p63 exerts spatio-temporal control of palatal epithelial cell fate to prevent cleft palate.

Cleft palate is a common congenital disorder that affects up to 1 in 2500 live births and results in considerable morbidity to affected individuals and their families. The aetiology of cleft palate is complex with both genetic and environmental factors implicated. Mutations in the transcription factor p63 are one of the major individual causes of cleft palate; however, the gene regulatory networks in which p63 functions remain only partially characterized. Our findings demonstrate that p63 functions as an essential regulatory molecule in the spatio-temporal control of palatal epithelial cell fate to ensure appropriate fusion of the palatal shelves. Initially, p63 induces periderm formation and controls its subsequent maintenance to prevent premature adhesion between adhesion-competent, intra-oral epithelia. Subsequently, TGFß3-induced down-regulation of p63 in the medial edge epithelia of the palatal shelves is a pre-requisite for palatal fusion by facilitating periderm migration from, and reducing the proliferative potential of, the midline epithelial seam thereby preventing cleft palate.

RNA-seq based transcriptomic map reveals new insights into mouse salivary gland development and maturation.

BACKGROUND: Mouse models have served a valuable role in deciphering various facets of Salivary Gland (SG) biology, from normal developmental programs to diseased states. To facilitate such studies, gene expression profiling maps have been generated for various stages of SG organogenesis. However these prior studies fall short of capturing the transcriptional complexity due to the limited scope of gene-centric microarray-based technology. Compared to microarray, RNA-sequencing (RNA-seq) offers unbiased detection of novel transcripts, broader dynamic range and high specificity and sensitivity for detection of genes, transcripts, and differential gene expression. Although RNA-seq data, particularly under the auspices of the ENCODE project, have covered a large number of biological specimens, studies on the SG have been lacking. RESULTS: To better appreciate the wide spectrum of gene expression profiles, we isolated RNA from mouse submandibular salivary glands at different embryonic and adult stages. In parallel, we processed RNA-seq data for 24 organs and tissues obtained from the mouse ENCODE consortium and calculated the average gene expression values. To identify molecular players and pathways likely to be relevant for SG biology, we performed functional gene enrichment analysis, network construction and hierarchal clustering of the RNA-seq datasets obtained from different stages of SG development and maturation, and other mouse organs and tissues. Our bioinformatics-based data analysis not only reaffirmed known modulators of SG morphogenesis but revealed novel transcription factors and signaling pathways unique to mouse SG biology and function. Finally we demonstrated that the unique SG gene signature obtained from our mouse studies is also well conserved and can demarcate features of the human SG transcriptome that is different from other tissues. CONCLUSIONS: Our RNA-seq based Atlas has revealed a high-resolution cartographic view of the dynamic transcriptomic landscape of the mouse SG at various stages. These RNA-seq datasets will complement pre-existing microarray based datasets, including the Salivary Gland Molecular Anatomy Project by offering a broader systems-biology based perspective rather than the classical gene-centric view. Ultimately such resources will be valuable in providing a useful toolkit to better understand how the diverse cell population of the SG are organized and controlled during development and differentiation.

TGFß3 regulates periderm removal through ΔNp63 in the developing palate.

The periderm is a flat layer of epithelium created during embryonic development. During palatogenesis, the periderm forms a protective layer against premature adhesion of the oral epithelia, including the palate. However, the periderm must be removed in order for the medial edge epithelia (MEE) to properly adhere and form a palatal seam. Improper periderm removal results in a cleft palate. Although the timing of transforming growth factor ß3 (TGFß3) expression in the MEE coincides with periderm degeneration, its role in periderm desquamation is not known. Interestingly, murine models of knockout (-/-) TGFß3, interferon regulatory factor 6 (IRF6) (-/-), and truncated p63 (ΔNp63) (-/-) are born with palatal clefts because of failure of the palatal shelves to adhere, suggesting that these genes regulate palatal epithelial differentiation. However, despite having similar phenotypes in null mouse models, no studies have analyzed the possible association between the TGFß3 signaling cascade and the IRF6/ΔNp63 genes during palate development. Recent studies indicate that regulation of ΔNp63, which depends on IRF6, facilitates epithelial differentiation. We performed biochemical analysis, gene activity and protein expression assays with palatal sections of TGFß3 (-/-), ΔNp63 (-/-), and wild-type (WT) embryos, and primary MEE cells from WT palates to analyze the association between TGFß3 and IRF6/ΔNp63. Our results suggest that periderm degeneration depends on functional TGFß3 signaling to repress ΔNp63, thereby coordinating periderm desquamation. Cleft palate occurs in TGFß3 (-/-) because of inadequate periderm removal that impedes palatal seam formation, while cleft palate occurs in ΔNp63 (-/-) palates because of premature fusion.

