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.

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.

miR-424(322)/503 is a breast cancer tumor suppressor whose loss promotes resistance to chemotherapy.

The female mammary gland is a very dynamic organ that undergoes continuous tissue remodeling during adulthood. Although it is well established that the number of menstrual cycles and pregnancy (in this case transiently) increase the risk of breast cancer, the reasons are unclear. Growing clinical and experimental evidence indicates that improper involution plays a role in the development of this malignancy. Recently, we described the miR-424(322)/503 cluster as an important regulator of mammary epithelial involution after pregnancy. Here, through the analysis of ∼3000 primary tumors, we show that miR-424(322)/503 is commonly lost in a subset of aggressive breast cancers and describe the genetic aberrations that inactivate its expression. Furthermore, through the use of a knockout mouse model, we demonstrate for the first time that loss of miR-424(322)/503 promotes breast tumorigenesis in vivo. Remarkably, we found that loss of miR-424(322)/503 promotes chemoresistance due to the up-regulation of two of its targets: BCL-2 and insulin-like growth factor-1 receptor (IGF1R). Importantly, targeted therapies blocking the aberrant activity of these targets restore sensitivity to chemotherapy. Overall, our studies reveal miR-424(322)/503 as a tumor suppressor in breast cancer and provide a link between mammary epithelial involution, tumorigenesis, and the phenomenon of chemoresistance.

Hair follicles' transit-amplifying cells govern concurrent dermal adipocyte production through Sonic Hedgehog.

Growth and regeneration of one tissue within an organ compels accommodative changes in the surrounding tissues. However, the molecular nature and operating logic governing these concurrent changes remain poorly defined. The dermal adipose layer expands concomitantly with hair follicle downgrowth, providing a paradigm for studying coordinated changes of surrounding lineages with a regenerating tissue. Here, we discover that hair follicle transit-amplifying cells (HF-TACs) play an essential role in orchestrating dermal adipogenesis through secreting Sonic Hedgehog (SHH). Depletion of Shh from HF-TACs abrogates both dermal adipogenesis and hair follicle growth. Using cell type-specific deletion of Smo, a gene required in SHH-receiving cells, we found that SHH does not act on hair follicles, adipocytes, endothelial cells, and hematopoietic cells for adipogenesis. Instead, SHH acts directly on adipocyte precursors, promoting their proliferation and their expression of a key adipogenic gene, peroxisome proliferator-activated receptor γ (Pparg), to induce dermal adipogenesis. Our study therefore uncovers a critical role for TACs in orchestrating the generation of both their own progeny and a neighboring lineage to achieve concomitant tissue production across lineages.

The mesenchymal transcription factor SNAI-1 instructs human liver specification.

Epithelial-mesenchymal transition (EMT) and the mesenchymal-epithelial transition (MET) are processes required for embryo organogenesis. Liver develops from the epithelial foregut endoderm from which the liver progenitors, hepatoblasts, are specified. The migrating hepatoblasts acquire a mesenchymal phenotype to form the liver bud. In mid-gestation, hepatoblasts mature into epithelial structures: the hepatocyte cords and biliary ducts. While EMT has been associated with liver bud formation, nothing is known about its contribution to hepatic specification. We previously established an efficient protocol from human embryonic stem cells (hESC) to generate hepatic cells (Hep cells) resembling the hepatoblasts expressing alpha-fetoprotein (AFP) and albumin (ALB). Here we show that Hep cells express both epithelial (EpCAM and E-cadherin) and mesenchymal (vimentin and SNAI-1) markers. Similar epithelial and mesenchymal hepatoblasts were identified in human and mouse fetal livers, suggesting a conserved interspecies phenotype. Knock-down experiments demonstrated the importance of SNAI-1 in Hep cell hepatic specification. Moreover, ChIP assays revealed direct binding of SNAI-1 in the promoters of AFP and ALB genes consistent with its transcriptional activator function in hepatic specification. Altogether, our hESC-derived Hep cell cultures reveal the dual mesenchymal and epithelial phenotype of hepatoblast-like cells and support the unexpected transcriptional activator role of SNAI-1 in hepatic specification.

Cbx4: A new guardian of p63's domain of epidermal control.

Epigenetic regulators are essential for cell lineage choices during development. In this issue, Mardaryev et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201506065) show that Polycomb subunit Cbx4 acts downstream of transcriptional regulator p63 to maintain epidermal progenitor identity and proliferation in the developing epidermis via Polycomb-dependent and -independent SUMO E3 ligase activities.

Terminal epidermal differentiation is regulated by the interaction of Fra-2/AP-1 with Ezh2 and ERK1/2.

