Biology subjects

Kurumizaka, H.

Publications and source records attributed to Kurumizaka, H..

17 recordsLinked to original sources

bioRxiv2026

MacroH2A1 and H3K27me3 target cGAS to facultative heterochromatin to restrain innate immune activation

The DNA sensor cGAS is largely sequestered on chromatin in an inactive form, but how this state is regulated remains unclear. We show that cGAS preferentially binds nucleosomes containing the histone variant macroH2A1 (mH2A1) marked by H3K27me3 and H3K9me3. Cryo-EM reveals that cGAS selectively engages the mH2A1/H2B acidic patch within heterotypic nucleosomes. Depleting mH2A1.1 or blocking H3K27me3 deposition releases cGAS from chromatin and activates the cGAS-STING-IRF3 pathway. Genome-wide profiling reveals two cGAS-bound chromatin compartments: chromosome-arm sites co-occupied by mH2A1.1, methylated CpG islands, and CTCF, where cGAS and mH2A1.1 binding are interdependent; and centromeric regions (14% of peaks), which lack DNA methylation and mH2A1.1 dependence. These findings establish mH2A1 and H3K27me3 as determinants of cGAS chromatin tethering, linking nuclear cGAS regulation to facultative heterochromatin and opening new avenues for modulating cGAS-STING signaling.

cell biology↗

bioRxiv2026

Structural basis for site-specific histone H3 acetylation-dependent regulation of RNAPII transcription through nucleosomes

Acetylation of histone H3 regulates chromatin dynamics and transcription, but how specific acetylation sites affect RNA polymerase II (RNAPII) transcription through nucleosomes remains unclear. Here, we found that H3 acetylation at Lys56 and Lys122 markedly enhances RNAPII transcription through nucleosomes, whereas acetylation at Lys64 has little effect. To elucidate the structural basis for these functional differences, we determined cryo-electron microscopy (cryo-EM) structures of nucleosomes bearing site-specific acetylation at H3K56, H3K64, or H3K122. The cryo-EM structures revealed that H3K56ac and H3K122ac locally weaken histone-DNA interactions at the DNA entry/exit region and near the dyad, respectively, while H3K64ac induces no detectable structural changes. These structural differences correlate with the observed transcriptional outcomes, indicating that acetylation at H3K56 and H3K122, but not H3K64, alleviates the nucleosomal barrier to RNAPII progression. Our findings provide direct structural evidence that specific acetylations within the histone fold domain of H3 finetune nucleosome dynamics to facilitate RNAPII transcription.

molecular biology↗

bioRxiv2026

Genome compartments guide protamine replacement and genome stability during spermiogenesis

Compartment-scale genome organisation persists in mammalian sperm, yet how histone-protamine replacement is orchestrated in space and time during spermiogenesis remains unclear. Here we combine stage-resolved purification of mouse spermatids with spike-in-normalised ATAC-seq and PRM1 CUT&Tag to map chromatin accessibility and protamine incorporation across spermiogenesis. We uncover a transient, genome-wide hyper-accessible phase coincident with replacement that is uncoupled from transcription and suppressed in catalytic PHF7-mutant spermatids. PRM1 loading initiates within accessible A-compartment chromatin and later spreads across both A- and B-compartments, whereas protamine-null mice demonstrated that PRM1/ PRM2 deficiency selectively destabilises A-compartment closure. Sequencing of short DNA fragments from Prm1/Prm2 dosage-reduced epididymal sperm reveals early enrichment of breakage at protamine-targeted A-compartment regions, which dissipates as fragmentation becomes genome-wide during epididymal transit. Together, our data place histone-protamine replacement within a compartment-centred framework that links 3D genome domains to the timing, targeting and integrity of the sperm genome.

genomics↗

bioRxiv2026

Molecular Crowding-Driven Nucleosome Interactions Revealed Through Single-Molecule Optical Tweezers

