Bridging tissue repair and epithelial carcinogenesis: epigenetic memory and field cancerization.

The epigenome coordinates spatial-temporal specific gene expression during development and in adulthood, for the maintenance of homeostasis and upon tissue repair. The upheaval of the epigenetic landscape is a key event in the onset of many pathologies including tumours, where epigenetic changes cooperate with genetic aberrations to establish the neoplastic phenotype and to drive cell plasticity during its evolution. DNA methylation, histone modifiers and readers or other chromatin components are indeed often altered in cancers, such as carcinomas that develop in epithelia. Lining the surfaces and the cavities of our body and acting as a barrier from the environment, epithelia are frequently subjected to acute or chronic tissue damages, such as mechanical injuries or inflammatory episodes. These events can activate plasticity mechanisms, with a deep impact on cells' epigenome. Despite being very effective, tissue repair mechanisms are closely associated with tumour onset. Here we review the similarities between tissue repair and carcinogenesis, with a special focus on the epigenetic mechanisms activated by cells during repair and opted by carcinoma cells in multiple epithelia. Moreover, we discuss the recent findings on inflammatory and wound memory in epithelia and describe the epigenetic modifications that characterise them. Finally, as wound memory in epithelial cells promotes carcinogenesis, we highlight how it represents an early step for the establishment of field cancerization.

Multiplicity of stem cell memories of inflammation and tissue repair in epithelia.

Adaptation enables cells to change their behavior in response to transient stimuli. While adaptive programs of immune cells have been widely described, it has recently emerged that epithelial cells also acquire memories in vivo. Here, we discuss and classify the adaptations identified in epithelia and describe the associated long-term consequences.

Comparison of antiviral responses in two bat species reveals conserved and divergent innate immune pathways.

Bats host a range of disease-causing viruses without displaying clinical symptoms. The mechanisms behind this are a continuous source of interest. Here, we studied the antiviral response in the Egyptian fruit bat and Kuhl's pipistrelle, representing two subordinal clades. We profiled the antiviral response in fibroblasts using RNA sequencing and compared bat with primate and rodent responses. Both bats upregulate similar genes; however, a subset of these genes is transcriptionally divergent between them. These divergent genes also evolve rapidly in sequence, have specific promoter architectures, and are associated with programs underlying tolerance and resistance. Finally, we characterized antiviral genes that expanded in bats, with duplicates diverging in sequence and expression. Our study reveals a largely conserved antiviral program across bats and points to a set of genes that rapidly evolve through multiple mechanisms. These can contribute to bat adaptation to viral infection and provide directions to understanding the mechanisms behind it.

Molecular and spatial landmarks of early mouse skin development.

A wealth of specialized cell populations within the skin facilitates its hair-producing, protective, sensory, and thermoregulatory functions. How the vast cell-type diversity and tissue architecture develops is largely unexplored. Here, with single-cell transcriptomics, spatial cell-type assignment, and cell-lineage tracing, we deconstruct early embryonic mouse skin during the key transitions from seemingly uniform developmental precursor states to a multilayered, multilineage epithelium, and complex dermal identity. We identify the spatiotemporal emergence of hair-follicle-inducing, muscle-supportive, and fascia-forming fibroblasts. We also demonstrate the formation of the panniculus carnosus muscle (PCM), sprouting blood vessels without pericyte coverage, and the earliest residence of mast and dendritic immune cells in skin. Finally, we identify an unexpected epithelial heterogeneity within the early single-layered epidermis and a signaling-rich periderm layer. Overall, this cellular and molecular blueprint of early skin development-which can be explored at https://kasperlab.org/tools-establishes histological landmarks and highlights unprecedented dynamic interactions among skin cells.

Tissue memory relies on stem cell priming in distal undamaged areas.

Epithelial cells that participated in wound repair elicit a more efficient response to future injuries, which is believed to be locally restricted. Here we show that cell adaptation resulting from a localized tissue damage has a wide spatial impact at a scale not previously appreciated. We demonstrate that a specific stem cell population, distant from the original injury, originates long-lasting wound memory progenitors residing in their own niche. Notably, these distal memory cells have not taken part in the first healing but become intrinsically pre-activated through priming. This cell state, maintained at the chromatin and transcriptional level, leads to an enhanced wound repair that is partially recapitulated through epigenetic perturbation. Importantly wound memory has long-term harmful consequences, exacerbating tumourigenesis. Overall, we show that sub-organ-scale adaptation to injury relies on spatially organized memory-dedicated progenitors, characterized by an actionable cell state that establishes an epigenetic field cancerization and predisposes to tumour onset.

DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells.

