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.

nudt7 gene depletion causes transcriptomic change in early development of zebrafish.

The Nudt family has been identified as enzymes performing Coenzyme A to 3'5'-ADP + 4'-phospho pantetheine catalysis. The members of this family have been shown to be particularly involved in lipid metabolism, while their involvement in gene regulation through regulating transcription or mRNA metabolism has also been suggested. Here, we focused on peroxisomal NUDT7, possessing enzymatic activity similar to that of its paralog, peroxisomal NUDT19, which is involved in mRNA degradation. No reports have been published about the Nudt family in zebrafish. Our transcriptomic data showed that the Nudt family members are highly expressed around zygotic gene activation (ZGA) in developing zebrafish embryos. Therefore, we confirmed the computational prediction that the products of the nudt7 gene in zebrafish were localized in the peroxisome and highly expressed in early embryogenesis. The depletion of nudt7 genes by the CRISPR/Cas9 system did not affect development; however, it decreased the rate of transcription in ZGA. In addition, H3K27ac ChIP-seq analysis demonstrated that this decrease in transcription was correlated with the genome-wide decrease of H3K27ac level. This study suggests that peroxisomal Nudt7 functions in regulating transcription in ZGA via formation of the H3K27ac domain in active chromatin.

Unusual nucleosome formation and transcriptome influence by the histone H3mm18 variant.

Histone H3mm18 is a non-allelic H3 variant expressed in skeletal muscle and brain in mice. However, its function has remained enigmatic. We found that H3mm18 is incorporated into chromatin in cells with low efficiency, as compared to H3.3. We determined the structures of the nucleosome core particle (NCP) containing H3mm18 by cryo-electron microscopy, which revealed that the entry/exit DNA regions are drastically disordered in the H3mm18 NCP. Consistently, the H3mm18 NCP is substantially unstable in vitro. The forced expression of H3mm18 in mouse myoblast C2C12 cells markedly suppressed muscle differentiation. A transcriptome analysis revealed that the forced expression of H3mm18 affected the expression of multiple genes, and suppressed a group of genes involved in muscle development. These results suggest a novel gene expression regulation system in which the chromatin landscape is altered by the formation of unusual nucleosomes with a histone variant, H3mm18, and provide important insight into understanding transcription regulation by chromatin.

Locus-specific induction of gene expression from heterochromatin loci during cellular senescence.

Senescence is a fate-determined state, accompanied by reorganization of heterochromatin. Although lineage-appropriate genes can be temporarily repressed through facultative heterochromatin, stable silencing of lineage-inappropriate genes often involves the constitutive heterochromatic mark, histone H3 lysine 9 trimethylation (H3K9me3). The fate of these heterochromatic genes during senescence is unclear. In the present study, we show that a small number of lineage-inappropriate genes, exemplified by the LCE2 skin genes, are derepressed during senescence from H3K9me3 regions in fibroblasts. DNA FISH experiments reveal that these gene loci, which are condensed at the nuclear periphery in proliferative cells, are decompacted during senescence. Decompaction of the locus is not sufficient for LCE2 expression, which requires p53 and C/EBPß signaling. NLRP3, which is predominantly expressed in macrophages from an open topologically associated domain (TAD), is also derepressed in senescent fibroblasts due to the local disruption of the H3K9me3-rich TAD that contains it. NLRP3 has been implicated in the amplification of inflammatory cytokine signaling in senescence and aging, highlighting the functional relevance of gene induction from 'permissive' H3K9me3 regions in senescent cells.

Modeling population size independent tissue epigenomes by ChIL-seq with single thin sections.

Recent advances in genome-wide technologies have enabled analyses using small cell numbers of even single cells. However, obtaining tissue epigenomes with cell-type resolution from large organs and tissues still remains challenging, especially when the available material is limited. Here, we present a ChIL-based approach for analyzing the diverse cellular dynamics at the tissue level using high-depth epigenomic data. "ChIL for tissues" allows the analysis of a single tissue section and can reproducibly generate epigenomic profiles from several tissue types, based on the distribution of target epigenomic states, tissue morphology, and number of cells. The proposed method enabled the independent evaluation of changes in cell populations and gene activation in cells from regenerating skeletal muscle tissues, using a statistical model of RNA polymerase II distribution on gene loci. Thus, the integrative analyses performed using ChIL can elucidate in vivo cell-type dynamics of tissues.

