SETD2 epidermal deficiency promotes cutaneous wound healing via activation of AKT/mTOR Signalling

ObjectivesCutaneous wound healing is one of the major medical problems worldwide. Epigenetic modifiers have been identified as important players in skin development, homeostasis and wound repair. SET domain–containing 2 (SETD2) is the only known histone H3K36 tri‐methylase; however, its role in skin wound healing remains unclear.Materials and MethodsTo elucidate the biological role of SETD2 in wound healing, conditional gene targeting was used to generate epidermis‐specific Setd2‐deficient mice. Wound‐healing experiments were performed on the backs of mice, and injured skin tissues were collected and analysed by haematoxylin and eosin (H&E) and immunohistochemical staining. In vitro, CCK8 and scratch wound‐healing assays were performed on Setd2‐knockdown and Setd2‐overexpression human immortalized keratinocyte cell line (HaCaT). In addition, RNA‐seq and H3K36me3 ChIP‐seq analyses were performed to identify the dysregulated genes modulated by SETD2. Finally, the results were validated in functional rescue experiments using AKT and mTOR inhibitors (MK2206 and rapamycin).ResultsEpidermis‐specific Setd2‐deficient mice were successfully established, and SETD2 deficiency resulted in accelerated re‐epithelialization during cutaneous wound healing by promoting keratinocyte proliferation and migration. Furthermore, the loss of SETD2 enhanced the scratch closure and proliferation of keratinocytes in vitro. Mechanistically, the deletion of Setd2 resulted in the activation of AKT/mTOR signalling pathway, while the pharmacological inhibition of AKT and mTOR with MK2206 and rapamycin, respectively, delayed wound closure.ConclusionsOur results showed that SETD2 loss promoted cutaneous wound healing via the activation of AKT/mTOR signalling.     

Cathepsin K deficiency promotes alveolar bone regeneration by promoting jaw bone marrow mesenchymal stem cells proliferation and differentiation via glycolysis pathway

ObjectivesTo clarify the possible role and mechanism of Cathepsin K (CTSK) in alveolar bone regeneration mediated by jaw bone marrow mesenchymal stem cells (JBMMSC).Materials and MethodsTooth extraction models of Ctsk knockout mice (Ctsk ^‐/‐) and their wildtype (WT) littermates were used to investigate the effect of CTSK on alveolar bone regeneration. The influences of deletion or inhibition of CTSK by odanacatib (ODN) on proliferation and osteogenic differentiation of JBMMSC were assessed by CCK‐8, Western blot and alizarin red staining. To explore the differently expressed genes, RNA from WT and Ctsk^‐/‐ JBMMSC was sent to RNA‐seq. ECAR, glucose consumption and lactate production were measured to identify the effect of Ctsk deficiency or inhibition on glycolysis. At last, we explored whether Ctsk deficiency or inhibition promoted JBMMSC proliferation and osteogenic differentiation through glycolysis.ResultsWe found out that Ctsk knockout could promote alveolar bone regeneration in vivo. In vitro, we confirmed that both Ctsk knockout and inhibition by ODN could promote proliferation of JBMMSC, up‐regulate expression of Runx2 and ALP, and enhance matrix mineralization. RNA‐seq results showed that coding genes of key enzymes in glycolysis were significantly up‐regulated in Ctsk^‐/‐ JBMMSC, and Ctsk deficiency or inhibition could promote glycolysis in JBMMSC. After blocking glycolysis by 3PO, the effect of Ctsk deficiency or inhibition on JBMMSC’s regeneration was blocked subsequently.ConclusionsOur findings revealed that Ctsk knockout or inhibition could promote alveolar bone regeneration by enhancing JBMMSC regeneration via glycolysis. These results shed new lights on the regulatory mechanism of CTSK on bone regeneration.     