Altered epidermal differentiation characterizes numerous skin diseases affecting >25% of the human population. Here we identified Fra-2/AP-1 as a key regulator of terminal epidermal differentiation. Epithelial-restricted, ectopic expression of Fra-2 induced expression of epidermal differentiation genes located within the epidermal differentiation complex (EDC). Moreover, in a papilloma-prone background, a reduced tumor burden was observed due to precocious keratinocyte differentiation by Fra-2 expression. Importantly, loss of Fra-2 in suprabasal keratinocytes is sufficient to cause skin barrier defects due to reduced expression of differentiation genes. Mechanistically, Fra-2 binds and transcriptionally regulates EDC gene promoters, which are co-occupied by the transcriptional repressor Ezh2. Fra-2 remains transcriptionally inactive in nondifferentiated keratinocytes, where it was found monomethylated and dimethylated on Lys104 and interacted with Ezh2. Upon keratinocyte differentiation, Fra-2 is C-terminally phosphorylated on Ser320 and Thr322 by ERK1/2, leading to transcriptional activation. Thus, the induction of epidermal differentiation by Fra-2 is controlled by a dual mechanism involving Ezh2-dependent methylation and activation by ERK1/2-dependent phosphorylation.

The microRNA 424/503 cluster reduces CDC25A expression during cell cycle arrest imposed by transforming growth factor ß in mammary epithelial cells.

Recently, we demonstrated that the microRNA 424(322)/503 [miR-424(322)/503] cluster is transcriptionally controlled by transforming growth factor ß (TGF-ß) in the mammary epithelium. Induction of this microRNA cluster impacts mammary epithelium fate by regulating apoptosis and insulin-like growth factor 1 (IGF1) signaling. Here, we expanded our finding to demonstrate that miR-424(322)/503 is an integral component of the cell cycle arrest mediated by TGF-ß. Mechanistically, we showed that after TGF-ß exposure, increased levels of miR-424(322)/503 reduce the expression of the cell cycle regulator CDC25A. miR-424(322)/503-dependent posttranscriptional downregulation of CDC25A cooperates with previously described transcriptional repression of the CDC25A promoter and proteasome-mediated degradation to reduce the levels of CDC25A expression and to induce cell cycle arrest. We also provide evidence that the TGF-ß/miR-424(322)/503 axis is part of the mechanism that regulates the proliferation of hormone receptor-positive (HR(+)) mammary epithelial cells in vivo.

The miR-424(322)/503 cluster orchestrates remodeling of the epithelium in the involuting mammary gland.

The mammary gland is a very dynamic organ that undergoes continuous remodeling. The critical regulators of this process are not fully understood. Here we identify the microRNA cluster miR-424(322)/503 as an important regulator of epithelial involution after pregnancy. Through the generation of a knockout mouse model, we found that regression of the secretory acini of the mammary gland was compromised in the absence of miR-424(322)/503. Mechanistically, we show that miR-424(322)/503 orchestrates cell life and death decisions by targeting BCL-2 and IGF1R (insulin growth factor-1 receptor). Furthermore, we demonstrate that the expression of this microRNA cluster is regulated by TGF-ß, a well-characterized regulator of mammary involution. Overall, our data suggest a model in which activation of the TGF-ß pathway after weaning induces the transcription of miR-424(322)/503, which in turn down-regulates the expression of key genes. Here, we unveil a previously unknown, multilayered regulation of epithelial tissue remodeling coordinated by the microRNA cluster miR-424(322)/503.

Epigenetic regulation of skin: focus on the Polycomb complex.

Chromatin regulators have recently emerged as key players in the control of tissue development and tumorigenesis. One specific chromatin regulator, the Polycomb complex, has been shown to regulate the identity of embryonic stem cells, but its role in controlling fates of multipotent progenitors in developing tissues is still largely unknown. Recent findings have revealed that this complex plays a critical role in control of skin stem cell renewal and differentiation. Moreover, the expression of Polycomb complex components is often aberrant in skin diseases, including skin cancers. This review will detail recent findings on Polycomb control of skin and highlight critical unknown questions.

EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair.

