Biology subjects

Shinkai, Y.

Publications and source records attributed to Shinkai, Y..

13 recordsLinked to original sources

bioRxiv2025

Resf1 is required for proper placental development and configuration of trophoblast cell-specific heterochromatin

Resf1 (Retroelement silencing factor 1) is involved in retroelement silencing cooperated with H3K9 methyltransferase SETDB1 by regulating H3K9 methylation in mouse embryonic stem cells (mESCs). However, it remains unknown whether Resf1 functions in retroelement silencing in vivo, and has a role in development. Here, we established Resf1-deficient mice, which exhibited developmental delay, partial embryonic lethality, and placental defects. Notably, retroelements were also upregulated in the Resf1-deficient placenta, correlating with increased expression of nearby genes. To further assess whether Resf1 functions within the trophoblast lineage, we generated Resf1-deficient trophoblast stem cell (TSC) lines. Both undifferentiated TSCs and differentiated TSCs (D-TSCs) display increased retroelement expression along with elevated levels of genes associated with placental development. Moreover, Resf1-deficient TSCs exhibit compromised maintenance of H3K9me3 domains in a manner independent of SETDB1. Collectively, our findings reveal that Resf1 plays multifaceted roles beyond retroelement silencing, underscoring its importance in development and its critical function in trophoblast lineage regulation.

developmental biology↗

bioRxiv2025

Chromatin landscape of budding yeast acquiring H3K9 methylation and its reader molecule HP1

Histone H3 lysine 9 (H3K9) methylation and heterochromatin protein 1 (HP1) are well conserved heterochromatin epigenome and its reader molecule. However, the details of how importance of them in heterochromatin formation still remain unclear. One of the reasons is the redundancy aspects, as there are multiple H3K9 methylation reader molecules, including HP1, and HP1 itself functions as a hub recruiting various effector molecules. To overcome those issues, we took a synthetic biology approach and introduced H3K9 methylation and HP1 into budding yeast Saccharomyces cerevisiae, which does not have this system, and examined its impact on transcription and chromatin compaction. We observed that the mammalian H3K9 methyltransferase can induce genome-wide H3K9 di- and tri-methylation (H3K9me2,3) in the budding yeast which mainly in the gene body region, and HP1 accumulates over the H3K9 methylated regions. The forced expression of H3K9 methyltransferase and HP1 had little impact on transcription. Furthermore, Micro-C-seq analysis revealed no significant effects on the chromatin 3D structure. These results suggest that although H3K9 methylation and recruitment of HP1 play essential roles in the epigenetic regulation of heterochromatin, they alone are not sufficient to alter the higher-order chromatin structure, at least in the gene body regions in budding yeast.

synthetic biology↗

bioRxiv2025

Mitochondrial translation termination, recycling, reinitiation, and rescue for in-frame and out-of-frame contexts

Because mitochondria diverged from a bacterial ancestor during evolution, the mitochondrial protein synthesis system includes both mRNAs and translation factors with unique characteristics. However, the molecular mechanisms underlying translation termination, recycling, and quality control remain unclear. Here, via high-resolution mitochondrial Ribo-Seq and Disome-Seq, we revealed: the specificity of release factors for different kinds of stop codons; the role of mtRF1 in vertebrates, which do not have noncanonical stop codons in their main ORFs; the recycling-coupled translation of internal ORFs; and the rescue of mitoribosomes in the early elongation stage. mtRF1L is a universal release factor that recognizes all stop codons, whereas mtRF1 recognizes only AGA/AGG noncanonical stop codons. Additionally, mtRF1 terminates the translation of out-of-frame ORFs that end with AGA/AGG. We also found that mtRRF and mtIF3 are required for mitoribosome recycling on stop codons and for the reinitiation of internal ORF translation. Mitoribosomes that stall at start codons are major substrates of the rescue factors ICT1, mtRF-R, and mtRES1. Moreover, HEMK1-mediated methylation of release factors enhances the termination reaction on stop codons. Our results provide insights into the mitoribosome dynamics that are associated with the completion of protein synthesis.

molecular biology↗

bioRxiv2025

Trichostatin A Treatment Has Little Impact on Nuclear compartments in Cells Depleted of H3K9me, H3K27me3, and uH2A

