Transcription-coupled changes in genomic region proximities during transcriptional bursting.

The orchestration of our genes heavily relies on coordinated communication between enhancers and promoters, yet the mechanisms behind this dynamic interplay during active transcription remain unclear. Here, we investigated enhancer-promoter (E-P) interactions in relation to transcriptional bursting in mouse embryonic stem cells using sequential DNA/RNA/immunofluorescence-fluorescence in situ hybridization analyses. Our data reveal that the active state of specific genes is characterized by specific proximities between different genomic regions and the accumulation of transcriptional regulatory factors.

Label-free ghost cytometry for manufacturing of cell therapy products.

Automation and quality control (QC) are critical in manufacturing safe and effective cell and gene therapy products. However, current QC methods, reliant on molecular staining, pose difficulty in in-line testing and can increase manufacturing costs. Here we demonstrate the potential of using label-free ghost cytometry (LF-GC), a machine learning-driven, multidimensional, high-content, and high-throughput flow cytometry approach, in various stages of the cell therapy manufacturing processes.

Image Analysis Protocol for DNA/RNA/Immunofluorescence (IF)-seqFISH Data.

Imaging-based spatial multi-omics technologies facilitate the analysis of higher-order genomic structures, gene transcription, and the localization of proteins and posttranslational modifications (PTMs) at the single-allele level, thereby enabling detailed observations of biological phenomena, including transcription machinery within cells and tissues. This chapter details the principles of such technologies, with a focus on DNA/RNA/immunofluorescence (IF) sequential fluorescence in situ hybridization (seqFISH). A comprehensive step-by-step protocol for image analysis is provided, covering image preprocessing, spot detection, and data visualization.

Inter-cellular mRNA Transfer Alters Human Pluripotent Stem Cell State.

Inter-cellular transmission of mRNA is being explored in mammalian species using immortal cell lines (1-3). Here, we uncover an inter-cellular mRNA transfer phenomenon that allows for the adaptation and reprogramming of human primed pluripotent stem cells (hPSCs). This process is induced by the direct cell contact-mediated coculture with mouse embryonic stem cells (mESCs) under the condition impermissible for human primed PSC culture.

The relationship between nanoscale genome organization and gene expression in mouse embryonic stem cells during pluripotency transition.

During early development, gene expression is tightly regulated. However, how genome organization controls gene expression during the transition from naïve embryonic stem cells to epiblast stem cells is still poorly understood. Using single-molecule microscopy approaches to reach nanoscale resolution, we show that genome remodeling affects gene transcription during pluripotency transition.

Precise immunofluorescence canceling for highly multiplexed imaging to capture specific cell states.

Cell states are regulated by the response of signaling pathways to receptor ligand-binding and intercellular interactions. High-resolution imaging has been attempted to explore the dynamics of these processes and, recently, multiplexed imaging has profiled cell states by achieving a comprehensive acquisition of spatial protein information from cells. However, the specificity of antibodies is still compromised when visualizing activated signals.

Organization of transcription and 3D genome as revealed by live-cell imaging.

Higher-order genomic structures play a critical role in regulating gene expression by influencing the spatial proximity of promoters and enhancers. Live-cell imaging studies have demonstrated that three-dimensional genome structures undergo dynamic changes over time. Transcription is also dynamic, with genes frequently switching between active and inactive states.

Fluorescent indicator displacement assay for the discovery of UGGAA repeat-targeted small molecules.

We report that a selective fluorescent indicator NBD-NCD for UGGAA repeats resulted in fluorescence quenching upon binding to RNA and recovered the fluorescence by displacing NBD-NCD with UGGAA repeat-targeted small molecules. The fluorescent indicator displacement assay using NBD-NCD can detect the interaction of small molecules with UGGAA repeats.

Construction and Evaluation of Zinc Finger Nucleases.

Zinc finger nucleases (ZFNs) are programmable nucleases that have contributed significantly to past genome-editing research. They are now utilized much less owing to the advent of transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats and CRISPR-associated protein system (CRISPR-Cas). These new methods allow for easier generation of reagents that target genomic sequences of interest and are less labor-intensive than ZFNs at targeting desired sequences.

STREAMING-tag system reveals spatiotemporal relationships between transcriptional regulatory factors and transcriptional activity.

Transcription is a dynamic process. To detect the dynamic relationship among protein clusters of RNA polymerase II and coactivators, gene loci, and transcriptional activity, we insert an MS2 repeat, a TetO repeat, and inteins with a selection marker just downstream of the transcription start site. By optimizing the individual elements, we develop the Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system.

STREAMING-Tag System: Technology to Enable Visualization of Transcriptional Activity and Subnuclear Localization of Specific Endogenous Genes.

The Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system enables imaging of nuclear localization as well as the transcription activity of a specific endogenous gene at sub-100-nm resolution in living cells. The use of this system combined with imaging of epigenome states enables a detailed analysis of the impact of epigenome status on transcriptional dynamics. In this chapter, we describe a method for quantifying distances between Nanog gene and clusters of cofactor BRD4 using the STREAMING-tag system in mouse embryonic stem cells.

Ribosomal protein L5 facilitates rDNA-bundled condensate and nucleolar assembly.

The nucleolus is the site of ribosome assembly and formed through liquid-liquid phase separation. Multiple ribosomal DNA (rDNA) arrays are bundled in the nucleolus, but the underlying mechanism and significance are unknown. In the present study, we performed high-content screening followed by image profiling with the wndchrm machine learning algorithm.

iPSC reprogramming-mediated aneuploidy correction in autosomal trisomy syndromes.