Polycomb protein group (PcG)-dependent trimethylation on H3K27 (H3K27me3) regulates identity of embryonic stem cells (ESCs). How H3K27me3 governs adult SCs and tissue development is unclear. Here, we conditionally target H3K27 methyltransferases Ezh2 and Ezh1 to address their roles in mouse skin homeostasis. Postnatal phenotypes appear only in doubly targeted skin, where H3K27me3 is abolished, revealing functional redundancy in EZH1/2 proteins. Surprisingly, while Ezh1/2-null hair follicles (HFs) arrest morphogenesis and degenerate due to defective proliferation and increased apoptosis, epidermis hyperproliferates and survives engraftment. mRNA microarray studies reveal that, despite these striking phenotypic differences, similar genes are up-regulated in HF and epidermal Ezh1/2-null progenitors. Featured prominently are (1) PcG-controlled nonskin lineage genes, whose expression is still significantly lower than in native tissues, and (2) the PcG-regulated Ink4a/Inkb/Arf locus. Interestingly, when EZH1/2 are absent, even though Ink4a/Arf/Ink4b genes are fully activated in HF cells, they are only partially so in epidermal progenitors. Importantly, transduction of Ink4b/Ink4a/Arf shRNAs restores proliferation/survival of Ezh1/2-null HF progenitors in vitro, pointing toward the relevance of this locus to the observed HF phenotypes. Our findings reveal new insights into Polycomb-dependent tissue control, and provide a new twist to how different progenitors within one tissue respond to loss of H3K27me3.

Cyfip1 is a putative invasion suppressor in epithelial cancers.

Identification of bona fide tumor suppressors is often challenging because of the large number of genetic alterations present in most human cancers. To evaluate candidate genes present within chromosomal regions recurrently deleted in human cancers, we coupled high-resolution genomic analysis with a two-stage genetic study using RNA interference (RNAi). We found that Cyfip1, a subunit of the WAVE complex, which regulates cytoskeletal dynamics, is commonly deleted in human epithelial cancers. Reduced expression of CYFIP1 is commonly observed during invasion of epithelial tumors and is associated with poor prognosis in this setting. Silencing of Cyfip1 disturbed normal epithelial morphogenesis in vitro and cooperated with oncogenic Ras to produce invasive carcinomas in vivo. Mechanistically, we have linked alterations in WAVE-regulated actin dynamics with impaired cell-cell adhesion and cell-ECM interactions. Thus, we propose Cyfip1 as an invasion suppressor gene.

Ezh2 orchestrates gene expression for the stepwise differentiation of tissue-specific stem cells.

Although in vitro studies of embryonic stem cells have identified polycomb repressor complexes (PRCs) as key regulators of differentiation, it remains unclear as to how PRC-mediated mechanisms control fates of multipotent progenitors in developing tissues. Here, we show that an essential PRC component, Ezh2, is expressed in epidermal progenitors but diminishes concomitant with embryonic differentiation and with postnatal decline in proliferative activity. We show that Ezh2 controls proliferative potential of basal progenitors by repressing the Ink4A-Ink4B locus and tempers the developmental rate of differentiation by preventing premature recruitment of AP1 transcriptional activator to the structural genes that are required for epidermal differentiation. Together, our studies reveal that PRCs control epigenetic modifications temporally and spatially in tissue-restricted stem cells. They maintain their proliferative potential and globally repressing undesirable differentiation programs while selectively establishing a specific terminal differentiation program in a stepwise fashion.

The proteasome regulatory particle alters the SAGA coactivator to enhance its interactions with transcriptional activators.

Promoter recruitment of the Saccharomyces cerevisiae SAGA histone acetyltransferase complex is required for RNA polymerase II-dependent transcription of several genes. SAGA is targeted to promoters through interactions with sequence-specific DNA binding transcriptional activators and facilitates preinitiation-complex assembly and transcription. Here, we show that the 19S proteasome regulatory particle (19S RP) alters SAGA to stimulate its interaction with transcriptional activators. The ATPase components of the 19S RP are required for stimulation of SAGA/activator interactions and enhance SAGA recruitment to promoters. Proteasomal ATPases genetically interact with SAGA, and their inhibition reduces global histone H3 acetylation levels and SAGA recruitment to target promoters in vivo. These results indicate that the 19S RP modulates SAGA complex using its ATPase components, thereby facilitating subsequent transcription events at promoters.

Proteasomal ATPases link ubiquitylation of histone H2B to methylation of histone H3.

In Saccharomyces cerevisiae, methylation of histone H3 at active genes is an epigenetic mark that distinguishes active from silent chromatin and functions as a short-term "memory" of recent transcription. Methylation of H3 at lysine residues K4 and K79 depends on ubiquitylation of histone H2B, but the mechanisms linking H2B ubiquitylation to H3 methylation are unknown. Here, we demonstrate that proteasomal ATPases Rpt4 and Rpt6 function to connect these two histone modifications. We show that recruitment of proteasome subunits to chromatin depends on H2B ubiquitylation and that mutations in Rpt4 and Rpt6 disrupt H3 methylation at K4 and K79 but leave H2B ubiquitylation intact. Consistent with their role in H3 methylation, we also find that mutations in Rpt4 and 6-but not components of the 20S proteasome-disrupt telomeric gene silencing. These data reveal that proteasome subunits function in epigenetic gene regulation by linking chromatin modifications that establish the histone code.