Nan Yang

Skeletal muscle is a complex heterogeneous tissue and characterizing its cellular heterogeneity and transcriptional and epigenetic signatures are important for understanding the details of its ontogeny. In our study, we applied scRNA‐seq and scATAC‐seq to investigate the cell types, molecular features, transcriptional and epigenetic regulation, and patterns of developing bovine skeletal muscle from gestational, lactational and adult stages. Detailed molecular analyses were used to dissect cellular heterogeneity, and we deduced the differentiation trajectory of myogenic cells and uncovered their dynamic gene expression profiles. SCENIC analysis was performed to demonstrate key regulons during cell fate decisions. We explored the future expression states of these heterogeneous cells by RNA velocity analysis and found extensive networks of intercellular communication using the toolkit CellChat. Moreover, the transcriptomic and chromatin accessibility modalities were confirmed to be highly concordant, and integrative analysis of chromatin accessibility and gene expression revealed key transcriptional regulators acting during myogenesis. In bovine skeletal muscle, by scRNA‐seq and scATAC‐seq analysis, different cell types such as adipocytes, endothelial cells, fibroblasts, lymphocytes, monocytes, pericyte cells and eight skeletal myogenic subpopulations were identified at the three developmental stages. The pseudotime trajectory exhibited a distinct sequential ordering for these myogenic subpopulations and eight distinct gene clusters were observed according to their expression pattern. Moreover, specifically expressed TFs (such as MSC, MYF5, MYOD1, FOXP3, ESRRA, BACH1, SIX2 and ATF4) associated with muscle development were predicted, and likely future transcriptional states of individual cells and the developmental dynamics of differentiation among neighbouring cells were predicted. CellChat analysis on the scRNA‐seq data set then classified many ligand–receptor pairs among these cell clusters, which were further categorized into significant signalling pathways, including BMP, IGF, WNT, MSTN, ANGPTL, TGFB, TNF, VEGF and FGF. Finally, scRNA‐seq and scATAC‐seq results were successfully integrated to reveal a series of specifically expressed TFs that are likely to be candidates for the promotion of cell fate transition during bovine skeletal muscle development. Overall, our results outline a single‐cell dynamic chromatin/transcriptional landscape for normal bovine skeletal muscle development; these provide an important resource for understanding the structure and function of mammalian skeletal muscle, which will promote research into its biology.

Significance: Tissue fibrotic diseases, for example of the liver and lung, represent a huge unmet medical need. In this study, using single-cell RNA sequencing, cytometry by time of flight (CyTOF), tissue imaging, and functional assays, we identify a complex vascular niche in Dupuytren’s disease (DD), a common localized fibrotic condition of the palm, where early-disease-stage tissue can be accessed readily. We uncover a population of myofibroblast precursors within the pericyte compartment and demonstrate that the endothelium instructs the differentiation of functionally distinct stromal cells, thereby orchestrating discrete microenvironments in the fibrotic milieu. Together, these findings provide a basis for the concept of targeting blood vessel signaling to control the progression of human fibrosis.

Fibrotic diseases remain a major cause of morbidity and mortality, yet there are few effective therapies. The underlying pathology of all fibrotic conditions is the activity of myofibroblasts. Using cells from freshly excised disease tissue from patients with Dupuytren’s disease (DD), a localized fibrotic disorder of the palm, we sought to identify new therapeutic targets for fibrotic disease. We hypothesized that the persistent activity of myofibroblasts in fibrotic diseases might involve epigenetic modifications. Using a validated genetics-led target prioritization algorithm (Pi) of genome wide association studies (GWAS) data and a broad screen of epigenetic inhibitors, we found that the acetyltransferase CREBBP/EP300 is a major regulator of contractility and extracellular matrix production via control of H3K27 acetylation at the profibrotic genes, ACTA2 and COL1A1. Genomic analysis revealed that EP300 is highly enriched at enhancers associated with genes involved in multiple profibrotic pathways, and broad transcriptomic and proteomic profiling of CREBBP/EP300 inhibition by the chemical probe SGC-CBP30 identified collagen VI (Col VI) as a prominent downstream regulator of myofibroblast activity. Targeted Col VI knockdown results in significant decrease in profibrotic functions, including myofibroblast contractile force, extracellular matrix (ECM) production, chemotaxis, and wound healing. Further evidence for Col VI as a major determinant of fibrosis is its abundant expression within Dupuytren’s nodules and also in the fibrotic foci of idiopathic pulmonary fibrosis (IPF). Thus, Col VI may represent a tractable therapeutic target across a range of fibrotic disorders. Significance Fibrosis remains a major unmet medical need as therapeutic targets discovered in animal models have failed to translate. A major challenge for identifying novel targets is the limited availability of early-stage human disease tissue. Here, we utilize Dupuytren’s disease (DD), a common localized fibrotic disorder, to evaluate the impact of epigenetic regulation of myofibroblasts and identify potential tractable targets in human fibrosis. We demonstrate that the epigenetic regulator CREBBP/EP300 is a critical determinant of the profibrotic phenotype. Furthermore, we identify collagen VI to be a key downstream target of CREBBP/EP300 and reveal valuable insights in the role it plays in key profibrotic functions, including contractile force, chemotaxis, and wound healing, and hence its potential as a therapeutic target.

