Synovial Sarcoma Chromatin Dynamics Reveal a Continuum in SS18:SSX Reprograming.

Synovial sarcoma (SyS) is an aggressive soft-tissue malignancy characterized by a pathognomonic chromosomal translocation leading to the formation of the SS18::SSX fusion oncoprotein. SS18::SSX associates with mammalian BAF complexes suggesting deregulation of chromatin architecture as the oncogenic driver in this tumour type. To examine the epigenomic state of SyS we performed comprehensive multi-omics analysis on 52 primary pre-treatment human SyS tumours. Our analysis revealed a continuum of epigenomic states across the cohort at fusion target genes independent of rare somatic genetic lesions. We identify cell-of-origin signatures defined by enhancer states and reveal unexpected relationships between H2AK119Ub1 and active marks. The number of bivalent promoters, dually marked by the repressive H3K27me3 and activating H3K4me3 marks, has strong prognostic value and outperforms tumor grade in predicting patient outcome. Finally, we identify SyS defining epigenomic features including H3K4me3 expansion associated with striking promoter DNA hypomethylation in which SyS displays the lowest mean methylation level of any sarcoma subtype. We explore these distinctive features as potential vulnerabilities in SyS and identify H3K4me3 inhibition as a promising therapeutic strategy.

Single-cell analysis of mesenchymal cells in permeable neural vasculature reveals novel diverse subpopulations of fibroblasts.

BACKGROUND: In the choroid plexus and pituitary gland, vasculature is known to have a permeable, fenestrated phenotype which allows for the free passage of molecules in contrast to the blood brain barrier observed in the rest of the CNS. The endothelium of these compartments, along with secretory, neural-lineage cells (choroid epithelium and pituitary endocrine cells) have been studied in detail, but less attention has been given to the perivascular mesenchymal cells of these compartments. METHODS: The Hic1CreERT2 Rosa26LSL-TdTomato mouse model was used in conjunction with a PdgfraH2B-EGFP mouse model to examine mesenchymal cells, which can be subdivided into Pdgfra+ fibroblasts and Pdgfra- pericytes within the choroid plexus (CP) and pituitary gland (PG), by histological, immunofluorescence staining and single-cell RNA-sequencing analyses. RESULTS: We found that both CP and PG possess substantial populations of distinct Hic1+ mesenchymal cells, including an abundance of Pdgfra+ fibroblasts. Within the pituitary, we identified distinct subpopulations of Hic1+ fibroblasts in the glandular anterior pituitary and the neurosecretory posterior pituitary. We also identified multiple distinct markers of CP, PG, and the meningeal mesenchymal compartment, including alkaline phosphatase, indole-n-methyltransferase and CD34. CONCLUSIONS: Novel, distinct subpopulations of mesenchymal cells can be found in permeable vascular interfaces, including the CP, PG, and meninges, and make distinct contributions to both organs through the production of structural proteins, enzymes, transporters, and trophic molecules.

Cell diversity and plasticity during atrioventricular heart valve EMTs.

Epithelial-to-mesenchymal transitions (EMTs) of both endocardium and epicardium guide atrioventricular heart valve formation, but the cellular complexity and small scale of this tissue have restricted analyses. To circumvent these issues, we analyzed over 50,000 murine single-cell transcriptomes from embryonic day (E)7.75 hearts to E12.5 atrioventricular canals. We delineate mesenchymal and endocardial bifurcation during endocardial EMT, identify a distinct, transdifferentiating epicardial population during epicardial EMT, and reveal the activation of epithelial-mesenchymal plasticity during both processes. In Sox9-deficient valves, we observe increased epithelial-mesenchymal plasticity, indicating a role for SOX9 in promoting endothelial and mesenchymal cell fate decisions. Lastly, we deconvolve cell interactions guiding the initiation and progression of cardiac valve EMTs. Overall, these data reveal mechanisms of emergence of mesenchyme from endocardium or epicardium at single-cell resolution and will serve as an atlas of EMT initiation and progression with broad implications in regenerative medicine and cancer biology.