Molecular crowding causes the compaction of chromatin fibers, contributing to the formation of the nuclear architecture. However, the molecular mechanism of compaction under crowded conditions is not yet fully understood. In this study, we employed the single-molecule optical tweezer method to investigate the effect of molecular crowding on chromatin structure. Force-extension experiments on a 12-mer polynucleosome in the presence of different sizes and concentrations of polyethylene glycol (PEG) as a crowding agent showed that at low concentrations of low-molecular-weight (MW) PEG, the compaction of the polynucleosome was not significant. In this respect, nucleosomes predominantly remained separated, while DNA-histone interactions within individual nucleosomes were slightly stabilized. In contrast, high concentrations of high-MW PEG significantly promote internucleosomal interactions, leading to highly compact polynucleosome conformations. Under these conditions, approximately 30 pN of force was required to disrupt the internucleosomal interactions and release DNA; this force was 36% higher than that required for DNA unwrapping in the absence of PEG. These findings suggest that molecular crowding impacts cellular processes by mechanically regulating chromatin accessibility for regulatory proteins and the passage of motor molecules such as RNA polymerase. Significance StatementChromatin condensation is closely related to biological processes such as transcription and replication. Molecular crowding has recently attracted attention as a factor regulating chromatin condensation. In this study, we used the optical tweezer method to analyze the molecular mechanisms underlying chromatin condensation. We found that high-molecular weight and high-concentration crowders (polyethylene glycol) induced significant compaction, which involved internucleosomal interactions that markedly reduced DNA accessibility. Our results suggest that molecular crowding not only alters the condensation state, but also mechanically regulates chromatin accessibility.

biophysics↗

bioRxiv2025

Structural basis of asymmetric transcription through a composite nucleosome formed by a hexasome and an octasome

The overlapping dinucleosome (OLDN), a composite chromatin particle consisting of a hexasome and a canonical octasome, forms immediately downstream of transcription start sites, likely through chromatin remodeling activity, and has been proposed to act as a transient regulatory intermediate during transcription. However, how RNA polymerase II (RNAPII) engages with and transcribes through this unusual structure remains unclear. Here, we reconstituted OLDNs in vitro and performed transcription assays with RNAPII. We found that transcription efficiency was markedly higher when RNAPII initiated from the hexasome side than from the octasome side. Cryo-electron microscopy further revealed that transcription from the hexasome side induced pronounced conformational rearrangements, in which RNAPII progression dramatically opened the hexasome-octasome interface. These results uncover the mechanism by which RNAPII senses the intrinsic transcriptional polarity of OLDNs and suggest that OLDNs function as dynamic, directionally sensitive regulators of transcription elongation.

molecular biology↗

bioRxiv2025

Locations of consecutive G·C base-pairs direct genomic nucleosome positioning

The nucleosome is a structural and functional subunit of chromatin, and its positioning and dynamics in the eukaryotic genomes serves as a key platform for gene regulation. Here, we determine the positions of fully wrapped nucleosomes across the yeast genome by chemical mapping through the histone H2A-A122C residue, which cleaves near the DNA entry/exit sites. This approach reveals the most refined sequence-dependent profile of nucleosomes reported to date. Comparison of H2A-A122C with the H3-Q85C and H4-S47C methods clearly shows a sequence preference for the chemical cleavage site, and mapping results suggest that the nucleosome locations are dynamic in vivo. More importantly, we find that the depletion of CC and GG dinucleotides at nucleotide positions -11 to -9 and +9 to +11 bp, respectively, from the nucleosome dyad position (0) is inextricably associated with the enrichment of AA/AT/TA/TT dinucleotides in both yeast and mouse genomes. Introducing consecutive C*G base pairs to the corresponding sites in the 601 sequence shifts the nucleosomes to less frequent positions without altering thermal stability, implying a structural constraint imposed by DNA sequence. Thus, CC and GG dinucleotides in the major groove blocks at superhelix locations (SHL) -1.0 and +1.0, respectively, destabilize histone-DNA interactions, serving as intrinsic determinants of nucleosome positioning in eukaryotic genomes.

molecular biology↗

bioRxiv2025

Structural basis of RNA polymerase II transcription on the H3-H4 octasome

The H3-H4 octasome is a nucleosome-like particle in which two DNA gyres are wrapped around each H3-H4 tetramer disk, forming a clamshell-like configuration. In the present study, we performed in vitro RNAPII transcription assays with the H3-H4 octasome and found that RNAPII transcribed the H3-H4 octasome more efficiently than the nucleosome. RNAPII paused at only one position, superhelical location (SHL) -4 in the H3-H4 octasome, in contrast to pausing at the SHL(-5), SHL(-2), and SHL(-1) positions in the nucleosome. Cryo-electron microscopy analysis revealed that two H3-H4 tetramer disks are retained when the RNAPII paused at the SHL(-4) position of the H3-H4 octasome. However, when RNAPII reached the SHL(-0.5) position, five base pairs before the dyad position of the H3-H4 octasome, the proximal H3-H4 tetramer was disassembled but the distal H3-H4 tetramer still remained on the DNA. Therefore, RNAPII efficiently transcribes the H3-H4 octasome by stepwise H3-H4 tetramer disassembly.

