Search

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 β-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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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.

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, TGFβ3-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.

Atopic dermatitis (AD) is the most common inflammatory skin disease with no well-delineated cause or effective cure. Here we show that the p53 family member p63, specifically the ΔNp63, isoform has a key role in driving keratinocyte activation in AD. We find that overexpression of ΔNp63 in transgenic mouse epidermis results in a severe skin phenotype that shares many of the key clinical, histological and molecular features associated with human AD. This includes pruritus, epidermal hyperplasia, aberrant keratinocyte differentiation, enhanced expression of selected cytokines and chemokines and the infiltration of large numbers of inflammatory cells including type 2  T-helper cells - features that are highly representative of AD dermatopathology. We further demonstrate several of these mediators to be direct transcriptional targets of ΔNp63 in keratinocytes. Of particular significance are two p63 target genes, IL-31 and IL-33, both of which are key players in the signaling pathways implicated in AD. Importantly, we find these observations to be in good agreement with elevated levels of ΔNp63 in skin lesions of human patients with AD. Our studies reveal an important role for ΔNp63 in the pathogenesis of AD and offer new insights into its etiology and possible therapeutic targets.

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 β3 (TGFβ3) expression in the MEE coincides with periderm degeneration, its role in periderm desquamation is not known. Interestingly, murine models of knockout (-/-) TGFβ3, 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 TGFβ3 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 TGFβ3 (-/-), ΔNp63 (-/-), and wild-type (WT) embryos, and primary MEE cells from WT palates to analyze the association between TGFβ3 and IRF6/ΔNp63. Our results suggest that periderm degeneration depends on functional TGFβ3 signaling to repress ΔNp63, thereby coordinating periderm desquamation. Cleft palate occurs in TGFβ3 (-/-) because of inadequate periderm removal that impedes palatal seam formation, while cleft palate occurs in ΔNp63 (-/-) palates because of premature fusion., (© 2014 Wiley Periodicals, Inc.)

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., (Published 2013. This article is a U.S. Government work and is in the public domain in the USA.)

Somatic cells can be converted into induced pluripotent stem cells (iPSCs) by forced expression of various combinations of transcription factors, but the molecular mechanisms of reprogramming are poorly understood. Specifically, evidence that the reprogramming process can take many distinct routes only begins to emerge. It is definitively established that p53 deficiency greatly enhances reprogramming, revealing p53's barrier function for induced pluripotency, but the role of its homologs p63 and p73 are unknown. Here we report that in stark contrast to p53, p73 has no role in reprogramming. However, p63 is an enabling (rather than a barrier) factor for Oct4, Sox2 and Klf4 (OSK) and Oct4 and Sox2 (OS), but not for Oct4 and Klf4 (OK) reprogramming of mouse embryonic fibroblasts. Specifically, p63 is essential during reprogramming for maximum efficiency, albeit not for the ability to reprogram per se, and is dispensable for maintaining stability and pluripotency of established iPSC colonies. ΔNp63, but not TAp63, is the principal isoform involved. Loss of p63 can affect reprogramming via several mechanisms such as reduced expression of mesenchymal-epithelial transition and pluripotency genes, hypoproliferation and loss of the most reprogrammable cell populations. During OSK and OS reprogramming, different mechanisms seem to be critical, such as regulation of epithelial and pluripotency genes in OSK reprogramming versus regulation of proliferation in OS reprogramming. Finally, our data reveal three different routes of reprogramming by OSK, OS or OK, based on their differential p63 requirements for iPSC efficiency and pluripotency marker expression. This supports the concept that many distinct routes of reprogramming exist.

