Biology subjects

Narita, T.

Publications and source records attributed to Narita, T..

9 recordsLinked to original sources

bioRxiv2025

The SPATA5-SPATA5L1-CINP-C1ORF109 complex is essential for cytoplasmic pre-60S ribosome maturation

The ribosome is a universally conserved and essential protein complex, but its biogenesis in mammals is more complex than in single-celled eukaryotes. To explore this added complexity, we conducted a protein-protein interaction screen in human cells. This led to the identification of the eumetazoan-specific SPATA5-SPATA5L1-CINP-C1ORF109 (55LCC) complex as a key regulator of ribosome biogenesis. Structural analyses using cryo-EM and X-ray crystallography defined the architecture of 55LCC. Functional studies following acute depletion revealed that each component is essential for pre-60S maturation. Swapping endogenous 55LCC components with mutant versions pinpointed critical functional interactions and showed that SPATA5s ATPase activity is more important than SPATA5L1s. Our findings support that SPATA5 evolved from the solitary yeast ATPase Drg1 into the multiprotein 55LCC complex in metazoans. This work provides insights into the complexity of ribosome biogenesis and lays the foundation for deeper exploration of 55LCCs role in pre-60S maturation.

biochemistry↗

bioRxiv2024

Mammalian enhancers and GWASs act proximally and seldom skip active genes

Enhancers play a critical role in regulating transcription. Nearly 90% of human genetic variants identified in genome-wide association studies (GWAS) are located in distal regions, underscoring the importance of enhancers in human development, diseases, and traits. It is widely suggested that mammalian enhancers frequently skip active genes, and thus, linear proximity is a poor predictor of their targets. A key unresolved question is how often mammalian enhancers skip proximal active genes to specifically target distal genes. Genome-wide enhancer-promoter mapping shows that enhancers frequently bypass active genes, while ultra-deep locus-specific analyses reveal extensive multi-way interactions between enhancers and promoters, forming nested microcompartments. The functional significance of these seemingly contrasting phenomena remains unclear. Here, we compared hundreds of enhancer-target gene pairs identified using enhancer-promoter chromatin contact maps, enhancer-promoter RNA interaction data, and genome-scale CRISPR interference (CRISPRi) perturbations. Our findings reveal limited overlap between active gene-skipping enhancer-gene pairs identified through physical interaction mapping and CRISPRi. Additionally, promoters involved in multi-way enhancer interactions are not co-regulated by shared coactivators. Notably, gene-skipping and non-skipping enhancers identified via CRISPRi differ fundamentally in chromatin features, gene activation strength, false discovery rates, target gene distance, coactivator requirements, and cell-type specificity of target genes. These results suggest that gene-skipping enhancer-promoter interactions observed in chromatin and RNA-based analyses do not reliably predict functional enhancer-gene relationships. We propose that linear enhancer-promoter proximity and coactivator dependency offer a simple, scalable, and cost-effective method for genome-wide prediction of enhancer and GWAS targets, with accuracy comparable to state-of-the-art experimental techniques. While enhancers can skip active genes, such deliberate skipping appears to be the exception rather than the rule.

genomics↗

bioRxiv2024

A unified model of gene expression control by cohesin and CTCF

Cohesin and CTCF fold vertebrate genomes into loops and topologically associating domains (TADs). The genome folding by cohesin and CTCF is considered crucial for enhancer-promoter communication and gene regulation. However, the extent of cohesin and CTCF-dependent looping in global enhancer function and their potential non-architectural roles in gene activation have remained unclear. Here, we demonstrate that the acute removal of cohesin or CTCF in mouse cells dysregulates hundreds of genes, albeit subtly. Among various possible enhancer types, cohesin almost exclusively facilitates gene activation by one enhancer type--the CBP/p300-dependent enhancers. Interestingly, CTCF plays a dual role, operating both with and without cohesin, and promoting gene activation both in an enhancer-dependent and independent manner. By anchoring cohesin loops, CTCF directs enhancers to specific target genes and prevents their mistargeting. Independently of cohesin and enhancers, CTCF acts as a transcriptional activator or repressor, depending on its precise binding position near promoters. Acting as a canonical transcription activator, CTCF directly activates hundreds of housekeeping genes, including those essential for mammalian cell proliferation. Mechanistically, promoter-bound CTCF controls DNA accessibility and RNA polymerase II recruitment. The transcriptional activator function of CTCF appears unique to vertebrates and is shared by its vertebrate-specific paralog, CTCFL, despite CTCFLs inability to anchor cohesin loops. These findings reveal the scope of cohesin and CTCF in gene regulation, delineate their shared and unique functions, define a specific class of enhancers using cohesin-mediated looping, and establish a crucial function of CTCF in gene regulation independent of its architectural role. The work reconciles conflicting views and provides a unified model for gene regulation by cohesin and CTCF.

