Cell-autonomous and redundant roles of Hey1 and HeyL in muscle stem cells: HeyL requires Hes1 to bind diverse DNA sites.

The undifferentiated state of muscle stem (satellite) cells (MuSCs) is maintained by the canonical Notch pathway. Although three bHLH transcriptional factors, Hey1, HeyL and Hes1, are considered to be potential effectors of the Notch pathway exerting anti-myogenic effects, neither HeyL nor Hes1 inhibits myogenic differentiation of myogenic cell lines. Furthermore, whether these factors work redundantly or cooperatively is unknown. Here, we showed cell-autonomous functions of Hey1 and HeyL in MuSCs using conditional and genetic null mice. Analysis of cultured MuSCs revealed anti-myogenic activity of both HeyL and Hes1. We found that HeyL forms heterodimeric complexes with Hes1 in living cells. Moreover, our ChIP-seq experiments demonstrated that, compared with HeyL alone, the HeyL-Hes1 heterodimer binds with high affinity to specific sites in the chromatin, including the binding sites of Hey1. Finally, analyses of myogenin promoter activity showed that HeyL and Hes1 act synergistically to suppress myogenic differentiation. Collectively, these results suggest that HeyL and Hey1 function redundantly in MuSCs, and that HeyL requires Hes1 for effective DNA binding and biological activity.

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.

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.

Targeted inhibition of EPAS1-driven IL-31 production by a small-molecule compound.

BACKGROUND: IL-31 is a major pruritogen associated with atopic dermatitis (AD). Although a specific antibody for IL-31 receptor has been shown to alleviate pruritus in patients with AD, therapeutic approaches to inhibition of IL-31 production remain unexploited. IL-31 production by TH cells critically depends on the transcription factor EPAS1, which mediates IL31 promoter activation in collaboration with SP1. OBJECTIVE: We aimed at developing small-molecule inhibitors that selectively block IL-31 production by TH cells. METHODS: We generated the reporter cell line that inducibly expressed EPAS1 in the presence of doxycycline to mediate Il31 promoter activation, and we screened 9600 chemical compounds. The selected compounds were further examined by using TH cells from a spontaneous mouse model of AD and TH cells from patients with AD. RESULTS: We have identified 4-(2-(4-isopropylbenzylidene)hydrazineyl)benzoic acid (IPHBA) as an inhibitor of IL31 induction. Although IPHBA did not affect nonspecific T-cell proliferation, IPHBA inhibited antigen-induced IL-31 production by TH cells from both an AD mouse model and patients with AD without affecting other cytokine production and hypoxic responses. In line with this, itch responses induced by adoptive transfer of IL-31-producing TH cells were attenuated when mice were orally treated with IPHBA. Mechanistically, IPHBA inhibited the association between EPAS1 and SP1, resulting in defective recruitment of both transcription factors to the specific sites of the IL31 promoter. We also determined the structure-activity relationship of IPHBA by synthesizing and analyzing 201 analogous compounds. CONCLUSION: IPHBA could be a potential drug leading to inhibition of EPAS1-driven IL-31 production.

Temporal regulation of chromatin during myoblast differentiation.

The commitment to and execution of differentiation programmes involves a significant change in gene expression in the precursor cell to facilitate development of the mature cell type. In addition to being regulated by lineage-determining and auxiliary transcription factors that drive these changes, the structural status of the chromatin has a considerable impact on the transcriptional competence of differentiation-specific genes, which is clearly demonstrated by the large number of cofactors and the extraordinary complex mechanisms by which these genes become activated. The terminal differentiation of myoblasts to myotubes and mature skeletal muscle is an excellent system to illustrate these points. The MyoD family of closely related, lineage-determining transcription factors directs, largely through targeting to chromatin, a cascade of cooperating transcription factors and enzymes that incorporate or remove variant histones, post-translationally modify histones, and alter nucleosome structure and positioning via energy released by ATP hydrolysis. The coordinated action of these transcription factors and enzymes prevents expression of differentiation-specific genes in myoblasts and facilitates the transition of these genes from transcriptionally repressed to activated during the differentiation process. Regulation is achieved in both a temporal as well as spatial manner, as at least some of these factors and enzymes affect local chromatin structure at myogenic gene regulatory sequences as well as higher-order genome organization. Here we discuss the transition of genes that promote myoblast differentiation from the silenced to the activated state with an emphasis on the changes that occur to individual histones and the chromatin structure present at these loci.

Chd2 regulates chromatin for proper gene expression toward differentiation in mouse embryonic stem cells.

