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. Specifically, after exit from the naïve pluripotency state, chromatin becomes less compacted, and the OCT4 transcription factor has lower mobility and is more bound to its cognate sites. In epiblast cells, the active transcription hallmark, H3K9ac, decreases within the Oct4 locus, correlating with reduced accessibility of OCT4 and, in turn, with reduced expression of Oct4 nascent RNAs. Despite the high variability in the distances between active pluripotency genes, distances between Nodal and Oct4 decrease during epiblast specification. In particular, highly expressed Oct4 alleles are closer to nuclear speckles during all stages of the pluripotency transition, while only a distinct group of highly expressed Nodal alleles are in close proximity to Oct4 when associated with a nuclear speckle in epiblast cells. Overall, our results provide new insights into the role of the spatiotemporal genome remodeling during mouse pluripotency transition and its correlation with the expression of key pluripotency genes.

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. Here, we develop Precise Emission Canceling Antibodies (PECAbs) that have cleavable fluorescent labeling. PECAbs enable high-specificity sequential imaging using hundreds of antibodies, allowing for reconstruction of the spatiotemporal dynamics of signaling pathways. Additionally, combining this approach with seq-smFISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system can serve as a comprehensive platform for analyzing complex cell processes.

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. Recent observations suggest that the formation of condensates, composed of transcription-related factors, RNA, and RNA-binding proteins, around genes can regulate transcription. Advancements in technology have facilitated the visualization of the intricate spatiotemporal relationship between higher-order genomic structures, condensate formation, and transcriptional activity in living cells.

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. However, fundamental ZFN patents have expired, enabling a wide range of their distribution for clinical and industrial applications. This article introduces a ZFN construction protocol that uses bacterial one-hybrid (B1H) selection and single-strand annealing (SSA) assay.

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.

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. Clusters of RNA polymerase II and BRD4 are observed proximal to the transcription start site of Nanog when the gene is transcribed in mouse embryonic stem cells. In contrast, clusters of MED19 and MED22 tend to be located near the transcription start site, even without transcription activity. Thus, the STREAMING-tag system reveals the spatiotemporal relationships between transcriptional activity and protein clusters near the gene. This powerful tool is useful for quantitatively understanding transcriptional regulation in living cells.

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. However, chromosome therapy has been developed to correct the extra chromosome in cultured patient cells, and it was recently reported that during reprogramming into iPSCs, fibroblasts from a Down syndrome patient lost the extra chromosome 21 due to a phenomenon called trisomy-biased chromosome loss. To gain preliminary insights into the underlying mechanism of trisomy rescue during the early stages of reprogramming, we reprogrammed skin fibroblasts from patients with trisomy syndromes 21, 18, 13, and 9 to iPSC, and evaluated the genomes of the individual iPSC colonies by molecular cytogenetic techniques. We report the spontaneous correction from trisomy to disomy upon cell reprogramming in at least one cell line examined from each of the trisomy syndromes, and three possible combinations of chromosomes were selected in the isogenic trisomy-rescued iPSC clones. Single nucleotide polymorphism analysis showed that the trisomy-rescued clones exhibited either heterodisomy or segmental uniparental isodisomy, ruling out the possibility that two trisomic chromosomes were lost simultaneously and the remaining one was duplicated, suggesting instead that one trisomic chromosome was lost to generate disomic cells. These results demonstrated that trisomy rescue may be a phenomenon with random loss of the extra chromosome and subsequent selection for disomic iPSCs, which is analogous to the karyotype correction in early preimplantation embryos. Our finding is relevant for elucidating the mechanisms of autonomous karyotype correction and future application in basic and clinical research on aneuploidy disorders.