In the mammalian nucleus, heterochromatin is segregated from transcriptionally active euchromatic regions (A compartments), forming large, condensed, and inactive nuclear compartments (B compartments). However, the mechanisms underlying its spatial organization remain incompletely understood. We previously demonstrated that simultaneous depletion of H3K9 methylation, H3K27me3, and H2A monoubiquitination in immortalized mouse embryonic fibroblasts (iMEFs) leads to heterochromatin weakening and alterations in nuclear compartments. However, the overall pattern of A/B compartments remains largely preserved even under these conditions, suggesting the involvement of unknown factors. Since histone deacetylation is a key factor in transcriptional repression, we investigated the impact of histone deacetylase (HDAC) inhibition on nuclear compartments by treating cells depleted of H3K9 methylation, H3K27me3, and H2A K119 monoubiquitylation (H2AK119ub/uH2A) with the HDAC inhibitor Trichostatin A (TSA). We performed Hi-C analysis to assess the nuclear compartment changes. Our results showed that, although TSA treatment globally increased H3K27ac levels, the difference in H3K27ac enrichment between A/B compartments remained evident, and nuclear compartments were minimally affected. These findings suggest that factors other than HDACs maintain nuclear compartmentalization when repressive chromatin modifications are depleted.

molecular biology↗

bioRxiv2025

H3K27 and H3K9 Methylation Mask Potential CTCF Binding Sites to Maintain 3D Genome Integrity

The three-dimensional (3D) genome structure is essential for gene regulation and various genomic functions. CTCF plays a key role in organizing Topologically Associated Domains (TADs) and promoter-enhancer loops, contributing to proper cell differentiation and development. Although CTCF binds the genome with high sequence specificity, its binding sites are dynamically regulated during development, and aberrant CTCF binding is linked to diseases such as cancer and neurological disorders, and aging. However, the mechanisms controlling CTCF binding remain unclear. Here, we investigated the role of repressive chromatin modifications in CTCF binding using H3K9 methyltransferase-deficient immortalized mouse embryonic fibroblasts (iMEFs) and H3K27 methyltransferase EZH1/2 inhibitor. We found that H3K9 and H3K27 methylation regulate CTCF binding at distinct genomic regions, and their simultaneous loss induces drastic changes in CTCF binding. These changes were associated with alterations in 3D genome architecture and gene expression, suggesting that repressive chromatin modifications preserve proper chromatin organization by preventing aberrant CTCF binding. Additionally, while CTCF binding sites repressed by H3K9 methylation were bound by CTCF in early mouse embryos, those repressed by both H3K9 and H3K27 methylation remained inaccessible, with early embryonic-specific H3K27 methylation forming at these sites. These findings implicate that H3K27 methylation prevents abnormal CTCF binding in early embryos, ensuring proper genome organization during development.

molecular biology↗

bioRxiv2025

Actin Painting: a multicolor fluorescence staining method highlighting cell type-specific differences in the composition of filamentous actin architectures.

Actin is a major structural component of the cytoskeleton in eukaryotic cells, and filamentous actin (F-actin) forms a variety of types of cellular structures. Many actin probes have been developed to visualize F-actin architectures in eukaryotic cells. However, it is known that double-stained images of F-actin obtained by two different types of actin probes often show partial inconsistencies. While developing actin probes, we observed that the merged images of three distinct actin probes, each labeled with a different fluorescent dye (red, green and blue), enabled various F-actin architectures to be distinguished based on color. This differentiation arises from slight variations in the distribution of each actin probe, and results in a unique color combination of actin probes that reflects the relative intensities of the three fluorescent dyes. In this report, we introduce a new cell staining method, named Actin Painting, which exploits the unique affinity of actin-binding molecules in each actin probe to F-actin architectures. This technique allows the classification of various cell types based on their specific actin cytoskeleton.

cell biology↗

bioRxiv2025

H3K27me3 and the PRC1-H2AK119ub pathway cooperatively maintain heterochromatin and transcriptional silencing after the loss of H3K9 methylation

