Genomic transcription factor binding site selection is edited by the chromatin remodeling factor CHD4.

Biologically precise enhancer licensing by lineage-determining transcription factors enables activation of transcripts appropriate to biological demand and prevents deleterious gene activation. This essential process is challenged by the millions of matches to most transcription factor binding motifs present in many eukaryotic genomes, leading to questions about how transcription factors achieve the exquisite specificity required. The importance of chromatin remodeling factors to enhancer activation is highlighted by their frequent mutation in developmental disorders and in cancer. Here, we determine the roles of CHD4 in enhancer licensing and maintenance in breast cancer cells and during cellular reprogramming. In unchallenged basal breast cancer cells, CHD4 modulates chromatin accessibility. Its depletion leads to redistribution of transcription factors to previously unoccupied sites. During cellular reprogramming induced by the pioneer factor GATA3, CHD4 activity is necessary to prevent inappropriate chromatin opening. Mechanistically, CHD4 promotes nucleosome positioning over GATA3 binding motifs to compete with transcription factor-DNA interaction. We propose that CHD4 acts as a chromatin proof-reading enzyme that prevents unnecessary gene expression by editing chromatin binding activities of transcription factors.

Plap-1 lineage tracing and single-cell transcriptomics reveal cellular dynamics in the periodontal ligament.

Periodontal tissue supports teeth in the alveolar bone socket via fibrous attachment of the periodontal ligament (PDL). The PDL contains periodontal fibroblasts and stem/progenitor cells, collectively known as PDL cells (PDLCs), on top of osteoblasts and cementoblasts on the surface of alveolar bone and cementum, respectively. However, the characteristics and lineage hierarchy of each cell type remain poorly defined. This study identified periodontal ligament associated protein-1 (Plap-1) as a PDL-specific extracellular matrix protein. We generated knock-in mice expressing CreERT2 and GFP specifically in Plap-1-positive PDLCs. Genetic lineage tracing confirmed the long-standing hypothesis that PDLCs differentiate into osteoblasts and cementoblasts. A PDL single-cell atlas defined cementoblasts and osteoblasts as Plap-1-Ibsp+Sparcl1+ and Plap-1-Ibsp+Col11a2+, respectively. Other populations, such as Nes+ mural cells, S100B+ Schwann cells, and other non-stromal cells, were also identified. RNA velocity analysis suggested that a Plap-1highLy6a+ cell population was the source of PDLCs. Lineage tracing of Plap-1+ PDLCs during periodontal injury showed periodontal tissue regeneration by PDLCs. Our study defines diverse cell populations in PDL and clarifies the role of PDLCs in periodontal tissue homeostasis and repair.

High-mobility group box-1 peptide ameliorates bronchopulmonary dysplasia by suppressing inflammation and fibrosis in a mouse model.

BACKGROUND: This study aimed to examine the effect of the HMGB1 peptide on Bronchopulmonary dysplasia (BPD)-related lung injury in a mouse model. RESULTS: HMGB1 peptide ameliorates lung injury by suppressing the release of inflammatory cytokines and decreasing soluble collagen levels in the lungs. Single-cell RNA sequencing showed that the peptide suppressed the hyperoxia-induced inflammatory signature in macrophages and the fibrotic signature in fibroblasts. These changes in the transcriptome were confirmed using protein assays. CONCLUSION: Systemic administration of HMGB1 peptide exerts anti-inflammatory and anti-fibrotic effects in a mouse model of BPD. This study provides a foundation for the development of new and effective therapies for BPD.

Generation of a recessive dystrophic epidermolysis bullosa mouse model with patient-derived compound heterozygous mutations.