A global analysis of the complex landscape of isoforms and regulatory networks of p63 in human cells and tissues.

BACKGROUND: The transcription factor p63 belongs to the p53/p63/p73 family and plays key functional roles during normal epithelial development and differentiation and in pathological states such as squamous cell carcinomas. The human TP63 gene, located on chromosome 3q28 is driven by two promoters that generate the full-length transactivating (TA) and N-terminal truncated (ΔN) isoforms. Furthermore alternative splicing at the C-terminus gives rise to additional α, ß, γ and likely several other minor variants. Teasing out the expression and biological function of each p63 variant has been both the focus of, and a cause for contention in the p63 field. RESULTS: Here we have taken advantage of a burgeoning RNA-Seq based genomic data-sets to examine the global expression profiles of p63 isoforms across commonly utilized human cell-lines and major tissues and organs. Consistent with earlier studies, we find ΔNp63 transcripts, primarily that of the ΔNp63α isoforms, to be expressed in most cells of epithelial origin such as those of skin and oral tissues, mammary glands and squamous cell carcinomas. In contrast, TAp63 is not expressed in the majority of normal cell-types and tissues; rather it is selectively expressed at moderate to high levels in a subset of Burkitt's and diffuse large B-cell lymphoma cell lines. We verify this differential expression pattern of p63 isoforms by Western blot analysis, using newly developed ΔN and TA specific antibodies. Furthermore using unsupervised clustering of human cell lines, tissues and organs, we show that ΔNp63 and TAp63 driven transcriptional networks involve very distinct sets of molecular players, which may underlie their different biological functions. CONCLUSIONS: In this study we report comprehensive and global expression profiles of p63 isoforms and their relationship to p53/p73 and other potential transcriptional co-regulators. We curate publicly available data generated in part by consortiums such as ENCODE, FANTOM and Human Protein Atlas to delineate the vastly different transcriptomic landscapes of ΔNp63 and TAp63. Our studies help not only in dispelling prevailing myths and controversies on p63 expression in commonly used human cell lines but also augur new isoform- and cell type-specific activities of p63.

Exome sequence identifies RIPK4 as the Bartsocas-Papas syndrome locus.

Pterygium syndromes are complex congenital disorders that encompass several distinct clinical conditions characterized by multiple skin webs affecting the flexural surfaces often accompanied by craniofacial anomalies. In severe forms, such as in the autosomal-recessive Bartsocas-Papas syndrome, early lethality is common, complicating the identification of causative mutations. Using exome sequencing in a consanguineous family, we identified the homozygous mutation c.1127C>A in exon 7 of RIPK4 that resulted in the introduction of the nonsense mutation p.Ser376X into the encoded ankyrin repeat-containing kinase, a protein that is essential for keratinocyte differentiation. Subsequently, we identified a second mutation in exon 2 of RIPK4 (c.242T>A) that resulted in the missense variant p.Ile81Asn in the kinase domain of the protein. We have further demonstrated that RIPK4 is a direct transcriptional target of the protein p63, a master regulator of stratified epithelial development, which acts as a nodal point in the cascade of molecular events that prevent pterygium syndromes.

ΔNp63 knockout mice reveal its indispensable role as a master regulator of epithelial development and differentiation.

