Biology subjects

Yamamoto, T.

Publications and source records attributed to Yamamoto, T..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Pythia: Non-random DNA repair allows predictable CRISPR/Cas9 integration and gene editing

CRISPR-based genome engineering holds enormous promise for basic science and therapeutic applications. Integrating and editing DNA sequences is still challenging in many cellular contexts, largely due to insufficient control of the repair process. We find that repair at the genome-cargo interface is predictable by deep-learning models and adheres to sequence context specific rules. Based on in silico predictions, we devised a strategy of triplet base-pair repeat repair arms that correspond to microhomologies at double-strand breaks (trimologies), which facilitated integration of large cargo (>2 kb) and protected the targeted locus and transgene from excessive damage. Successful integrations occurred in >30 loci in human cells and in in vivo models. Germline transmissible transgene integration in Xenopus, and endogenous tagging of tubulin in adult mice brains demonstrated integration during early embryonic cleavage and in non-dividing differentiated cells. Further, optimal repair arms for single- or double nucleotide edits were predictable, and facilitated small edits in vitro and in vivo using oligonucleotide templates. We provide a design-tool (Pythia, pythia-editing.org) to optimize custom integration, tagging or editing strategies. Pythia will facilitate genomic integration and editing for experimental and therapeutic purposes for a wider range of target cell types and applications.

molecular biology↗

bioRxiv2024

GPRC6A as a novel kokumi receptor responsible for enhanced taste preferences by ornithine

The concept of "kokumi," which refers to an enhanced and more delicious flavor of food, has recently generated considerable interest in food science. However, kokumi has not been well studied in gustatory physiology, and the underlying neuroscientific mechanisms remain largely unexplored. Our previous research demonstrated that ornithine (L-ornithine), which is abundant in shijimi clams, enhanced taste preferences in mice. The present study aimed to build on these findings and investigate the mechanisms responsible for kokumi in rats. In two-bottle preference tests, the addition of ornithine, at a low concentration that did not increase the favorability of this substance alone, enhanced the animals preferences for umami, sweet, fatty, salty, and bitter solutions, with the intake of monosodium glutamate showing the most significant increase. Additionally, a mixture of umami and ornithine synergistically induced significant responses in the chorda tympani nerve, which transmits taste information to the brain from the anterior part of the tongue. The observed preference enhancement and increase in taste-nerve response were abolished by antagonists of the G-protein-coupled receptor family C group 6 subtype A (GPRC6A). Furthermore, immunohistochemical analysis indicated that GPRC6A was expressed in a subset of type II taste cells in rat fungiform papillae. These results provide new insights into flavor-enhancement mechanisms, confirming that ornithine is a kokumi substance and GPRC6A is a novel kokumi receptor.

neuroscience↗

bioRxiv2024

Comparative analysis of trans-chromosomic rodent models reveals improved somatic hypermutation and class-switch recombination in rats

Humanized rodent models, especially humanization of genetic/genomic components involved in immunity have significantly advanced our understanding of human immune system. Here, we utilized trans-chromosomic (Tc) technology to generate a TC-mAb rat model that stably harbors a mouse artificial chromosome carrying full-length human immunoglobulin (Ig) heavy and kappa light chain genes (IGHK-NAC) in a rat Ig knockout background. In contrast with TC-mAb mice, serum human IgG concentration was found higher than IgM. Number of lymphocytes was recovered, and B cell population in the spleen was normal. Remarkably, repertoire analysis revealed similarities between the model and human PBMCs; somatic hypermutation and class-switch recombination also more closely resembled humans. Furthermore, immunization resulted in generation of antigen-specific human antibodies. Collectively, our strategy to generate both rat and mouse models through introduction of the identical IGHK-NAC offers unprecedented opportunities to comprehensively evaluate genomic regulation and its outcomes associated with genomic sequences and host-derived protein factors.

