Biology subjects

Tsukiyama, T.

Publications and source records attributed to Tsukiyama, T..

15 recordsLinked to original sources

bioRxiv2026

Adolescent prodromal phenotypes of psychiatric disorders: insights from a novel DISC1-perturbed monkey model

Neurodevelopmental deficits frequently lead to mild but significant adolescent social and cognitive changes, representing prodromal manifestations of adult-onset psychiatric disorders in a cross-disease manner. Although experimental medicine utilizes animal models to address the mechanisms, rodent brains lack critical structures for human higher brain functions such as the dorsolateral prefrontal cortex. To fill this translational gap, we generated monkeys with an impaired DISC1 gene locus, targeting the multifunctional protein essential for neurodevelopment and involved in transdiagnostic mental health risks. Here we analyze the F0 animals by minimizing mosaicism. At ages equivalent to human adolescence, these monkeys exhibited blunted responsiveness to social stimuli and increased internal drives, mirroring altered social behaviors in the prodromal stage in humans. Resting-state functional MRI data indicated disrupted global functional connectivity and reorganization of network topology, marked by a shift in the dominance of brain hubs from prefrontal regions to sensory areas. This primate model provides a unique platform for studying psychiatric disorders, such as schizophrenia, across the developmental trajectory.

neuroscience↗

bioRxiv2026

Chromatin architecture sets origin licensing capacity

Replication origin licensing enables complete and faithful genome duplication, yet how chromatin regulates this process in vivo remains unclear. Using MCM-ChEC-seq to track helicase loading from metaphase through G1 in budding yeast, we find that licensing occurs in a rapid, synchronous burst at mitotic exit and then reaches an early plateau despite continued permissive cell-cycle conditions and persistent ORC binding at origins. Here we show that this plateau is imposed by chromatin architecture at replication origins, which limits the extent of origin licensing. Histone H3K56 acetylation marks newly replicated chromatin and is removed at S-phase exit by the deacetylases Hst3 and Hst4. Persistent H3K56ac severely impairs MCM loading without affecting ORC occupancy, indicating that chromatin limits licensing at the helicase-loading step. Strikingly, deletion or catalytic inactivation of the chromatin remodeler Isw2 increases licensing by approximately 40% in wild-type cells and fully suppresses the licensing defect in hst3{Delta} hst4{Delta} mutants, identifying Isw2 as a physiological inhibitor of origin licensing. Isw2-dependent nucleosome repositioning narrows the origin nucleosome-depleted region and restricts helicase loading. Together, these findings show that chromatin architecture at replication origins sets licensing capacity. Newly replicated chromatin transiently adopts an Isw2-dependent inhibitory configuration that is relieved, but not completely eliminated, by post-replicative chromatin maturation. Genome-wide licensing thus reflects integration of chromatin-imposed licensing capacity with cell cycle-dependent control of licensing timing.

genetics↗

bioRxiv2025

Dynamic evolution of EZHIP, an inhibitor of the Polycomb Repressive Complex 2 in mammals