Deregulation of the hedgehog (HH) pathway results in overexpression of the GLI target BCL2 and is an initiating event in specific tumor types including basal cell carcinoma of the skin. Regulation of the HH pathway during keratinocyte differentiation is not well understood. We measured HH pathway activity in response to differentiation stimuli in keratinocytes. An upregulation of suppressor of fused (SUFU), a negative regulator of the HH pathway, lowered HH pathway activity and was accompanied by loss of BCL2 expression associated with keratinocyte differentiation. We used in vitro and in vivo models to demonstrate that ΔNp63α, a crucial regulator of epidermal development, activates SUFU transcription in keratinocytes. Increasing SUFU protein levels inhibited GLI-mediated gene activation in suprabasal keratinocytes and promoted differentiation. Loss of SUFU expression caused deregulation of keratinocyte differentiation and BCL2 overexpression. Using in vivo murine models, we also provide evidence of GLI-mediated regulation of the TP63 pathway. p63 expression appears essential to establish an optimally functioning HH pathway. These observations present a regulatory mechanism by which SUFU acts as an interacting node between the HH and TP63 pathways to mediate differentiation and maintain epidermal homeostasis. Disruption of this regulatory node can be an important contributor to multistep carcinogenesis.

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.

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.

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.

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.

Head and neck squamous cell carcinoma (HNSCC) and many other epithelial malignancies exhibit increased proliferation, invasion, and inflammation, concomitant with aberrant nuclear activation of TP53 and NF-κB family members ΔNp63, cRel, and RelA. However, the mechanisms of cross-talk by which these transcription factors coordinate gene expression and the malignant phenotype remain elusive. In this study, we showed that ΔNp63 regulates a cohort of genes involved in cell growth, survival, adhesion, and inflammation, which substantially overlaps with the NF-κB transcriptome. ΔNp63 with cRel and/or RelA are recruited to form novel binding complexes on p63 or NF-κB/Rel sites of multitarget gene promoters. Overexpressed ΔNp63- or TNF-α-induced NF-κB and inflammatory cytokine interleukin-8 (IL-8) reporter activation depended on RelA/cRel regulatory binding sites. Depletion of RelA or ΔNp63 by small interfering RNA (siRNA) significantly inhibited NF-κB-specific, or TNF-α-induced IL-8 reporter activation. ΔNp63 siRNA significantly inhibited proliferation, survival, and migration by HNSCC cells in vitro. Consistent with these data, an increase in nuclear ΔNp63, accompanied by increased proliferation (Ki-67) and adhesion (β4 integrin) markers, and induced inflammatory cell infiltration was observed throughout HNSCC specimens, when compared with the basilar pattern of protein expression and minimal inflammation seen in nonmalignant mucosa. Furthermore, overexpression of ΔNp63α in squamous epithelial cells in transgenic mice leads to increased suprabasilar cRel, Ki-67, and cytokine expression, together with epidermal hyperplasia and diffuse inflammation, similar to HNSCC. Our study reveals ΔNp63 as a master transcription factor that, in coordination with NF-κB/Rels, orchestrates a broad gene program promoting epidermal hyperplasia, inflammation, and the malignant phenotype of HNSCC., (©2011 AACR)

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.

The p63 transcription factor has a pivotal role in epithelial morphogenesis. Multiple transcripts of the TP63 gene are generated because of alternative promoter usage and splicing. DeltaNp63alpha is the predominant isoform of p63 observed during epithelial morphogenesis and in human cancers. Loss of DeltaNp63alpha expression has been shown to promote invasiveness in a subset of human cancer cell lines. Here, we studied whether the regulation of VDR by DeltaNp63alpha controls the invasiveness of an epidermoid cancer cell line. We demonstrate that VDR expression is induced by all p63 isoforms, including DeltaNp63alpha. Endogenous DeltaNp63alpha protein was observed to bind to the VDR promoter, and silencing of endogenous DeltaNp63alpha resulted in diminished VDR expression. Although silencing of p63 inhibits VDR expression leading to an increase in cell migration, overexpression of p63 or VDR results in reduced cell migration as a result of increased VDR expression. Therefore, it is conceivable that p63 inhibits cell invasion by regulating VDR expression. Finally, we observed that expression of p63 and VDR overlaps in the wild-type mouse skin, but a reduced or complete absence of VDR expression was observed in skin from p63-null mice and in p63-null mouse embryonic fibroblasts. In conclusion, we demonstrate a direct transcriptional regulation of VDR by DeltaNp63alpha. Our results highlight a crucial role for VDR in p63-mediated biological functions.

