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.

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.

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.

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.

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.

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.

p63 and Its Target Follistatin Maintain Salivary Gland Stem/Progenitor Cell Function through TGF-ß/Activin Signaling.

Multipotent ΔNp63-positive cells maintain all epithelial cell lineages of the embryonic and adult salivary gland (SG). However, the molecular mechanisms by which ΔNp63 regulates stem/progenitor (SP) cell populations in the SG remains elusive. To understand the role of ΔNp63 in directing cell fate choices in this gland, we have generated ΔNp63-deleted adult mice and primary salivary cell cultures to probe alterations in SP cell differentiation and function. In parallel, we have leveraged RNA-seq and ChIP-seq-based characterization of the ΔNp63-driven cistrome and scRNA-seq analysis to molecularly interrogate altered SG cellular identities and differentiation states dependent on ΔNp63. Our studies reveal that ablation of ΔNp63 results in a loss of the SP cell population and skewed differentiation that is mediated by Follistatin-dependent dysregulated TGF-ß/Activin signaling. These findings offer new revelations into the SP cell gene regulatory networks that are likely to be relevant for normal or diseased SG states.

Transcriptomic and Network Analysis of Minor Salivary Glands of Patients With Primary Sjögren's Syndrome.

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterized primarily by immune-mediated destruction of exocrine tissues, such as those of the salivary and lacrimal glands, resulting in the loss of saliva and tear production, respectively. This disease predominantly affects middle-aged women, often in an insidious manner with the accumulation of subtle changes in glandular function occurring over many years. Patients commonly suffer from pSS symptoms for years before receiving a diagnosis. Currently, there is no effective cure for pSS and treatment options and targeted therapy approaches are limited due to a lack of our overall understanding of the disease etiology and its underlying pathology. To better elucidate the underlying molecular nature of this disease, we have performed RNA-sequencing to generate a comprehensive global gene expression profile of minor salivary glands from an ethnically diverse cohort of patients with pSS. Gene expression analysis has identified a number of pathways and networks that are relevant in pSS pathogenesis. Moreover, our detailed integrative analysis has revealed a primary Sjögren's syndrome molecular signature that may represent important players acting as potential drivers of this disease. Finally, we have established that the global transcriptomic changes in pSS are likely to be attributed not only to various immune cell types within the salivary gland but also epithelial cells which are likely playing a contributing role. Overall, our comprehensive studies provide a database-enriched framework and resource for the identification and examination of key pathways, mediators, and new biomarkers important in the pathogenesis of this disease with the long-term goals of facilitating earlier diagnosis of pSS and to mitigate or abrogate the progression of this debilitating disease.

Independent amylase gene copy number bursts correlate with dietary preferences in mammals.

The amylase gene (AMY), which codes for a starch-digesting enzyme in animals, underwent several gene copy number gains in humans (Perry et al., 2007), dogs (Axelsson et al., 2013), and mice (Schibler et al., 1982), possibly along with increased starch consumption during the evolution of these species. Here, we present comprehensive evidence for AMY copy number expansions that independently occurred in several mammalian species which consume diets rich in starch. We also provide correlative evidence that AMY gene duplications may be an essential first step for amylase to be expressed in saliva. Our findings underscore the overall importance of gene copy number amplification as a flexible and fast evolutionary mechanism that can independently occur in different branches of the phylogeny.

A new Elf5CreERT2-GFP BAC transgenic mouse model for tracing Elf5 cell lineages in adult tissues.

Elf5 is a transcription factor known to regulate critical developmental processes and has been shown to act as a tumour suppressor in multiple cancers. Elf5 knockout mice are embryonically lethal, limiting in vivo studies pertaining to its function. Moreover, haploinsufficiency of Elf5 limits the use of current mouse models to investigate adult tissue distribution of Elf5. Here, we successfully generated Elf5CreERT2-GFP bacterial artificial chromosome (BAC) transgenic mice and show that Elf5+ cells are present in several adult tissues, where its expression was previously not known. Our study demonstrates the unique distribution of Elf5+ cells in multiple adult organs, which will facilitate future studies investigating the function of Elf5 in these tissues during homeostasis, repair and cancer.

Activation of Myd88-Dependent TLRs Mediates Local and Systemic Inflammation in a Mouse Model of Primary Sjögren's Syndrome.

