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Salivary gland (SG) development, maturation, and homeostasis require coordinated roles of transcription factors (TFs) that dictate specific cell identities and fate. The ETS family of proteins are important transcriptional drivers of diverse cell lineages, tissue development, and differentiation programs and hence are also likely to play an important role in the SG. Here we have leveraged genomic and epigenomic data of the SG to examine the expression profile of ETS genes and identified 2 closely related paralogs, Elf5 and Ehf, that are highly expressed in distinct epithelial subpopulations. By using a well-defined mouse knockout model of Elf5, we show that Elf5, despite its enriched expression in the acinar cells, is functionally dispensable for maintaining the homeostatic state of the adult SG epithelium. The lack of a discernible phenotype of the Elf5-null SG might be due to possible functional redundancy with Ehf or other ETS factors. To probe this possibility and to examine the specific consequences of Ehf loss in the SG, we used CRISPR-Cas9 to generate mice in which the DNA-binding ETS domain of Ehf is disrupted due to an insertion mutation. We demonstrate that the Ehf mutant (EhfMut) mice exhibit a distinct cellular phenotype with decreased granular convoluted tubules that are accompanied by an increased accumulation of the intercalated Sox9-positive ductal cell population. Interestingly, the ductal phenotype of the EhfMut animals is highly pronounced in males, reaffirming the established sexual dimorphism of the SG that exists in rodents. Our results show that unlike Elf5, Ehf plays a nonredundant role in directing ductal cell differentiation of the SG and highlights the phenotypic subtlety in mutant mice of closely related TFs and the importance of careful consideration of cell type-specific studies.

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 Elf5 Cre ERT 2- 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., (© 2019 Federation of European Biochemical Societies.)

The Cancer Genome Atlas (TCGA) network study of 12 cancer types (PanCancer 12) revealed frequent mutation of TP53, and amplification and expression of related TP63 isoform ΔNp63 in squamous cancers. Further, aberrant expression of inflammatory genes and TP53/p63/p73 targets were detected in the PanCancer 12 project, reminiscent of gene programs comodulated by cREL/ΔNp63/TAp73 transcription factors we uncovered in head and neck squamous cell carcinomas (HNSCCs). However, how inflammatory gene signatures and cREL/p63/p73 targets are comodulated genome wide is unclear. Here, we examined how the inflammatory factor tumor necrosis factor-α (TNF-α) broadly modulates redistribution of cREL with ΔNp63α/TAp73 complexes and signatures genome wide in the HNSCC model UM-SCC46 using chromatin immunoprecipitation sequencing (ChIP-seq). TNF-α enhanced genome-wide co-occupancy of cREL with ΔNp63α on TP53/p63 sites, while unexpectedly promoting redistribution of TAp73 from TP53 to activator protein-1 (AP-1) sites. cREL, ΔNp63α and TAp73 binding and oligomerization on NF-κB-, TP53- or AP-1-specific sequences were independently validated by ChIP-qPCR (quantitative PCR), oligonucleotide-binding assays and analytical ultracentrifugation. Function of the binding activity was confirmed using TP53-, AP-1- and NF-κB-specific REs or p21, SERPINE1 and IL-6 promoter luciferase reporter activities. Concurrently, TNF-α regulated a broad gene network with cobinding activities for cREL, ΔNp63α and TAp73 observed upon array profiling and reverse transcription-PCR. Overlapping target gene signatures were observed in squamous cancer subsets and in inflamed skin of transgenic mice overexpressing ΔNp63α. Furthermore, multiple target genes identified in this study were linked to TP63 and TP73 activity and increased gene expression in large squamous cancer samples from PanCancer 12 TCGA by CircleMap. PARADIGM inferred pathway analysis revealed the network connection of TP63 and NF-κB complexes through an AP-1 hub, further supporting our findings. Thus, inflammatory cytokine TNF-α mediates genome-wide redistribution of the cREL/p63/p73, and AP-1 interactome, to diminish TAp73 tumor suppressor function and reciprocally activate NF-κB and AP-1 gene programs implicated in malignancy.

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, β, 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.