Recessive dystrophic epidermolysis bullosa (RDEB) is an intractable genetic disease of the skin caused by mutations in the COL7A1 gene. The majority of patients with RDEB harbor compound heterozygous mutations-two distinct mutations on each chromosome-without any apparent hotspots in the COL7A1 mutation pattern. This situation has made it challenging to establish a reliable RDEB mouse model with mutations that accurately mimic the genomic background of patients. Here, we established an RDEB mouse model harboring patient-type mutations in a compound heterozygous manner, using the CRISPR-based genome-editing technology i-GONAD. We selected two mutations, c.5818delC and E2857X, that have frequently been identified in cohorts of Japanese patients with RDEB. These mutations were introduced into the mouse genome at locations corresponding to those identified in patients. Mice homozygous for the 5818delC mutation developed severe RDEB-like phenotypes and died immediately after birth, whereas E2857X homozygous mice did not have a shortened lifespan compared to wild-type mice. Adult E2857X homozygous mice showed hair abnormalities, syndactyly, and nail dystrophy; these findings indicate that E2857X is indeed pathogenic in mice. Mice with the c.5818delC/E2857X compound heterozygous mutation presented an intermediate phenotype between the c.5818delC and E2857X homozygous mice. Single-cell RNA sequencing further clarified that the intrafollicular keratinocytes in c.5818delC/E2857X compound heterozygous mice exhibited abnormalities in cell cycle regulation. The proposed strategy to produce compound heterozygous mice, in addition to the established mouse line, will facilitate research on RDEB pathogenesis to develop a cure for this devastating disease.

Synthesized HMGB1 peptide prevents the progression of inflammation, steatosis, fibrosis, and tumor occurrence in a non-alcoholic steatohepatitis mouse model.

AIM: Non-alcoholic steatohepatitis (NASH) with fibrosis eventually leads to cirrhosis and hepatocellular carcinoma. Thus, the development of therapies other than dietary restriction and exercise, particularly those that suppress steatosis and fibrosis of the liver and have a long-term beneficial effect, is necessary. We aimed to evaluate the therapeutic effects of the HMGB1 peptide synthesized from box A using the melanocortin-4 receptor-deficient (Mc4r-KO) NASH model mouse. METHODS: We performed short- and long-term administration of this peptide and evaluated the effects on steatosis, fibrosis, and carcinogenesis using Mc4r-KO mice. We also analyzed the direct effect of this peptide on macrophages and hepatic stellate cells in vitro and performed lipidomics and metabolomics techniques to evaluate the effect. RESULTS: Although this peptide did not show direct effects on macrophages and hepatic stellate cells in vitro, in the short-term administration model, we could confirm the reduction of liver damage, steatosis, and fibrosis progression. The results of lipidomics and metabolomics suggested that the peptide might ameliorate NASH by promoting lipolysis via the activation of fatty acid ß-oxidation and improving insulin resistance. In the long-term administration model, this peptide prevented progression to cirrhosis but retained the steatosis state, that is, the peptide prevents the progression to "burnt-out NASH." This peptide inhibited carcinogenesis by about one-third. CONCLUSION: This HMGB1 peptide can reduce liver damage, improve fibrosis and steatosis, and inhibit carcinogenesis, suggesting that the peptide would be a new treatment candidate for NASH and can contribute to the long-term prognosis for patients with NASH.

Single-cell transcriptome analysis of fractional CO2 laser efficiency in treating a mouse model of alopecia.

OBJECTIVES: Hair loss, including alopecia, is a common dermatological issue worldwide. At present, the application of fractional carbon dioxide (CO2 ) laser in the treatment of alopecia has been documented; however, the results vary between reports. These varying results may be due to the limited knowledge of cellular action in laser-irradiated skin. The objective of this study was to investigate the molecular and cellular mechanisms of laser treatment under effective conditions for hair cycle initiation. METHODS: A fractional CO2 laser was applied and optimized to initiate the hair cycle in a mouse model of alopecia. Several cellular markers were analyzed in the irradiated skin using immunofluorescence staining. Cellular populations and their comprehensive gene expression were analyzed using single-cell RNA sequencing and bioinformatics. RESULTS: The effective irradiation condition for initiating the hair cycle was found to be 15 mJ energy/spot, which generates approximately 500 µm depth columns, but does not penetrate the dermis, only reaching approximately 1 spot/mm2 . The proportion of macrophage clusters significantly increased upon irradiation, whereas the proportion of fibroblast clusters decreased. The macrophages strongly expressed C-C chemokine receptor type 2 (Ccr2), which is known to be a key signal for injury-induced hair growth. CONCLUSIONS: We found that fractional CO2 laser irradiation recruited Ccr2 positive macrophages, and induced hair regrowth in a mouse alopecia model. These findings may contribute to the development of stable and effective fractional laser irradiation conditions for human alopecia treatment.