Chromatin reorganization is necessary for pluripotent stem cells, including embryonic stem cells (ESCs), to acquire lineage potential. However, it remains unclear how ESCs maintain their characteristic chromatin state for appropriate gene expression upon differentiation. Here, we demonstrate that chromodomain helicase DNA-binding domain 2 (Chd2) is required to maintain the differentiation potential of mouse ESCs. Chd2-depleted ESCs showed suppressed expression of developmentally regulated genes upon differentiation and subsequent differentiation defects without affecting gene expression in the undifferentiated state. Furthermore, chromatin immunoprecipitation followed by sequencing revealed alterations in the nucleosome occupancy of the histone variant H3.3 for developmentally regulated genes in Chd2-depleted ESCs, which in turn led to elevated trimethylation of the histone H3 lysine 27. These results suggest that Chd2 is essential in preventing suppressive chromatin formation for developmentally regulated genes and determines subsequent effects on developmental processes in the undifferentiated state.

Periostin Promotes Scar Formation through the Interaction between Pericytes and Infiltrating Monocytes/Macrophages after Spinal Cord Injury.

Scar formation is a prominent pathological feature of traumatic central nervous system (CNS) injury, which has long been implicated as a major impediment to the CNS regeneration. However, the factors affecting such scar formation remain to be elucidated. We herein demonstrate that the extracellular matrix protein periostin (POSTN) is a key player in scar formation after traumatic spinal cord injury (SCI). Using high-throughput RNA sequencing data sets, we found that the genes involved in the extracellular region, such as POSTN, were significantly expressed in the injured spinal cord. The expression of POSTN peaked at 7 days after SCI, predominantly in the scar-forming pericytes. Notably, we found that genetic deletion of POSTN in mice reduced scar formation at the lesion site by suppressing the proliferation of the pericytes. Conversely, we found that recombinant POSTN promoted the migration capacity of the monocytes/macrophages and increased the expression of tumor necrosis factor-α from the monocytes/macrophages in vitro, which facilitated the proliferation of pericytes. Furthermore, we revealed that the pharmacological blockade of POSTN suppressed scar formation and improved the long-term functional outcome after SCI. Our findings suggest a potential mechanism whereby POSTN regulates the scar formation after SCI and provide significant evidence that POSTN is a promising therapeutic target for CNS injury.

Crystal Structure and Characterization of Novel Human Histone H3 Variants, H3.6, H3.7, and H3.8.

Non-allelic histone variants are considered as epigenetic factors that regulate genomic DNA functions in eukaryotic chromosomes. In this study, we identified three new human histone H3 variants (named H3.6, H3.7, and H3.8), which were previously annotated as pseudogenes. H3.6 and H3.8 conserve the H3.3-specific amino acid residues, but H3.7 shares the specific amino acid residues with H3.1. We successfully reconstituted the nucleosome containing H3.6 in vitro and determined its crystal structure. In the H3.6 nucleosome, the H3.6-specific Val62 residue hydrophobically contacts the cognate H4 molecule, but its contact area is smaller than that of the corresponding H3.3 Ile62 residue. The thermal stability assay revealed that the H3.6 nucleosome is substantially unstable, as compared to the H3.3 nucleosome. Interestingly, mutational analysis demonstrated that the H3.6 Val62 residue is fully responsible for the H3.6 nucleosome instability, probably because of the weakened hydrophobic interaction with H4. We also reconstituted the nucleosome containing H3.8, but its thermal stability was quite low. In contrast, purified H3.7 failed to form nucleosomes in vitro. The identification and characterization of these novel human histone H3 variants provide important new insights into understanding the epigenetic regulation of the human genome.

Incorporation of histone H3.1 suppresses the lineage potential of skeletal muscle.

Lineage potential is triggered by lineage-specific transcription factors in association with changes in the chromatin structure. Histone H3.3 variant is thought to play an important role in the regulation of lineage-specific genes. To elucidate the function of H3.3 in myogenic differentiation, we forced the expression of GFP-H3.1 to alter the balance between H3.1 and H3.3 in mouse C2C12 cells that could be differentiated into myotubes. GFP-H3.1 replaced H3.3 in the regulatory regions of skeletal muscle (SKM) genes and induced a decrease of H3K4 trimethylation (H3K4me3) and increase of H3K27 trimethylation (H3K27me3). Similar results were obtained by H3.3 knockdown. In contrast, MyoD-dependent H3.3 incorporation into SKM genes in fibroblasts induced an increase of H3K4me3 and H3K27me3. In mouse embryos, a bivalent modification of H3K4me3 and H3K27me3 was formed on H3.3-incorporated SKM genes before embryonic skeletal muscle differentiation. These results suggest that lineage potential is established through a selective incorporation of specific H3 variants that governs the balance of histone modifications.

Spatial re-organization of myogenic regulatory sequences temporally controls gene expression.