The Polycomb Repressive Complex 2 (PRC2) is an ancient, conserved chromatin-interacting complex that controls gene expression, facilitating differentiation and cellular identity during development. Its regulation is critical in most eukaryotes. EZHIP was recently characterized as a PRC2 inhibitor and oncohistone mimic in mammals. Although EZHIP expression is typically restricted to the germline, its aberrant expression in pediatric brain tumors inhibits PRC2-mediated H3K27 methylation, driving disease progression. To gain a deeper understanding of its normal functions, we systematically examined EZHIP evolution across mammals using comparative genomics, synteny analyses, and motif discovery. Extending previous work, we find that EZHIP originated on the X chromosome and was retained there in most placental mammals, except in Afrotheria. In addition to the highly conserved H3K27M-like histone mimic motif, our motif analyses reveal six previously unidentified EZHIP motifs, including a putative nuclear localization signal, a serine-enriched region, and tandem repeats that are largely well-conserved in placental mammals. We hypothesize that these motifs are also critical to EZHIPs functions, including in PRC2 interaction and inhibition. We show that EZHIP has evolved under strong diversifying selection in primates and underwent dynamic expansions and losses across species. Some paralogs, such as EZHIP2 in primates, also evolved under positive selection. Based on its evolutionary attributes and germ-cell expression, we propose that EZHIP arose and evolved rapidly due to inter-parental conflict over fetal development in utero in placental mammals. Our work provides a foundation for further investigations into EZHIPs multifaceted roles in mammalian reproduction and disease. SignificanceHistone modifications regulate genes to orchestrate cell differentiation and development. Their precise regulation is essential for normal development and growth. The Polycomb Repressive Complex 2 (PRC2) deposits the repressive H3K27 methylation mark, whereas EZHIP antagonizes PRC2 to de-repress gene silencing mediated by H3K27 methylation. While aberrant EZHIP activity in cancers is well documented, its normal germline functions, evolutionary origins, and divergence remain unclear. We show that EZHIP is a placental, X-linked innovation that diversified through positive selection and recurrent gene duplications, yielding germline-specific paralogs. We discover conserved motifs that may have critical roles in PRC2 control. Our findings provide an evolutionary model - parental control over in utero reproduction - that drove the origin and innovation of EZHIPs in placental mammals.

evolutionary biology↗

bioRxiv2024

Sir2 is required for the quiescence-specific condensed three-dimensional chromatin structure of rDNA

Quiescence in Saccharomyces cerevisiae is a reversible G0 crucial for long-term survival under nutrient-deprived conditions. During quiescence, the genome is hypoacetylated and chromatin undergoes significant compaction. However, the 3D structure of the ribosomal DNA (rDNA) locus in this state is not well understood. Here, we report that the rDNA locus in quiescent cells forms a distinct condensed loop-like structure, different from structures observed during the mitotic cell cycle. Deletion of SIR2 disrupts this structure, causing it to collapse into a small dot and resulting in quiescence entry and exit defects. In contrast, Sir2 affects rDNA structure only modestly in G2/M phase. In the absence of Sir2, occupancy of both RNA Polymerase II and histone H3 increase at the rDNA locus during quiescence and through quiescence exit, further indicating gross defects in chromatin structure. Together, these results uncover a previously undescribed rDNA chromatin structure specific to quiescent cells and underscore the importance of Sir2 in facilitating the transition between cellular states.

molecular biology↗

bioRxiv2024

Atlas of Cynomolgus Macaque Hematopoiesis

Self-renewal and differentiation are inherent properties of hematopoietic stem cells (HSCs) that are necessary to support hematopoiesis; however, the underlying mechanisms, especially in human, remain unclear. Here, using the cynomolgus macaque as a surrogate model, we develop a new gating strategy to isolate with high purity transplantable cynomolgus HSCs and generated a single-cell transcriptomic map of cynomolgus HSCs and progenitor cells -- that covering gestational periods previously not analyzed in human. We show that hematopoietic cells from the late-1st to early-3rd trimester fetal liver and late-2nd trimester and thereafter bone marrow have repopulating potential, closely mimicking humans. Unexpectedly however, we found unlike in human, cynomolgus HSCs express CD38 but not CD33, indicating that these cellular counterparts are molecularly distinct. Our transcriptomic analysis reveals the presence of a direct differentiation pathway from HSCs to megakaryocyte lineages, lineage-primed multipotent progenitors and also identified putative HSC surface markers. Taken together, our comprehensive dataset highlights not only the utility of cynomolgus monkeys as model systems to study hematopoiesis but also their potential for translational applications.

cell biology↗

bioRxiv2023

Acidic media promotes quiescence entry in Saccharomyces cerevisiae

Quiescence is a conserved cellular state wherein cells cease proliferation and remain poised to re-enter the cell cycle when conditions are appropriate. Budding yeast is a powerful model for studying cellular quiescence. In this work, we demonstrate that the pH of the YPD media strongly affects quiescence entry efficiency in Saccharomyces cerevisiae. Adjusting media pH to 5.5 significantly improves quiescence entry efficiency compared to unadjusted YPD media. Thermotolerance of the produced quiescence yeast are similar, suggesting the media pH influences the quantity of quiescent cells more than quality of quiescence reached.