The correct establishment of DNA methylation patterns during mouse early development is essential for cell fate specification. However, the molecular targets as well as the mechanisms that determine the specificity of the de novo methylation machinery during differentiation are not completely elucidated. Here we show that the DNMT3B-dependent DNA methylation of key developmental regulatory regions at epiblast-like cells (EpiLCs) provides an epigenetic priming that ensures flawless commitment at later stages. Using in vitro stem cell differentiation and loss of function experiments combined with high-throughput genome-wide bisulfite-, bulk-, and single cell RNA-sequencing we dissected the specific role of DNMT3B in cell fate. We identify DNMT3B-dependent regulatory elements on the genome which, in Dnmt3b knockout (3BKO), impair the differentiation into meso-endodermal (ME) progenitors and redirect EpiLCs towards the neuro-ectodermal lineages. Moreover, ectopic expression of DNMT3B in 3BKO re-establishes the DNA methylation of the master regulator Sox2 super-enhancer, downmodulates its expression, and restores the expression of ME markers. Taken together, our data reveal that DNMT3B-dependent methylation at the epiblast stage is essential for the priming of the meso-endodermal lineages and provide functional characterization of the de novo DNMTs during EpiLCs lineage determination.

Inflammaging is driven by upregulation of innate immune receptors and systemic interferon signaling and is ameliorated by dietary restriction.

Aging is characterized by a chronic low-grade inflammation known as inflammaging in multiple tissues, representing a risk factor for age-related diseases. Dietary restriction (DR) is the best-known non-invasive method to ameliorate aging in many organisms. However, the molecular mechanism and the signaling pathways that drive inflammaging across different tissues and how they are modulated by DR are not yet understood. Here we identify a multi-tissue gene network regulating inflammaging. This network is characterized by chromatin opening and upregulation in the transcription of innate immune system receptors and by activation of interferon signaling through interferon regulatory factors, inflammatory cytokines, and Stat1-mediated transcription. DR ameliorates aging-induced alterations of chromatin accessibility and RNA transcription of the inflammaging gene network while failing to rescue those alterations on the rest of the genome. Our results present a comprehensive understanding of the molecular network regulating inflammation in aging and DR and provide anti-inflammaging therapeutic targets.

Single-Cell Sequencing for Everybody.

Over the past 7 years, single-cell sequencing has become very popular. For this reason, many laboratories of different biological disciplines that span from neurobiology to developmental biology from immunology to tumor biology have been approaching this technique. For someone new to this field that wants to investigate heterogeneity in what appears to be a single-cell population, the choice of the best protocol can be difficult, due to the high abundance of available protocols, instruments, and options. For this reason, here we describe the Smart-seq2 protocol for full-length mRNA sequencing of single cell. This protocol can be easily optimized in every molecular biology laboratory provided with standard laboratory equipment. The protocol is suitable for many different cell types, and the cost per cell is relatively small, allowing a good balance between costs and transcript coverage.

Wnt/ß-Catenin Signaling Stabilizes Hemidesmosomes in Keratinocytes.

Hemidesmosomes (HDs) are adhesion complexes that promote epithelial-stromal attachment in stratified and complex epithelia, including the epidermis. In various biological processes, such as differentiation and migration of epidermal keratinocytes during wound healing or carcinoma invasion, quick assembly and disassembly of HDs are prerequisites. In this study, we show that inhibition of Wnt/ß-catenin signaling disturbs HD organization in keratinocytes. Screening with inhibitors identified the depletion of HD components and HD-like structures through Wnt inhibition, but keratinocyte differentiation was not affected. Wnt inhibition significantly diminished plectin and type XVII collagen expression in the basal side of Wnt-inhibited cells and the dermo-epidermal junction of the Wnt-inactive murine basal epidermis. Similar to Wnt inhibition, PLEC-knockout cells or cells with plectin-type XVII collagen binding defects showed type XVII collagen reduction in the basal side of the cells, implying the possible involvement of Wnt/ß-catenin signaling in HD assembly. Atypical protein kinase C inhibition ameliorated the phenotypes of Wnt-inhibited cells. These findings show that Wnt/ß-catenin signaling regulates the localization of HD components in keratinocytes and that the atypical protein kinase C pathway is involved in Wnt inhibition‒induced HD disarrangement. Our study suggests that the Wnt signaling pathway could be a potential therapeutic target for treating HD-defective diseases, such as epidermolysis bullosa.

Hair follicle stem cell progeny heal blisters while pausing skin development.