bioengineering↗

bioRxiv2024

Post-transcriptional regulation by the CCR4-NOT deadenylase complex maintains redox homeostasis in insulin biosynthesis in mouse pancreatic β cells

Pancreatic {beta} cells synthesize insulin to maintain glucose homeostasis. In diabetes, elevated blood glucose and insulin resistance compel {beta} cells to produce insulin, and hence {beta} cells are vulnerable to oxidative stress by glucotoxicity. In insulin biosynthesis, the conversion of proinsulin to insulin is initiated by forming disulfide bonds in proinsulin for oxidative protein folding. Insulin content and insulin secretion in {beta} cells are decreased by deletion of CNOT7, a catalytic subunit of the CCR4-NOT complex, and accompanied by increased proinsulin, implying the impaired conversion of proinsulin to insulin. We found that PRDX4 essential for disulfide bond formation in proinsulin, is reduced in Cnot7-KO {beta} cells. Moreover, protein expression of CNOT8, a paralog of CNOT7, is increased in Cnot7-KO {beta} cells and binds to Prdx4 mRNA via MSI2. Here, we demonstrate the post-transcriptional regulation of Prdx4 mRNA by the CCR4-NOT complex to maintain oxidative-reductive homeostasis in insulin biosynthesis. HIGHLIGHTSO_LIInsulin content and secretion are decreased in Cnot7-KO pancreatic {beta} cells. C_LIO_LIPRDX4, needed for proinsulin folding, is decreased in Cnot7-KO pancreatic islets. C_LIO_LIThe conversion of proinsulin to insulin is impaired in Cnot7-KO {beta} cells. C_LIO_LICNOT8, but not CNOT7, interacts with MSI2 that binds to Prdx4 mRNA C_LI

physiology↗

bioRxiv2024

Task-guided Generative Adversarial Networks for Synthesizing and Augmenting Structural Connectivity Matrices for Connectivity-Based Prediction

Recent machine learning techniques have improved connectome-based predictions by modelling complex dependencies between brain connectivity and cognitive traits. However, they typically require large datasets that are costly and time-consuming to collect. To address this, we propose Task-guided GAN II, a novel data augmentation method that uses generative adversarial networks (GANs) to expand sample sizes in connectome-based prediction tasks. Our method incorporates a task-guided branch within the Wasserstein GAN framework, specifically designed to synthesize structural connectivity matrices and improve prediction accuracy by capturing task-relevant features. We evaluated Task-guided GAN II on the prediction of fluid intelligence using the NIMH Health Research Volunteer Dataset. Results showed that data augmentation improved prediction accuracy. To further assess whether augmentation can substitute for increasing real-world sample sizes, we conducted additional validation using the HCP WU-Minn S1200 dataset. Task-guided GAN II improved prediction performance with limited real data, with gains of up to twofold augmentation observed. However, excessive augmentation did not result in further improvements, suggesting that augmentation complements, but does not fully replace, real data augmentation. These results suggest that Task-guided GAN II is a promising tool for harnessing small datasets in human connectomics research, improving predictive modelling where large-scale data collection is impractical.

neuroscience↗

bioRxiv2023

Transcription-coupled changes in higher-order genomic structure and transcription hub viscosity prolong enhancer-promoter connectivity

The orchestration of our genes heavily relies on a coordinated communication between enhancers and promoters, yet how this dynamic interplay remains elusive while transcription is active. Here, we investigated enhancer-promoter (E-P) interactions and relative to transcriptional bursting in mouse embryonic stem cells using sequential DNA/RNA/immunofluorescence (IF)-FISH analyses and computational simulations. Our data reveal that the active state of specific genes is characterized by higher-order genomic structures and local condensates of transcriptional regulatory factors, leading to an elevation in local viscosity that highly stabilizes the duration of E-P interactions. Our study underscores the pivotal role of viscosity in transcriptional dynamics and paves the way for a more nuanced understanding of gene-specific regulatory mechanisms.