During skeletal muscle differentiation, the activation of some tissue-specific genes occurs immediately while others are delayed. The molecular basis controlling temporal gene regulation is poorly understood. We show that the regulatory sequences, but not other regions of genes expressed at late times of myogenesis, are in close physical proximity in differentiating embryonic tissue and in differentiating culture cells, despite these genes being located on different chromosomes. Formation of these inter-chromosomal interactions requires the lineage-determinant MyoD and functional Brg1, the ATPase subunit of SWI/SNF chromatin remodeling enzymes. Ectopic expression of myogenin and a specific Mef2 isoform induced myogenic differentiation without activating endogenous MyoD expression. Under these conditions, the regulatory sequences of late gene loci were not in close proximity, and these genes were prematurely activated. The data indicate that the spatial organization of late genes contributes to temporal regulation of myogenic transcription by restricting late gene expression during the early stages of myogenesis.

Chd5 Regulates MuERV-L/MERVL Expression in Mouse Embryonic Stem Cells Via H3K27me3 Modification and Histone H3.1/H3.2.

Chd5 is an essential factor for neuronal differentiation and spermatogenesis and is a known tumor suppressor. H3K27me3 and H3K4un are modifications recognized by Chd5; however, it remains unclear how Chd5 remodels chromatin structure. We completely disrupted the Chd5 locus using the CRISPR-Cas9 system to generate a 52 kbp long deletion and analyzed Chd5 function in mouse embryonic stem cells. Our findings show that Chd5 represses murine endogenous retrovirus-L (MuERV-L/MERVL), an endogenous retrovirus-derived retrotransposon, by regulating H3K27me3 and H3.1/H3.2 function.

Chd2 interacts with H3.3 to determine myogenic cell fate.

Cell differentiation is mediated by lineage-determining transcription factors. We show that chromodomain helicase DNA-binding domain 2 (Chd2), a SNF2 chromatin remodelling enzyme family member, interacts with MyoD and myogenic gene regulatory sequences to specifically mark these loci via deposition of the histone variant H3.3 prior to cell differentiation. Directed and genome-wide analysis of endogenous H3.3 incorporation demonstrates that knockdown of Chd2 prevents H3.3 deposition at differentiation-dependent, but not housekeeping, genes and inhibits myogenic gene activation. The data indicate that MyoD determines cell fate and facilitates differentiation-dependent gene expression through Chd2-dependent deposition of H3.3 at myogenic loci prior to differentiation.

Distribution of histone H4 modifications as revealed by a panel of specific monoclonal antibodies.

Post-translational histone modifications play a critical role in genome functions such as epigenetic gene regulation and genome maintenance. The tail of the histone H4 N-terminus contains several amino acids that can be acetylated and methylated. Some of these modifications are known to undergo drastic changes during the cell cycle. In this study, we generated a panel of mouse monoclonal antibodies against histone H4 modifications, including acetylation at K5, K8, K12, and K16, and different levels of methylation at K20. Their specificity was evaluated by ELISA and immunoblotting using synthetic peptide and recombinant proteins that harbor specific modifications or amino acid substitutions. Immunofluorescence confirmed the characteristic distributions of target modifications. An H4K5 acetylation (H4K5ac)-specific antibody CMA405 reacted with K5ac only when the neighboring K8 was unacetylated. This unique feature allowed us to detect newly assembled H4, which is diacetylated at K5 and K12, and distinguish it from hyperacetylated H4, where K5 and K8 are both acetylated. Chromatin immunoprecipiation combined with deep sequencing (ChIP-seq) revealed that acetylation of both H4K8 and H4K16 were enriched around transcription start sites. These extensively characterized and highly specific antibodies will be useful for future epigenetics and epigenome studies.

Relationship between the risk for a shrimp allergy and freshness or cooking.

Tropomyosins are defined as risk factors for shrimp allergy. However, their concentration in different preparations has not been clarified. We quantified the tropomyosin concentration in shrimp meat, which was cooked using several methods or was stored under various conditions. The results demonstrated that shrimp meat from various preparations and storage conditions maintained tropomyosin concentrations that were sufficient to cause food allergies.

A co-localization model of paired ChIP-seq data using a large ENCODE data set enables comparison of multiple samples.

Deep sequencing approaches, such as chromatin immunoprecipitation by sequencing (ChIP-seq), have been successful in detecting transcription factor-binding sites and histone modification in the whole genome. An approach for comparing two different ChIP-seq data would be beneficial for predicting unknown functions of a factor. We propose a model to represent co-localization of two different ChIP-seq data. We showed that a meaningful overlapping signal and a meaningless background signal can be separated by this model. We applied this model to compare ChIP-seq data of RNA polymerase II C-terminal domain (CTD) serine 2 phosphorylation with a large amount of peak-called data, including ChIP-seq and other deep sequencing data in the Encyclopedia of DNA Elements (ENCODE) project, and then extracted factors that were related to RNA polymerase II CTD serine 2 in HeLa cells. We further analyzed RNA polymerase II CTD serine 7 phosphorylation, of which their function is still unclear in HeLa cells. Our results were characterized by the similarity of localization for transcription factor/histone modification in the ENCODE data set, and this suggests that our model is appropriate for understanding ChIP-seq data for factors where their function is unknown.