Injury in adult tissue generally reactivates developmental programs to foster regeneration, but it is not known whether this paradigm applies to growing tissue. Here, by employing blisters, we show that epidermal wounds heal at the expense of skin development. The regenerated epidermis suppresses the expression of tissue morphogenesis genes accompanied by delayed hair follicle (HF) growth. Lineage tracing experiments, cell proliferation dynamics, and mathematical modeling reveal that the progeny of HF junctional zone stem cells, which undergo a morphological transformation, repair the blisters while not promoting HF development. In contrast, the contribution of interfollicular stem cell progeny to blister healing is small. These findings demonstrate that HF development can be sacrificed for the sake of epidermal wound regeneration. Our study elucidates the key cellular mechanism of wound healing in skin blistering diseases.

Fibrotic enzymes modulate wound-induced skin tumorigenesis.

Fibroblasts are a major component of the microenvironment of most solid tumours. Recent research elucidated a large heterogeneity and plasticity of activated fibroblasts, indicating that their role in cancer initiation, growth and metastasis is complex and context-dependent. Here, we performed genome-wide expression analysis comparing fibroblasts in normal, inflammatory and tumour-associated skin. Cancer-associated fibroblasts (CAFs) exhibit a fibrotic gene signature in wound-induced tumours, demonstrating persistent extracellular matrix (ECM) remodelling within these tumours. A top upregulated gene in mouse CAFs encodes for PRSS35, a protease capable of collagen remodelling. In human skin, we observed PRSS35 expression uniquely in the stroma of high-grade squamous cell carcinomas. Ablation of PRSS35 in mouse models of wound- or chemically-induced tumorigenesis resulted in aberrant collagen composition in the ECM and increased tumour incidence. Our results indicate that fibrotic enzymes expressed by CAFs can regulate squamous tumour initiation by remodelling the ECM.

Contribution of GATA6 to homeostasis of the human upper pilosebaceous unit and acne pathogenesis.

Although acne is the most common human inflammatory skin disease, its pathogenic mechanisms remain incompletely understood. Here we show that GATA6, which is expressed in the upper pilosebaceous unit of normal human skin, is down-regulated in acne. GATA6 controls keratinocyte proliferation and differentiation to prevent hyperkeratinisation of the infundibulum, which is the primary pathological event in acne. When overexpressed in immortalised human sebocytes, GATA6 triggers a junctional zone and sebaceous differentiation program whilst limiting lipid production and cell proliferation. It modulates the immunological repertoire of sebocytes, notably by upregulating PD-L1 and IL10. GATA6 expression contributes to the therapeutic effect of retinoic acid, the main treatment for acne. In a human sebaceous organoid model GATA6-mediated down-regulation of the infundibular differentiation program is mediated by induction of TGFß signalling. We conclude that GATA6 is involved in regulation of the upper pilosebaceous unit and may be an actionable target in the treatment of acne.

Mutant Lef1 controls Gata6 in sebaceous gland development and cancer.

Mutations in Lef1 occur in human and mouse sebaceous gland (SG) tumors, but their contribution to carcinogenesis remains unclear. Since Gata6 controls lineage identity in SG, we investigated the link between these two transcription factors. Here, we show that Gata6 is a ß-catenin-independent transcriptional target of mutant Lef1. During epidermal development, Gata6 is expressed in a subset of Sox9-positive Lef1-negative hair follicle progenitors that give rise to the upper SG Overexpression of Gata6 by in utero lentiviral injection is sufficient to induce ectopic sebaceous gland elements. In mice overexpressing mutant Lef1, Gata6 ablation increases the total number of skin tumors yet decreases the proportion of SG tumors. The increased tumor burden correlates with impaired DNA mismatch repair and decreased expression of Mlh1 and Msh2 genes, defects frequently observed in human sebaceous neoplasia. Gata6 specifically marks human SG tumors and also defines tumors with elements of sebaceous differentiation, including a subset of basal cell carcinomas. Our findings reveal that Gata6 controls sebaceous gland development and cancer.

Gene expression variability across cells and species shapes innate immunity.

As the first line of defence against pathogens, cells mount an innate immune response, which varies widely from cell to cell. The response must be potent but carefully controlled to avoid self-damage. How these constraints have shaped the evolution of innate immunity remains poorly understood. Here we characterize the innate immune response's transcriptional divergence between species and variability in expression among cells. Using bulk and single-cell transcriptomics in fibroblasts and mononuclear phagocytes from different species, challenged with immune stimuli, we map the architecture of the innate immune response. Transcriptionally diverging genes, including those that encode cytokines and chemokines, vary across cells and have distinct promoter structures. Conversely, genes that are involved in the regulation of this response, such as those that encode transcription factors and kinases, are conserved between species and display low cell-to-cell variability in expression. We suggest that this expression pattern, which is observed across species and conditions, has evolved as a mechanism for fine-tuned regulation to achieve an effective but balanced response.

Epidermal Wnt signalling regulates transcriptome heterogeneity and proliferative fate in neighbouring cells.