Transcriptome analysis of gene expression changes upon enzymatic dissociation in skeletal myoblasts.

Single-cell RNA-sequencing analysis is one of the most effective tools for understanding specific cellular states. The use of single cells or pooled cells in RNA-seq analysis requires the isolation of cells from a tissue or culture. Although trypsin or more recently cold-active protease (CAP) has been used for cell dissociation, the extent to which the gene expression changes are suppressed has not been clarified. To this end, we conducted detailed profiling of the enzyme-dependent gene expression changes in mouse skeletal muscle progenitor cells, focusing on the enzyme treatment time, amount and temperature. We found that the genes whose expression was changed by the enzyme treatment could be classified in a time-dependent manner and that there were genes whose expression was changed independently of the enzyme treatment time, amount and temperature. This study will be useful as reference data for genes that should be excluded or considered for RNA-seq analysis using enzyme isolation methods.

Epigenetic effects induced by the ectopic expression of Pax7 in 3T3-L1.

Although skeletal muscle cells and adipocytes are derived from the same mesoderm, they do not transdifferentiate in vivo and are strictly distinct at the level of gene expression. To elucidate some of the regulatory mechanisms underlying this strict distinction, Pax7, a myogenic factor, was ectopically expressed in 3T3-L1 adipose progenitor cells to perturb their adipocyte differentiation potential. Transcriptome analysis showed that ectopic expression of Pax7 repressed the expression of some adipocyte genes and induced expression of some skeletal muscle cell genes. We next profiled the epigenomic state altered by Pax7 expression using H3K27ac, an activating histone mark, and H3K27me3, a repressive histone mark, as indicators. Our results show that ectopic expression of Pax7 did not result in the formation of H3K27ac at loci of skeletal muscle-related genes, but instead resulted in the formation of H3K27me3 at adipocyte-related gene loci. These findings suggest that the primary function of ectopic Pax7 expression is the formation of H3K27me3, and muscle gene expression results from secondary regulation.

Genome-wide analysis of chromatin structure changes upon MyoD binding in proliferative myoblasts during the cell cycle.

MyoD, a myogenic differentiation protein, has been studied for its critical role in skeletal muscle differentiation. MyoD-expressing myoblasts have a potency to be differentiated with proliferation of ectopic cells. However, little is known about the effect on chromatin structure of MyoD binding in proliferative myoblasts. In this study, we evaluated the chromatin structure around MyoD-bound genome regions during the cell cycle by chromatin immunoprecipitation sequencing. Genome-wide analysis of histone modifications was performed in proliferative mouse C2C12 myoblasts during three phases (G1, S, G2/M) of the cell cycle. We found that MyoD-bound genome regions had elevated levels of active histone modifications, such as H3K4me1/2/3 and H3K27ac, compared with MyoD-unbound genome regions during the cell cycle. We also demonstrated that the elevated H3K4me2/3 modification level was maintained during the cell cycle, whereas the H3K27ac and H3K4me1 modification levels decreased to the same level as MyoD-unbound genome regions during the later phases. Immunoblot analysis revealed that MyoD abundance was high in the G1 phase then decreased in the S and G2/M phases. Our results suggest that MyoD binding formed selective epigenetic memories with H3K4me2/3 during the cell cycle in addition to myogenic gene induction via active chromatin formation coupled with transcription.

Long non-coding RNA MANCR is a target of BET bromodomain protein BRD4 and plays a critical role in cellular migration and invasion abilities of prostate cancer.

