Biology subjects

Asahina, M.

Publications and source records attributed to Asahina, M..

7 recordsLinked to original sources

bioRxiv2026

Osmotic-stress-inducible nuclear condensates restrict gene inducibility

Plants, as sessile organisms, have developed various mechanisms to respond to environmental stress conditions. The plant hormone abscisic acid (ABA) is necessary for the plant to adapt to osmotic stress conditions. However, the molecular mechanisms preceding ABA accumulation remain largely unknown. To isolate transcriptional complexes on the promoter region of NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3) encoding a rate-limiting enzyme in the ABA biosynthetic pathway in planta, we developed the insertional chromatin immunoprecipitation (iChIP) screen method. The identified ALBA proteins formed condensates through liquid-liquid phase separation (LLPS) in response to osmotic stress conditions. ALBA4 directly binds to stress-inducible genes, including NCED3, and suppresses their stress inducibility. Our results demonstrate how plants respond to osmotic stress at early timepoints before ABA biosynthesis through condensate formation as osmo-sensors.

plant biology↗

bioRxiv2025

The Binding Landscape of an Essential Nuclear Hormone Receptor in C. elegans

Transcription factors (TFs) control gene expression by binding to response elements but distinguishing functional binding sites from non-functional ones remains a major challenge in the field of transcriptional regulation. To probe transcription factor binding and function in a multicellular organism we characterized the binding and transcriptional landscape of NHR-25, a highly-conserved and essential transcription factor in C. elegans. Using CRISPR/Cas9, we tagged the essential nuclear hormone receptor, nhr-25, with GFP and FLAG at its endogenous locus in C. elegans and performed ChIP-seq. We found that NHR-25 binds with two distinct modes: proximal promoter binding and distal enhancer-type binding. Motif analysis of the ChIP-seq peaks showed that the NHR-25 binding motif depended on chromatin context and identified additional enriched motifs, suggesting other putative regulatory partners at composite response elements. By combining ChIP-seq with conditional RNAi knockdown, we characterized nhr-25s transcriptome and identified putative direct targets of NHR-25 binding. Through an 11-bp mutation of the summit of an identified NHR-25 occupied region, we demonstrated that a single response element was responsible for regulating the expression of multiple genes. Our findings provide a comprehensive view of an essential transcription factors functional binding landscape at native protein levels, highlighting the intricate nature of response element logic and demonstrating the critical role of a single regulatory element in regulating gene expression.

genomics↗

bioRxiv2023

Carotenoid metabolism negatively regulates auxin-mediated root growth

Root development is essential for plant survival. The lack of carotenoid biosynthesis in the phytoene desaturase 3 (pds3) mutant results in short primary roots (PR) and reduced lateral root (LR) formation. In this study, we show that short-term inhibition of PDS by fluridone suppressed PR growth in WT, but to a lesser extent in auxin mutants. Such an inhibition of PDS activity increased endogenous indole-3-acetic acid (IAA) levels, promoted auxin signaling, and partially complemented the PR growth of auxin deficient mutant, YUCCA 3 5 7 8 9 quadruple mutant (yucQ). The exogenous application of retinaldehyde (retinal), an apocarotenoid derived from {beta}-carotene, complemented the fluridone-induced suppression of root growth, as well as the short roots of the pds3 mutant. Retinal also partially complemented the auxin-induced suppression of root growth. These results suggest that retinal may play a role in regulating root growth by modulating endogenous auxin levels. One-sentence summaryThe short-term inhibition of carotenoid biosynthesis mediates the production of {beta}-carotene-derived apocarotenoid retinal which negatively regulates auxin levels and signaling to control root growth.

plant biology↗

bioRxiv2023

Uncovering the essential roles of human GCP 2 orthologs in Caenorhabditis elegans.

Human glutamate carboxypeptidase 2 (GCP2) from the M28B metalloprotease group is an important target for therapy in neurological disorders and an established tumor marker. However, its physiological functions remain unclear. To better understand general roles, we used the model organism Caenorhabditis elegans genetically manipulate its three existing orthologous genes and evaluate the impact on worm physiology. The results of gene knockout studies showed that C. elegans GCP2 orthologs affect the pharyngeal physiology, reproduction, and structural integrity of the organism. Promoter-driven GFP expression revealed distinct localization for each of the three gene paralogs, with gcp-2.1 being most abundant in muscles, intestine, and pharyngeal interneurons, gcp-2.2 restricted to the phasmid neurons, and gcp-2.3 located in the excretory cell. This study provides new insight into the unique phenotypic effects of GCP2 gene knockouts in C. elegans, and the specific tissue localizations. We believe that elucidation of particular roles in a non-mammalian organism can help to explain important questions linked to human GCP2 physiology and in extension to GCP2 involvement in pathophysiological processes.