Production of a Monoclonal Antibody for Histone H2b Isoform H2b3b.

H2b3b is one of the histone H2b isoforms that differs from canonical H2b by five to six amino acids. Previously, we identified H3t as the testis-specific histone H3 variant located in histone cluster 3, which is also the site of H2b3b. In this study, we produced monoclonal antibodies against H2b3b, using the iliac rat lymph node method for rat antibody and the immunochamber method for rabbit antibody. Immunoblot analysis confirmed that our antibodies could specifically discriminate between H2b3b and canonical H2b. Moreover, immunostaining revealed colocalization with a testicular stem cell marker, Plzf, but not with a meiotic marker, Sycp. This indicated that H2b3b is expressed in spermatogenic cells before meiosis. Our monoclonal antibodies enable further studies to reveal specific functions of H2b3b during spermatogenesis. We also hope that the established method will lead to the production of antibodies that can identify other H2b isoforms.

Plasma cell differentiation is regulated by the expression of histone variant H3.3.

The differentiation of B cells into plasma cells is associated with substantial transcriptional and epigenetic remodeling. H3.3 histone variant marks active chromatin via replication-independent nucleosome assembly. However, its role in plasma cell development remains elusive. Herein, we show that during plasma cell differentiation, H3.3 is downregulated, and the deposition of H3.3 and chromatin accessibility are dynamically changed. Blockade of H3.3 downregulation by enforced H3.3 expression impairs plasma cell differentiation in an H3.3-specific sequence-dependent manner. Mechanistically, enforced H3.3 expression inhibits the upregulation of plasma cell-associated genes such as Irf4, Prdm1, and Xbp1 and maintains the expression of B cell-associated genes, Pax5, Bach2, and Bcl6. Concomitantly, sustained H3.3 expression prevents the structure of chromatin accessibility characteristic for plasma cells. Our findings suggest that appropriate H3.3 expression and deposition control plasma cell differentiation.

The anti-tumor effect of trifluridine via induction of aberrant mitosis is unaffected by mutations modulating p53 activity.

The fluorinated thymidine analog trifluridine (FTD) is a chemotherapeutic drug commonly used to treat cancer; however, the mechanism by which FTD induces cytotoxicity is not fully understood. In addition, the effect of gain-of-function (GOF) missense mutations of the TP53 gene (encoding p53), which promote cancer progression and chemotherapeutic drug resistance, on the chemotherapeutic efficacy of FTD is unclear. Here, we revealed the mechanisms by which FTD-induced aberrant mitosis and contributed to cytotoxicity in both p53-null and p53-GOF missense mutant cells. In p53-null mutant cells, FTD-induced DNA double-stranded breaks, single-stranded DNA accumulation, and the associated DNA damage responses during the G2 phase. Nevertheless, FTD-induced DNA damage and the related responses were not sufficient to trigger strict G2/M checkpoint arrest. Thus, these features were carried over into mitosis, resulting in chromosome breaks and bridges, and subsequent cytokinesis failure. Improper mitotic exit eventually led to cell apoptosis, caused by the accumulation of extensive DNA damage and the presence of micronuclei encapsulated in the disrupted nuclear envelope. Upon FTD treatment, the behavior of the p53-GOF-missense mutant, isogenic cell lines, generated by CRISPR/Cas9 genome editing, was similar to that of p53-null mutant cells. Thus, our data suggest that FTD treatment overrode the effect on gene expression induced by p53-GOF mutants and exerted its anti-tumor activity in a manner that was independent of the p53 function.

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.

Contribution of structural reversibility to the heat stability of the tropomyosin shrimp allergen.

Tropomyosins are common heat-stable crustacean allergens. However, their heat stability and their effects on antigenicity have not been clarified. We purified tropomyosin in this study from raw kuruma prawns (Marsupenaeus japonicus) without heat processing. SDS-PAGE of the purified protein showed a band at approximately 35 kDa that cross-reacted with IgE from the serum of a shrimp-allergic patient, identifying it as Pen j 1. The circular dichroism spectrum of native Pen j 1 revealed the common α-helical structure of tropomyosins which easily collapsed upon heating to 80 °C. However, there were no insoluble aggregates after heating, and the protein regained its native CD spectral pattern after cooling to 25 °C. There was no significant difference in total IgG production between mice sensitized with native and heated Pen j 1. These results suggest that heat-denatured Pen j 1 refolded upon cooling and maintained its antigenicity following the heat treatment.