H4-mono- to tetra-acetylated nucleosome core particles (NCPs), and solved the crystal structures of the H4-K5/K8/K12/K16-tetra-acetylated NCP and unmodified NCP at 2.4 Å and 2.2 Å resolutions, respectively. The structure of the H4-tetra-acetylated NCP resembled that of the unmodified NCP, and the DNA wrapped the histone octamer as precisely as in the unmodified NCP. However, the B-factors were significantly increased for the peripheral DNAs near the N-terminal tail of the intra- or inter-nucleosomal H4. In contrast, the B-factors were negligibly affected by the H4 tetra-acetylation in histone core residues, including those composing the acidic patch, and at H4-R23, which interacts with the acidic patch of the neighboring NCP. The present study revealed that the H4 tetra-acetylation impairs NCP self-association by changing the interactions of the H4 tail with DNA, and is the first demonstration of crystallization quality NCPs reconstituted with genuine PTMs.
Project description:The nucleosome core particle (NCP) is the fundamental building block of chromatin which compacts approximately 146 bp of DNA around a core histone protein octamer. The effects of NCP packaging on long-range DNA charge transport reactions have not been adequately assessed to date. Here we study DNA hole transport reactions in a 157 bp DNA duplex (AQ-157TG) incorporating multiple repeats of the DNA TG-motif, a strong NCP positioning sequence and a covalently attached Anthraquinone photooxidant. Following a thorough biophysical characterization of the structure of AQ-157TG NCPs by Exonuclease III and hydroxyl radical footprinting, we compared the dynamics of DNA charge transport in ultraviolet-irradiated free and NCP-incorporated AQ-157TG. Compaction into a NCP changes the charge transport dynamics in AQ-157TG drastically. Not only is the overall yield of oxidative lesions decreased in the NCPs, but the preferred sites of oxidative damage change as well. This NCP-dependent attenuation of DNA charge transport is attributed to DNA-protein interactions involving the folded histone core since removal of the histone tails did not perturb the charge transport dynamics in AQ-157TG NCPs.
Project description:We engineered nucleosome core particles (NCPs) with two site-specific cysteine crosslinks that increase the stability of the particle. The first disulfide was introduced between the two copies of H2A via an H2A-N38C point mutation, effectively crosslinking the two H2A/H2B heterodimers together to stabilize the histone octamer against H2A/H2B dimer dissociation. The second crosslink was engineered between an R40C point mutation on the N-terminal tail of H3 and the NCP DNA ends by the introduction of a convertible nucleotide. This crosslink maintains the nucleosome DNA in a fixed translational setting relative to the histone octamer and prevents dilution-driven dissociation. The X-ray crystal structures of NCPs containing the disulfides in isolation and in combination were determined. Both disulfides stabilize the structure of the NCP without disturbing the overall structure. Nucleosomes containing these modifications will be advantageous for biochemical and structural studies as a consequence of their greater resistance to dissociation during high dilution in purification, elevated salt for crystallization and vitrification for cryogenic electron microscopy.
Project description:The modulation of DNA accessibility by nucleosomes is a fundamental mechanism of gene regulation in eukaryotes. The nucleosome core particle (NCP) consists of 147 bp of DNA wrapped around a symmetric octamer of histone proteins. The dynamics of DNA packaging and unpackaging from the NCP affect all DNA-based chemistries, but depend on many factors, including DNA positioning sequence, histone variants and modifications. Although the structure of the intact NCP has been studied by crystallography at atomic resolution, little is known about the structures of the partially unwrapped, transient intermediates relevant to nucleosome dynamics in processes such as transcription, DNA replication and repair. We apply a new experimental approach combining contrast variation with time-resolved small angle X-ray scattering (TR-SAXS) to determine transient structures of protein and DNA constituents of NCPs during salt-induced disassembly. We measure the structures of unwrapping DNA and monitor protein dissociation from Xenopus laevis histones reconstituted with two model NCP positioning constructs: the Widom 601 sequence and the sea urchin 5S ribosomal gene. Both constructs reveal asymmetric release of DNA from disrupted histone cores, but display different patterns of protein dissociation. These kinetic intermediates may be biologically important substrates for gene regulation.
Project description:Nucleosome is the basic structural unit of chromatin, and its dynamics plays critical roles in the regulation of genome functions. However, how the nucleosome structure is regulated by histone variants in vivo is still largely uncharacterized. Here, by employing Micrococcal nuclease (MNase) digestion of crosslinked chromatin followed by chromatin immunoprecipitation (ChIP) and paired-end sequencing (MNase-X-ChIP-seq), we mapped unwrapping states of nucleosomes containing histone variant H2A.Z in mouse embryonic stem (ES) cells. We found that H2A.Z nucleosomes are more enriched with unwrapping states compared with canonical nucleosomes. Interestingly, +1 H2A.Z nucleosomes with 30-80 bp DNA is correlated with less active genes compared with +1 H2A.Z nucleosomes with 120-140 bp DNA. We confirmed the unwrapping of H2A.Z nucleosomes under native condition by re-ChIP of H2A.Z and H2A after CTCF CUT&RUN in mouse ES cells. Importantly, we found that depletion of H2A.Z results in decreased unwrapping of H3.3 nucleosomes and increased CTCF binding. Taken together, through MNase-X-ChIP-seq, we showed that histone variant H2A.Z regulates nucleosome unwrapping in vivo and that its function in regulating transcription or CTCF binding is correlated with unwrapping states of H2A.Z nucleosomes.