Tenascin-C expressing touch dome keratinocytes exhibit characteristics of all epidermal lineages.

The touch dome (TD) keratinocytes are specialized epidermal cells that intimately associate with the light touch sensing Merkel cells (MCs). The TD keratinocytes function as a niche for the MCs and can induce de novo hair follicles upon stimulation; however, how the TD keratinocytes are maintained during homeostasis remains unclear. scRNA-seq identified a specific TD keratinocyte marker, Tenascin-C (TNC). Lineage tracing of Tnc-expressing TD keratinocytes revealed that these cells maintain themselves as an autonomous epidermal compartment and give rise to MCs upon injury. Molecular characterization uncovered that, while the transcriptional and chromatin landscape of the TD keratinocytes is remarkably similar to that of the interfollicular epidermal keratinocytes, it also shares certain molecular signatures with the hair follicle keratinocytes. Our study highlights that the TD keratinocytes in the adult skin have molecular characteristics of keratinocytes of diverse epidermal lineages.

Defining the fate trajectory of eccrine gland formation.

Eccrine glands secrete water onto the surface of human skin to regulate body temperature. In this issue of Developmental Cell, Dingwall et al. dissect the transcriptional signature of developing eccrine glands, and they also uncover a unique dermal niche that is responsible for promoting eccrine gland developmental progression.

Cleavage Under Targets & Release Using Nuclease (CUT&RUN) of Histone Modifications in Epidermal Stem Cells of Adult Murine Skin.

Cleavage Under Targets & Release Using Nuclease (CUT&RUN) has swiftly become the preferred procedure over the past few years for genomic mapping and detecting interactions between chromatin and its bound proteins. CUT&RUN is now being widely used for characterizing the epigenetic landscape in many cell types as it utilizes far less cell numbers when compared to Chromatin Immunoprecipitation-sequencing (ChIP-seq), thereby making it a powerful tool for researchers working with limited material. This protocol has been specifically optimized for detecting histone modifications in fluorescence-activated cell sorting (FACS)-isolated epidermal stem cells from adult mice.

Updates in SJS/TEN: collaboration, innovation, and community.

Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis (SJS/TEN) is a predominantly drug-induced disease, with a mortality rate of 15-20%, that engages the expertise of multiple disciplines: dermatology, allergy, immunology, clinical pharmacology, burn surgery, ophthalmology, urogynecology, and psychiatry. SJS/TEN has an incidence of 1-5/million persons per year in the United States, with even higher rates globally. One of the challenges of SJS/TEN has been developing the research infrastructure and coordination to answer questions capable of transforming clinical care and leading to improved patient outcomes. SJS/TEN 2021, the third research meeting of its kind, was held as a virtual meeting on August 28-29, 2021. The meeting brought together 428 international scientists, in addition to a community of 140 SJS/TEN survivors and family members. The goal of the meeting was to brainstorm strategies to support the continued growth of an international SJS/TEN research network, bridging science and the community. The community workshop section of the meeting focused on eight primary themes: mental health, eye care, SJS/TEN in children, non-drug induced SJS/TEN, long-term health complications, new advances in mechanisms and basic science, managing long-term scarring, considerations for skin of color, and COVID-19 vaccines. The meeting featured several important updates and identified areas of unmet research and clinical need that will be highlighted in this white paper.

Merkel Cell Polyomavirus T Antigen-Mediated Reprogramming in Adult Merkel Cell Progenitors.