The transcription factor p63 is important in the development of the skin as p63-null mice exhibit striking defects in embryonic epidermal morphogenesis. Understanding the mechanisms that underlie this phenotype is complicated by the existence of multiple p63 isoforms, including TAp63 and ΔNp63. To investigate the role of ΔNp63 in epidermal morphogenesis we generated ΔNp63 knock-in mice in which the ΔNp63-specific exon is replaced by GFP. Homozygous ΔNp63(gfp/gfp) animals exhibit severe developmental anomalies including truncated forelimbs and the absence of hind limbs, largely phenocopying existing knockouts in which all p63 isoforms are deleted. ΔNp63-null animals show a poorly developed stratified epidermis comprising isolated clusters of disorganized epithelial cells. Despite the failure to develop a mature stratified epidermis, the patches of ΔNp63-null keratinocytes are able to stratify and undergo a program of terminal differentiation. However, we observe premature expression of markers associated with terminal differentiation, which is unique to ΔNp63-null animals and not evident in the skin of mice lacking all p63 isoforms. We posit that the dysregulated and accelerated keratinocyte differentiation phenotype is driven by significant alterations in the expression of key components of the Notch signaling pathway, some of which are direct transcriptional targets of ΔNp63 as demonstrated by ChIP experiments. The analysis of ΔNp63(gfp/gfp) knockout mice reaffirms the indispensable role of the ΔN isoform of p63 in epithelial biology and confirms that ΔNp63-null keratinocytes are capable of committing to an epidermal cell lineage, but are likely to suffer from diminished renewal capacity and an altered differentiation fate.

Epidermal overexpression of transgenic ΔNp63 promotes type 2 immune and myeloid inflammatory responses and hyperplasia via NF-κB activation.

ΔNp63 is known to be critical in skin development and cancer; however, how it triggers proliferation and inflammation in vivo remains to be elucidated. Here, we find that induced ΔNp63 expression in skin of transgenic mice (TG) results in a hyperproliferative epidermis coupled with inflammatory infiltrates. In situ, infiltrating cells include CD45(+) leukocytes, CD19(+) B lymphocytes, CD3(+) T lymphocytes, CD4(+) T helper, CD25(+)/Foxp3(+) Treg, Ly6B(+) neutrophils, S-100(+) dendritic cells, and macrophages bearing CD11b(+), F4/80(+), CD68(+), and CD206(+) M2 type markers. Transcriptional profiling of TG skin revealed increased gene expression involved in inflammation and immune responses, including Th2/M2 cytokines and chemokines. These genes were co-regulated by ΔNp63 and NF-κB RelA or cRel, and enhanced by TNF-α. Elevated cRel, RelA, and IKKs were observed in TG mouse skin and human squamous carcinomas with ΔNp63 overexpression. Thus, our findings unveil a missing link connecting overexpressed ΔNp63 with aberrant NF-κB activation, pro-inflammatory and type 2 cytokines and chemokines, and host infiltrates during skin inflammation and hyperplasia. Our findings provide a missing link between ΔNp63 overexpression and NF-κB-mediated inflammation, of potential relevance to the pathogenesis of squamous carcinoma.

Family matters: sibling rivalry and bonding between p53 and p63 in cancer.

The p53 family (p53, p63 and p73) is intimately linked with an overwhelming number of cellular processes during normal physiological as well as pathological conditions including cancer. The fact that these proteins are expressed in myriad isoforms, each with unique biochemical properties and distinct effects on tumorigenesis, complicates their study. A case in point is Squamous Cell Carcinoma (SCC) where p53 is often mutated and the ΔNp63 isoform is overexpressed. Given that p53 and p63 can hetero-dimerize, bind to quite similar DNA elements and share common co-factors, any alterations in their individual expression levels, activity and/or mutation can severely disrupt the family equilibrium. The burgeoning genomics data sets and new additions to the experimental toolbox are offering crucial insights into the complex role of the p53 family in SCC, but more mechanistic studies are needed.

Brg1 determines urothelial cell fate during ureter development.

Developing and adult ureters express the epigenetic regulator Brg1, but the role of Brg1 in ureter development is not well understood. We conditionally ablated Brg1 in the developing ureter using Hoxb7-Cre and found that Brg1 expression is upstream of p63, Pparγ, and sonic hedgehog (Shh) expression in the ureteral epithelium. In addition, epithelial stratification in the basal cells required Brg1-dependent p63 expression, whereas terminal differentiation of the umbrella cells required Brg1-dependent Pparγ expression. Furthermore, the loss of ureteric Brg1 resulted in failure of Shh expression, which correlated with reduced smooth muscle cell development and hydroureter. Taken together, we conclude that Brg1 expression unifies three aspects of ureter development: maintenance of the basal cell population, guidance for terminal differentiation of urothelial cells, and proper investment of ureteral smooth muscle cells.