genomics↗

bioRxiv2024

Modeling nascent transcription from chromatin landscape and structure

BackgroundDifferent cell types and their associated functionalities emerge from a single genomic sequence when certain regions are expressed while others remain silenced. Modeling gene expression and its potential malfunctioning in different cellular contexts is hence pivotal to understand both development and disease. ResultsWe present the Chromatin Landscape and Structure to Expression Regressor (CLASTER), an epigenetic-based deep neural network that can integrate different data modalities describing the chromatin landscape and its 3D structure. CLASTER effectively translates them into nascent transcription levels measured by EU-seq at a kilobasepair resolution. Our predictions reached a Pearson correlation with targets above r=0.86 at both bin and gene levels, without relying on DNA sequence nor explicitly extracted chromatin features. The model mostly used the information found within 10 kbp of the predicted locus, even when a wide genomic region of 1 Mbp was available. Explicit modeling of long-range interactions using multi-headed attention and high-resolution chromatin contact maps had little impact on model performance, despite the model correctly identifying elements in these inputs influencing nascent transcription. The trained model served then as a platform to predict the transcriptional impact of simulated epigenetic silencing perturbations. ConclusionsOur results point towards a rather local, integrative and combinatorial paradigm of gene regulation, where changes in the chromatin environment surrounding a gene shape its context-specific transcription. We conclude that the predominant locality and limitations of current machine learning approaches might emerge as a genuine signature of genomic organization, having broad implications for future modeling approaches.

bioinformatics↗

bioRxiv2023

A systems-scale, integrated view of the ubiquitylation site occupancy and dynamics

Ubiquitylation regulates virtually all proteins and biological processes in a cell. However, the global site-specific occupancy (stoichiometry) and turnover rate of ubiquitylation have never been quantified. Here, we present the first integrated picture of ubiquitylation site occupancy and half-life. Ubiquitylation occupancy spans four orders of magnitude, but the median ubiquitylation site occupancy is three orders of magnitude lower than that of phosphorylation. The occupancy, turnover rate, and the regulation of sites by proteasome inhibitors show strong interrelationships. These properties can discriminate signaling-relevant sites from the sites involved in proteasomal degradation. The sites strongly upregulated by proteasome inhibitors have a longer half-life, and the half-life increases with increasing protein length. Importantly, a previously unknown surveillance mechanism rapidly deubiquitylates all ubiquitin-specific E1 and E2 enzymes and protects them against bystander ubiquitylation accumulation. This work reveals general principles of ubiquitylation-dependent governance and offers conceptual insights into the dynamic regulation of the cell. HighlightsO_LIUbiquitylation site occupancy is 3 orders of magnitude lower than phosphorylation C_LIO_LIThe highest 80% and the lowest 20% occupancy sites have distinct properties C_LIO_LIHigh occupancy sites are concentrated in the cytoplasmic domains of SLC proteins C_LIO_LIA dedicated mechanism prevevents ubiquitylation accumulation in E1s and E2s C_LI

biochemistry↗

bioRxiv2023

A mechanism of melanogenesis mediated by E-cadherin downregulation and its involvement in solar lentigines

BackgroundIntensive studies have revealed pleiotropic melanocytic factors for age spot formation. In particular, dysfunctional keratinocyte differentiation is thought to be an upstream cause of age spot formation. Although keratinocyte differentiation is mediated by a cell-cell contact factor, E-cadherin, its involvement in age spots remains unknown. To find the origin of age spots and an integrated solution, we focused on E-cadherin. MethodsImmunofluorescent staining with cutaneous tissues and cultured cells was performed. Keratinocytes treated with siRNAs were cocultured with melanocytes. With the supernatants of the keratinocyte culture, secretion factors were identified using proteomic analysis. For the activity of melanogenesis and the ingredient screening, a quantitative PCR was performed. For the behavioral analysis of melanocytes, time-lapse imaging of melanocytes was done by confocal laser scanning microscopy. ResultsIn age spots, E-cadherin expression in the epidermis was downregulated, suggesting that E-cadherin is implicated in age spot formation. E-cadherin knockdown (E-cad-KD) keratinocytes not only promoted the secretion of melanocytic/inflammatory factors, but also increased melanogenesis by upregulating the expression of melanogenesis factors. Furthermore, live imaging showed E-cadherin downregulation detained melanocyte dynamics and accelerated melanin-uptake. Finally, we identified Rosa multiflora fruit extract as a solution for upregulating E-cadherin in keratinocytes. ConclusionOur findings showed that E-cadherin downregulation triggers various downstream melanocytic processes such as secretion of melanocytic factors and melanogenesis. Additionally, we showed that Rosa multiflora fruit extract upregulates E-cadherin expression in keratinocytes.