Aberrant gene activation in synovial sarcoma relies on SSX specificity and increased PRC1.1 stability.

The SS18-SSX fusion drives oncogenic transformation in synovial sarcoma by bridging SS18, a member of the mSWI/SNF (BAF) complex, to Polycomb repressive complex 1 (PRC1) target genes. Here we show that the ability of SS18-SSX to occupy H2AK119ub1-rich regions is an intrinsic property of its SSX C terminus, which can be exploited by fusion to transcriptional regulators beyond SS18. Accordingly, SS18-SSX recruitment occurs in a manner that is independent of the core components and catalytic activity of BAF. Alternative SSX fusions are also recruited to H2AK119ub1-rich chromatin and reproduce the expression signatures of SS18-SSX by engaging with transcriptional activators. Variant Polycomb repressive complex 1.1 (PRC1.1) acts as the main depositor of H2AK119ub1 and is therefore required for SS18-SSX occupancy. Importantly, the SSX C terminus not only depends on H2AK119ub1 for localization, but also further increases it by promoting PRC1.1 complex stability. Consequently, high H2AK119ub1 levels are a feature of murine and human synovial sarcomas. These results uncover a critical role for SSX-C in mediating gene deregulation in synovial sarcoma by providing specificity to chromatin and further enabling oncofusion binding by enhancing PRC1.1 stability and H2AK119ub1 deposition.

Biallelic Dicer1 Mutations in the Gynecologic Tract of Mice Drive Lineage-Specific Development of DICER1 Syndrome-Associated Cancer.

DICER1 is an RNase III enzyme essential for miRNA biogenesis through cleaving precursor-miRNA hairpins. Germline loss-of-function DICER1 mutations underline the development of DICER1 syndrome, a rare genetic disorder that predisposes children to cancer development in organs such as lung, gynecologic tract, kidney, and brain. Unlike classical tumor suppressors, the somatic "second hit" in DICER1 syndrome-associated cancers does not fully inactivate DICER1 but impairs its RNase IIIb activity only, suggesting a noncanonical two-hit hypothesis. Here, we developed a genetically engineered conditional compound heterozygous Dicer1 mutant mouse strain that fully recapitulates the biallelic DICER1 mutations in DICER1 syndrome-associated human cancers. Crossing this tool strain with tissue-specific Cre strains that activate Dicer1 mutations in gynecologic tract cells at two distinct developmental stages revealed that embryonic biallelic Dicer1 mutations caused infertility in females by disrupting oviduct and endometrium development and ultimately drove cancer development. These multicystic tubal and intrauterine tumors histologically resembled a subset of DICER1 syndrome-associated human cancers. Molecular analysis uncovered accumulation of additional oncogenic events (e.g., aberrant p53 expression, Kras mutation, and Myc activation) in murine Dicer1 mutant tumors and validated miRNA biogenesis defects in 5P miRNA strand production, of which, loss of let-7 family miRNAs was identified as a putative key player in transcriptomic rewiring and tumor development. Thus, this DICER1 syndrome-associated cancer model recapitulates the biology of human cancer and provides a unique tool for future investigation and therapeutic development. SIGNIFICANCE: Generation of a Dicer1 mutant mouse model establishes the oncogenicity of missense mutations in the DICER1 RNase IIIb domain and provides a faithful model of DICER1 syndrome-associated cancer for further investigation.

Exogenously delivered iPSCs disrupt the natural repair response of endogenous MPCs after bone injury.