molecular biology↗

bioRxiv2023

Role of the medial agranular cortex in unilateral spatial neglect

Unilateral spatial neglect (USN) results from impaired attentional networks and can affect various sensory modalities, such as visual and somatosensory. The rodent medial agranular cortex (AGm), located in the medial part of the forebrain from rostral to caudal direction, is considered a region associated with spatial attention. The AGm selectively receives multisensory input with the rostral AGm receiving somatosensory input and caudal part receiving visual input. Our previous study showed slower recovery from neglect with anterior AGm lesion using the somatosensory neglect assessment. Conversely, the functional differences in spatial attention across the entire AGm locations (anterior, intermediate, and posterior parts) are unknown. Here, we investigated the relationship between the severity of neglect and various locations across the entire AGm in a mouse stroke model using a newly developed program-based analysis method that does not require human intervention. Among the various lesion positions, acute severity was higher with the lesion in the intermediate rostrocaudal position. On the other hand, the recovery from USN-like behavior after this phase tended to be slower in cases with more rostral lesions in the AGm. Additionally, no motor paralysis was observed in any of the mice with lesions in each AGm. These results suggest that the intermediate rostrocaudal position of the AGm may significantly influence selection of the direction, regardless of the areas to which it is connected. On the contrary, recovery from USN-like behavior may be dependent on the areas to which it is connected. HighlightsLesion of the rodent medial agranular cortex (AGm) results in unilateral spatial neglect (USN). In the acute phase, the severity was higher with lesions in the intermediate AGm position. Recovery from somatosensory USN tended to be slower with rostral AGm lesions. Recovery from USN may depend on sensory modalities associated with the connected areas. Our results revealed location-dependent differences in attentional functions within the AGm.

animal behavior and cognition↗

bioRxiv2023

Sclerostin modulates the degree of mineralization and the stiffness profile of the fibrocartilaginous enthesis for mechanical tissue integrity

Fibrocartilaginous entheses consist of four graded tissue layers including tendon, the unmineralized and mineralized fibrocartilage, and subchondral bone with varying degrees of stiffness. Here we examined the functional role of sclerostin that is expressed in mature mineralized fibrochondrocytes. Following rapid mineralization of the unmineralized fibrocartilage and parallel replacement of epiphyseal hyaline cartilage by bone, the unmineralized fibrocartilage re-expanded after a decline in alkaline phosphatase activity at the mineralization front. Sclerostin was co-expressed with osteocalcin in the bottom of the mineralized fibrocartilage adjacent to subchondral bone. In Scx deficient mice with less mechanical loading due to defects of the Achilles tendon, the number of sclerostin+ fibrochondrocytes was significantly reduced in the defective enthesis where chondrocyte maturation was markedly impaired in both fibrocartilage and hyaline cartilage. Loss of the Sost gene, coding for sclerostin, caused increased mineral density in the mineralized zones of the fibrocartilaginous enthesis. Atomic force microscopy analysis revealed the higher stiffness of fibrocartilage. These lines of evidence suggest that sclerostin in mature mineralized fibrochondrocytes acts as a modulator for mechanical tissue integrity of the fibrocartilaginous enthesis.

developmental biology↗

bioRxiv2023

Assembly of continuous high-resolution draft genome sequence of Hemicentrotus pulcherrimus using long-read sequencing

The update of the draft genome assembly of sea urchin, Hemicentrotus pulcherrimus, which is widely studied in East Asia as a model organism of early development, was performed using Oxford nanopore long-read sequencing. The updated assembly provided [~]600 Mb genome sequences divided into 2,163 contigs with N50 = 516 kb. BUSCO completeness score and transcriptome model mapping ratio (TMMR) of the present assembly were obtained as 96.5 % and 77.8 % respectively. These results were more continuous with higher resolution than those by the previous version of H. pulcerrimus draft genome, HpulGenome_v1, where the number of scaffolds = 16,251 with a total of [~]100 Mb gaps, N50 = 143 kb, BUSCO completeness score = 86.1 %, and TMMR = 40.61. The obtained genome contained 36,055 gene models that were consistent with those in other echinoderms. Additionally, two tandem repeat sequences of early histone gene locus containing 47 copies and 34 copies of all histone genes, and 185 of the homologous sequences of the interspecifically conserved region of the Ars insulator, ArsInsC, were obtained. These results provide further advance for genome-wide research of development, gene regulation, and intranuclear structural dynamics of multicellular organisms using Hemicentrotus pulcherrimus.