microbiology↗

bioRxiv2023

Apical-driven cell sorting optimised for tissue geometry ensures robust patterning

Tissue patterning coordinates morphogenesis, cell dynamics and fate specification. Understanding how these processes are coupled to achieve precision despite their inherent variability remains a challenge. Here, we investigate how salt-and-pepper epiblast and primitive endoderm (PrE) cells sort and robustly pattern the inner cell mass (ICM) of mammalian blastocysts. Quantifying cellular dynamics and mechanics together with simulations show a key role for the autonomously acquired apical polarity of mouse PrE cells in coupling cell fate and dynamics in tissue contexts. Specifically, apical polarity forms actin protrusions and is required for Rac1-dependent migration towards the ICM surface, where PrE cells are trapped due to decreased tension at their apical domain, while depositing an extracellular matrix gradient, breaking the tissue-level symmetry and collectively guiding their own migration. Tissue size perturbations and comparison with monkey blastocysts further demonstrate that the fixed proportion of PrE/epiblast cells is optimal and robust to variability in embryo size.

developmental biology↗

bioRxiv2023

Temporal variability and cell mechanics control robustness in mammalian embryogenesis

How living systems achieve precision in form and function despite their intrinsic stochasticity is a fundamental yet open question in biology. Here, we establish a quantitative morphomap of pre-implantation embryogenesis in mouse, rabbit and monkey embryos, which reveals that although blastomere divisions desynchronise passively without compensation, 8-cell embryos still display robust 3D structure. Using topological analysis and genetic perturbations in mouse, we show that embryos progressively change their cellular connectivity to a preferred topology, which can be predicted by a simple physical model where noise and actomyosin-driven compaction facilitate topological transitions lowering surface energy. This favours the most compact embryo packing at the 8- and 16-cell stage, thus promoting higher number of inner cells. Impairing mitotic desynchronisation reduces embryo packing compactness and generates significantly more cell mis-allocation and a lower proportion of inner-cell-mass-fated cells, suggesting that stochasticity in division timing contributes to achieving robust patterning and morphogenesis.

developmental biology↗

bioRxiv2021

Novel classes and evolutionary turnover of histone H2B variants in the mammalian germline

Histones and their post-translational modifications facilitate diverse chromatin functions in eukaryotes. Core histones (H2A, H2B, H3, and H4) package genomes after DNA replication. In contrast, variant histones promote specialized chromatin functions, including DNA repair, genome stability, and epigenetic inheritance. Previous studies have identified only a few H2B variants in animals; their roles and evolutionary origins remain largely unknown. Here, using phylogenomic analyses, we reveal the presence of five H2B variants broadly present in mammalian genomes. In addition to three previously described variants (H2B.1, subH2B, and H2B.W), we identify and describe two new variants, H2B.L and H2B.N. Four of these five H2B variants originated in mammals, whereas H2B.L arose prior to the last common ancestor of bony vertebrates. We find that though mammalian H2B variants are subject to high gene turnover, most are broadly retained in mammals, including humans. Despite an overall signature of purifying selection, H2B variants evolve more rapidly than core H2B with considerable divergence in sequence and length. All five H2B variants are expressed in the germline. H2B.L and H2B.N are predominantly expressed in oocytes, an atypical expression site for mammalian histone variants. Our findings suggest that H2B variants likely encode potentially redundant but vital functions via unusual chromatin packaging or non-chromatin functions in mammalian germline cells. Our discovery of novel histone variants highlights the advantages of comprehensive phylogenomic analyses and provides unique opportunities to study how innovations in chromatin function evolve.