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. We revealed that cells lacking a specific 60S ribosomal protein set exhibited common nucleolar disintegration. The depletion of RPL5 (also known as uL18), the liquid-liquid phase separation facilitator, was most effective, and resulted in an enlarged and un-separated sub-nucleolar compartment. Single-molecule tracking analysis revealed less-constrained mobility of its components. rDNA arrays were also unbundled. These results were recapitulated by a coarse-grained molecular dynamics model. Transcription and processing of ribosomal RNA were repressed in these aberrant nucleoli. Consistently, the nucleoli were disordered in peripheral blood cells from a Diamond-Blackfan anemia patient harboring a heterozygous, large deletion in RPL5 Our combinatorial analyses newly define the role of RPL5 in rDNA array bundling and the biophysical properties of the nucleolus, which may contribute to the etiology of ribosomopathy.

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. Here, we further dissect mechanisms of RF clustering and transcription activation in mouse embryonic stem cells. RF recruitment into clusters involves specific molecular recognition of cognate DNA and chromatin-binding sites, suggesting underlying cis-element clustering. Strikingly, imaging of tagged genomic loci, with ≤1 kilobase and ~20-nanometer precision, in live cells, reveals distal enhancer clusters over the extended locus in frequent close proximity to target genes-within RF-clustering distances. These high-interaction-frequency enhancer-cluster 'superclusters' create nano-environments wherein clustered RFs activate target genes, providing a structural framework for relating genome organization, focal RF accumulation and transcription activation.

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. Informatics analyses and functional assays revealed that transcriptional bursting kinetics was regulated by a combination of promoter- and gene body-binding proteins, including the polycomb repressive complex 2 and transcription elongation factors. Furthermore, large-scale CRISPR-Cas9-based screening identified that the Akt/MAPK signaling pathway regulated bursting kinetics by modulating transcription elongation efficiency. These results uncovered the key molecular mechanisms underlying transcriptional bursting and cell-to-cell gene expression noise in mammalian 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. Here, we report that peroxisomes are essential for the transport of cholesterol into the ciliary membrane. Zellweger syndrome (ZS) is a peroxisome-deficient hereditary disorder with several ciliopathy-related features and cells from these patients showed a reduced cholesterol level in the ciliary membrane. Reverse genetics approaches revealed that the GTP exchange factor Rabin8, the Rab GTPase Rab10, and the microtubule minus-end-directed kinesin KIFC3 form a peroxisome-associated complex to control the movement of peroxisomes along microtubules, enabling communication between peroxisomes and ciliary pocket membranes. Our findings suggest that insufficient ciliary cholesterol levels may underlie ciliopathies.

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. The tau MTBR also forms the core of the neuropathological filaments identified in AD brain and other tauopathies. Multiple approaches are being taken to limit tau pathology, including immunotherapy with anti-tau antibodies. Given its key structural role within fibrils, specifically targetting the MTBR with a therapeutic antibody to inhibit tau seeding and aggregation may be a promising strategy to provide disease-modifying treatment for AD and other tauopathies. Therefore, a monoclonal antibody generating campaign was initiated with focus on the MTBR. Herein we describe the pre-clinical generation and characterisation of E2814, a humanised, high affinity, IgG1 antibody recognising the tau MTBR. E2814 and its murine precursor, 7G6, as revealed by epitope mapping, are antibodies bi-epitopic for 4R and mono-epitopic for 3R tau isoforms because they bind to sequence motif HVPGG. Functionally, both antibodies inhibited tau aggregation in vitro. They also immunodepleted a variety of MTBR-containing tau protein species. In an in vivo model of tau seeding and transmission, attenuation of deposition of sarkosyl-insoluble tau in brain could also be observed in response to antibody treatment. In AD brain, E2814 bound different types of tau filaments as shown by immunogold labelling and recognised pathological tau structures by immunohistochemical staining. Tau fragments containing HVPGG epitopes were also found to be elevated in AD brain compared to PSP or control. Taken together, the data reported here have led to E2814 being proposed for clinical development.

PHi-C: deciphering Hi-C data into polymer dynamics.