Whether Merkel cells regenerate in adult skin and from which progenitor cells they regenerate are a subject of debate. Understanding Merkel cell regeneration is of interest to the study of Merkel cell carcinoma, a rare neuroendocrine skin cancer hypothesized to originate in a Merkel cell progenitor transformed by Merkel cell polyomavirus small and large T antigens. We sought to understand what the adult Merkel cell progenitors are and whether they can give rise to Merkel cell carcinoma. We used lineage tracing to identify SOX9-expressing cells (SOX9+ cells) as Merkel cell progenitors in postnatal murine skin. Merkel cell regeneration from SOX9+ progenitors occurs rarely in mature skin unless in response to minor mechanical injury. Merkel cell polyomavirus small T antigen and functional imitation of large T antigen in SOX9+ cells enforced neuroendocrine and Merkel cell lineage reprogramming in a subset of cells. These results identify SOX9+ cells as postnatal Merkel cell progenitors that can be reprogrammed by Merkel cell polyomavirus T antigens to express neuroendocrine markers.

Methyltransferase-independent function of enhancer of zeste homologue 2 maintains tumorigenicity induced by human oncogenic papillomavirus and polyomavirus.

Merkel cell polyomavirus (MCV) and high-risk human papillomavirus (HPV) are human tumor viruses that cause Merkel cell carcinoma (MCC) and oropharyngeal squamous cell carcinoma (OSCC), respectively. HPV E7 and MCV large T (LT) oncoproteins target the retinoblastoma tumor suppressor protein (pRb) through the conserved LxCxE motif. We identified enhancer of zeste homolog 2 (EZH2) as a common host oncoprotein activated by both viral oncoproteins through the pRb binding motif. EZH2 is a catalytic subunit of the polycomb 2 (PRC2) complex that trimethylates histone H3 at lysine 27 (H3K27me3). In MCC tissues EZH2 was highly expressed, irrespective of MCV status. Loss-of-function studies revealed that viral HPV E6/E7 and T antigen expression are required for Ezh2 mRNA expression and that EZH2 is essential for HPV(+)OSCC and MCV(+)MCC cell growth. Furthermore, EZH2 protein degraders reduced cell viability efficiently and rapidly in HPV(+)OSCC and MCV(+)MCC cells, whereas EZH2 histone methyltransferase inhibitors did not affect cell proliferation or viability within the same treatment period. These results suggest that a methyltransferase-independent function of EZH2 contributes to tumorigenesis downstream of two viral oncoproteins, and that direct targeting of EZH2 protein expression could be a promising strategy for the inhibition of tumor growth in HPV(+)OSCC and MCV(+)MCC patients.

Polycomb repressive complex 2 in adult hair follicle stem cells is dispensable for hair regeneration.

Hair follicle stem cells (HFSCs) are multipotent cells that cycle through quiescence and activation to continuously fuel the production of hair follicles. Prior genome mapping studies had shown that tri-methylation of histone H3 at lysine 27 (H3K27me3), the chromatin mark mediated by Polycomb Repressive Complex 2 (PRC2), is dynamic between quiescent and activated HFSCs, suggesting that transcriptional changes associated with H3K27me3 might be critical for proper HFSC function. However, functional in vivo studies elucidating the role of PRC2 in adult HFSCs are lacking. In this study, by using in vivo loss-of-function studies we show that, surprisingly, PRC2 plays a non-instructive role in adult HFSCs and loss of PRC2 in HFSCs does not lead to loss of HFSC quiescence or changes in cell identity. Interestingly, RNA-seq and immunofluorescence analyses of PRC2-null quiescent HFSCs revealed upregulation of genes associated with activated state of HFSCs. Altogether, our findings show that transcriptional program under PRC2 regulation is dispensable for maintaining HFSC quiescence and hair regeneration.

Polycomb Repressive Complex(es) and Their Role in Adult Stem Cells.

Populations of resident stem cells (SCs) are responsible for maintaining, repairing, and regenerating adult tissues. In addition to having the capacity to generate all the differentiated cell types of the tissue, adult SCs undergo long periods of quiescence within the niche to maintain themselves. The process of SC renewal and differentiation is tightly regulated for proper tissue regeneration throughout an organisms' lifetime. Epigenetic regulators, such as the polycomb group (PcG) of proteins have been implicated in modulating gene expression in adult SCs to maintain homeostatic and regenerative balances in adult tissues. In this review, we summarize the recent findings that elucidate the composition and function of the polycomb repressive complex machinery and highlight their role in diverse adult stem cell compartments.