Promoting bone healing including fracture non-unions are promising targets for bone tissue engineering due to the limited success of current clinical treatment methods. There has been significant research on the use of stem cells with and without biomaterial scaffolds to treat bone fractures due to their promising regenerative capabilities. However, the relative roles of exogenous vs. endogenous stem cells and their overall contribution to in vivo fracture repair is not well understood. The purpose of this study was to determine the interaction between exogenous and endogenous stem cells during bone healing. This study was conducted using a standardized burr-hole bone injury model in a mesenchymal progenitor cell (MPC) lineage-tracing mouse under normal homeostatic and osteoporotic conditions. Burr-hole injuries were treated with a collagen-I biomaterial loaded with and without labelled induced pluripotent stem cells (iPSCs). Using lineage-tracing, the roles of exogenous and endogenous stem cells during bone healing were examined. It was observed that treatment with iPSCs resulted in muted healing compared to untreated controls in intact mice post-injury. When the cell populations were examined histologically, iPSC-treated burr-hole defects presented with a dramatic reduction in endogenous MPCs and cell proliferation throughout the injury site. However, when the ovaries were removed and an osteoporotic-like phenotype induced in the mice, iPSCs treatment resulted in increased bone formation relative to untreated controls. In the absence of iPSCs, endogenous MPCs demonstrated robust proliferative and osteogenic capacity to undertake repair and this behaviour was disrupted in the presence of iPSCs which instead took on an osteoblast fate but with little proliferation. This study clearly demonstrates that exogenously delivered cell populations can impact the normal function of endogenous stem/progenitor populations during the normal healing cascade. These interactions need to be better understood to inform cell and biomaterial therapies to treat fractures.

Hic1 identifies a specialized mesenchymal progenitor population in the embryonic limb responsible for bone superstructure formation.

The musculoskeletal system relies on the integration of multiple components with diverse physical properties, such as striated muscle, tendon, and bone, that enable locomotion and structural stability. This relies on the emergence of specialized, but poorly characterized, interfaces between these various elements during embryonic development. Within the appendicular skeleton, we show that a subset of mesenchymal progenitors (MPs), identified by Hic1, do not contribute to the primary cartilaginous anlagen but represent the MP population, whose progeny directly contribute to the interfaces that connect bone to tendon (entheses), tendon to muscle (myotendinous junctions), and the associated superstructures. Furthermore, deletion of Hic1 leads to skeletal defects reflective of deficient muscle-bone coupling and, consequently, perturbation of ambulation. Collectively, these findings show that Hic1 identifies a unique MP population that contributes to a secondary wave of bone sculpting critical to skeletal morphogenesis.

Cellular taxonomy of Hic1+ mesenchymal progenitor derivatives in the limb: from embryo to adult.

Tissue development and regeneration rely on the cooperation of multiple mesenchymal progenitor (MP) subpopulations. We recently identified Hic1 as a marker of quiescent MPs in multiple adult tissues. Here, we describe the embryonic origin of appendicular Hic1+ MPs and demonstrate that they arise in the hypaxial somite, and migrate into the developing limb at embryonic day 11.5, well after limb bud initiation. Time-resolved single-cell-omics analyses coupled with lineage tracing reveal that Hic1+ cells generate a unique MP hierarchy, that includes both recently identified adult universal fibroblast populations (Dpt+, Pi16+ and Dpt+ Col15a1+) and more specialised mesenchymal derivatives such as, peri and endoneurial cells, pericytes, bone marrow stromal cells, myotenocytes, tenocytes, fascia-resident fibroblasts, with limited contributions to chondrocytes and osteocytes within the skeletal elements. MPs endure within these compartments, continue to express Hic1 and represent a critical reservoir to support post-natal growth and regeneration.

Proteoglycan 4 (PRG4) treatment enhances wound closure and tissue regeneration.