ClC‐c regulates the proliferation of intestinal stem cells via the EGFR signalling pathway in Drosophila

ObjectivesAdult stem cells uphold a delicate balance between quiescent and active states, which is crucial for tissue homeostasis. Whereas many signalling pathways that regulate epithelial stem cells have been reported, many regulators remain unidentified.Materials and MethodsFlies were used to generate tissue‐specific gene knockdown and gene knockout. qRT‐PCR was used to assess the relative mRNA levels. Immunofluorescence was used to determine protein localization and expression patterns. Clonal analyses were used to observe the phenotype. RNA‐seq was used to screen downstream mechanisms.ResultsHere, we report a member of the chloride channel family, ClC‐c, which is specifically expressed in Drosophila intestinal stem/progenitor cells and regulates intestinal stem cell (ISC) proliferation under physiological conditions and upon tissue damage. Mechanistically, we found that the ISC loss induced by the depletion of ClC‐c in intestinal stem/progenitor cells is due to inhibition of the EGFR signalling pathway.ConclusionOur findings reveal an ISC‐specific function of ClC‐c in regulating stem cell maintenance and proliferation, thereby providing new insights into the functional links among the chloride channel family, ISC proliferation and tissue homeostasis.     

Periplaneta Americana extract inhibits osteoclastic differentiation in vitro

ObjectivesPeriplaneta americana extract (PAE) is proven to be promising in treating fever, wound healing, liver fibrosis, and cardiovascular disease. However, the role of PAE in skeletal disorders remains unclear. This study investigated whether PAE regulates osteoclastic differentiation in vitro via the culture using RAW264.7 cells and bone marrow derived macrophages (BMDMs).Materials and MethodsRAW264.7 cells and BMDMs were cultured and induced for osteoclastic differentiation supplementing with different concentrations of PAE (0, 0.1, 1, and 10 mg/mL). Cell counting kit‐8 (CCK‐8) assay was used to detect the cytotoxicity and cell proliferation. TRAP staining, actin ring staining, real‐time quantitative PCR (RT‐qPCR), and bone resorption activity test were performed to detect osteoclastic differentiation. RT‐qPCR and enzyme‐linked immunosorbent assay (ELISA) were conducted to assay the expression and secretion of inflammatory factors. RNA sequencing (RNA‐seq) and western blot analysis were carried out to uncover the underlying mechanism.ResultsCCK‐8 results showed that 10 mg/mL and a lower concentration of PAE did not affect cell proliferation. RT‐qPCR analysis verified that PAE down‐regulated the osteoclastic genes Nfatc1, Ctsk, and Acp5 in macrophages. Moreover, PAE restrained the differentiation, formation, and function of osteoclasts. Besides, RT‐qPCR and ELISA assays showed that PAE decreased inflammatory genes expression and reduced the secretion of inflammatory factors, including IL1β, IL6, and TNFα. Subsequent RNA‐seq analysis identified possible genes and signaling pathways of PAE‐mediated osteoclastogenesis suppression.ConclusionsOur study indicates that PAE has inhibitive effects on osteoclastogenesis and may be a potential therapeutic alternative for bone diseases.

Periplaneta americana extract (PAE), the animal medicine material extracted from the insects Periplaneta americana, is proven to possess a variety of pharmacological functions. However, the role of PAE in skeletal disorders remains unclear. In this study, we found that PAE decreased osteoclast genes expression Nfatc1, Ctsk, and Acp5 in macrophages. Besides, PAE restrained the differentiation, formation, and function of osteoclasts. Moreover, PAE suppressed the LPS‐induced inflammation. Subsequent RNA‐seq analysis identified the signaling pathways of PAE‐mediated osteoclastogenesis suppression. Our study indicated that PAE has inhibitive effects on osteoclastic differentiation and may be a potential therapeutic Chinese medicine for bone diseases.     