evolutionary biology↗

bioRxiv2021

RSC primes the quiescent genome for hypertranscription upon cell cycle re-entry

Quiescence is a reversible G0 state essential for differentiation, regeneration, stem cell renewal, and immune cell activation. Necessary for long-term survival, quiescent chromatin is compact, hypoacetylated, and transcriptionally inactive. How transcription activates upon cell-cycle re-entry is undefined. Here we report robust, widespread transcription within the first minutes of quiescence exit. During quiescence, the chromatin-remodeling enzyme RSC was already bound to the genes induced upon quiescence exit. RSC depletion caused severe quiescence exit defects: a global decrease in RNA polymerase II (Pol II) loading, Pol II accumulation at transcription start sites, initiation from ectopic upstream loci, and aberrant antisense transcription. These phenomena were due to a combination of highly robust Pol II transcription and severe chromatin defects in the promoter regions and gene bodies. Together, these results uncovered multiple mechanisms by which RSC facilitates initiation and maintenance of large-scale, rapid gene expression despite a globally repressive chromatin state.

molecular biology↗

bioRxiv2020

Chromosomal-scale De novo Genome Assemblies of Cynomolgus Macaque and Common Marmoset

Cynomolgus macaque (Macaca fascicularis) and common marmoset (Callithrix jacchus) have been widely used in human biomedical research. Their genomes were sequenced and assembled initially using short-read sequences, with the advent of massively parallel sequencing. However, the resulting contig sequences tended to remain fragmentary, and long-standing primate genome assemblies used the human genome as a reference for ordering and orienting the assembled fragments into chromosomes. Here we performed de novo genome assembly of these two species without any human genome-based bias observed in the genome assemblies released earlier. Firstly we assembled PacBio long reads, and the resultant contigs were scaffolded with Hi-C data. The scaffolded sequences obtained were further refined based on assembly results of alternate de novo assemblies and Hi-C contact maps by resolving identified inconsistencies. The final assemblies achieved N50 lengths of 149 Mb and 137 Mb for cynomolgus macaque and common marmoset, respectively, and the numbers of scaffolds longer than 10Mb are equal to their chromosome numbers. The high fidelity of our assembly is ascertained by concordance to the BAC-end read pairs observed for common marmoset, as well as a high resemblance of their karyotypic organization. Our assembly of cynomolgus macaque outperformed all the available assemblies of this species in terms of contiguity. The chromosome-scale genome assemblies produced in this study are valuable resources for non-human primate models and provide an important baseline in human biomedical research.

genomics↗

bioRxiv2020

Chromatin Fiber Folding Represses Transcription and Loop Extrusion in Quiescent Cells

Determining the conformation of chromatin in cells at the nucleosome level and its relationship to cellular processes has been a central challenge in biology. We show that in quiescent yeast, widespread transcriptional repression coincides with the local compaction of chromatin fibers into structures that are less condensed and more heteromorphic than canonical 30-nanometer forms. Acetylation or substitution of H4 tail residues decompacts fibers and leads to global transcriptional de-repression. Fiber decompaction also increases the rate of loop extrusion by condensin. These findings establish a role for H4 tail-dependent local chromatin fiber folding in regulating transcription and loop extrusion in cells. They also demonstrate the physiological relevance of canonical chromatin fiber folding mechanisms even in the absence of regular 30-nanometer structures.

molecular biology↗

bioRxiv2018

SWI/SNF coordinates transcriptional activation through Rpd3-mediated histone hypoacetylation during quiescence entry

Whether or not a cell chooses to divide is a tightly regulated and extremely important decision. Cells from yeast to human are able to reversibly exit the cell cycle in response to environmental changes such as nutritional changes or removal of growth cues to become quiescent. An inappropriate response to environmental cues can result in overproliferation which can lead to cancer, or a failure to proliferate which can result in developmental defects, premature aging and defects in wound healing. While many of the cell signaling pathways involved in regulating cellular quiescence have been identified, how these pathways translate their messages into transcriptional outputs is not well characterized. We previously showed that the histone deacetylase Rpd3 mediates global histone deacetylation and transcription repression upon quiescence entry. How the activation of quiescence-specific genes occurs in the midst of this transcriptionally repressive environment is not well understood. We show that the SWI/SNF chromatin remodeling complex activates quiescence specific genes to promote entry into quiescence. We additionally show that SWI/SNF binding early during quiescence entry is important for facilitating localization of the transcriptional activator Gis1, as well as histone H4 hypoacetylation in coding regions later on. The increase in H4 acetylation that we observe at Snf2-regulated genes upon Snf2 depletion corresponds to a decrease in promoter-bound Rpd3, suggesting that Snf2 remodels chromatin not only to facilitate activator binding, but also the binding of Rpd3. These observations provide mechanistic insight as to how quiescence-specific genes can be activated in the face of global deacetylation and transcription repression.