Evolutionarily conserved circadian clocks generate 24-hour rhythms in physiology and behaviour that adapt organisms to their daily and seasonal environments. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is the principal co-ordinator of the cell-autonomous clocks distributed across all major tissues. The importance of robust daily rhythms is highlighted by experimental and epidemiological associations between circadian disruption and human diseases. BMAL1 (a bHLH-PAS domain-containing transcription factor) is the master positive regulator within the transcriptional-translational feedback loops (TTFLs) that cell-autonomously define circadian time. It drives transcription of the negative regulators Period and Cryptochrome alongside numerous clock output genes, and thereby powers circadian time-keeping. Because deletion of Bmal1 alone is sufficient to eliminate circadian rhythms in cells and the whole animal it has been widely used as a model for molecular disruption of circadian rhythms, revealing essential, tissue-specific roles of BMAL1 in, for example, the brain, liver and the musculoskeletal system. Moreover, BMAL1 has clock-independent functions that influence ageing and protein translation. Despite the essential role of BMAL1 in circadian time-keeping, direct measures of its intra-cellular behaviour are still lacking. To fill this knowledge-gap, we used CRISPR Cas9 to generate a mouse expressing a knock-in fluorescent fusion of endogenous BMAL1 protein (Venus::BMAL1) for quantitative live imaging in physiological settings. The Bmal1^Venus mouse model enabled us to visualise and quantify the daily behaviour of this core clock factor in central (SCN) and peripheral clocks, with single-cell resolution that revealed its circadian expression, anti-phasic to negative regulators, nuclear-cytoplasmic mobility and molecular abundance. Cell-autonomous circadian clocks are transcriptional/translational feedback loops that co-ordinate almost all mammalian physiology and behaviour. Although their genetic basis is well understood, we are largely ignorant of the natural behaviour of clock proteins and how they work within these loops. This is particularly true for the essential transcriptional activator BMAL1. To address this, we created and validated a mouse carrying a fully functional knock-in allele that encodes a fluorescent fusion of BMAL1 (Venus::BMAL1). Quantitative live imaging in tissue explants and cells, including the central clock of the suprachiasmatic nucleus (SCN), revealed the circadian expression, nuclear-cytoplasmic mobility, fast kinetics and surprisingly low molecular abundance of endogenous BMAL1, providing significant quantitative insights into the intracellular mechanisms of circadian timing at single-cell resolution.

Objective: To investigate the impact of JTXK granule on the miRNA expression profiles in hepatic tissue of diabetic mice, and to explore the molecular targets and associated signaling pathways of JTXK granule in its anti-diabetic effect. Methods: Eight mice were randomly selected as normal group fed with chow diet. Then high fat diet was used to induce diabetic model, and the mice were subsequently divided into JTXK-treated group (J group, n = 6) and model group (M group, n = 6). After 8 weeks’ intervention we examined the fasting blood glucose and observed the histopathologic changes in hepatic tissue between these two groups. Next we screened the differentially expressed miRNAs between the two groups using microRNA sequencing analysis. Finally, miRNA target gene prediction, GO and KEGG analysis were applied to explore the function of DEMs. Results: The blood glucose level in J group was significantly lower than M group (P < 0.05). The results from H&E staining showed that the arrangement and structure of hepatocytes from J group were basically normal with fewer ballooning degeneration and less inflammatory cell infiltration. Furthermore, a total of 33 significantly differentiated miRNAs were detected in comparison between the two groups (| log2(fold change) | >0.3, P < 0.05). MiRNA–mRNA analysis showed that mmu-miR-30a-5p, mmu-miR-23b-5p, mmu-miR-199a-5p, mmu-miR-425-5p, and mmu-miR-214-3p are closely related to inflammatory response, histological changes and insulin signal transduction in liver. In addition, KEGG analysis showed that the DEMs were closely related to Ras and insulin signaling pathway. Conclusion: JTXK granule exerts anti-diabetic effect in hepatic tissue of diabetic mice by modulating miRNAs and mRNAs network.