UV-induced reduction in Polycomb repression promotes epidermal pigmentation.

Ultraviolet (UV) radiation is a prime environmental stressor that our epidermis is exposed to on a daily basis. To avert UV-induced damage, epidermal stem cells (EpSCs) become pigmented via a process of heterotypic interaction between melanocytes and EpSCs; however, the molecular mechanisms of this interaction are not well understood. In this study, we show that the function of a key chromatin regulator, the Polycomb complex, was reduced upon UV exposure in human and mouse epidermis. Genetic ablation of key Polycomb subunits in murine EpSCs, mimicking depletion upon UV exposure, results in an increased number of epidermal melanocytes and subsequent epidermal pigmentation. Genome-wide transcriptional and chromatin studies show that Polycomb regulates the expression of UV-responsive genes and identifies type II collagen (COL2A1) as a critical secreted regulator of melanogenesis and epidermal pigmentation. Together, our findings show how UV exposure induces Polycomb-mediated changes in EpSCs to affect melanocyte behavior and promote epidermal pigmentation.

Polycomb complexes redundantly maintain epidermal stem cell identity during development.

Polycomb repressive complex 1 (PRC1) and PRC2 are critical epigenetic developmental regulators. PRC1 and PRC2 largely overlap in their genomic binding and cooperate to establish repressive chromatin domains demarcated by H2AK119ub and H3K27me3. However, the functional contribution of each complex to gene repression has been a subject of debate, and understanding of its physiological significance requires further studies. Here, using the developing murine epidermis as a paradigm, we uncovered a previously unappreciated functional redundancy between Polycomb complexes. Coablation of PRC1 and PRC2 in embryonic epidermal progenitors resulted in severe defects in epidermal stratification, a phenotype not observed in the single PRC1-null or PRC2-null epidermis. Molecular dissection indicated a loss of epidermal identity that was coupled to a strong derepression of nonlineage transcription factors, otherwise repressed by either PRC1 or PRC2 in the absence of its counterpart. Ectopic expression of subsets of PRC1/2-repressed nonepidermal transcription factors in wild-type epidermal stem cells was sufficient to suppress epidermal identity genes, highlighting the importance of functional redundancy between PRC1 and PRC2. Altogether, our studies show how PRC1 and PRC2 function as two independent counterparts, thereby providing a repressive safety net that protects and preserves lineage identity.

Regulatory mechanisms governing epidermal stem cell function during development and homeostasis.

Cell divisions and cell-fate decisions require stringent regulation for proper tissue development and homeostasis. The mammalian epidermis is a highly organized tissue structure that is sustained by epidermal stem cells (ESCs) that balance self-renewal and cell-fate decisions to establish a protective barrier, while replacing dying cells during homeostasis and in response to injury. Extensive work over past decades has provided insights into the regulatory mechanisms that control ESC specification, self-renewal and maintenance during different stages of the lifetime of an organism. In this Review, we discuss recent findings that have furthered our understanding of key regulatory features that allow ESCs to establish a functional barrier during development and to maintain tissue homeostasis in adults.

Chromatin Immunoprecipitation of Low Number of FACS-Purified Epidermal Cells.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a method designed to detect interactions between chromatin and the proteins bound to it. This method has been widely used for characterizing epigenetic landscapes in many cell types; however, a limiting factor has been the requirement of a high number of cells. Here, we describe a protocol for ChIP in epidermal cells from a newborn mouse, purified by fluorescence-activated cell sorting (FACS). This protocol has been optimized specifically for prefixed, low cell numbers, resulting in enough immunoprecipitated DNA suitable for genome-wide analysis.

Activity of PRC1 and Histone H2AK119 Monoubiquitination: Revising Popular Misconceptions.