Trisomy 21, 18, and 13 are the major autosomal aneuploidy disorders in humans. They are mostly derived from chromosome non-disjunction in maternal meiosis, and the extra trisomic chromosome can cause several congenital malformations. Various genes on the trisomic chromosomes are intricately involved in the development of disease, and fundamental treatments have not yet been established.

Single-gene imaging links genome topology, promoter-enhancer communication and transcription control.

Transcription activation by distal enhancers is essential for cell-fate specification and maintenance of cellular identities. How long-range gene regulation is physically achieved, especially within complex regulatory landscapes of non-binary enhancer-promoter configurations, remains elusive. Recent nanoscopy advances have quantitatively linked promoter kinetics and ~100- to 200-nm-sized clusters of enhancer-associated regulatory factors (RFs) at important developmental genes.

Genome-wide kinetic properties of transcriptional bursting in mouse embryonic stem cells.

Transcriptional bursting is the stochastic activation and inactivation of promoters, contributing to cell-to-cell heterogeneity in gene expression. However, the mechanism underlying the regulation of transcriptional bursting kinetics (burst size and frequency) in mammalian cells remains elusive. In this study, we performed single-cell RNA sequencing to analyze the intrinsic noise and mRNA levels for elucidating the transcriptional bursting kinetics in mouse embryonic stem cells.

Insufficiency of ciliary cholesterol in hereditary Zellweger syndrome.

Primary cilia are antenna-like organelles on the surface of most mammalian cells that receive sonic hedgehog (Shh) signaling in embryogenesis and carcinogenesis. Cellular cholesterol functions as a direct activator of a seven-transmembrane oncoprotein called Smoothened (Smo) and thereby induces Smo accumulation on the ciliary membrane where it transduces the Shh signal. However, how cholesterol is supplied to the ciliary membrane remains unclear.

Pre-clinical characterisation of E2814, a high-affinity antibody targeting the microtubule-binding repeat domain of tau for passive immunotherapy in Alzheimer's disease.

Tau deposition in the brain is a pathological hallmark of many neurodegenerative disorders, including Alzheimer's disease (AD). During the course of these tauopathies, tau spreads throughout the brain via synaptically-connected pathways. Such propagation of pathology is thought to be mediated by tau species ("seeds") containing the microtubule binding region (MTBR) composed of either three repeat (3R) or four repeat (4R) isoforms.

Role of dynamic nuclear deformation on genomic architecture reorganization.

Higher-order genomic architecture varies according to cell type and changes dramatically during differentiation. One of the remarkable examples of spatial genomic reorganization is the rod photoreceptor cell differentiation in nocturnal mammals. The inverted nuclear architecture found in adult mouse rod cells is formed through the reorganization of the conventional architecture during terminal differentiation.

Real-Time Observation of Localization and Expression (ROLEX) System for Live Imaging of the Transcriptional Activity and Nuclear Position of a Specific Endogenous Gene.

Long genomic DNA is folded in a cell-type-specific manner and stored in the cell nucleus. The higher-order structure of genomic DNA is thought to be important for DNA transcription, repair, and replication. Recent advancements in live cell imaging techniques that enable the labeling of specific genomic loci and RNA have made it possible to capture the dynamic relationships between higher-order genomic structure and gene expression.

Single-Molecule Nanoscopy Elucidates RNA Polymerase II Transcription at Single Genes in Live Cells.

Transforming the vast knowledge from genetics, biochemistry, and structural biology into detailed molecular descriptions of biological processes inside cells remains a major challenge-one in sore need of better imaging technologies. For example, transcription involves the complex interplay between RNA polymerase II (Pol II), regulatory factors (RFs), and chromatin, but visualizing these dynamic molecular transactions in their native intracellular milieu remains elusive. Here, we zoom into single tagged genes using nanoscopy techniques, including an active target-locking, ultra-sensitive system that enables single-molecule detection in addressable sub-diffraction volumes, within crowded intracellular environments.

Early Relowering of Serum Sodium Concentration Overcomes Disturbances in Consciousness during Hyponatremia Overcorrection and Prevents Osmotic Demyelination Syndrome.

A 79-year-old woman without any cerebral hernia symptoms was hospitalized with hyponatremia. After syndrome of inappropriate antidiuretic hormone induced by drugs was diagnosed and water restriction implemented, the patient became comatose during overcorrection caused by the generation of a large volume of electrolyte-free urine. Once the serum sodium concentration was immediately relowered by the administration of desmopressin and 5% glucose solution, the patient's level of consciousness improved dramatically without osmotic demyelination syndrome (ODS) developing.

Existence of NEU1 sialidase on mouse thymocytes whose natural substrate is CD5.

Membrane-bound sialidases in the mouse thymus are unique and mysterious because their activity at pH 6.5 is equal to or higher than that in the acidic region. The pH curve like this has never been reported in membrane-bound form.

Dynamic changes in the interchromosomal interaction of early histone gene loci during development of sea urchin.

The nuclear positioning and chromatin dynamics of eukaryotic genes are closely related to the regulation of gene expression, but they have not been well examined during early development, which is accompanied by rapid cell cycle progression and dynamic changes in nuclear organization, such as nuclear size and chromatin constitution. In this study, we focused on the early development of the sea urchin and performed three-dimensional fluorescence hybridization of gene loci encoding early histones (one of the types of histone in sea urchin). There are two non-allelic early histone gene loci per sea urchin genome.

Construction and Evaluation of Zinc Finger Nucleases.

Zinc-finger nucleases (ZFNs) are programmable nucleases that have opened the door to the genome editing era. The construction of ZFNs recognizing a target sequence of interest is laborious, and has not been widely used recently. However, key ZFN patents are expiring over the next 2-4 years, enabling a wide range of deployments for clinical and industrial applications.