Androgen receptor (AR)-negative castration-resistant prostate cancer (CRPC) is highly aggressive and is resistant to most of the current therapies. Bromodomain and extra terminal domain (BET) protein BRD4 binds to super-enhancers (SEs) that drive high expression of oncogenes in many cancers. A BET inhibitor, JQ1, has been found to suppress the malignant phenotypes of prostate cancer cells, however, the target genes of JQ1 remain largely unknown. Here we show that SE-associated genes specific for AR-negative CRPC PC3 cells include genes involved in migration and invasion, and that JQ1 impairs migration and invasion of PC3 cells. We identified a long non-coding RNA, MANCR, which was markedly down-regulated by JQ1, and found that BRD4 binds to the MANCR locus. MANCR knockdown led to a significant decrease in migration and invasion of PC3 cells. Furthermore, RNA sequencing analysis revealed that expression of the genes involved in migration and invasion was altered by MANCR knockdown. In summary, our data demonstrate that MANCR plays a critical role in migration and invasion of PC3 cells.

NOTCH-mediated non-cell autonomous regulation of chromatin structure during senescence.

Senescent cells interact with the surrounding microenvironment achieving diverse functional outcomes. We have recently identified that NOTCH1 can drive 'lateral induction' of a unique senescence phenotype in adjacent cells by specifically upregulating the NOTCH ligand JAG1. Here we show that NOTCH signalling can modulate chromatin structure autonomously and non-autonomously. In addition to senescence-associated heterochromatic foci (SAHF), oncogenic RAS-induced senescent (RIS) cells exhibit a massive increase in chromatin accessibility. NOTCH signalling suppresses SAHF and increased chromatin accessibility in this context. Strikingly, NOTCH-induced senescent cells, or cancer cells with high JAG1 expression, drive similar chromatin architectural changes in adjacent cells through cell-cell contact. Mechanistically, we show that NOTCH signalling represses the chromatin architectural protein HMGA1, an association found in multiple human cancers. Thus, HMGA1 is involved not only in SAHFs but also in RIS-driven chromatin accessibility. In conclusion, this study identifies that the JAG1-NOTCH-HMGA1 axis mediates the juxtacrine regulation of chromatin architecture.

Multiple expression cassette exchange via TP901-1, R4, and Bxb1 integrase systems on a mouse artificial chromosome.

The site-specific excision of a target DNA sequence for genetic knockout or lineage tracing is a powerful tool for investigating biological systems. Currently, site-specific recombinases (SSRs), such as Cre or Flp recombination target cassettes, have been successfully excised or inverted by a single SSR to regulate transgene expression. However, the use of a single SSR might restrict the complex control of gene expression. This study investigated the potential for expanding the multiple regulation of transgenes using three different integrase systems (TP901-1, R4, and Bxb1). We designed three excision cassettes that expressed luciferase, where the luciferase expression could be exchanged to a fluorescent protein by site-specific recombination. Individual cassettes that could be regulated independently by a different integrase were connected in tandem and inserted into a mouse artificial chromosome (MAC) vector in Chinese hamster ovary cells. The transient expression of an integrase caused the targeted luciferase activity to be lost and fluorescence was activated. Additionally, the integrase system enabled the specific excision of targeted DNA sequences without cross-reaction with the other recombination targets. These results suggest that the combined use of these integrase systems in a defined locus on a MAC vector permits the multiple regulation of transgene expression and might contribute to genomic or cell engineering.

NOTCH1 mediates a switch between two distinct secretomes during senescence.

Senescence, a persistent form of cell-cycle arrest, is often associated with a diverse secretome, which provides complex functionality for senescent cells within the tissue microenvironment. We show that oncogene-induced senescence is accompanied by a dynamic fluctuation of NOTCH1 activity, which drives a TGF-ß-rich secretome, while suppressing the senescence-associated pro-inflammatory secretome through inhibition of C/EBPß. NOTCH1 and NOTCH1-driven TGF-ß contribute to 'lateral induction of senescence' through a juxtacrine NOTCH-JAG1 pathway. In addition, NOTCH1 inhibition during senescence facilitates upregulation of pro-inflammatory cytokines, promoting lymphocyte recruitment and senescence surveillance in vivo. As enforced activation of NOTCH1 signalling confers a near mutually exclusive secretory profile compared with typical senescence, our data collectively indicate that the dynamic alteration of NOTCH1 activity during senescence dictates a functional balance between these two distinct secretomes: one representing TGF-ß and the other pro-inflammatory cytokines, highlighting that NOTCH1 is a temporospatial controller of secretome composition.