ΔNp63 regulates Sfrp1 expression to direct salivary gland branching morphogenesis.

Branching morphogenesis is a complex process shared by many organs including the lungs, kidney, prostate, as well as several exocrine organs including the salivary, mammary and lacrimal glands. This critical developmental program ensures the expansion of an organ's surface area thereby maximizing processes of cellular secretion or absorption. It is guided by reciprocal signaling from the epithelial and mesenchymal cells. While signaling pathways driving salivary gland branching morphogenesis have been relatively well-studied, our understanding of the underlying transcriptional regulatory mechanisms directing this program, is limited. Here, we performed in vivo and ex vivo studies of the embryonic mouse submandibular gland to determine the function of the transcription factor ΔNp63, in directing branching morphogenesis. Our studies show that loss of ΔNp63 results in alterations in the differentiation program of the ductal cells which is accompanied by a dramatic reduction in branching morphogenesis that is mediated by dysregulation of WNT signaling. We show that ΔNp63 modulates WNT signaling to promote branching morphogenesis by directly regulating Sfrp1 expression. Collectively, our findings have revealed a novel role for ΔNp63 in the regulation of this critical process and offers a better understanding of the transcriptional networks involved in branching morphogenesis.

Tlr7 drives sex- and tissue-dependent effects in Sjögren's disease.

Primary Sjögren's disease (pSD) is a systemic autoimmune disease that has the strongest female predilection of all autoimmune diseases. The underlying mechanisms that govern this sexual dimorphism, however, remain poorly understood. We hypothesized that pSD females would exhibit more robust disease as compared to males, and that Tlr7 controls distinct disease manifestations in males and females. Using a well-established pSD mouse model, we harvested exocrine glands, and pulmonary and renal tissue from males and females and quantified the inflammation present. We then collected salivary glands, spleens, and cervical lymph nodes and performed flow cytometry to assess immune populations implicated in disease. We also harvested sera to examine total and autoreactive antibodies. Our data revealed that pSD mice displayed sex-biased disease, as pSD females showed decreased dacryoadenitis, but increased nephritis as compared to males. Moreover, females exhibited increased proportions of germinal center B cells and CD4+ activated/memory T cells in the periphery. Additionally, salivary gland immune populations were altered in a sex-dependent manner in pSD. Females with pSD also displayed elevated total and autoreactive IgG as compared to males. Additionally, splenic B cell Tlr7 expression was increased in females. We next generated pSD mice that lacked Tlr7 systemically and found that ablation of Tlr7 was primarily protective in pSD females, while Tlr7-deficient pSD males showed heightened disease. Thus, pSD mice display sex-biased disease and these dichotomous manifestations are governed by Tlr7 activation. This study identifies Tlr7 as a druggable target for pSD, and highlights the importance of studying pSD disease mechanisms in both sexes.

Abnormal hair follicle development and altered cell fate of follicular keratinocytes in transgenic mice expressing DeltaNp63alpha.

The transcription factor p63 plays an essential role in epidermal morphogenesis. Animals lacking p63 fail to form many ectodermal organs, including the skin and hair follicles. Although the indispensable role of p63 in stratified epithelial skin development is well established, relatively little is known about this transcriptional regulator in directing hair follicle morphogenesis. Here, using specific antibodies, we have established the expression pattern of DeltaNp63 in hair follicle development and cycling. DeltaNp63 is expressed in the developing hair placode, whereas in mature hair its expression is restricted to the outer root sheath (ORS), matrix cells and to the stem cells of the hair follicle bulge. To investigate the role of DeltaNp63 in hair follicle morphogenesis and cycling, we have utilized a Tet-inducible mouse model system with targeted expression of this isoform to the ORS of the hair follicle. DeltaNp63 transgenic animals display dramatic defects in hair follicle development and cycling, eventually leading to severe hair loss. Strikingly, expression of DeltaNp63 leads to a switch in cell fate of hair follicle keratinocytes, causing them to adopt an interfollicular epidermal (IFE) cell identity. Moreover, DeltaNp63 transgenic animals exhibit a depleted hair follicle stem-cell niche, which further contributes to the overall cycling defects observed in the mutant animals. Finally, global transcriptome analysis of transgenic skin identified altered expression levels of crucial mediators of hair morphogenesis, including key members of the Wnt/beta-catenin signaling pathway, which, in part, account for these effects. Our data provide evidence supporting a role for DeltaNp63alpha in actively suppressing hair follicle differentiation and directing IFE cell lineage commitment.