genomics↗

bioRxiv2023

UGGT1/2-mediated reglucosylation of N-glycan competes with ER-associated degradation of unstable and misfolded glycoproteins

How the fate (folding versus degradation) of glycoproteins is determined in the endoplasmic reticulum (ER) is an intriguing question. Monoglucosylated glycoproteins are recognized by lectin chaperones to facilitate their folding, whereas glycoproteins exposing well-trimmed mannoses are subjected to glycoprotein ER-associated degradation (gpERAD); we have elucidated how mannoses are sequentially trimmed by EDEM family members (George et al., 2020, 2021 eLife). Although reglucosylation by UGGT was previously reported to have no effect on substrate degradation, here we directly tested this notion using cells with genetically disrupted UGGT1/2. Strikingly, the results showed that UGGT1 delayed the degradation of misfolded substrates and unstable glycoproteins including ATF6. An experiment with a point mutant of UGGT1 indicated that the glucosylation activity of UGGT1 was required for the inhibition of early glycoprotein degradation. These and overexpression-based competition experiments suggested that the fate of glycoproteins is determined by a tug-of-war between structure formation by UGGT1 and degradation by EDEMs. We further demonstrated the physiological importance of UGGT1, since ATF6 cannot function properly without UGGT1. Thus, our work strongly suggests that UGGT1 is a central factor in ER protein quality control via regulation of both glycoprotein folding and degradation.

cell biology↗

bioRxiv2023

NSUN5 is essential for proper Hippo signaling in mouse preimplantation development

AbstractNOL1/NOP2/Sun domain family, member 5 (NSUN5) is an enzyme belonging to the 5-methylcytosine (m5C) writer family that modifies rRNA and mRNA. By re-analyzing published RNA-sequencing data, we found that Nsun5 expression was increased at the 2-cell stage during mouse preimplantation development. It has been reported that m5C regulates the stability of rRNA and mRNA. Therefore, NSUN5 may affect the expression of genes required for the development and differentiation of early mouse embryos via its role in modifying RNA. The Hippo signaling pathway has been identified as a critical regulator of the segregation of inner cell mass and trophectoderm lineages during mouse embryogenesis. According to these findings, we hypothesized that NSUN5 controls cell differentiation by regulating the expression of components of the Hippo signaling pathway in mouse early embryos. Using Nsun5-specific small interfering RNAs, we efficiently knocked down Nsun5 expression in mouse preimplantation embryos, resulting in impairments in early development, including reduced blastocyst formation, smaller size, and impaired hatching from the zona pellucida. Nsun5 depletion also led to decreased cell numbers, suggesting impaired cell proliferation. Furthermore, Nsun5 knockdown embryos exhibited reduced yes-associated protein 1 (YAP1) nuclear translocation during the morula stage, potentially affecting cell differentiation. The imbalance in pluripotent and trophectoderm lineages was evident with a significant reduction in the ratio of CDX2-positive cells to OCT4-positive cells. Nsun5 depletion impacted the Hippo signaling pathway, increasing the expression of the key genes Lats1 and Lats2 during the morula stage. Our findings underscore the essential role of Nsun5 in early embryonic development by affecting cell proliferation, YAP1 nuclear translocation, and the Hippo pathway. Understanding the roles of Nsun5 in mouse early embryos will provide insights into complex regulatory networks in development, with implications for reproductive medicine and fertility research.

developmental biology↗

bioRxiv2023

Epithelial cell chirality emerges through the dynamic concentric pattern of actomyosin cytoskeleton