Proteins That Read DNA Methylation.

Covalent modification of DNA via deposition of a methyl group at the 5' position on cytosine residues alters the chemical groups available for interaction in the major groove of DNA. This modification, thereby, alters the affinity and specificity of DNA-binding proteins; some of them favor interaction with methylated DNA, and others disfavor it. Molecular recognition of cytosine methylation by proteins often initiates sequential regulatory events that impact gene expression and chromatin structure. The known methyl-DNA-binding proteins have unique domains responsible for DNA methylation recognition: (1) the methyl-CpG-binding domain (MBD), (2) the SET- and RING finger-associated domain (SRA), and (3) some of TF families, such as the C2H2 zinc finger domain, basic helix-loop-helix (bHLH), basic leucine-zipper (bZIP), and homeodomain proteins. Structural analyses have revealed that each domain has a characteristic methylated DNA-binding pattern, and the difference in the recognition mechanisms renders the DNA methylation mark able to transmit complicated biological information. Recent genetic and genomic studies have revealed novel functions of methyl-DNA-binding proteins. These emerging data have also provided glimpses into how methyl-DNA-binding proteins possess unique features and, presumably, functions. In this chapter, we summarize structural and biochemical analyses elucidating the mechanisms for recognition of DNA methylation and correlate this information with emerging genomic and functional data.

A Novel Quantitative Evaluation of Bone Formation After Opening Wedge High Tibial Osteotomy Using Tomosynthesis.

This study aimed to establish and validate a novel evaluation method using digital tomosynthesis to quantify bone formation in the gap after opening wedge high tibial osteotomy (OW-HTO). We retrospectively analyzed bone formation in the gap in 22 patients who underwent OW-HTO using digital tomosynthesis at 1, 2, 3, 6, 9, and 12 months postoperatively. Bone formation was semi-quantitatively assessed using the modified van Hemert's score and density measurements on digital tomosynthesis images. The gap filling value (GFV) was calculated as the ratio of the intensities of the opening gap and the tibial shaft. In addition, the relationship between the modified van Hemert's score and GFV was evaluated. The reproducibility of GFV had an interclass correlation coefficient (ICC [1,2]) of 0.958 for intraobserver reliability and an ICC (2,1) of 0.975 for interobserver reliability. The GFV increased in a time-dependent manner and was moderately correlated with the modified van Hemert's score (r = 0.630, p < 0.001). The GFV plateaued at 6 months postoperatively. In addition, the GFV was higher in patients with a modified van Hemert's score of 2 than in patients with a modified van Hemert's score of 3 (p = 0.008). The GFVs obtained using digital tomosynthesis can be used to assess postoperative bone formation in the opening gap after OW-HTO with high accuracy and reproducibility.

Controlled induction of immune tolerance by mesenchymal stem cells transferred by maternal microchimerism.

Feto-maternal immune tolerance is established during pregnancy; however, its mechanism and maintenance remain underexplored. Here, we investigated whether mesenchymal stem/stromal cells (MSCs) as non-inherited maternal antigens (NIMAs) transferred by maternal microchimerism could induce immune tolerance. We showed that MSCs had a potential equivalent to hematopoietic stem and progenitor cells (HSPCs) to induce immune tolerance and that MSCs were essential to induce tolerance to MSC-specific antigens. Furthermore, we demonstrated that MSCs as NIMAs transferred by maternal microchimerism could induce robust immune tolerance that can be further enhanced using a drug. Our data shed light on induction of immune tolerance and serve as a foundation to develop new therapies using maternally derived cells for autoimmune or genetic diseases.

Contribution of PDGFRα lineage cells in adult mouse hematopoiesis.