Abnormal microglial polarization induced by Arid1a deletion leads to neuronal differentiation deficits

ObjectiveMicroglia, the prototypical innate immune cells of the central nervous system (CNS), are highly plastic and assume their phenotypes dependent on intrinsically genetic, epigenetic regulation or extrinsically microenvironmental cues. Microglia has been recognized as key regulators of neural stem/progenitor cells (NSPCs) and brain functions. Chromatin accessibility is implicated in immune cell development and functional regulation. However, it is still unknown whether and how chromatin remodelling regulates the phenotypic plasticity of microglia and exerts what kind of effects on NSPCs.MethodsWe investigated the role of chromatin accessibility in microglia by deleting chromatin remodelling gene Arid1a using microglia‐specific Cx3cr1‐cre and Cx3cr1‐CreERT2 mice. RNA‐seq and ATAC‐seq were performed to dissect the molecular mechanisms. In addition, we examined postnatal M1/M2 microglia polarization and analysed neuronal differentiation of NSPCs. Finally, we tested the effects of microglial Arid1a deletion on mouse behaviours.ResultsIncreased chromatin accessibility upon Arid1a ablation resulted in enhanced M1 microglial polarization and weakened M2 polarization, which led to abnormal neurogenesis and anxiety‐like behaviours. Switching the polarization state under IL4 stimulation could rescue abnormal neurogenesis, supporting an essential role for chromatin remodeler ARID1A in balancing microglial polarization and brain functions.ConclusionsOur study identifies ARID1A as a central regulator of microglia polarization, establishing a mechanistic link between chromatin remodelling, neurogenesis and mouse behaviours, and highlights the potential development of innovative therapeutics exploiting the innate regenerative capacity of the nervous system.

Microglia are thought to play an essential role in differentiation and maturation of neural precursor cells into neurons. As the prototypical innate immune cells of the central nervous system (CNS), the balance of microglial polarization is regulated by genetic or epigenetic modulators. Our study showed enhancement of chromatin accessibility by the chromatin remodelling gene Arid1a deletion led to abnormal development of microglia, which shows heightened M1 microglial polarization and weakened M2 polarization. In addition, irregular polarization led to abnormal neurogenesis with a significant decrease of neural stem/progenitor cell differentiation into neurons.     

CellRegMap: a statistical framework for mapping context‐specific regulatory variants using scRNA‐seq

Single‐cell RNA sequencing (scRNA‐seq) enables characterizing the cellular heterogeneity in human tissues. Recent technological advances have enabled the first population‐scale scRNA‐seq studies in hundreds of individuals, allowing to assay genetic effects with single‐cell resolution. However, existing strategies to analyze these data remain based on principles established for the genetic analysis of bulk RNA‐seq. In particular, current methods depend on a priori definitions of discrete cell types, and hence cannot assess allelic effects across subtle cell types and cell states. To address this, we propose the Cell Regulatory Map (CellRegMap), a statistical framework to test for and quantify genetic effects on gene expression in individual cells. CellRegMap provides a principled approach to identify and characterize genotype–context interactions of known eQTL variants using scRNA‐seq data. This model‐based approach resolves allelic effects across cellular contexts of different granularity, including genetic effects specific to cell subtypes and continuous cell transitions. We validate CellRegMap using simulated data and apply it to previously identified eQTL from two recent studies of differentiating iPSCs, where we uncover hundreds of eQTL displaying heterogeneity of genetic effects across cellular contexts. Finally, we identify fine‐grained genetic regulation in neuronal subtypes for eQTL that are colocalized with human disease variants.     