biochemistry↗

bioRxiv2020

Cell-cell adhesion in plant grafting is facilitated by β-1,4-glucanases

Plant grafting is conducted for vegetative propagation in plants, whereby a piece of living tissue is attached to another tissue through establishment of cell-cell adhesion. Plant grafting has a long history in agriculture and has been applied to improve crop traits for thousands of years1. Plant grafting has mostly relied on the natural ability of a plant for wound healing. However, the compatibility of cell-cell adhesion typically limits graft combinations to closely related species2-4, and the mechanism by which cell-cell adhesion of injured tissues is established is largely unknown. Here, we show that a subclade of {beta}-1,4-glucanases secreted into the extracellular region facilitates cell-cell adhesion near the graft interface. Nicotiana shows a propensity for cell-cell adhesion with a diverse range of angiosperms, including vegetables, fruit trees, and monocots, in which cell wall reconstruction was promoted in a similar manner to conventional intrafamily grafting5-7. Using transcriptomic approaches, we identified a specific clade of {beta}-1,4-glucanases that is upregulated during grafting in successful graft combinations but not in incompatible grafts and precedes graft adhesion in inter- and intrafamily grafts. Grafting was facilitated with an overexpressor of the {beta}-1,4-glucanase and, using Nicotiana stem as an interscion, we produced tomato fruits on rootstocks from other plant families. Our results demonstrate that the mechanism of cell-cell adhesion is partly conserved in plants and is a potential target to enhance plant grafting techniques.

plant biology↗

bioRxiv2020

Genetic exploration of a nuclear receptor transcriptional regulatory complex

Metazoan transcriptional regulatory factors (TFs) bind to genomic response elements and assemble with co-regulators into transcriptional regulatory complexes (TRCs) whose composition, structure and activities are gene-, cell- and physiological-context specific. Each TRC is a "regulatory logic module," integrating incoming signaling information, which defines context and thereby recruits a distinct combination of co-regulators that together specify outgoing regulatory activity. Analyzing TRCs unique to every context is daunting, yet justified by their properties as self-contained regulatory modules. As proof-of-concept, we performed a forward genetic screen in C. elegans carrying a synthetic simple response element for nuclear receptor NHR-25 upstream of a fluorescent reporter gene. We isolated independent mutations in uba-2, a component of the sumoylation signaling machinery, and in lir-2, which we demonstrated to be a novel co-regulator, interacting with NHR-25 through LxxLL motifs and modulating target gene expression. Our studies establish that an unbiased genetic screen readily identifies both afferent and efferent components that specify TRC function, and suggest that screening natural response elements of interest could illuminate molecular mechanisms of both context-specificity and transcriptional regulation.

genetics↗

bioRxiv2019

Definition of alleles and altered regulatory motifs across Cas9-edited cell populations

BackgroundGenetic alteration of candidate response elements at their native chromosomal loci is the only valid determinant of their potential transcriptional regulatory activities. Targeted DNA cleavage by Cas9 coupled with cellular repair processes can produce arrays of alleles that can be defined by massively parallel sequencing by synthesis (SBS), presenting an opportunity to generate and survey edited cell populations that include informative alterations. Such editing efforts commonly rely on subclonal enrichment to isolate cells with preferred genotypic properties at target loci; short nucleotide adducts (indices/barcodes) allow PCR-amplified molecules from diverse sample sources to be pooled, sequenced, and demultiplexed to resolve source-specific content. Not widely available, however, are capabilities for barcoding thousands of clones, or for automated analysis of individual candidate regulatory loci PCR-amplified and sequenced from a genetically heterogeneous population--specifically, imputation of discrete genotype(s) by allele definition and abundance, and identification of altered regulatory factor binding motifs.\n\nResultsWe describe a panel of 192 8-nucleotide barcode primers compatible with Illumina(R) sequencing platforms, and the application of these barcodes to genotypic analysis of Cas9-edited clones. Permutations of the ninety-six i7 (read 1) and ninety-six i5 (read 2) barcodes allow unique labeling of up to 9,216 distinct samples. We created three independent Python scripts: SampleSheet.py automates construction of Illumina(R) Sample Sheets encoding up to 9,216 barcode:sample relationships; ImputedGenotypes.py defines alleles and imputes genotypes from demultiplexed fastq files; CollatedMotifs.py flags transcription factor recognition motif matches altered in alleles relative to a reference sequence.\n\nConclusionsCode-enabled definition of alleles and regulatory motifs in sequenced, demultiplexed amplicons facilitates evaluation of genetic diversity in up to 9,216 distinct samples. Here, we demonstrate the utility of three scripts in analysis of cell populations targeted by Cas9 for disruption of glucocorticoid receptor (GR) binding sites near FKBP5, a GR-regulated gene in the human adenocarcinoma cell line A549. SampleSheet.py, ImputedGenotypes.py, and CollatedMotifs.py operate independently and are broadly applicable beyond the case described here.

molecular biology↗