Elf5 regulates mammary gland stem/progenitor cell fate by influencing notch signaling.

The transcription factor E74-like factor 5 (Elf5) functions downstream of the prolactin receptor signaling pathway and plays an important role in mammary gland development. Using conditional mouse knockouts, we have previously shown that Elf5-null mammary glands exhibit a complete failure of alveologenesis during pregnancy. The Elf5-null developmental phenotype is mediated through alteration in the expression of several critical genes involved in alveologenesis, particularly those belonging to the JAK/STAT pathway. Here, we demonstrate that in addition to regulating terminal differentiation of alveolar cells, Elf5 also plays a critical role in determining cell fate and in regulating the stem/progenitor function of the mammary epithelium. Targeted deletion of Elf5 in the mammary glands leads to accumulation of cell types with dual luminal/basal properties such as coexpression of K8 and K14 and an increase in CD61(+) luminal progenitor population during pregnancy. Further interrogation suggests that the abnormal increase in K14(+) K8(+) cells may represent the CD61(+) luminal progenitors blocked in differentiation. Remarkably, Elf5 deficiency in mammary epithelium also triggers an increase of adult mammary stem activity as evidenced by the accumulation of mammary stem cell (MaSC)-enriched cell population in both pregnant and virgin mice and further confirmed by mammosphere and transplantation assays. Additional support for this phenotype comes from the enriched MaSC gene signature based on transcriptomic analysis of the Elf5-null mammary gland. Finally, our biochemical studies suggest that Elf5 loss leads to hyperactivation of the Notch signaling pathway, which might constitute in part, the underlying molecular mechanism for the altered cell lineage decisions in Elf5-null mammary epithelial cells.

A Systemic and Integrated Analysis of p63-Driven Regulatory Networks in Mouse Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity and is linked to tobacco exposure, alcohol consumption, and human papillomavirus infection. Despite therapeutic advances, a lack of molecular understanding of disease etiology, and delayed diagnoses continue to negatively affect survival. The identification of oncogenic drivers and prognostic biomarkers by leveraging bulk and single-cell RNA-sequencing datasets of OSCC can lead to more targeted therapies and improved patient outcomes. However, the generation, analysis, and continued utilization of additional genetic and genomic tools are warranted. Tobacco-induced OSCC can be modeled in mice via 4-nitroquinoline 1-oxide (4NQO), which generates a spectrum of neoplastic lesions mimicking human OSCC and upregulates the oncogenic master transcription factor p63. Here, we molecularly characterized established mouse 4NQO treatment-derived OSCC cell lines and utilized RNA and chromatin immunoprecipitation-sequencing to uncover the global p63 gene regulatory and signaling network. We integrated our p63 datasets with published bulk and single-cell RNA-sequencing of mouse 4NQO-treated tongue and esophageal tumors, respectively, to generate a p63-driven gene signature that sheds new light on the role of p63 in murine OSCC. Our analyses reveal known and novel players, such as COTL1, that are regulated by p63 and influence various oncogenic processes, including metastasis. The identification of new sets of potential biomarkers and pathways, some of which are functionally conserved in human OSCC and can prognosticate patient survival, offers new avenues for future mechanistic studies.

An integrated genomic approach identifies follistatin as a target of the p63-epidermal growth factor receptor oncogenic network in head and neck squamous cell carcinoma.

