Biology subjects

Osakabe, A.

Publications and source records attributed to Osakabe, A..

6 recordsLinked to original sources

bioRxiv2025

Antagonistic histone H2A variants and autonomous heterochromatin formation shape epigenomic patterns in Arabidopsis

Heterochromatin formation is pivotal in many eukaryotes with repetitive sequences, such as transposable elements (TEs). However, in plants, where the known de novo DNA methylation mechanism (RdDM) targets euchromatin, how heterochromatin is formed in a region-specific manner remains unclear. We previously reported an RdDM-independent de novo establishment of H3K9me and non-CpG methylation, both of which localize in heterochromatin. Here we show that the mutually exclusive histone H2A variants, H2A.W and H2A.Z, function as guides to initiate heterochromatin formation; H2A.W and H2A.Z promotes and inhibits heterochromatin establishment, respectively, especially in chromosomal arm regions with dispersed TEs. In contrast, pericentromeric TEs demonstrate autonomous heterochromatin formation, less dependently on these H2A variants. Furthermore, H2A.Z protects protein-coding genes from ectopic heterochromatin formation, possibly by preventing its spreading. We propose that the genome indexing mechanism driven by H2A variants, as well as the autonomous formation of pericentromeric heterochromatin, shapes proper epigenomic patterns in Arabidopsis.

genomics↗

bioRxiv2025

MBD8 is required for LDL2-mediated transcriptional repression downstream of H3K9me2 in Arabidopsis

Histone H3 lysine 9 methylation (H3K9me) is a conserved epigenetic mark for transcriptional gene repression. In Arabidopsis, H3K9 dimethylation (H3K9me2) promotes the removal of a transcription-associated mark, H3K4 monomethylation (H3K4me1), and this process within genes requires the histone demethylase LDL2. However, how LDL2 specifically functions in genes that accumulate H3K9me2 remains unknown. Here, we show that methyl-CpG-binding domain protein 8 (MBD8) binds to LDL2 and plays a role in H3K4me1 removal and transcriptional gene repression downstream of H3K9me2. MBD8 preferentially binds GC-poor DNA, regardless of its cytosine methylation status. Interestingly, LDL2 protein levels were significantly reduced in the mbd8 mutant background, suggesting that MBD8 stabilizes the LDL2 protein, facilitating H3K4me1 removal and the transcriptional repression of H3K9me-marked genes. The domain necessary for the MBD8-LDL2 interaction is conserved among plants. We propose that MBD proteins have diverse functions beyond their methylated DNA-binding capabilities.

plant 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↗

bioRxiv2023

Histone variants shape the chromatin states in Arabidopsis

How different intrinsic sequence variation or regulatory modifications of histones regulate nucleosome interactions with transcription remain unclear. By contrast with H3 and H2B variants, H2A variants occupy specific domains of chromatin in Arabidopsis thaliana. Broad domains of chromatin are affected by the loss of remodelers that affect the deposition or the exchange of H2A variants. Notably, the chromatin remodeler DECREASED IN DNA METHYLATION (DDM1) is required to maintain enrichment in all markers of constitutive heterochromatin including DNA methylation, H3K9me1/2 and the variant H2A.W. To test the importance of histone variants in the organization of chromatin we investigated how histone variants and histone modifications assemble in the Arabidopsis thaliana genome and showed that a limited number of chromatin states divide euchromatin and heterochromatin into several subdomains. We found that histone variants are as significant as histone modifications in determining the composition of chromatin states. Particularly strong associations were observed between H2A variants and specific combinations of histone modifications. To study the role of H2A variants in organizing chromatin states we determined the role the chromatin remodeler DECREASED IN DNA METHYLATION (DDM1) in the organization of chromatin states. We showed that the loss of DDM1 prevented the exchange of the histone variant H2A.Z to H2A.W in constitutive heterochromatin, resulting in significant effects on the definition and distribution of chromatin states in and outside of heterochromatin. We thus propose that dynamic exchanges of histone variants control the organization of histone modifications into chromatin states, acting as molecular landmarks.

genomics↗

bioRxiv2022

The histone variant H2A.W cooperates with chromatin modifications and linker histone H1 to maintain transcriptional silencing of transposons in Arabidopsis

Transposable elements (TEs) are marked by a complex array of chromatin modifications, but a central unifying mechanism for how they are silenced remains elusive. Histone H3 Lysine 9 methylation (H3K9me) is an important component of heterochromatin in most eukaryotes, including plants. In flowering plants, the specialized histone variant H2A.W occupies nucleosomes found at TE sequences. This variant is deposited by the chromatin remodeler DDM1 and confers specific biophysical properties to the nucleosomes. Here we use genetic and genomic strategies to evaluate the role of H2A.W in transposon silencing in Arabidopsis. Compared with mutants lacking either H2A.W or H3K9me, the combined loss of both H2A.W and H3K9me causes a dramatic increase in both the number of expressed TEs and their expression levels. Synergistic effects are also observed when H2A.W is lost in combination with histone H1 or CH methylation. Collectively, these TEs are also upregulated in mutants lacking DDM1, which are impaired in H2A.W deposition and lose heterochromatic marks. We conclude that H2A.W acts in combination with different elements of heterochromatin to maintain silencing across a large spectrum of TEs present primarily in pericentric heterochromatin in Arabidopsis. In mammals, the DDM1 ortholog LSH deposits macroH2A to heterochromatin and silences TEs. We thus propose that specialized H2A variants localized to heterochromatin interact with a complex array of histone modifications to silence TEs in eukaryotes.

genomics↗

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↗