The wound healing response is one of most primitive and conserved physiological responses in the animal kingdom, as restoring tissue integrity/homeostasis can be the difference between life and death. Wound healing in mammals is mediated by immune cells and inflammatory signaling molecules that regulate tissue resident cells, including local progenitor cells, to mediate closure of the wound through formation of a scar. Proteoglycan 4 (PRG4), a protein found throughout the animal kingdom from fish to elephants, is best known as a glycoprotein that reduces friction between articulating surfaces (e.g. cartilage). Previously, PRG4 was also shown to regulate the inflammatory and fibrotic response. Based on this, we asked whether PRG4 plays a role in the wound healing response. Using an ear wound model, topical application of exogenous recombinant human (rh)PRG4 hastened wound closure and enhanced tissue regeneration. Our results also suggest that rhPRG4 may impact the fibrotic response, angiogenesis/blood flow to the injury site, macrophage inflammatory dynamics, recruitment of immune and increased proliferation of adult mesenchymal progenitor cells (MPCs) and promoting chondrogenic differentiation of MPCs to form the auricular cartilage scaffold of the injured ear. These results suggest that PRG4 has the potential to suppress scar formation while enhancing connective tissue regeneration post-injury by modulating aspects of each wound healing stage (blood clotting, inflammation, tissue generation and tissue remodeling). Therefore, we propose that rhPRG4 may represent a potential therapy to mitigate scar and improve wound healing.

Proteoglycan 4 is present within the dura mater and produced by mesenchymal progenitor cells.

Mesenchymal progenitor cells (MPCs) have been recently identified in human and murine epidural fat and have been hypothesized to contribute to the maintenance/repair/regeneration of the dura mater. MPCs can secrete proteoglycan 4 (PRG4/lubricin), and this protein can regulate tissue homeostasis through bio-lubrication and immunomodulatory functions. MPC lineage tracing reporter mice (Hic1) and human epidural fat MPCs were used to determine if PRG4 is expressed by these cells in vivo. PRG4 expression co-localized with Hic1+ MPCs in the dura throughout skeletal maturity and was localized adjacent to sites of dural injury. When Hic1+ MPCs were ablated, PRG4 expression was retained in the dura, yet when Prx1+ MPCs were ablated, PRG4 expression was completely lost. A number of cellular processes were impacted in human epidural fat MPCs treated with rhPRG4, and human MPCs contributed to the formation of epidural fat, and dura tissues were xenotransplanted into mouse dural injuries. We have shown that human and mouse MPCs in the epidural/dura microenvironment produce PRG4 and can contribute to dura homeostasis/repair/regeneration. Overall, these results suggest that these MPCs have biological significance within the dural microenvironment and that the role of PRG4 needs to be further elucidated.

Inflammation-Induced Metastatic Colonization of the Lung Is Facilitated by Hepatocyte Growth Factor-Secreting Monocyte-Derived Macrophages.

A rate-limiting step for circulating tumor cells to colonize distant organ sites is their ability to locate a microenvironmental niche that supports their survival and growth. This can be achieved by features intrinsic to the tumor cells and/or by the conditioning of a "premetastatic" niche. To determine if pulmonary inflammation promotes the latter, we initiated models for inflammatory asthma, hypersensitivity pneumonitis, or bleomycin-induced sterile inflammation before introducing tumor cells with low metastatic potential into the circulation. All types of inflammation increased the end-stage metastatic burden of the lungs 14 days after tumor cell inoculation without overtly affecting tumor extravasation. Instead, the number and size of early micrometastatic lesions found within the interstitial tissues 96 hours after tumor cell inoculation were increased in the inflamed lungs, coincident with increased tumor cell survival and the presence of nearby inflammation-induced monocyte-derived macrophages (MoDM; CD11b+CD11c+). Remarkably, the adoptive transfer of these MoDM was sufficient to increase lung metastasis in the absence of inflammation. These inflammation-induced MoDM secrete a number of growth factors and cytokines, one of which is hepatocyte growth factor (HGF), that augmented tumor cell survival under conditions of stress in vitro. Importantly, blocking HGF signaling with the cMET inhibitor capmatinib abolished inflammation-induced early micrometastatic lesion formation in vivo. These findings indicate that inflammation-induced MoDM and HGF in particular increase the efficiency of early metastatic colonization in the lung by locally preconditioning the microenvironment. IMPLICATIONS: Inflammation preconditions the distant site microenvironment to increase the metastatic potential of tumor cells that arrive there.

Murine Limb Bud Organ Cultures for Studying Musculoskeletal Development.