cell biology↗

bioRxiv2022

The logic of native enhancer-promoter compatibility and cell-type-specific gene expression variation

Cis-regulatory enhancers are essential for differential expression of developmental and housekeeping genes. However, the specificity of native mammalian enhancers and how it shapes cell-type-specific gene expression landscapes remain largely unknown. We show that endogenous enhancers are broadly compatible with the promoters of developmental and housekeeping genes. Broad enhancer compatibility affords retrofitting new regulatory capabilities to housekeeping genes that evolved before the advent of enhancers. This enables cell-type-specific tuning of ubiquitously expressed genes. Segregation between enhancer-dependent and -independent type regulation is blurred. Within the same cell type, a single promoter can be activated by enhancers and non-enhancer promoter-regulatory elements (PREs). It is the tunable and integrated strengths of enhancers and PREs that quantitatively shape gene expression landscapes, within and across cell types. Our findings have broad implications for understanding cell-type-specific quantitative gene expression variation, as well as the emergence and rewiring of gene regulatory networks in disease and organismal evolution.

genomics↗

bioRxiv2022

A unique H2B acetylation signature marks active enhancers and predicts their target genes

Chromatin features are widely used for genome-scale mapping of enhancers. However, discriminating active enhancers from other cis-regulatory elements, predicting enhancer strength, and identifying their target genes remains challenging. Here we establish histone H2B N-terminus multisite lysine acetylation (H2BNTac) as a genuine signature of active enhancers. H2BNTac prominently marks candidate active enhancers and their target promoters and discriminates them from ubiquitously active promoters. Two mechanisms afford the distinct H2BNTac specificity. (1) Unlike H3K27ac, H2BNTac is specifically catalyzed by CBP/p300. (2) H2A-H2B, but not H3-H4, are rapidly exchanged through transcription-induced nucleosome remodeling. H2BNTac-positive candidate enhancers show a high validation rate in orthogonal enhancer activity assays, and a vast majority of endogenously active enhancers are marked by H2BNTac and H3K27ac. Notably, H2BNTac intensity predicts enhancer strength and outperforms the current state-of-the-art models in predicting enhancer target genes. These findings have broad implications for generating fine-grained enhancer maps and modeling enhancer-dependent gene regulation.

genomics↗

bioRxiv2021

Myosin phosphatase target subunit 1 governs integrity of the embryonic gut epithelium to circumvent atresia development in medaka, Oryzias latipes

Congenital intestinal atresia (IA) is a birth defect characterised by the absence or closure of part of the intestine. Although genetic factors are implicated, mechanistic understanding has been hindered by the lack of suitable animal models. Here, we describe a medaka (Oryzias latipes) mutant, generated by N-ethyl-N-nitrosourea (ENU) mutagenesis, that develops IA during embryogenesis. Positional cloning identified a nonsense mutation in mypt1, encoding myosin phosphatase target subunit 1. Mutant embryos exhibited ectopic accumulation of F-actin and phosphorylated myosin regulatory light chain (Mrlc) in the intestinal epithelium, consistent with disrupted actomyosin regulation. These cytoskeletal abnormalities were accompanied by epithelial disorganisation without notable alterations in cell proliferation, motility, or apoptosis. Inhibition of myh11a, encoding smooth muscle (SM) myosin heavy chain, ameliorated the IA phenotype but Blebbistatin treatment completely rescued the defect, suggesting a non-contractile role prior to SM maturation. Together, these findings demonstrate that mypt1 loss disrupts intestinal morphogenesis through actomyosin dysregulation. Given the recent clinical identification of IA associated with MYPT1 mutations, this medaka model offers a valuable platform to investigate the developmental and molecular basis of MYPT1-associated IA in human.

developmental biology↗