molecular biology↗

bioRxiv2018

Condensin-dependent chromatin condensation represses transcription globally during quiescence

Quiescence is a stress-resistant state in which cells reversibly exit the mitotic cell cycle and suspend most cellular processes. Quiescence is essential for stem cell maintenance and its misregulation is implicated in tumor formation. One of the conserved hallmarks of quiescent cells, from Saccharomyces cerevisiae to humans, is highly condensed chromatin. Here, we use Micro-C XL to map chromatin contacts at single-nucleosome resolution genome-wide to elucidate mechanisms and functions of condensed chromatin in quiescent S. cerevisiae cells. We describe previously uncharacterized chromatin domains on the order of 10-60 kilobases that in quiescent cells are formed by condensin-mediated chromatin loops. Conditional depletion of condensin prevents chromatin condensation during quiescence entry and leads to widespread transcriptional de-repression. We further demonstrate that condensin-dependent chromatin compaction is conserved in quiescent human fibroblasts. We propose that condensin-dependent condensation of chromatin represses transcription throughout the quiescent cell genome.

molecular biology↗

bioRxiv2018

Isw2 and Ino80 chromatin remodeling factors regulate chromatin, replication, and copy number at the yeast ribosomal DNA locus

In the budding yeast Saccharomyces cerevisiae, ribosomal RNA genes are encoded in a highly repetitive tandem array referred to as the ribosomal DNA (rDNA) locus. The yeast rDNA is the site of a diverse set of DNA-dependent processes, including transcription of ribosomal RNAs by RNA Polymerases I and III, transcription of non-coding RNAs by RNA Polymerase II, DNA replication initiation, replication fork blocking, and recombination-mediated regulation of rDNA repeat copy number. All of this takes place in the context of chromatin, but relatively little is known about the roles played by ATP-dependent chromatin remodeling factors at the yeast rDNA. In this work, we report that the Isw2 and Ino80 chromatin remodeling factors are targeted to this highly repetitive locus. We characterize for the first time their function in modifying local chromatin structure, finding that loss of these factors affects the occupancy of nucleosomes in the 35S ribosomal RNA gene and the positioning of nucleosomes flanking the ribosomal origin of replication. In addition, we report that Isw2 and Ino80 promote efficient firing of the ribosomal origin of replication and facilitate the regulated increase of rDNA repeat copy number. This work significantly expands our understanding of the importance of ATP-dependent chromatin remodeling for rDNA biology.\n\nAuthor SummaryTo satisfy high cellular demand for ribosomes, genomes contain many copies of the genes encoding the RNA components of ribosomes. In the budding yeast Saccharomyces cerevisiae, these ribosomal RNA genes are located in the \"ribosomal DNA locus\", a highly repetitive array that contains approximately 150 copies of the same unit, in contrast to the single copies that suffice for most genes. This repetitive quality creates unique regulatory needs. Chromatin structure, the packaging and organization of DNA, is a critical determinant of DNA-dependent processes throughout the genome. ATP-dependent chromatin remodeling factors are important regulators of chromatin structure, and yet relatively little is known about how members of this class of protein affect DNA organization or behavior at the rDNA. In this work, we show that the Isw2 and Ino80 chromatin remodeling factors regulate two features of chromatin structure at the rDNA, the occupancy and the positioning of nucleosomes. In addition, we find that these factors regulate two critical processes that function uniquely at this locus: DNA replication originating from within the rDNA array, and the regulated increase of rDNA repeat copy number.

molecular biology↗