Translating the effects of mTOR on secretory senescence.

Cellular senescence is often accompanied by the production of secreted proteins that mediate the diverse effects of senescence on the tissue microenvironment. The mammalian target of rapamycin (mTOR), a master regulator of protein synthesis, is now shown to control the senescence-associated secretory phenotype by modulating gene transcription and mRNA translation and stabilization.

Phenotype specific analyses reveal distinct regulatory mechanism for chronically activated p53.

The downstream functions of the DNA binding tumor suppressor p53 vary depending on the cellular context, and persistent p53 activation has recently been implicated in tumor suppression and senescence. However, genome-wide information about p53-target gene regulation has been derived mostly from acute genotoxic conditions. Using ChIP-seq and expression data, we have found distinct p53 binding profiles between acutely activated (through DNA damage) and chronically activated (in senescent or pro-apoptotic conditions) p53. Compared to the classical 'acute' p53 binding profile, 'chronic' p53 peaks were closely associated with CpG-islands. Furthermore, the chronic CpG-island binding of p53 conferred distinct expression patterns between senescent and pro-apoptotic conditions. Using the p53 targets seen in the chronic conditions together with external high-throughput datasets, we have built p53 networks that revealed extensive self-regulatory 'p53 hubs' where p53 and many p53 targets can physically interact with each other. Integrating these results with public clinical datasets identified the cancer-associated lipogenic enzyme, SCD, which we found to be directly repressed by p53 through the CpG-island promoter, providing a mechanistic link between p53 and the 'lipogenic phenotype', a hallmark of cancer. Our data reveal distinct phenotype associations of chronic p53 targets that underlie specific gene regulatory mechanisms.

Antigen-specific in vitro immunization: a source for human monoclonal antibodies.

Human monoclonal antibody has great potential for treatment of various diseases utilizing their specificity against antigens. We have shown an in vitro immunization (IVI) protocol inducing antigen-specific immune responses in human peripheral blood mononuclear cells (PBMCs) for efficient production of human monoclonal antibodies. By using IVI method antigen specific antibody genes can be efficiently obtained because of increasing production of antigen-specific antibodies from in vitro immunized PBMCs. This IVI protocol will be widely applied for combination with several display methods and enhance the production of human monoclonal antibodies.

A rapid screening and production method using a novel mammalian cell display to isolate human monoclonal antibodies.

Antibody display methods are increasingly being used to produce human monoclonal antibodies for disease therapy. Rapid screening and isolation of specific human antibody genes are valuable for producing human monoclonal antibodies showing high specificity and affinity. In this report, we describe a novel mammalian cell display method in which whole human IgG is displayed on the cell surface of CHO cells. Cells expressing antigen-specific human monoclonal IgGs with high affinity on the cell surface after normal folding and posttranscriptional modification were screened using a cell sorter. The membrane-type IgG-expressing CHO cells were then converted to IgG-secreting cells by transfection with a plasmid coding Cre recombinase. This mammalian cell display method was applied to in vitro affinity maturation of monoclonal C9 IgG specific to the human high-affinity IgE receptor (FcεRIα). The CDR3 of the C9 heavy chain variable region gene was randomly mutated and inserted into pcDNA5FRT/IgG. A C9 IgG (CDRH3r)-expressing CHO cell display library consisting of 1.1×10(6) independent clones was constructed. IgG-displaying cells showing high reactivity to FcεRIα antigen were screened by the cell sorter, resulting in the establishment of a CHO cell line producing with higher reactivity than the parent C9 IgG.

Redistribution of the Lamin B1 genomic binding profile affects rearrangement of heterochromatic domains and SAHF formation during senescence.