The biological signals that coordinate the three-dimensional outgrowth and patterning of the vertebrate limb bud have been well delineated. These include a number of vital embryonic signaling pathways, including the fibroblast growth factor, WNT, transforming growth factor, and hedgehog. Collectively these signals converge on multiple progenitor populations to drive the formation of a variety of tissues that make up the limb musculoskeletal system, such as muscle, tendon, cartilage, stroma, and bone. The basic mechanisms regulating the commitment and differentiation of diverse limb progenitor populations has been successfully modeled in vitro using high density primary limb mesenchymal or micromass cultures. However, this approach is limited in its ability to more faithfully recapitulate the assembly of progenitors into organized tissues that span the entire musculoskeletal system. Other biological systems have benefitted from the development and availability of three-dimensional organoid cultures which have transformed our understanding of tissue development, homeostasis and regeneration. Such a system does not exist that effectively models the complexity of limb development. However, limb bud organ cultures while still necessitating the use of collected embryonic tissue have proved to be a powerful model system to elucidate the molecular underpinning of musculoskeletal development. In this methods article, the derivation and use of limb bud organ cultures from murine limb buds will be described, along with strategies to manipulate signaling pathways, examine gene expression and for longitudinal lineage tracking.

Abortive γδTCR rearrangements suggest ILC2s are derived from T-cell precursors.

Innate lymphoid cells (ILCs) are a recently identified subset of leukocytes that play a central role in pathogen surveillance and resistance, modulation of immune response, and tissue repair. They are remarkably similar to CD4+ T-helper subsets in terms of function and transcription factors required for their development but are distinguished by their lack of antigen-specific receptors. Despite their similarities, the absence of a surface T-cell receptor (TCR) and presence of ILCs and precursors in adult bone marrow has led to speculation that ILCs and T cells develop separately from lineages that branch at the point of precursors within the bone marrow. Considering the common lineage markers and effector cytokine profiles shared between ILCs and T cells, it is surprising that the status of the TCR loci in ILCs was not fully explored at the time of their discovery. Here, we demonstrate that a high proportion of peripheral tissue ILC2s have TCRγ chain gene rearrangements and TCRδ locus deletions. Detailed analyses of these loci show abundant frameshifts and premature stop codons that would encode nonfunctional TCR proteins. Collectively, these data argue that ILC2 can develop from T cells that fail to appropriately rearrange TCR genes, potentially within the thymus.

Distinct Regulatory Programs Control the Latent Regenerative Potential of Dermal Fibroblasts during Wound Healing.

Dermal fibroblasts exhibit considerable heterogeneity during homeostasis and in response to injury. Defining lineage origins of reparative fibroblasts and regulatory programs that drive fibrosis or, conversely, promote regeneration will be essential for improving healing outcomes. Using complementary fate-mapping approaches, we show that hair follicle mesenchymal progenitors make limited contributions to wound repair. In contrast, extrafollicular progenitors marked by the quiescence-associated factor Hic1 generated the bulk of reparative fibroblasts and exhibited functional divergence, mediating regeneration in the center of the wound neodermis and scar formation in the periphery. Single-cell RNA-seq revealed unique transcriptional, regulatory, and epithelial-mesenchymal crosstalk signatures that enabled mesenchymal competence for regeneration. Integration with scATAC-seq highlighted changes in chromatin accessibility within regeneration-associated loci. Finally, pharmacological modulation of RUNX1 and retinoic acid signaling or genetic deletion of Hic1 within wound-activated fibroblasts was sufficient to modulate healing outcomes, suggesting that reparative fibroblasts have latent but modifiable regenerative capacity.

Development and function of smooth muscle cells is modulated by Hic1 in mouse testis.