Although numerous putative oncogenes have been associated with the etiology of head and neck squamous cell carcinoma (HNSCC), the mechanisms by which these oncogenes and their downstream targets mediate tumor progression have not been fully elucidated. We performed an integrative analysis to identify a crucial set of targets of the oncogenic transcription factor p63 that are common across multiple transcriptomic datasets obtained from HNSCC patients, and representative cell line models. Notably, our analysis revealed FST which encodes follistatin, a secreted glycoprotein that inhibits the transforming growth factor TGFß/activin signaling pathways, to be a direct transcriptional target of p63. In addition, we found that FST expression is also driven by epidermal growth factor receptor EGFR signaling, thus mediating a functional link between the TGF-ß and EGFR pathways. We show through loss- and gain-of-function studies that FST predominantly imparts a tumor-growth and migratory phenotype in HNSCC cells. Furthermore, analysis of single-cell RNA sequencing data from HNSCC patients unveiled cancer cells as the dominant source of FST within the tumor microenvironment and exposed a correlation between the expression of FST and its regulators with immune infiltrates. We propose FST as a prognostic biomarker for patient survival and a compelling candidate mediating the broad effects of p63 on the tumor and its associated microenvironment.

ΔNp63 maintains the fidelity of the myoepithelial cell lineage and directs cell differentiation programs in the murine salivary gland.

Salivary glands consist of several epithelial cell types of distinct lineages and functional characteristics that are established by directed differentiation programs of resident stem and progenitor cells. We have shown that ΔNp63, a crucial transcriptional regulator of stem/progenitor cells, is enriched in both the basal and myoepithelial cell (MEC) populations and that ΔNp63 positive cells maintain all the descendent epithelial cell lineages of the adult mouse salivary glands (mSGs). Although this pivotal role of ΔNp63 in driving the broader epithelial cell fate and identity in the mSG has been demonstrated, how ΔNp63 functions specifically in the commitment and differentiation of the MEC population is less understood. Using multiple genetic mouse models that allow for cell tracing, we show that ΔNp63 is critical in maintaining and renewing MECs, in part through the transcriptional regulation of Acta2 gene expression, a defining marker of this cell population. We demonstrate that during adult mSG homeostasis, ΔNp63 enriched MECs function as bipotent progenitor cells that maintain not only the MEC population, but also the distinctly different ductal cell lineages. The fidelity of this process is dependent on ΔNp63 expression, since MEC-specific ablation of ΔNp63 results in altered MEC differentiation and affects cellular plasticity resulting in aberrant differentiation of the intercalated ducts and acinar cells. In contrast, we find that the contribution of MECs to ductal and acinar cell regeneration following severe injury is independent of ΔNp63. Our observations offer new insights into cellular mechanisms driving MEC fate choices and differentiation programs in the context of salivary gland homeostasis and in response to injury and regeneration. Long term, these findings have implications for better treatment of salivary gland dysfunction through stem cell-based approaches.

DeltaNp63 regulates stem cell dynamics in the mammalian olfactory epithelium.

The ability of the olfactory epithelium (OE) to regenerate after injury is mediated by at least two populations of presumed stem cells-globose basal cells (GBCs) and horizontal basal cells (HBCs). Of the two, GBCs are molecularly and phenotypically analogous to the olfactory progenitors of the embryonic placode (OPPs). In contrast, HBCs are a reserve stem cell population that appears later in development and requires activation by severe epithelial damage before contributing to epithelial reconstitution. Neither HBC emergence nor the mechanism of activation after injury is understood. Here we show that the transcription factor p63 (Trp63), which is expressed selectively by adult HBCs, is required for HBC differentiation. The first evidence of HBC differentiation is the expression of p63 by cells that closely resemble embryonic OPPs and adult GBCs by morphology and expression of the transcription factors Sox2, Ascl1, and Hes1. HBC formation is delayed in Ascl1 knock-out OE and is completely abrogated in p63-null mice. Strikingly, other cell types of the OE form normally in the p63 knock-out OE. The role of p63 in HBC differentiation appears to be conserved in the regenerating rat OE, where HBCs disappear and then reappear after tissue lesion. Finally, p63 protein is downregulated in HBCs activated by lesion to become multipotent progenitor cells. Together, our data identify a novel mechanism for the generation of a reserve stem cell population and suggest that a p63-dependent molecular switch is responsible for activating reserve stem cells when they are needed.

EHF is a novel regulator of cellular redox metabolism and predicts patient prognosis in HNSCC.