Polycomb group proteins are evolutionary conserved chromatin-modifying complexes, essential for the regulation of developmental and cell-identity genes. Polycomb-mediated transcriptional regulation is provided by two multi-protein complexes known as Polycomb repressive complex 1 (PRC1) and 2 (PRC2). Recent studies positioned PRC1 as a foremost executer of Polycomb-mediated transcriptional control. Mammalian PRC1 complexes can form multiple sub-complexes that vary in their core and accessory subunit composition, leading to fascinating and diverse transcriptional regulatory mechanisms employed by PRC1 complexes. These mechanisms include PRC1-catalytic activity toward monoubiquitination of histone H2AK119, a well-established hallmark of PRC1 complexes, whose importance has been long debated. In this review, the central roles that PRC1-catalytic activity plays in transcriptional repression are emphasized and the recent evidence supporting a role for PRC1 complexes in gene activation is discussed.

Conversion of Sox2-dependent Merkel cell carcinoma to a differentiated neuron-like phenotype by T antigen inhibition.

Viral cancers show oncogene addiction to viral oncoproteins, which are required for survival and proliferation of the dedifferentiated cancer cell. Human Merkel cell carcinomas (MCCs) that harbor a clonally integrated Merkel cell polyomavirus (MCV) genome have low mutation burden and require viral T antigen expression for tumor growth. Here, we showed that MCV+ MCC cells cocultured with keratinocytes undergo neuron-like differentiation with neurite outgrowth, secretory vesicle accumulation, and the generation of sodium-dependent action potentials, hallmarks of a neuronal cell lineage. Cocultured keratinocytes are essential for induction of the neuronal phenotype. Keratinocyte-conditioned medium was insufficient to induce this phenotype. Single-cell RNA sequencing revealed that T antigen knockdown inhibited cell cycle gene expression and reduced expression of key Merkel cell lineage/MCC marker genes, including HES6, SOX2, ATOH1, and KRT20 Of these, T antigen knockdown directly inhibited Sox2 and Atoh1 expression. MCV large T up-regulated Sox2 through its retinoblastoma protein-inhibition domain, which in turn activated Atoh1 expression. The knockdown of Sox2 in MCV+ MCCs mimicked T antigen knockdown by inducing MCC cell growth arrest and neuron-like differentiation. These results show Sox2-dependent conversion of an undifferentiated, aggressive cancer cell to a differentiated neuron-like phenotype and suggest that the ontology of MCC arises from a neuronal cell precursor.

Polycomb Repressive Complex 1 Controls Maintenance of Fungiform Papillae by Repressing Sonic Hedgehog Expression.

How tissue patterns are formed and maintained are fundamental questions. The murine tongue epithelium, a paradigm for tissue patterning, consists of an array of specialized fungiform papillae structures that harbor taste cells. The formation of fungiform papillae is preceded by pronounced spatial changes in gene expression, in which taste cell genes such as Shh, initially diffused in lingual epithelial progenitors, become restricted to taste cells when their specification progresses. However, the requirement of spatial restriction of taste cell gene expression for patterning and formation of fungiform papillae is unknown. Here, we show that a chromatin regulator, Polycomb repressive complex (PRC) 1, is required for proper maintenance of fungiform papillae by repressing Shh and preventing ectopic SHH signaling in non-taste cells. Ablation of SHH signaling in PRC1-null non-taste cells rescues the maintenance of taste cells. Altogether, our studies exemplify how epigenetic regulation establishes spatial gene expression patterns necessary for specialized niche structures.

Epigenetic gene regulation, chromatin structure, and force-induced chromatin remodelling in epidermal development and homeostasis.