The chirality of tissues and organs is essential for their proper function and development. Tissue-level chirality derives from the chirality of individual cells that comprise the tissue, and cellular chirality is considered to emerge through the organization of chiral molecules within the cell. However, the principle of how molecular chirality leads to cellular chirality remains unresolved. To address this fundamental question, we experimentally studied the chiral behaviors of isolated epithelial cells derived from a carcinoma line and developed a theoretical understanding of how their behaviors arise from molecular-level chirality. We first found that the nucleus rotates and the cytoplasm circulates robustly in a clockwise direction. During the rotation, actin and myosin IIA are organized into stress fibers with a vortex-like chiral orientation at the ventral side of the cell periphery, simultaneously forming thin filaments with a concentric orientation at the dorsal level of the cell. Surprisingly, we found that the intracellular rotation is driven by the concentric pattern of actomyosin filaments on the dorsal surface of the cell, not by the vortex-like chiral stress fibers. To elucidate how the concentric actomyosin filaments induce chiral rotation, we analyzed a theoretical model developed based on the theory of active chiral fluid, and revealed that the observed cell-scale unidirectional rotation is driven by the molecular-scale chirality of actomyosin filaments even in the absence of cell-scale chiral orientational order. Our study thus provides novel mechanistic insights into how the molecular chirality is organized into the cellular chirality and an important step towards understanding left-right symmetry breaking in tissues and organs.

cell biology↗

bioRxiv2023

Neutralizing antibody responses and cellular responses against severe acute respiratory syndrome coronavirus 2 omicron subvariant BA.5 after an mRNA severe acute respiratory syndrome coronavirus 2 vaccine dose in kidney transplant recipients

We examined the anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein IgG antibody and neutralizing antibody titers and cellular immunity in 73 uninfected recipients and 17 uninfected healthy controls who received three doses of a coronavirus 2019 mRNA vaccine. Neutralizing antibody titers were evaluated using GFP-carrying recombinant SARS-CoV-2 with spike protein of B.1.1, omicron BA.1, or BA.5. For cellular immunity, peripheral blood mononuclear cells were stimulated with peptides corresponding to spike protein antigens of B.1.1, BA.1, and BA.5; spike-specific CD4/CD8 memory T cells were evaluated using intracellular cytokine staining. The median IgG antibody titers were 7.8 AU/mL in recipients and 143.0 AU/mL in healthy controls (p < 0.0001). Neutralizing antibody titers against all three viral variants were significantly lower in recipients (p < 0.0001). The number of spike-specific CD8 + memory T cells significantly decreased in recipients (p < 0.0001). Twenty recipients and seven healthy controls additionally received a bivalent omicron-containing booster vaccine, and IgG antibody and neutralizing antibody titers increased in both groups; however, the increase was significantly lower in recipients. Recipients did not gain sufficient immunity with a third dose of vaccine, suggesting a need to explore methods other than vaccines.

immunology↗

bioRxiv2023

The reciprocal regulation between mitochondrial-associated membranes and Notch signaling in skeletal muscle atrophy