Head and Neck Squamous Cell Carcinoma (HNSCC) is a heterogeneous disease with relatively high morbidity and mortality rates. The lack of effective therapies, high recurrence rates and drug resistance driven in part, by tumor heterogeneity, contribute to the poor prognosis for patients diagnosed with this cancer. This problem is further exacerbated by the fact that key regulatory factors contributing to the disease diversity remains largely elusive. Here, we have identified EHF as an important member of the ETS family of transcription factors that is highly expressed in normal oral tissues, but lost during HNSCC progression. Interestingly, HNSCC tumors and cell lines exhibited a dichotomy of high and low EHF expression, and patients whose tumors retained EHF expression showed significantly better prognosis, suggesting a potential tumor suppressive role for EHF. To address this, we have performed gain and loss of function studies and leveraged bulk and single-cell cancer genomic datasets to identify global EHF targets by RNA-sequencing (RNA-seq) and Chromatin Immunoprecipitation and next generation sequencing (ChIP-seq) experiments of HNSCC cell lines. These mechanistic studies have revealed that EHF, acts as a regulator of a broad spectrum of metabolic processes, specifically targeting regulators of redox homeostasis such as NRF2 and SOX2. Our immunostaining results confirm the mutually exclusive expression patterns of EHF and SOX2 in HNSCC tumors and suggest a possible role for these two factors in establishing discrete metabolic states within the tumor microenvironment. Taken together, EHF may serve as a novel prognostic marker for classifying HNSCC patients for actionable and targeted therapeutic intervention.

Dynamic life of a skin keratinocyte: an intimate tryst with the master regulator p63.

Skin keratinocytes form a tightly knit and layered epithelium at the surface of the body protecting the body from the outside environment. The formation and maintenance of skin epidermis is governed by dynamic and well-coordinated processes of cell proliferation, differentiation, and self-renewal. Such important cell fate decisions are made possible in part by transcription factors, which activate and repress unique sets of genes in a temporal and spatial pattern. The Tp63 gene encodes for multiple isoforms for one such transcription factor that serves as a key regulator of epidermal development and differentiation. The crucial function of p63 is epitomized by the phenotype of p63 knockout mice--in the absence of p63, there is a profound block in the development of skin epidermis and all related appendages such as hair follicles. Human syndromes resulting from Tp63 gene mutations phenocopy the p63 knockout phenotype, highlighting the evolutionarily conserved function of this factor in epithelial biology. Although the function of p63 as an important hub in transcriptional and signaling networks of keratinocytes is well established, the underlying molecular mechanisms of p63 action is continually redefined with the development of new genetic models and more extensive biochemical analysis. In this review the biological role of deltaNp63, the predominant isoform that is expressed in skin keratinocytes has been described. Results from transgenic animal models that have shed new information on the function of deltaNp63 in the epidermis and hair follicles have been discussed. Further, the molecular mechanisms that maintain the fine-tuned expression of deltaNp63 in skin keratinocytes are also described.

Transcriptomic and Single-Cell Analysis Reveals Regulatory Networks and Cellular Heterogeneity in Mouse Primary Sjögren's Syndrome Salivary Glands.

Sjögren's Syndrome (SS) is a chronic autoimmune disease of unknown etiology which primarily affects the salivary and lacrimal glands resulting in the loss of secretory function. Treatment options for SS have been hampered due to the lack of a better understanding of the underlying gene regulatory circuitry and the interplay between the myriad pathological cellular states that contribute to salivary gland dysfunction. To better elucidate the molecular nature of SS, we have performed RNA-sequencing analysis of the submandibular glands (SMG) of a well-established primary Sjögren's Syndrome (pSS) mouse model. Our comprehensive examination of global gene expression and comparative analyses with additional SS mouse models and human datasets, have identified a number of important pathways and regulatory networks that are relevant in SS pathobiology. To complement these studies, we have performed single-cell RNA sequencing to examine and identify the molecular and cellular heterogeneity of the diseased cell populations of the mouse SMG. Interrogation of the single-cell transcriptomes has shed light on the diversity of immune cells that are dysregulated in SS and importantly, revealed an activated state of the salivary gland epithelial cells that contribute to the global immune mediated responses. Overall, our broad studies have not only revealed key pathways, mediators and new biomarkers, but have also uncovered the complex nature of the cellular populations in the SMG that are likely to drive the progression of SS. These newly discovered insights into the underlying molecular mechanisms and cellular states of SS will better inform targeted therapeutic discoveries.