molecular biology↗

bioRxiv2024

Structural basis of transcription-coupled H3K36 trimethylation by Set2 and RNAPII elongation complex in the nucleosome

Trimethylation of the histone H3K36 residue (H3K36me3) plays an indispensable role in ensuring transcription fidelity by suppressing undesired cryptic transcription in chromatin. The H3K36me3 modification is accomplished by Set2/SETD2 during transcription elongation by the RNA polymerase II elongation complex (EC). Here we found that the Set2-mediated H3K36me3 deposition occurs primarily on the nucleosome reassembling behind the EC. Cryo-electron microscopy structures of the transcribing EC complexed with Set2 and the reassembled nucleosome revealed that Set2 is anchored by the Spt6 subunit of the EC and captures an H3 N-terminal tail of the nucleosome. Abrogation of the Set2-Spt6 interaction leads to defective transcription-coupled H3K36me3 deposition. These insights elucidate the structure-based mechanism of transcription-coupled H3K36me3 deposition in chromatin. One-Sentence SummaryCryo-EM structures of the EC-Set2-nucleosome complex reveal the mechanism of H3 Lys36 trimethylation in chromatin.

biochemistry↗

bioRxiv2024

Engineered acetylation patterns drive large-scale chromatin organization in vitro

Chromatin organization plays a crucial role in gene regulation [1, 2, 3], but disentangling the contributions of various epigenetic components to gene-scale chromatin structure remains challenging. While in vitro chromatin reconstitution enables controlled studies on the effect of bio-chemical factors on the structure, current methods are either limited to short arrays or lack control over histone modification patterns. Here we directly test how histone modification affects higher-order chromatin architecture by characterizing gene-scale reconstituted chromatin using single-molecule microscopy and in vitro Hi-C. We reconstitute 20-kilobase chromatin arrays with histone modification patterns controlled at 12-nucleosome resolution, achieving complete assembly of 96 nucleosomes in the designed order as confirmed by atomic force microscopy and longread sequencing. Observing end-to-end fluctuations of the reconstituted arrays, we find that increasing the density of acetylated nucleosomes leads to larger structural fluctuations with longer relaxation times, consistent with the predictions of a polymer model with hydrodynamic interactions. We demonstrate through in vitro Hi-C how acetylation reduces contact frequency between nucleosomes and induces open conformations. In heterogeneously modified arrays, differential contact probabilities between acetylated and unmodified regions lead to distinct structural domains. The results establish the physical principles by which histone modifications directly modulate chromatin architecture through altered nucleosome-nucleosome interactions, providing a quantitative framework for understanding and engineering genome organization.

biophysics↗

bioRxiv2023

ISWI chromatin remodeling complexes recruit NSD2 and H3K36me2 in pericentromeric heterochromatin

Histone H3 lysine36 dimethylation (H3K36me2) is generally distributed in the gene body and euchromatic intergenic regions. However, we found that H3K36me2 is enriched in pericentromeric heterochromatin in some mouse cell lines. We here revealed the mechanism of heterochromatin targeting of H3K36me2. Among several H3K36 methyltransferases, NSD2 was responsible for inducing heterochromatic H3K36me2. Depletion and overexpression analyses of NSD2-associating proteins revealed that NSD2 recruitment to heterochromatin was mediated through the imitation switch (ISWI) chromatin remodeling complexes, such as BAZ1B-SMARCA5 (WICH), which directly binds to AT-rich DNA via a BAZ1B domain containing AT-hook-like motifs. The abundance and stoichiometry of NSD2, SMARCA5, and BAZ1B or BAZ2A could determine the localization of H3K36me2 in different cell types. To explore the physiological role of heterochromatic H3K36me2, we analyzed mouse tissues and embryos. As a result, H3K36me2 was found in heterochromatin at the 2- to 4-cell stages of mouse preimplantation embryos, suggesting its involvement in developmental regulation. SummaryThe authors discovered histone H3K36me2, which is believed to be enriched in potentially active genomic regions, is also located in transcriptionally inactive regions called heterochromatin in some cell types. The detailed molecular mechanism of its heterochromatin targeting is now revealed.