In mammalian testis, contractile peritubular myoid cells (PMCs) regulate the transport of sperm and luminal fluid, while secreting growth factors and extracellular matrix proteins to support the spermatogonial stem cell niche. However, little is known about the role of testicular smooth muscle cells during postnatal testicular development. Here we report age-dependent expression of hypermethylated in cancer 1 (Hic1; also known as ZBTB29) in testicular smooth muscle cells, including PMCs and vascular smooth muscle cells, in the mouse. Postnatal deletion of Hic1 in smooth muscle cells led to their increased proliferation and resulted in dilatation of seminiferous tubules, with increased numbers of PMCs. These seminiferous tubules contained fewer Sertoli cells and more spermatogonia, and fibronectin was not detected in their basement membrane. The expression levels of genes encoding smooth muscle contractile proteins, Acta2 and Cnn1, were downregulated in the smooth muscle cells lacking Hic1, and the seminiferous tubules appeared to have reduced contractility. These data imply a role for Hic1 in determining the size of seminiferous tubules by regulating postnatal smooth muscle cell proliferation, subsequently affecting spermatogenesis in adulthood.

Loss of Thymine DNA Glycosylase Causes Dysregulation of Bile Acid Homeostasis and Hepatocellular Carcinoma.

Thymine DNA glycosylase (TDG) is a nuclear receptor coactivator that plays an essential role in the maintenance of epigenetic stability in cells. Here, we demonstrate that the conditional deletion of TDG in adult mice results in a male-predominant onset of hepatocellular carcinoma (HCC). TDG loss leads to a prediabetic state, as well as bile acid (BA) accumulation in the liver and serum of male mice. Consistent with these data, TDG deletion led to dysregulation of the farnesoid X receptor (FXR) and small heterodimer partner (SHP) regulatory cascade in the liver. FXR and SHP are tumor suppressors of HCC and play an essential role in BA and glucose homeostasis. These results indicate that TDG functions as a tumor suppressor of HCC by regulating a transcriptional program that protects against the development of glucose intolerance and BA accumulation in the liver.

Pathogenic Potential of Hic1-Expressing Cardiac Stromal Progenitors.

The cardiac stroma contains multipotent mesenchymal progenitors. However, lineage relationships within cardiac stromal cells are poorly defined. Here, we identified heart-resident PDGFRa+ SCA-1+ cells as cardiac fibro/adipogenic progenitors (cFAPs) and show that they respond to ischemic damage by generating fibrogenic cells. Pharmacological blockade of this differentiation step with an anti-fibrotic tyrosine kinase inhibitor decreases post-myocardial infarction (post-MI) remodeling and leads to improvement in cardiac function. In the undamaged heart, activation of cFAPs through lineage-specific deletion of the gene encoding the quiescence-associated factor HIC1 reveals additional pathogenic potential, causing fibrofatty infiltration within the myocardium and driving major pathological features pathognomonic in arrhythmogenic cardiomyopathy (AC). In this regard, cFAPs contribute to multiple pathogenic cell types within cardiac tissue and therapeutic strategies aimed at modifying their activity are expected to have tremendous benefit for the treatment of diverse cardiac diseases.

Hic1 Defines Quiescent Mesenchymal Progenitor Subpopulations with Distinct Functions and Fates in Skeletal Muscle Regeneration.

Many adult tissues contain resident stem cells, such as the Pax7+ satellite cells within skeletal muscle, that regenerate parenchymal elements following damage. Tissue-resident mesenchymal progenitors (MPs) also participate in regeneration, although their function and fate in this process are unclear. Here, we identify Hypermethylated in cancer 1 (Hic1) as a marker of MPs in skeletal muscle and further show that Hic1 deletion leads to MP hyperplasia. Single-cell RNA-seq and ATAC-seq analysis of Hic1+ MPs in skeletal muscle shows multiple subpopulations, which we further show have distinct functions and lineage potential. Hic1+ MPs orchestrate multiple aspects of skeletal muscle regeneration by providing stage-specific immunomodulation and trophic and mechanical support. During muscle regeneration, Hic1+ derivatives directly contribute to several mesenchymal compartments including Col22a1-expressing cells within the myotendinous junction. Collectively, these findings demonstrate that HIC1 regulates MP quiescence and identifies MP subpopulations with transient and enduring roles in muscle regeneration.