Platelet-derived growth factor receptor alpha (PDGFRα) is a dominant marker of mesodermal mesenchymal cells in mice. Previous studies demonstrated that PDGFRα-positive (PDGFRα+) mesodermal cells develop not only into mesenchymal cells but also into a subset of total hematopoietic cells (HCs) in the limited period during mouse embryogenesis. However, the precise characteristics of the PDGFRα lineage positive (PDGFRα Lin+) HCs in adult mouse hematopoiesis are largely unknown. In this study, we systematically evaluated the characteristics of PDGFRα Lin+ HCs in the bone marrow and peripheral blood using PDGFRα-CRE; ROSAtdTomato mice. Flow cytometry analysis revealed that PDGFRα Lin+ HCs accounted for approximately 20% of total HCs in both the bone marrow and peripheral blood in adult mice. Compositions of myeloid and lymphoid subpopulations among CD45+ mononuclear cells were almost identical in both PDGFRα Lin+ and PDGFRα Lin- cells. Single-cell RNA-sequencing analysis also demonstrated that the transcriptomic signatures of the PDGFRα Lin+ HCs in the peripheral blood largely overlapped with those of the PDGFRα Lin- HCs, suggesting equivalent functions of the PDGFRα Lin+ and PDGFRα Lin- HCs. Although pathophysiological activities of the PDGFRα Lin + HCs were not evaluated, our data clearly demonstrate a significant role of the PDGFRα Lin + HCs in physiological hematopoiesis in adult mice.

CHD4 and the NuRD complex directly control cardiac sarcomere formation.

Cardiac development relies on proper cardiomyocyte differentiation, including expression and assembly of cell-type-specific actomyosin subunits into a functional cardiac sarcomere. Control of this process involves not only promoting expression of cardiac sarcomere subunits but also repressing expression of noncardiac myofibril paralogs. This level of transcriptional control requires broadly expressed multiprotein machines that modify and remodel the chromatin landscape to restrict transcription machinery access. Prominent among these is the nucleosome remodeling and deacetylase (NuRD) complex, which includes the catalytic core subunit CHD4. Here, we demonstrate that direct CHD4-mediated repression of skeletal and smooth muscle myofibril isoforms is required for normal cardiac sarcomere formation, function, and embryonic survival early in gestation. Through transcriptomic and genome-wide analyses of CHD4 localization, we identified unique CHD4 binding sites in smooth muscle myosin heavy chain, fast skeletal α-actin, and the fast skeletal troponin complex genes. We further demonstrate that in the absence of CHD4, cardiomyocytes in the developing heart form a hybrid muscle cell that contains cardiac, skeletal, and smooth muscle myofibril components. These misexpressed paralogs intercalate into the nascent cardiac sarcomere to disrupt sarcomere formation and cause impaired cardiac function in utero. These results demonstrate the genomic and physiological requirements for CHD4 in mammalian cardiac development.

Cut-C: cleavage under tethered nuclease for conformational capture.

BACKGROUND: Deciphering the 3D structure of the genome is essential for elucidating the regulatory mechanisms of gene expression in detail. Existing methods, such as chromosome conformation capture (3C) and Hi-C have enabled the identification of novel aspects of chromatin structure. Further identification of protein-centric chromatin conformation is enabled by coupling the Hi-C procedure with a conventional chromatin immunoprecipitation assay. However, these methods are time-consuming and require independent methods for validation. RESULTS: To simultaneously identify protein-centric chromatin conformation and target protein localization, we have developed Cut-C, a method that combines antibody-mediated cleavage by tethered nuclease with chromosome conformation capture to identify chromatin interactions mediated by a protein of interest. Applying Cut-C to H3K4me3, a histone modification enriched at active gene promoters, we have successfully identified chromatin loops mediated by H3K4me3 along with the genome-wide distribution of H3K4me3. Cut-C also identified chromatin loops mediated by CTCF, validating the general applicability of the method. CONCLUSIONS: Cut-C identifies protein-centric chromatin conformations along with the genome-wide distribution of target proteins using simple procedures. The simplified protocol will improve the efficiency of analysing chromatin conformation using precious materials, such as clinical samples.

Transcriptionally distinct mesenchymal stem/stromal cells circulate in fetus.