Skeletal muscle atrophy and the inhibition of muscle regeneration are known to occur as a natural consequence of aging, yet the underlying mechanisms that lead to these processes in atrophic myofibers remain largely unclear. Our research has revealed that the maintenance of proper mitochondrial-associated endoplasmic reticulum membranes (MAM) is vital for preventing skeletal muscle atrophy in microgravity environments. We discovered that the deletion of the mitochondrial fusion protein Mitofusin2 (MFN2), which serves as a tether for MAM, in human iPS cells or the reduction of MAM in differentiated myotubes caused by microgravity interfered with myogenic differentiation process and an increased susceptibility to muscle atrophy, as well as the activation of the Notch signaling pathway. The atrophic phenotype of differentiated myotubes in microgravity and the regenerative capacity of Mfn2-deficient muscle stem cells in dystrophic mice were both ameliorated by treatment with the gamma-secretase inhibitor DAPT. Our findings demonstrate how the orchestration of mitochondrial morphology in differentiated myotubes and regenerating muscle stem cells plays a crucial role in regulating Notch signaling through the interaction of MAM. digestThis study investigated the link between Mfn2 and Notch signaling in skeletal muscle atrophy. We used a microgravity system to induce muscle atrophy and found that the loss of Mfn2 leads to decreased numbers of MAM and activation of Notch signaling and that treating MFN2-deficient human iPS cells with a gamma-secretase inhibitor DAPT improved their mitochondrial morphology and function. Additionally, Mfn2-deficient muscle stem cells in mice have a lower capacity to regenerate dystrophic muscles and DAPT treatment improves the regeneration of these cells. The study suggests that targeting the Notch signaling pathway with a gamma-secretase inhibitor could be a therapeutic option for skeletal muscle atrophy caused by defects in Mfn2.

developmental biology↗

bioRxiv2023

The Benchtop mesoSPIM: a next-generation open-source light-sheet microscope for large cleared samples

In 2015, we launched the mesoSPIM initiative (www.mesospim.org), an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of light-sheet microscopy. Here, we introduce the next-generation mesoSPIM ("Benchtop") with significantly increased field of view, improved resolution, higher throughput, more affordable cost and simpler assembly compared to the original version. We developed a new method for testing objectives, enabling us to select detection objectives optimal for light-sheet imaging with large-sensor sCMOS cameras. The new mesoSPIM achieves high spatial resolution (1.5 {micro}m laterally, 3.3 {micro}m axially) across the entire field of view, a magnification up to 20x, and supports sample sizes ranging from sub-mm up to several centimetres, while being compatible with multiple clearing techniques. The new microscope serves a broad range of applications in neuroscience, developmental biology, and even physics.

neuroscience↗

bioRxiv2023

Cognitive Dynamics Estimation: A whole-brain spatial regression paradigm for extracting the temporal dynamics of cognitive processes

Functional MRI (fMRI) has been instrumental in understanding how cognitive processes are spatially mapped in the brain, yielding insights about brain regions and functions. However, in case the orthogonality of behavioral or stimulus timing is not guaranteed, the estimated brain maps fail to dissociate each cognitive process, and the resultant maps become unstable. Also, the brain mapping exercise can not provide temporal information on the cognitive process. Here we propose a qualitatively different approach to fMRI analysis, named Cognitive Dynamics Estimation (CDE), that estimates how multiple cognitive processes change over time even when behavior or stimulus logs are unavailable. This method transposes the conventional brain mapping; the brain activity pattern at each time point is subject to regression analysis with data-driven maps of cognitive processes as regressors, resulting in the time series of cognitive processes. The estimated time series captured the fluctuation of intensity and timing of cognitive processes on a trial-by-trial basis, which conventional analysis could not capture. Notably, the estimated time series predicted participants cognitive ability to perform each psychological task. As an addition to our fMRI analytic toolkit, these results suggest the potential for CDE to elucidate underexplored cognitive phenomena, especially in the temporal domain. HighlightsWe propose a novel fMRI analysis that are equivalently effective to the brain mapping approach The temporal dynamics of multiple cognitive processes were captured on a trial-by-trial basis. This analysis can be applied data-driven even when the researchers do not have any hypothesis about what kind of cognitive processes are recruited in the task. This analysis can be applied even when the cognitive processes are strongly correlated in the temporal domain. The estimated temporal dynamics well predicted individuals cognitive ability required for each task.

neuroscience↗

bioRxiv2023

Inhibition of OCT4 Binding at the MYCN Locus Induces Neuroblastoma Cell Death Accompanied by Downregulation of Transcripts with High-Open Reading Frame Dominance