The skin epidermis is a constantly renewing stratified epithelium that provides essential protective barrier functions throughout life. Epidermal stratification is governed by a step-wise differentiation program that requires precise spatiotemporal control of gene expression. How epidermal self-renewal and differentiation are regulated remains a fundamental open question. Cell-intrinsic and cell-extrinsic mechanisms that modify chromatin structure and interactions have been identified as key regulators of epidermal differentiation and stratification. Here, we will review the recent advances in our understanding of how chromatin modifiers, tissue-specific transcription factors, and force-induced nuclear remodeling processes function to shape chromatin and to control epidermal tissue development and homeostasis.

Dissection of Merkel cell formation in hairy and glabrous skin reveals a common requirement for FGFR2-mediated signalling.

Merkel cells are mechanosensory cells involved in tactile discrimination. Merkel cells have been primarily studied in the murine back skin, where they are found in specialized structures called touch domes located around primary hair follicles. Yet, little is known about the morphogenesis of Merkel cells in areas of the skin devoid of hair, such as the glabrous paw skin. Here, we describe Merkel cell formation in the glabrous paw skin during embryogenesis. We first found in the glabrous paw skin that Merkel cells were specified at E15.5, 24 hours later, compared to in the back skin. Additionally, by performing lineage-tracing experiments, we found that unlike in the back skin, SOX9(+) cells do not give rise to Merkel cells in the glabrous paw skin. Finally, we compared the transcriptomes of Merkel cells in the back and the glabrous paw skin and showed that they are similar. Genetic and transcriptome studies showed that the formation of Merkel cells in both regions was controlled by similar regulators. Among them was FGFR2, an upstream factor of MAPK signalling that was reported to have a critical function in Merkel cell formation in the back skin. Here, we showed that FGFR2 is also required for Merkel cell development in the glabrous paw skin. Taken together, our results demonstrate that Merkel cells in the murine back skin and glabrous paw skin are similar, and even though their formation is controlled by a common genetic programme, their precursor cells might differ.

PRC1 preserves epidermal tissue integrity independently of PRC2.

Polycomb-repressive complex 1 (PRC1) and PRC2 are critical chromatin regulators of gene expression and tissue development. Here, we show that despite extensive genomic cobinding, PRC1 is essential for epidermal integrity, whereas PRC2 is dispensable. Loss of PRC1 resulted in blistering skin, reminiscent of human skin fragility syndromes. Conversely, PRC1 does not restrict epidermal stratification during skin morphogenesis, whereas PRC2 does. Molecular dissection demonstrated that PRC1 functions with PRC2 to silence/dampen expression of adhesion genes. In contrast, PRC1 promotes expression of critical epidermal adhesion genes independently of PRC2-mediated H3K27me3. Together, we demonstrate a functional link between epigenetic regulation and skin diseases.

Publisher Correction: FGF signalling controls the specification of hair placode-derived SOX9 positive progenitors to Merkel cells.

The originally published version of this Article contained an error in Figure 2. In panel e, the blue bar was incorrectly labelled 'KRT8(+)/TOMATO(-)'. Furthermore, during the process of preparing a correction, the publication date of the Article was inadvertently changed to June 20th 2018. Both of these errors have been corrected in the PDF and HTML versions of the Article.

FGF signalling controls the specification of hair placode-derived SOX9 positive progenitors to Merkel cells.

Merkel cells are innervated mechanosensory cells responsible for light-touch sensations. In murine dorsal skin, Merkel cells are located in touch domes and found in the epidermis around primary hairs. While it has been shown that Merkel cells are skin epithelial cells, the progenitor cell population that gives rise to these cells is unknown. Here, we show that during embryogenesis, SOX9-positive (+) cells inside hair follicles, which were previously known to give rise to hair follicle stem cells (HFSCs) and cells of the hair follicle lineage, can also give rise to Merkel Cells. Interestingly, while SOX9 is critical for HFSC specification, it is dispensable for Merkel cell formation. Conversely, FGFR2 is required for Merkel cell formation but is dispensable for HFSCs. Together, our studies uncover SOX9(+) cells as precursors of Merkel cells and show the requirement for FGFR2-mediated epithelial signalling in Merkel cell specification.