Umbilical cord blood contains mesenchymal stem/stromal cells (MSCs) in addition to hematopoietic stem cells, serving as an attractive tool for regenerative medicine. As umbilical cord blood originates from fetus, abundant MSCs are expected to circulate in fetus. However, the properties of circulating MSCs in fetus have not been fully examined. In the present study, we aimed to analyze circulating MSCs, marked by the expression of platelet-derived growth factor receptor α (PDGFRα), during fetal development. Using PDGFRα GFP knock-in mice, we quantified the number of circulating PDGFRα positive MSCs during development. We further performed whole transcriptome analysis of circulating MSCs at single cell levels. We found that abundant PDGFRα positive cells circulate in embryo and diminish immediately after birth. In addition, single cell RNA-sequencing revealed transcriptional heterogeneity of MSCs in fetal circulation. These data lay a foundation to analyze the function of circulating MSCs during development.

DNA Methylation Changes in Tbx3 in a Mouse Model Exposed to Polybrominated Diphenyl Ethers.

DE-71, a commercial mixture of polybrominated diphenyl ethers widely used in flame retardants, is a pervasive environmental contaminant due to its continuing release from waste material and its long half-life in humans. Although the genotoxic potential of DE-71 appears to be low based on bacterial mutagenicity, it remains a public health concern due to its reported involvement in tumor development. Molecular mechanisms by which DE-71 influences tumor incidence or progression remain understudied. We used liver carcinoma tissue from mice exposed to DE-71 to test the hypothesis that epigenetic alterations consistent with tumor development, specifically DNA methylation, result from long-term DE-71 exposure. We profiled DNA methylation status using the methylated-CpG island recovery assay coupled with microarray analysis of hepatocellular carcinoma DNA from animals exposed to DE-71. DE-71 exposure had little impact on global DNA methylation. However, we detected gene body-specific hypomethylation within the Tbx3 locus, a transcription factor important in liver tumorigenesis and in embryonic and cancer stem cell proliferation. This nonpromoter hypomethylation was accompanied by upregulation of Tbx3 mRNA and protein and by alterations in downstream cell cycle-associated marker expression. Thus, exposure to DE-71 may facilitate tumor development by inducing epigenetic programs that favor expansion of progenitor cell populations.

Proteins That Read DNA Methylation.

Covalent modification of DNA via deposition of a methyl group at the 5' position on cytosine residues alters the chemical groups available for interaction in the major groove of DNA. The information content inherent in this modification alters the affinity and the specificity of DNA binding; some proteins favor interaction with methylated DNA, and others disfavor it. Molecular recognition of cytosine methylation by proteins often initiates sequential regulatory events which impact gene expression and chromatin structure. The known methyl-DNA-binding proteins have unique domains responsible for DNA methylation recognition: (1) the methyl-CpG-binding domain (MBD), (2) the C2H2 zinc finger domain, and (3) the SET- and RING finger-associated (SRA) domain. Structural analyses have revealed that each domain has a characteristic methylated DNA-binding pattern, and this difference in the recognition mechanism renders the DNA methylation mark able to transmit complicated biological information. Recent genetic and genomic studies have revealed novel functions of methyl-DNA-binding proteins. These emerging data have also provided glimpses into how methyl-DNA-binding proteins possess unique features and, presumably, functions. In this review, we summarize structural and biochemical analyses elucidating the mechanism for recognition of DNA methylation and correlate this information with emerging genomic and functional data.

MBD3 localizes at promoters, gene bodies and enhancers of active genes.

The Mi-2/nucleosome remodeling and histone deacetylase (NuRD) complex is a multiprotein machine proposed to regulate chromatin structure by nucleosome remodeling and histone deacetylation activities. Recent reports describing localization of NuRD provide new insights that question previous models on NuRD action, but are not in complete agreement. Here, we provide location analysis of endogenous MBD3, a component of NuRD complex, in two human breast cancer cell lines (MCF-7 and MDA-MB-231) using two independent genomic techniques: DNA adenine methyltransferase identification (DamID) and ChIP-seq. We observed concordance of the resulting genomic localization, suggesting that these studies are converging on a robust map for NuRD in the cancer cell genome. MBD3 preferentially associated with CpG rich promoters marked by H3K4me3 and showed cell-type specific localization across gene bodies, peaking around the transcription start site. A subset of sites bound by MBD3 was enriched in H3K27ac and was in physical proximity to promoters in three-dimensional space, suggesting function as enhancers. MBD3 enrichment was also noted at promoters modified by H3K27me3. Functional analysis of chromatin indicated that MBD3 regulates nucleosome occupancy near promoters and in gene bodies. These data suggest that MBD3, and by extension the NuRD complex, may have multiple roles in fine tuning expression for both active and silent genes, representing an important step in defining regulatory mechanisms by which NuRD complex controls chromatin structure and modification status.