Amplification of MYCN is observed in high-risk neuroblastomas (NBs) and is associated with a poor prognosis. MYCN expression is directly regulated by multiple transcription factors, including OCT4, MYCN, CTCF, and p53 in NB. Our previous study showed that inhibition of p53 binding at the MYCN locus induces NB cell death. However, it remains unclear whether other transcription factors contribute to NB cell survival. In this study, we revealed that the inhibition of OCT4 binding at the MYCN locus, a critical site for the human-specific OCT4-MYCN positive feedback loop, induces caspase-2-mediated cell death in MYCN-amplified NB. We used the CRISPR/deactivated Cas9 (dCas9) technology to specifically inhibit transcription factors from binding to the MYCN locus in the MYCN-amplified NB cell lines CHP134 and IMR32. In both cell lines, the inhibition of OCT4 binding at the MYCN locus reduced MYCN activity. Differentially downregulated transcripts were associated with high-open reading frame (ORF) dominance score, which is associated with the translation efficiency of transcripts. These transcripts were enriched in splicing factors, including MYCN-target genes such as HNRNPA1 and PTBP1. Furthermore, transcripts with high-ORF dominance were significantly associated with genes whose high expression is associated with a poor prognosis of NB. In conclusion, the inhibition of OCT4 binding at the MYCN locus resulted in reduced MYCN activity, which in turn led to the downregulation of high-ORF dominance transcripts and subsequently induced caspase-2-mediated cell death in MYCN-amplified NB cells. Therefore, disruption of the human-specific OCT4-MYCN positive feedback loop may serve as an effective therapeutic strategy for MYCN-amplified NB. Contribution to the fieldNeuroblastoma (NB) is a childhood tumor. Amplification of MYCN is frequently observed in high-risk NBs and is linked to a poor prognosis. Multiple transcription factors, including OCT4, MYCN, CTCF, and p53, regulate MYCN expression by binding to the MYCN locus. This study investigated the contribution of these transcription factors in NB cell survival. We used CRISPR/deactivated Cas9 (dCas9) technology to specifically inhibit transcription factors from binding to the MYCN locus in MYCN-amplified NB cell lines. We found that the inhibition of OCT4 binding at the MYCN locus, a critical site for the human-specific OCT4-MYCN positive feedback loop, reduces MYCN activity and induces NB cell death. A detailed investigation of the molecular mechanisms of cell death revealed that the downregulated transcripts after suppressed MYCN activity were associated with high-open reading frame (ORF) dominance scores, which are associated with translation efficiency of transcripts. These transcripts were enriched in splicing factors, including MYCN-target genes such as HNRNPA1 and PTBP1. Reduced expression of these splicing factors altered the PKM mRNA splicing accompanied by the induction of p53-caspase-2-MDM2-mediated cell death. These findings suggest that disrupting the human-specific OCT4-MYCN positive feedback loop may serve as a promising therapeutic strategy for MYCN-amplified NB.

cancer biology↗

bioRxiv2023

MYC is essential for induction of major ZGA and subsequent preimplantation development

In mouse preimplantation development, zygotic genome activation (ZGA), which synthesizes new transcripts from the embryos, begins in the S phase of the one-cell stage, with major ZGA occurring especially at the late two-cell stage. Myc is a transcription factor expressed in parallel with ZGA, but its direct association with the major ZGA has not been clarified. In this study, we found that developmental arrest occurs at the two-cell stage when mouse embryos were treated with antisense oligos targeting Myc or inhibitors specific for MYC from the one-cell stage. In order to identify when MYC inhibition affected development, we applied time-limited inhibitor treatment, and found that inhibition of MYC at the two-cell, four-cell, and morula stages had no effect on preimplantation development, whereas treatment with the inhibitor at the early two-cell stage arrested development at the two-cell stage. Furthermore, transcriptome analysis revealed that when MYC function was inhibited, genes expressed in the major ZGA phase were suppressed. These results suggest that Myc is essential for the induction of major ZGA and its subsequent development. Revealing the function of Myc in preimplantation development is expected to contribute to advances in assisted reproductive technology.

developmental biology↗