Background: One major defining characteristic of the basal keratinocytes of the stratified epithelium is the expression of the keratin genes K5 and K14. The temporal and spatial expression of these two genes is usually tightly and coordinately regulated at the transcriptional level. This ensures the obligate pairing of K5 and K14 proteins to generate an intermediate filament (IF) network that is essential for the structure and function of the proliferative keratinocytes. Our previous studies have shown that the basal-keratinocyte restricted transcription factor p63 is a direct regulator of K14 gene., Methodology/principal Findings: Here we provide evidence that p63, specifically the DeltaN isoform also regulates the expression of the K5 gene by binding to a conserved enhancer within the 5' upstream region. By using specific antibodies against DeltaNp63, we show a concordance in the expression between basal keratins and DeltaNp63 proteins but not the TAp63 isoforms during early embryonic skin development. We demonstrate, that contrary to a previous report, transgenic mice expressing DeltaNp63 in lung epithelium exhibit squamous metaplasia with de novo induction of K5 and K14 as well as transdifferentiation to the epidermal cell lineage. Interestingly, the in vivo epidermal inductive properties of DeltaNp63 do not require the C-terminal SAM domain. Finally, we show that DeltaNp63 alone can restore the expression of the basal keratins and reinitiate the failed epidermal differentiation program in the skin of p63 null animals., Significance: DeltaNp63 is a critical mediator of keratinocyte stratification program and directly regulates the basal keratin genes.

Keratin14 (K14) is a prototypic marker of dividing basal keratinocytes where its gene is transcribed at high levels. Transcriptional regulation of K14 is governed by an evolutionarily conserved functional enhancer marked by DNase 1 hypersensitive sites present upstream of the gene. This enhancer is sufficient to confer epidermal-specific gene expression, which is mediated in part by binding of members of activator protein-2 (AP)-2, AP-1, Ets, and Sp1 families of transcription factors. Here we provide evidence that a keratinocyte-specific nuclear protein identified as deltaNp63 binds to a conserved motif within this enhancer. Interestingly, the selective expression profile of deltaNp63 in various cell lines correlates with both the nuclear complex and the expression of K14. Biochemical studies reveal that deltaNp63 can bind to a specific DNA sequence present in the K14 enhancer and this binding leads to transactivation. In addition, chromatin immunoprecipitation experiments with deltaNp63-specific antibodies demonstrate that the enhancer is occupied by deltaNp63 in cultured keratinocytes and in mouse skin epidermal cells in vivo. Finally, we show that ectopic expression of either p63 isoform (deltaN or TA) can induce de novo expression of K14. These studies provide a potential mechanism by which deltaNp63 directly governs the expression of K14 in a keratinocyte-specific manner.

p63, a homolog of the tumor suppressor p53, plays an important role in the formation of stratified epithelium such as those in the epidermis of the skin. The p63 gene gives rise to multiple functionally distinct protein isoforms, including the DeltaNp63 class of isoforms, which lacks the N-terminal transactivation domain and is synthesized from an internal promoter. DeltaNp63 proteins are the predominant isoforms expressed in keratinocytes and are thought to be important for maintenance of the proliferative capacity of these cells. Here, we have examined the transcriptional control mechanisms that govern the expression DeltaNp63 in keratinocytes. We first performed DNase I hypersensitive site mapping and demonstrated that the promoter region of DeltaNp63 is in open chromatin state in keratinocytes. To identify the cis-elements that regulate DeltaNp63, we have performed transient transfection assays in keratinocytes with several DeltaNp63 promoter constructs. This identified a short evolutionarily conserved fragment that harbors most of the transcriptional activity of the DeltaNp63 promoter. Biochemical studies of this element have revealed critical roles for CCAAT-box-binding factor (CBF/NF-Y) and Sp1/Sp3 family of proteins. In addition, our data suggest that DeltaNp63 is recruited to and can activate its own promoter, possibly through protein-protein interactions, thus providing an auto-regulatory loop of self-regulation. These studies support the notion that unique and distinct pathways control the expression of individual p53 family members and their various isoforms.