Inhibition of histone binding by supramolecular hosts.

The tandem PHD (plant homeodomain) fingers of the CHD4 (chromodomain helicase DNA-binding protein 4) ATPase are epigenetic readers that bind either unmodified histone H3 tails or H3K9me3 (histone H3 trimethylated at Lys9). This dual function is necessary for the transcriptional and chromatin remodelling activities of the NuRD (nucleosome remodelling and deacetylase) complex. In the present paper, we show that calixarene-based supramolecular hosts disrupt binding of the CHD4 PHD2 finger to H3K9me3, but do not affect the interaction of this protein with the H3K9me0 (unmodified histone H3) tail. A similar inhibitory effect, observed for the association of chromodomain of HP1γ (heterochromatin protein 1γ) with H3K9me3, points to a general mechanism of methyl-lysine caging by calixarenes and suggests a high potential for these compounds in biochemical applications. Immunofluorescence analysis reveals that the supramolecular agents induce changes in chromatin organization that are consistent with their binding to and disruption of H3K9me3 sites in living cells. The results of the present study suggest that the aromatic macrocyclic hosts can be used as a powerful new tool for characterizing methylation-driven epigenetic mechanisms.

PDGFRalpha-positive cells in bone marrow are mobilized by high mobility group box 1 (HMGB1) to regenerate injured epithelia.

The role of bone marrow cells in repairing ectodermal tissue, such as skin epidermis, is not clear. To explore this process further, this study examined a particular form of cutaneous repair, skin grafting. Grafting of full thickness wild-type mouse skin onto mice that had received a green fluorescent protein-bone marrow transplant after whole body irradiation led to an abundance of bone marrow-derived epithelial cells in follicular and interfollicular epidermis that persisted for at least 5 mo. The source of the epithelial progenitors was the nonhematopoietic, platelet-derived growth factor receptor α-positive (Lin(-)/PDGFRα(+)) bone marrow cell population. Skin grafts release high mobility group box 1 (HMGB1) in vitro and in vivo, which can mobilize the Lin(-)/PDGFRα(+) cells from bone marrow to target the engrafted skin. These data provide unique insight into how skin grafts facilitate tissue repair and identify strategies germane to regenerative medicine for skin and, perhaps, other ectodermal defects or diseases.

High-mobility group box 1 fragment ameliorates chronic pancreatitis induced by caerulein in mice.

BACKGROUND: Chronic pancreatitis (CP) is a progressive disease characterized by pancreatic fibrosis for which effective treatment options are lacking. Mesenchymal stem cells (MSCs) have shown potential for fibrosis treatment but face limitations in clinical application. The high-mobility group box 1 (HMGB1) fragment mobilizes MSCs from bone marrow into the blood and has emerged as a promising therapeutic agent for tissue regeneration in various pathological conditions. The aim of this study was to investigate the potential therapeutic effects of systemic administration of the HMGB1 fragment in a mouse model of CP. METHODS: A caerulein-induced CP mouse model was used, and the HMGB1 fragment was administered by tail vein injection. Parameters such as body weight, pancreatic tissue damage, fibrosis, inflammatory cytokine expression, and collagen-related gene expression were evaluated using various assays, including immunohistochemistry, real-time PCR, serum analysis, and single-cell transcriptome analysis. And the migration of MSCs to the pancreas was evaluated using the parabiosis model. RESULTS: Administration of the HMGB1 fragment was associated with significant improvements in pancreatic tissue damage and fibrosis. It suppressed the expression of inflammatory cytokines and activated platelet-derived growth factor receptor-α+ MSCs, leading to their accumulation in the pancreas. The HMGB1 fragment also shifted gene expression patterns associated with pancreatic fibrosis toward those of the normal pancreas. Systemic administration of the HMGB1 fragment demonstrated therapeutic efficacy in attenuating pancreatic tissue damage and fibrosis in a CP mouse model. CONCLUSION: These findings highlight the potential of the HMGB1 fragment as a therapeutic target for the treatment of CP.