Toll-like receptors (TLRs) are important mediators of chronic inflammation in numerous autoimmune diseases, although the role of these receptors in primary Sjögren's syndrome (pSS) remains incompletely understood. Previous studies in our laboratory established Myd88 as a crucial mediator of pSS, although the disease-relevant ligands and the upstream signaling events that culminate in Myd88 activation have yet to be established. The objective of this study was to identify specific Myd88-dependent TLR-related pathways that are dysregulated both locally and systemically in a mouse model of pSS [NOD.B10Sn-H2b /J (NOD.B10)]. We performed RNA-sequencing on spleens derived from NOD.B10 mice. We then harvested salivary tissue and spleens from Myd88-sufficient and deficient C57BL/10 (BL/10) and NOD.B10 mice and performed flow cytometry to determine expression of Myd88-dependent TLRs. We cultured splenocytes with TLR2 and TLR4 agonists and measured production of inflammatory mediators by ELISA. Next, we evaluated spontaneous and TLR4-mediated inflammatory cytokine secretion in NOD.B10 salivary tissue. Finally, we assessed spontaneous Myd88-dependent cytokine secretion by NOD.B10 salivary cells. We identified dysregulation of numerous TLR-related networks in pSS splenocytes, particularly those employed by TLR2 and TLR4. We found upregulation of TLRs in both the splenic and salivary tissue from pSS mice. In NOD.B10 splenic tissue, robust expression of B cell TLR1 and TLR2 required Myd88. Splenocytes from NOD.B10 mice were hyper-responsive to TLR2 ligation and the endogenous molecule decorin modulated inflammation via TLR4. Finally, we observed spontaneous secretion of numerous inflammatory cytokines and this was enhanced following TLR4 ligation in female NOD.B10 salivary tissue as compared to males. The spontaneous production of salivary IL-6, MCP-1 and TNFα required Myd88 in pSS salivary tissue. Thus, our data demonstrate that Myd88-dependent TLR pathways contribute to the inflammatory landscape in pSS, and inhibition of such will likely have therapeutic utility.

Genetic and scRNA-seq Analysis Reveals Distinct Cell Populations that Contribute to Salivary Gland Development and Maintenance.

Stem and progenitor cells of the submandibular salivary gland (SMG) give rise to, maintain, and regenerate the multiple lineages of mature epithelial cells including those belonging to the ductal, acinar, basal and myoepithelial subtypes. Here we have exploited single cell RNA-sequencing and in vivo genetic lineage tracing technologies to generate a detailed map of the cell fate trajectories and branch points of the basal and myoepithelial cell populations of the mouse SMG during embryonic development and in adults. Our studies show that the transcription factor p63 and alpha-smooth muscle actin (SMA) serve as faithful markers of the basal and myoepithelial cell lineages, respectively and that both cell types are endowed with progenitor cell properties. However, p63+ basal and SMA+ myoepithelial cells exhibit distinct cell fates by virtue of maintaining different cellular lineages during morphogenesis and in adults. Collectively, our results reveal the dynamic and complex nature of the diverse SMG cell populations and highlight the distinct differentiation potential of the p63 and SMA expressing subtypes in the stem and progenitor cell hierarchy. Long term these findings have profound implications towards a better understanding of the molecular mechanisms that dictate lineage commitment and differentiation programs during development and adult gland maintenance.

Single Cell and Open Chromatin Analysis Reveals Molecular Origin of Epidermal Cells of the Skin.

How embryonic progenitors coordinate cell fate specification and establish transcriptional and signaling competence is a fundamental question in developmental biology. Here, we show that transcription factor ΔNp63 profoundly changes the transcriptome and remodels thousands of open chromatin regions of Krt8+ progenitors during epidermal fate specification. ATAC-seq and single-cell RNA-seq reveal that ΔNp63-dependent programs govern epidermal lineage formation, and ΔNp63-independent programs, mediated by AP2 and AP1 transcription factors, promote epidermal differentiation and epithelial-to-mesenchymal transition. ΔNp63 promotes Wnt signaling by directly upregulating Wnt ligands, Frizzled receptors, and transcription factors. Deletion of ß-catenin in Krt8+ progenitors delays their maturation into Krt5+ progenitors. The lack of epidermal Wnt production in the absence of ΔNp63 also incapacitates Wnt activation in the underlying dermal cells. These findings reveal the remarkable changes of the transcriptome, open chromatin, and signaling pathways at the onset of skin development and uncover the molecular cascade for epidermal lineage formation.

Functional characterization and genomic studies of a novel murine submandibular gland epithelial cell line.

A better understanding of the normal and diseased biology of salivary glands (SG) has been hampered, in part, due to difficulties in cultivating and maintaining salivary epithelial cells. Towards this end, we have generated a mouse salivary gland epithelial cell (mSGc) culture system that is well-suited for the molecular characterization of SG cells and their differentiation program. We demonstrate that mSGc can be maintained for multiple passages without a loss of proliferation potential, readily form 3D-spheroids and importantly express a panel of well-established salivary gland epithelial cell markers. Moreover, mSGc 3D-spheroids also exhibit functional maturation as evident by robust agonist-induced intracellular calcium signaling. Finally, transcriptomic characterization of mSGc by RNA-seq and hierarchical clustering analysis with adult organ RNA-seq datasets reveal that mSGc retain most of the molecular attributes of adult mouse salivary gland. This well-characterized mouse salivary gland cell line will fill a critical void in the field by offering a valuable resource to examine various mechanistic aspects of mouse salivary gland biology.

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.

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.

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.