BACKGROUND: Canonical Wnt/beta-catenin signalling regulates self-renewal and lineage selection within the mammalian epidermis. Although the transcriptional response of keratinocytes that receive a Wnt signal is well characterized, little is known about the mechanism by which keratinocytes in proximity to the Wnt-receiving cell are co-opted to undergo a change in cell fate. RESULTS: To address this, we perform single-cell RNA-sequencing on mouse keratinocytes co-cultured with and without beta-catenin-activated neighbouring cells. We identify five distinct cell states in cultures that had not been exposed to the beta-catenin stimulus and show that the stimulus redistributes wild-type subpopulation proportions. Using temporal single-cell analysis, we reconstruct the cell fate change induced by Wnt activation from neighbouring cells. Gene expression heterogeneity is reduced in neighbouring cells and this effect is most dramatic for protein synthesis-associated genes. Changes in gene expression are accompanied by a shift to a more proliferative stem cell state. By integrating imaging and reconstructed sequential gene expression changes during the state transition we identify transcription factors, including Smad4 and Bcl3, that are responsible for effecting the transition in a contact-dependent manner. CONCLUSIONS: Our data indicate that non-cell autonomous Wnt/beta-catenin signalling decreases transcriptional heterogeneity. This furthers our understanding of how epidermal Wnt signalling orchestrates regeneration and self-renewal.

Locked and Loaded: Inflammation Training Prepares Skin Epithelial Stem Cells for Trauma.

Memory of a trauma and how to cope with it is useful for acting rapidly in the event of a second traumatic incident. Recently, Naik et al. (2017) reported in Nature that skin epithelial stem cells have this ability by maintaining long-term chromatin features acquired during the first assault.

Type XVII collagen coordinates proliferation in the interfollicular epidermis.

Type XVII collagen (COL17) is a transmembrane protein located at the epidermal basement membrane zone. COL17 deficiency results in premature hair aging phenotypes and in junctional epidermolysis bullosa. Here, we show that COL17 plays a central role in regulating interfollicular epidermis (IFE) proliferation. Loss of COL17 leads to transient IFE hypertrophy in neonatal mice owing to aberrant Wnt signaling. The replenishment of COL17 in the neonatal epidermis of COL17-null mice reverses the proliferative IFE phenotype and the altered Wnt signaling. Physical aging abolishes membranous COL17 in IFE basal cells because of inactive atypical protein kinase C signaling and also induces epidermal hyperproliferation. The overexpression of human COL17 in aged mouse epidermis suppresses IFE hypertrophy. These findings demonstrate that COL17 governs IFE proliferation of neonatal and aged skin in distinct ways. Our study indicates that COL17 could be an important target of anti-aging strategies in the skin.

Wounding induces dedifferentiation of epidermal Gata6+ cells and acquisition of stem cell properties.

The epidermis is maintained by multiple stem cell populations whose progeny differentiate along diverse, and spatially distinct, lineages. Here we show that the transcription factor Gata6 controls the identity of the previously uncharacterized sebaceous duct (SD) lineage and identify the Gata6 downstream transcription factor network that specifies a lineage switch between sebocytes and SD cells. During wound healing differentiated Gata6+ cells migrate from the SD into the interfollicular epidermis and dedifferentiate, acquiring the ability to undergo long-term self-renewal and differentiate into a much wider range of epidermal lineages than in undamaged tissue. Our data not only demonstrate that the structural and functional complexity of the junctional zone is regulated by Gata6, but also reveal that dedifferentiation is a previously unrecognized property of post-mitotic, terminally differentiated cells that have lost contact with the basement membrane. This resolves the long-standing debate about the contribution of terminally differentiated cells to epidermal wound repair.

A genome-wide screen identifies YAP/WBP2 interplay conferring growth advantage on human epidermal stem cells.

Individual human epidermal cells differ in their self-renewal ability. To uncover the molecular basis for this heterogeneity, we performed genome-wide pooled RNA interference screens and identified genes conferring a clonal growth advantage on normal and neoplastic (cutaneous squamous cell carcinoma, cSCC) human epidermal cells. The Hippo effector YAP was amongst the top positive growth regulators in both screens. By integrating the Hippo network interactome with our data sets, we identify WW-binding protein 2 (WBP2) as an important co-factor of YAP that enhances YAP/TEAD-mediated gene transcription. YAP and WPB2 are upregulated in actively proliferating cells of mouse and human epidermis and cSCC, and downregulated during terminal differentiation. WBP2 deletion in mouse skin results in reduced proliferation in neonatal and wounded adult epidermis. In reconstituted epidermis YAP/WBP2 activity is controlled by intercellular adhesion rather than canonical Hippo signalling. We propose that defective intercellular adhesion contributes to uncontrolled cSCC growth by preventing inhibition of YAP/WBP2.