Arid1a regulates neural stem/progenitor cell proliferation and differentiation during cortical development

ObjectiveNeurodevelopmental diseases are common disorders caused by the disruption of essential neurodevelopmental processes. Recent human exome sequencing and genome‐wide association studies have shown that mutations in the subunits of the SWI/SNF (BAF) complex are risk factors for neurodevelopmental diseases. Clinical studies have found that ARID1A (BAF250a) is the most frequently mutated SWI/SNF gene and its mutations lead to mental retardation and microcephaly. However, the function of ARID1A in brain development and its underlying mechanisms still remain elusive.MethodsThe present study used Cre/loxP system to generate an Arid1a conditional knockout mouse line. Cell proliferation, cell apoptosis and cell differentiation of NSPCs were studied by immunofluorescence staining. In addition, RNA‐seq and RT‐PCR were performed to dissect the molecular mechanisms of Arid1a underlying cortical neurogenesis. Finally, rescue experiments were conducted to evaluate the effects of Neurod1 or Fezf2 overexpression on the differentiation of NSPCs in vitro.ResultsConditional knockout of Arid1a reduces cortical thickness in the developing cortex. Arid1a loss of function inhibits the proliferation of radial glial cells, and increases cell death during late cortical development, and leads to dysregulated expression of genes associated with proliferation and differentiation. Overexpression of Neurod1 or Fezf2 in Arid1a cKO NSPCs rescues their neural differentiation defect in vitro.ConclusionsThis study demonstrates for the first time that Arid1a plays an important role in regulating the proliferation and differentiation of NSPCs during cortical development, and proposes several gene candidates that are worth to understand the pathological mechanisms and to develop novel interventions of neurodevelopment disorders caused by Arid1a mutations.     

Infusion of hESC derived Immunity‐and‐matrix regulatory cells improves cognitive ability in early‐stage AD mice

ObjectivesIn this study, we administered immunity‐and‐matrix regulatory cells (IMRCs) via tail vein (IV) and intracerebroventricular (ICV) injection to 3‐month‐old 5×FAD transgenic mice to assess the effects of IMRC transplantation on the behaviour and pathology of early‐stage Alzheimer''s disease (AD).Materials and methodsClinical‐grade human embryonic stem cell (hESC)‐derived IMRCs were produced under good manufacturing practice (GMP) conditions. Three‐month‐old 5×FAD mice were administered IMRCs via IV and ICV injection. After 3 months, the mice were subjected to behavioural tests and electrophysiological analysis to evaluate their cognitive function, memory ability and synaptic plasticity. The effect of IMRCs on amyloid‐beta (Aβ)‐related pathology was detected by thioflavin‐S staining and Western blot. Quantitative real‐time PCR, ELISA and immunostaining were used to confirm that IMRCs inhibit neuroinflammation. RNA‐seq analysis was performed to measure changes in gene expression and perform a pathway analysis in response to IMRC treatment.ResultsIMRC administration via tail vein injection significantly ameliorated cognitive deficits in early‐stage AD (5×FAD) mice. However, no significant change was observed in the characteristic pathology of AD in the ICV group. Plaque analysis revealed that IMRCs did not influence either plaque deposition or BACE1 expression. In addition, IMRCs inhibited inflammatory responses and reduced microglial activation in vivo.ConclusionsWe have shown that peripheral administration of IMRCs can ameliorate AD pathology and associated cognitive deficits.     

TATA and paused promoters active in differentiated tissues have distinct expression characteristics

Core promoter types differ in the extent to which RNA polymerase II (Pol II) pauses after initiation, but how this affects their tissue‐specific gene expression characteristics is not well understood. While promoters with Pol II pausing elements are active throughout development, TATA promoters are highly active in differentiated tissues. We therefore used a genomics approach on late‐stage Drosophila embryos to analyze the properties of promoter types. Using tissue‐specific Pol II ChIP‐seq, we found that paused promoters have high levels of paused Pol II throughout the embryo, even in tissues where the gene is not expressed, while TATA promoters only show Pol II occupancy when the gene is active. The promoter types are associated with different chromatin accessibility in ATAC‐seq data and have different expression characteristics in single‐cell RNA‐seq data. The two promoter types may therefore be optimized for different properties: paused promoters show more consistent expression when active, while TATA promoters have lower background expression when inactive. We propose that tissue‐specific genes have evolved to use two different strategies for their differential expression across tissues.