Circadian rhythms maintain tissue homeostasis during the 24-h day-night cycle. Cell-autonomous circadian clocks play fundamental roles in cell division, DNA damage responses and metabolism. Circadian disruptions have been proposed as a contributing factor for cancer initiation and progression, although definitive evidence for altered molecular circadian clocks in cancer is still lacking. In this study, we looked at circadian clocks in breast cancer. We isolated primary tumours and normal tissues from the same individuals who had developed breast cancer with no metastases. We assessed circadian clocks within primary cells of the patients by lentiviral expression of circadian reporters, and the levels of clock genes in tissues by qPCR. We histologically examined collagen organisation within the normal and tumour tissue areas, and probed the stiffness of the stroma adjacent to normal and tumour epithelium using atomic force microscopy. Epithelial ducts were disorganised within the tumour areas. Circadian clocks were altered in cultured tumour cells. Tumour regions were surrounded by stroma with an altered collagen organisation and increased stiffness. Levels of Bmal1 messenger RNA (mRNA) were significantly altered in the tumours in comparison to normal epithelia. Circadian rhythms are suppressed in breast tumour epithelia in comparison to the normal epithelia in paired patient samples. This correlates with increased tissue stiffness around the tumour region. We suggest possible involvement of altered circadian clocks in the development and progression of breast cancer.

The circadian clock is an autonomous molecular feedback loop inside almost every cell in the body. We have shown that the mammary epithelial circadian clock is regulated by the cellular microenvironment. Moreover, a stiff extracellular matrix dampens the oscillations of the epithelial molecular clock. Here, we extend this analysis to other tissues and cell types, and identify an inverse relationship between circadian clocks in epithelia and fibroblasts. Epithelial cells from mammary gland, lung and skin have significantly stronger oscillations of clock genes in soft 3D microenvironments, compared to stiff 2D environments. Fibroblasts isolated from the same tissues show the opposite response, exhibiting stronger oscillations and more prolonged rhythmicity in stiff microenvironments. RNA analysis identified that a subset of mammary epithelial clock genes, and their regulators, are upregulated in 3D microenvironments in soft compared to stiff gels. Furthermore, the same genes are inversely regulated in fibroblasts isolated from the same tissues. Thus, our data reveal for the first time an intrinsic difference in the regulation of circadian genes in epithelia and fibroblasts.

Objectives The circadian clocks are internal timing mechanisms that drive ∼24-hour rhythms in a tissue-specific manner. Many aspects of the physiology of the intervertebral disc (IVD) show clear diurnal rhythms. However, it is unknown whether IVD tissue contains functional circadian clocks and if so, how their dysregulation is implicated in IVD degeneration. Methods Clock gene dynamics in ex vivo IVD explants (from PER2:: luciferase (LUC) reporter mice) and human disc cells (transduced with lentivirus containing Per2::luc reporters) were monitored in real time by bioluminescence photon counting and imaging. Temporal gene expression changes were studied by RNAseq and quantitative reverse transcription (qRT)-PCR. IVD pathology was evaluated by histology in a mouse model with tissue-specific deletion of the core clock gene Bmal1. Results Here we show the existence of the circadian rhythm in mouse IVD tissue and human disc cells. This rhythm is dampened with ageing in mice and can be abolished by treatment with interleukin-1β but not tumour necrosis factor α. Time-series RNAseq revealed 607 genes with 24-hour patterns of expression representing several essential pathways in IVD physiology. Mice with conditional knockout of Bmal1 in their disc cells demonstrated age-related degeneration of IVDs. Conclusions We have established autonomous circadian clocks in mouse and human IVD cells which respond to age and cytokines, and control key pathways involved in the homeostasis of IVDs. Genetic disruption to the mouse IVD molecular clock predisposes to IVD degeneration. These results support the concept that disruptions to circadian rhythms may be a risk factor for degenerative IVD disease and low back pain.

Circadian clocks drive ∼24 h rhythms in tissue physiology. They rely on transcriptional/translational feedback loops driven by interacting networks of clock complexes. However, little is known about how cell-intrinsic circadian clocks sense and respond to their microenvironment. Here, we reveal that the breast epithelial clock is regulated by the mechano-chemical stiffness of the cellular microenvironment in primary cell culture. Moreover, the mammary clock is controlled by the periductal extracellular matrix in vivo, which contributes to a dampened circadian rhythm during ageing. Mechanistically, the tension sensing cell-matrix adhesion molecule, vinculin, and the Rho/ROCK pathway, which transduces signals provided by extracellular stiffness into cells, regulate the activity of the core circadian clock complex. We also show that genetic perturbation, or age-associated disruption of self-sustained clocks, compromises the self-renewal capacity of mammary epithelia. Thus, circadian clocks are mechano-sensitive, providing a potential mechanism to explain how ageing influences their amplitude and function.