cell biology↗

bioRxiv2023

Molecular and structural basis of the heterochromatin-specific chromatin remodeling activity by Arabidopsis DDM1

The chromatin remodeler DECREASE IN DNA METHYLATION 1 (DDM1) deposits the histone H2A variant H2A.W and silences transposons in Arabidopsis thaliana. However, the molecular mechanisms by which DDM1 specifically targets the nucleosome containing H2A.W and allows chromatin writers to access heterochromatin remained elusive. Here, we show that DDM1 promotes remodeling of the H2A.W nucleosome and requires interactions with the H2A.W-specific C-terminal tail. The cryo-EM structure of the DDM1-H2A.W nucleosome complex revealed that DDM1 binds to the N-terminal tail of H4 and the nucleosomal DNA. Comparison with the cryo-EM structure of the nucleosome containing H2A.W suggested that DDM1 increases the DNA end flexibility of nucleosomes. Based on these biochemical and structural results, we propose that the chromatin remodeling activity of DDM1 with the heterochromatin-specific H2A.W contributes to the maintenance of repressive epigenetic marks in heterochromatin by providing DNA methyltransferases with access to nucleosomal DNA.

molecular biology↗

bioRxiv2021

Distinctive nuclear zone for RAD51-mediated homologous recombinational DNA repair

Genome-based functions are inseparable from the dynamic higher-order architecture of the cell nucleus. In this context, the repair of DNA damage is coordinated by precise spatiotemporal controls that target and regulate the repair machinery required to maintain genome integrity. However, the mechanisms that pair damaged DNA with intact template for repair by homologous recombination (HR) without illegitimate recombination remain unclear. This report highlights the intimate relationship between nuclear architecture and HR in mammalian cells. RAD51, the key recombinase of HR, forms spherical foci in S/G2 phases spontaneously. Using super-resolution microscopy, we show that following induction of DNA double-strand breaks RAD51 foci at damaged sites elongate to bridge between intact and damaged sister chromatids; this assembly occurs within bundle-shaped distinctive nuclear zones, requires interactions of RAD51 with various factors, and precedes ATP-dependent events involved the recombination of intact and damaged DNA. We observed a time-dependent transfer of single-stranded DNA overhangs, generated during HR, into such zones. Our observations suggest that RAD51-mediated homologous pairing during HR takes place within the distinctive nuclear zones to execute appropriate recombination.

cell biology↗

bioRxiv2021

Cryo-electron microscopy structure of the H3-H4 octasome without histones H2A and H2B

The canonical nucleosome, which represents the predominant packaging unit in eukaryotic chromatin, has an octameric core made up of two histone H2A-H2B and H3-H4 dimers with ~147 base-pair (bp) DNA wrapping around it. Non-nucleosome particles with alterative histone stoichiometries and DNA wrapping configurations have been found, and they could profoundly influence genome architecture and function. Here we solved the structure of the H3-H4 octasome, which is a nucleosome-like particle with a core made up of four H3-H4 dimers. Two conformations, open and closed, are determined at 3.9 [A] and 3.6 [A] resolutions by cryo-electron microscopy, respectively. The H3-H4 octasome, made up of a di-tetrameric core, is wrapped by ~120 bp DNA in 1.5 negative superhelical turns. The symmetrical halves are connected by a unique H4-H4 interface along the dyad axis. In vivo crosslinking of cysteine probes placed at another unique H3-H3 interface demonstrated the existence of the H3-H4 octasome in cells.

biochemistry↗

bioRxiv2020

Modeling population size independent tissue epigenomes by ChIL-seq with single-thin sections

Recent advances in omics studies have enabled analysis at the single-cell level; however, methods for analyzing the whole cell of large organs and tissues remain challenging. Here, we developed a method named tsChIL to understand the diverse cellular dynamics at the tissue level using high-depth epigenomic data. tsChIL allowed the analysis of a single tissue section and could reproducibly acquire epigenomic profiles from several types of tissues, based on the distribution of target epigenomic states, tissue morphology, and number of cells. The proposed method enabled the independent evaluation of changes in cell populations and gene activation of cells in regenerating skeletal muscle tissues, using a statistical model of RNA polymerase II distribution on gene loci. Thus, the integrative analysis by tsChIL can elucidate in vivo cell-type dynamics of tissues.