Senescence is a stress-responsive form of stable cell cycle exit. Senescent cells have a distinct gene expression profile, which is often accompanied by the spatial redistribution of heterochromatin into senescence-associated heterochromatic foci (SAHFs). Studying a key component of the nuclear lamina lamin B1 (LMNB1), we report dynamic alterations in its genomic profile and their implications for SAHF formation and gene regulation during senescence. Genome-wide mapping reveals that LMNB1 is depleted during senescence, preferentially from the central regions of lamina-associated domains (LADs), which are enriched for Lys9 trimethylation on histone H3 (H3K9me3). LMNB1 knockdown facilitates the spatial relocalization of perinuclear H3K9me3-positive heterochromatin, thus promoting SAHF formation, which could be inhibited by ectopic LMNB1 expression. Furthermore, despite the global reduction in LMNB1 protein levels, LMNB1 binding increases during senescence in a small subset of gene-rich regions where H3K27me3 also increases and gene expression becomes repressed. These results suggest that LMNB1 may contribute to senescence in at least two ways due to its uneven genome-wide redistribution: first, through the spatial reorganization of chromatin and, second, through gene repression.

Extension of the lifespan of Caenorhabditis elegans by the use of electrolyzed reduced water.

Electrolyzed reduced water (ERW) has attracted much attention because of its therapeutic effects. In the present study, a new culture medium, which we designated Water medium, was developed to elucidate the effects of ERW on the lifespan of Caenorhabditis elegans. Wild-type C. elegans had a significantly shorter lifespan in Water medium than in conventional S medium. However, worms cultured in ERW-Water medium exhibited a significantly extended lifespan (from 11% to 41%) compared with worms cultured in ultrapure water-Water medium. There was no difference between the lifespans of worms cultured in ERW-S medium and ultrapure water-S medium. Nematodes cultured in ultrapure water-Water medium showed significantly higher levels of reactive oxygen species than those cultured in ultrapure water-S medium. Moreover, ERW-Water medium significantly reduced the ROS accumulation induced in the worms by paraquat, suggesting that ERW-Water medium extends the longevity of nematodes at least partly by scavenging ROS.

Production of human monoclonal antibodies against Fc(epsilon)RI(alpha) by a method combining in vitro immunization with phage display.

An in vitro immunization protocol using human peripheral blood mononuclear cells (PBMC) was developed to generate human antigen-specific antibodies. Monoclonal antibodies have great potential, and in particular, efficient acquirement of monoclonal antibodies against membrane proteins provides advantages. In this study, we tried to generate a human monoclonal antibody against the high affinity IgE receptor, Fc(epsilon)RI(alpha), using a method combining in vitro immunization and phage display. Heavy and light chain variable region genes were obtained from PBMC immunized in vitro with Fc(epsilon)RI(alpha)-expressed KU812F cells. Subsequently a combined phage antibody library 6 x 10(3) in the size was generated. Antigen-specific phage antibody clones were selected by panning with recombinant Fc(epsilon)RI(alpha) and recombined to produce human IgG format antibodies using CHO cells. The antibodies exhibited specific binding against Fc(epsilon)RI(alpha). These results suggest that one can obtain membrane protein-specific human monoclonal antibodies from a relatively small phage antibody library using in vitro immunized PBMCs.

Generation of human monoclonal antibodies against Propionibacterium acnes by applying the phage display method to human peripheral blood mononuclear cells immunized in vitro.

Propionibacterium acnes is a gram-positive, non-spore-forming, rod-shaped bacterium that is often detected in normal human skin flora. P. acnes has been associated with many diseases. In this study, we attempted to generate anti-P. acnes human monoclonal antibodies. A phage antibody library was first generated from human peripheral blood mononuclear cells immunized in vitro with P. acnes using the phage display method, and P. acnes-specific phage antibodies were obtained using solid phase panning. Antigen-specific variable region genes were then amplified and recombined into vectors expressing human IgG antibodies. The results indicated that the recombinant human IgG antibodies exhibited P. acnes-specific binding. This study demonstrates that the combined use of an in vitro immunization protocol and the phage display method enables the generation of human monoclonal antibodies against pathogenic bacteria and toxic antigens.