Genomes are spatiotemporally organized within the cell nucleus. Genome-wide chromosome conformation capture (Hi-C) technologies have uncovered the 3D genome organization. Furthermore, live-cell imaging experiments have revealed that genomes are functional in 4D. Although computational modeling methods can convert 2D Hi-C data into population-averaged static 3D genome models, exploring 4D genome nature based on 2D Hi-C data remains lacking. Here, we describe a 4D simulation method, PHi-C (polymer dynamics deciphered from Hi-C data), that depicts 4D genome features from 2D Hi-C data by polymer modeling. PHi-C allows users to interpret 2D Hi-C data as physical interaction parameters within single chromosomes. The physical interaction parameters can then be used in the simulations and analyses to demonstrate dynamic characteristics of genomic loci and chromosomes as observed in live-cell imaging experiments. PHi-C is available at https://github.com/soyashinkai/PHi-C.

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. However, the mechanisms underlying these changes remain largely unknown. Here, we found that the dynamic deformation of nuclei via actomyosin-mediated contractility contributes to chromocenter clustering and promotes genomic architecture reorganization during differentiation by conducting an in cellulo experiment coupled with phase-field modeling. Similar patterns of dynamic deformation of the nucleus and a concomitant migration of the nuclear content were also observed in rod cells derived from the developing mouse retina. These results indicate that the common phenomenon of dynamic nuclear deformation, which accompanies dynamic cell behavior, can be a universal mechanism for spatiotemporal genomic reorganization.

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. We have established the real-time observation of localization and expression (ROLEX) system for live imaging of the transcriptional state and nuclear position of a specific endogenous gene. In this chapter, I will introduce the detailed protocol of ROLEX imaging in mouse embryonic stem cells.

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. We image, track, and quantify Pol II with single-molecule resolution, unveiling its dynamics during the transcription cycle. Further probing multiple functionally linked events-RF-chromatin interactions, Pol II dynamics, and nascent transcription kinetics-reveals detailed operational parameters of gene-regulatory mechanisms hitherto-unseen in vivo. Our approach sets the stage for single-molecule studies of complex molecular processes in live cells.

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. This outcome suggests that, similar to the findings in rat models, relowering the serum sodium concentration as early as possible to counter a disturbance of consciousness during the overcorrection of hyponatremia prevents ODS.

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. To clarify this enzyme, we studied the sialidase activities of crude membrane fractions from immature-T, mature-T and non-T cells from C57BL/6 mice and from SM/J mice, a strain with a defect in NEU1 activity. Non-T cells from C57BL/6 mice had high activity at pH 6.5, but those from SM/J mice did not. Neu1 and Neu3 mRNA was shown by real-time PCR to be expressed in T cells and also in non-T cells, whereas Neu2 was expressed mainly in non-T cells and Neu4 was scarcely expressed. However, the in situ hybridization study on the localization of four sialidases in the thymus showed that Neu4 was clearly expressed. We then focused on a sialidase on the thymocyte surface because the possibility of the existence of a sialidase on thymocytes was suggested by peanut agglutinin (PNA) staining after incubation of the cells alone in PBS. This activity was inhibited by NEU1-selective sialidase inhibitor C9-butyl-amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid. The natural substrate for the cell surface sialidase was identified as clustered differentiation 5 (CD5) by PNA-blot analysis of anti-CD5 immunoprecipitate. We conclude that NEU1 exists on the cell surface of mouse thymocytes and CD5 is a natural substrate for it. Although this is not the main reaction of the membrane-bound thymus-sialidases, it must be important for the thymus.

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 Hemicentrotus pulcherrimus and performed three-dimensional fluorescence in situ 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. We found that during the morula stage, when the early histone gene expression levels are at their maximum, interchromosomal interactions were often formed between the early histone gene loci on separate chromosomes and that the gene loci were directed to locate to more interior positions. Furthermore, these interactions were associated with the active transcription of the early histone genes. Thus, such dynamic interchromosomal interactions may contribute to the efficient synthesis of early histone mRNA during the morula stage of sea urchin development.