molecular biology↗

bioRxiv2020

Tracking H3K27me3 and H4K20me1 dynamics during XCI reveals similarities in recruitment mechanism

During X chromosome inactivation (XCI), in female placental mammals, gene silencing is initiated by the Xist long-noncoding RNA. Xist accumulation at the X leads to enrichment of specific chromatin marks, including PRC2-dependent H3K27me3 and SETD8-dependent H4K20me1. However, the dynamics of this process in relation to Xist RNA accumulation remains unknown as is the molecular mechanism allowing for H4K20me1 enrichment. To follow XCI dynamics in living cells, we developed a genetically-encoded, H3K27me3-specific intracellular antibody, or H3K27me3-mintbody. By combining it with live-imaging of H4K20me1, the X chromosome and Xist RNA we uncover similarities in the initial accumulation dynamics of H3K27me3 and H4K20me1. Further ChIP-seq analysis confirmed concurrent accumulation of both marks during XCI albeit with distinct genomic distributions. Using a Xist B and C repeat mutant, which can silence the X but does not allow for H3K27me3 deposition, we also found a lack of H4K20me1 enrichment. Thus, these two marks accumulate at the X thanks to the same region of Xist and H4K20me1 in particular may have a role in the chromatin compaction that characterises facultative heterochromatin.

developmental biology↗

bioRxiv2019

Structural Studies of Overlapping Dinucleosomes in Solution

An overlapping dinucleosome (OLDN) is a structure composed of one hexasome and one octasome and appears to be formed through nucleosome collision promoted by nucleosome remodeling factor(s). In the present study, the solution structure of the OLDN was investigated through integration of small-angle X-ray and neutron scattering (SAXS and SANS, respectively), computer modeling, and molecular dynamics simulations. Starting from the crystal structure, we generated a conformational ensemble based on normal mode analysis, and searched for the conformations that well reproduced the SAXS and SANS scattering curves. We found that inclusion of histone tails, which are not observed in the crystal structure, greatly improved model quality. The obtained structural models suggest that OLDNs adopt a variety of conformations stabilized by histone tails situated at the interface between the hexasome and octasome, simultaneously binding to both the hexasomal and octasomal DNA. In addition, our models define a possible direction for the conformational changes or dynamics, which may provide important information that furthers our understanding of the role of chromatin dynamics in gene regulation.\n\nStatement of SignificanceOverlapping dinucleosomes (OLDNs) are intermediate structures formed through nucleosome collision promoted by nucleosome remodeling factor(s). To study the solution structure of OLDNs, a structural library containing a wide variety of conformations was prepared though simulations, and the structures that well reproduced the small angle X-ray and neutron scattering data were selected from the library. Simultaneous evaluation of the conformational variation in the global OLDN structures and in the histone tails is difficult using conventional MD simulations. We overcame this problem by combining multiple simulation techniques, and showed the importance of the histone tails for stabilizing the structures of OLDNs in solution.

biophysics↗

bioRxiv2019

Chromatin structure-dependent histone incorporation revealed by a genome-wide deposition assay

In eukaryotes, histone variant distribution within the genome is the key epigenetic feature. To understand how each histone variant is targeted to the genome, we developed a new method, in which epitope-tagged histone complexes are introduced into permeabilized cells and incorporated into their chromatin. We found that the incorporation of histones H2A and H2A.Z mainly occurred at less condensed chromatin (open), suggesting that the condensed chromatin (closed) is a barrier for histone incorporation. To overcome this barrier, H2A, but not H2A.Z, uses a replication-coupled deposition mechanism. This led to the recapitulation of the pre-existing chromatin structure: the genome-wide even distribution of H2A and the exclusion of H2A.Z from the closed chromatin. Intriguingly, an H2A.Z mutant with mutations in the developmentally essential region was incorporated into closed chromatin. Our study revealed that the combination of chromatin structure and DNA replication dictates the differential histone deposition for maintaining the epigenetic chromatin states.

biochemistry↗