Heterochromatin is a key architectural feature of eukaryotic chromosomes, essential for cell type-specific gene expression and genome stability. In the mammalian nucleus, heterochromatin is segregated from transcriptionally active euchromatic regions (A compartments), forming large, condensed, and inactive nuclear compartments (B compartments). However, the mechanisms underlying its spatial organization remain incompletely understood. Histone H3 lysine 9 and lysine 27 trimethylation (H3K9me3 and H3K27me3) are two major epigenetic modifications that enrich constitutive and facultative heterochromatin, respectively. Previously, we found that the redistribution of H3K27me3 following the loss of H3K9 methylation contributes to heterochromatin maintenance, while the simultaneous loss of both H3K27me3 and H3K9 methylation induces heterochromatin decondensation in mouse embryonic fibroblasts. However, nearly all B compartments were preserved despite the loss of these repressive chromatin modifications. These findings suggest that other factors are responsible for maintaining B compartments under these conditions. In this study, we explored the role of another repressive chromatin modification, PRC1-mediated H2A K119 monoubiquitylation (H2AK119ub/uH2A), in maintaining heterochromatin structure following the loss of H3K9/K27 methylation. We found that uH2A and H3K27me3 independently accumulate in the B compartments after the loss of H3K9 methylation in iMEFs and cooperatively maintain heterochromatin. Our data indicates that the PRC1-uH2A pathway contributes to maintain heterochromatin organization following the loss of H3K9/K27 methylation in mammalian cells.

molecular biology↗

bioRxiv2024

Conserved loop of a phase modifier endows protein condensates with fluidity

Dipeptide repeats (DPRs) that are gene products from abnormal hexanucleotide repeat expansion in C9orf72 trigger amyotrophic lateral sclerosis (ALS) through unknown mechanism. This study highlights, importin Karyopherin{beta}2 (Kap{beta}2), which is responsible for nuclear transport and phase modification of RNA-binding proteins (RBPs), as a major DPR target. We demonstrate DPR accumulation in the nucleus via Kap{beta}2-mediated transport, which results in dose-dependent toxicity observed in nematode and yeast models. In vitro interaction studies exploiting chemical probe arrays and biophysical measurements reveal multivalent DPR binding to Kap{beta}2, including at the conserved acidic loop. Refractive index and fluorescence imaging coupled with biochemical assays unveiled that binding of excess DPRs to the acidic loop turns a phase modifier Kap{beta}2 into phase disrupter, resulting more condensed and viscous RBP condensates. Our findings provides molecular insight into C9orf72-ALS related to age and repeat expansion.

biochemistry↗

bioRxiv2023

Growth inhibitory factor/metallothionein-3 is a sulfane sulfur-binding protein

Cysteine-bound sulfane sulfur atoms in proteins have received much attention as key factors in cellular redox homeostasis. However, the role of sulfane sulfur in zinc regulation has been underinvestigated. We report here that cysteine-bound sulfane sulfur atoms serve as ligands to hold and release zinc ions in growth inhibitory factor (GIF)/metallothionein-3 (MT-3) with an unexpected C-S-S-Zn structure. Oxidation of such a zinc/persulfide cluster in Zn7GIF/MT-3 results in the release of zinc ions, and intramolecular tetrasulfide bridges in apo-GIF/MT-3 efficiently undergo S-S bond cleavage by thioredoxin to regenerate Zn7GIF/MT-3. Three-dimensional molecular modeling confirmed the critical role of the persulfide group in the thermostability and Zn-binding affinity of GIF/MT-3. The present discovery raises the fascinating possibility that the function of other Zn-binding proteins is controlled by sulfane sulfur.

biochemistry↗

bioRxiv2022

OVGP1 is an oviductal fluid factor essential particularly for early embryonic development in golden hamsters

The mammalian oviductal lumen is a specialized chamber that provides an environment that strictly regulates fertilization an early embryogenesis, the regulatory mechanisms to gametes/zygote are still largely unknown. In this report, we studied the oviductal regulation of early embryonic development using Ovgp1 (a gene encoding an oviductal humoral factor, OVGP1)-knockout (KO) hamsters. The experimental results revealed the following: 1) Female Ovgp1-KO hamsters fail to produce any litters at all; 2) In the oviducts from KO animal, fertilized eggs are sometimes identified, but their morphology shows abnormal features; 3) The number of implantations in the KO females is evidently low; 4) Even if implantations occur, the embryos develop abnormally and eventually become embryonic lethal; and 5) Ovgp1-KO females transferred to wild-type females produce KO egg-derived litters, but the reverse experiment does not. These results suggest that OVGP1-mediated physiological events are crucial for early embryonic development in vivo. This animal model shows that the fate of the fertilized egg is not only genetically determined, but that the surrounding oviductal microenvironment plays a pivotal role in normal embryonic development. Summary statementDeficiency an oviductal humoral factor (OVGP1) caused female infertility in the golden hamsters. The presence or absence of OVGP1 has significant physiological effects on early embryonic development in vivo.

developmental biology↗

bioRxiv2021

METTL18-mediated histidine methylation on RPL3 modulates translation elongation for proteostasis maintenance

Protein methylation occurs predominantly on lysine and arginine residues, but histidine also serves as a substrate for the modification. However, a limited number of enzymes responsible for this modification have been reported. Moreover, the biological role of histidine methylation has remained poorly understood. Here, we report that human METTL18 is a histidine methyltransferase for the ribosomal protein RPL3 and that the modification specifically slows ribosome traverse on tyrosine codons, allowing the proper folding of synthesized proteins. By performing an in vitro methylation assay with a methyl donor analog and quantitative mass spectrometry, we found that His245 of RPL3 is methylated at the {tau}-N position by METTL18. Structural comparison of the modified and unmodified ribosomes showed stoichiometric modification and suggested a role in translation tuning. Indeed, genome-wide ribosome profiling revealed suppressed ribosomal translocation at tyrosine codons by RPL3 methylation. Because the slower elongation provides enough time for nascent protein folding, RPL3 methylation protects cells from the cellular aggregation of Tyr-rich proteins. Our results reveal histidine methylation as an example of a "ribosome code" that ensures proteome integrity in cells.

molecular biology↗

bioRxiv2021

Transcriptional states of retroelement-inserted regions and KRAB zinc finger protein association regulate DNA methylation of retroelements in human male germ cells

DNA methylation, repressive histone modifications, and PIWI-interacting RNAs are essential for controlling retroelement silencing in mammalian germ lines. Dysregulation of retroelement silencing is associated with male sterility. Although retroelement silencing mechanisms have been extensively studied in mouse germ cells, little progress has been made in humans. Here, we show that the Kruppel-associated box domain zinc finger proteins (KRAB-ZFPs) are associated with DNA methylation of retroelements in human primordial germ cells (hPGCs), and hominoid-specific retroelement SINE-VNTR-Alus (SVA) is subjected to transcription-directed de novo DNA methylation during human spermatogenesis. Furthermore, we show that the degree of de novo DNA methylation in SVAs varies among human individuals, which confers a significant inter-individual epigenetic variation in sperm. Collectively, our results provide potential molecular mechanisms for the regulation of retroelements in human male germ cells.

molecular biology↗

bioRxiv2020

Functional correlation of H3K9me2 and nuclear compartment formation

BackgroundHistone H3 lysine 9 dimethylation (H3K9me2) is a highly conserved silencing epigenetic mark. Chromatin marked with H3K9me2 forms large domains in mammalian cells and correlates well with lamina-associated domains and the B compartment. However, the role of H3K9me2 in 3-dimensional (3D) genome organization remains unclear. ResultsWe investigated the genome-wide H3K9me2 distribution, the transcriptome and 3D genome organization in mouse embryonic stem cells (mESCs) upon the inhibition or depletion of H3K9 methyltransferases (MTases) G9a/GLP, SETDB1, and SUV39H1/2. We found that H3K9me2 is regulated by these five MTases; however, H3K9me2 and transcription in the A and B compartments were largely regulated by different sets of the MTases: H3K9me2 in the A compartments were mainly regulated by G9a/GLP and SETDB1, while H3K9me2 in the B compartments were regulated by all five H3K9 MTases. Furthermore, decreased H3K9me2 correlated with the changes to the more active compartmental state that accompanied transcriptional activation. ConclusionOur data showed that H3K9me2 domain formation is functionally linked to 3D genome organization.

molecular biology↗