Emerging evidence suggests that cancer is populated and maintained by tumour-initiating cells (TICs) with stem-like properties similar to those of adult tissue stem cells. Despite recent advances, the molecular regulatory mechanisms that may be shared between normal and malignant stem cells remain poorly understood. Here we show that the ΔNp63 isoform of the Trp63 transcription factor promotes normal mammary stem cell (MaSC) activity by increasing the expression of the Wnt receptor Fzd7, thereby enhancing Wnt signalling. Importantly, Fzd7-dependent enhancement of Wnt signalling by ΔNp63 also governs tumour-initiating activity of the basal subtype of breast cancer. These findings establish ΔNp63 as a key regulator of stem cells in both normal and malignant mammary tissues and provide direct evidence that breast cancer TICs and normal MaSCs share common regulatory mechanisms.

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., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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 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., (Copyright © 2012 AlphaMed Press.)

The Tp63 gene encodes for multiple isoforms of the p63 transcription factor, a member of the p53 family of proteins. Much like its more famous sibling, the biological role of p63 is quite complex, with wide-ranging effects on development, differentiation, and cellular lineage choices. The crucial function of p63 is epitomized by the striking phenotype of p63 knockout mice. These animals have a profound block in the development of stratified epithelia and aplasia of multiple ectodermal appendages, as well as orofacial clefting and limb defects. Remarkably, a similar spectrum of phenotypic alterations is observed in human syndromes resulting from Tp63 gene mutations. p63 is an important hub in the transcriptional and signaling networks of epithelial cells; thus, it is not surprising that dysregulation of this transcription factor is associated with squamous cell carcinoma. Finally, as a testament to the growing repertoire of p63-associated diseases, autoantibodies to p63 are associated with chronic ulcerative stomatitis, an oral immunologically mediated disease. Over the past decade, our understanding of the broad biologic and pathophysiological roles of p63 has grown significantly. In this review, we discuss the molecular attributes of Tp63 and the clinical consequences of Tp63 dysregulation, particularly as it pertains to oral tissues.

Inflammation-induced activation of proto-oncogenic NF-κB/REL and dysfunction of tumor suppressor TP53/p63/p73 family transcription factors are key events in cancer progression. How inflammatory signaling coordinates dysregulation of these two transcription factor families during oncogenesis remains incompletely understood. Here, we observed that oncoprotein c-REL and tumor suppressor TAp73 are coexpressed and complex with ΔNp63α in the nucleus of a subset of head and neck squamous cell carcinoma (HNSCC) cell lines with mutant (mt)TP53. TNF-α, a proinflammatory cytokine, promoted c-REL nuclear translocation, c-REL/ΔNp63α interaction, and dissociation of TAp73 from ΔNp63α and the nucleus to the cytoplasm, whereas c-REL siRNA knockdown attenuated this effect. Overexpression of c-REL or a c-REL κB-site DNA-binding mutant enhanced protein interaction with ΔNp63α and TAp73 dissociation, implicating c-REL/ΔNp63α-specific interactions in these effects. We discovered that TNF-α or genetic alteration of c-REL expression inversely modulates ΔNp63α/TAp73 interactions on distinct p63 DNA-binding sites, including those for key growth arrest and apoptotic genes p21WAF1, NOXA, and PUMA. Functionally, c-REL repressed these genes and the antiproliferative effects of TNF-α or TAp73. Conversely, c-REL siRNA depletion enhanced TAp73 promoter interaction and expression of genes mediating growth arrest and apoptosis. Similar to TNF-α-treated HNSCC lines, human HNSCC tumors and hyperplastic squamous epithelia of transgenic mice overexpressing ΔNp63α that exhibit inflammation also show increased nuclear c-REL/ΔNp63α and cytoplasmic TAp73 localization. These findings unveil a novel and reversible dynamic mechanism whereby proinflammatory cytokine TNF-α-induced c-REL/ΔNp63α interactions inactivate tumor suppressor TAp73 function, promoting TNF-α resistance and cell survival in cancers with mtTP53., (©2011 AACR.)

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.

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)

Background: The ETS transcription factor Elf5 (also known as ESE-2) is highly expressed in the mammary gland and plays an important role in its development and differentiation. Indeed studies in mice have illustrated an essential role for Elf5 in directing alveologenesis during pregnancy. Although the molecular mechanisms that underlie the developmental block in Elf5 null mammary glands are beginning to be unraveled, this investigation has been hampered by limited information about the identity of Elf5-target genes. To address this shortcoming, in this study we have performed ChIP-cloning experiments to identify the specific genomic segments that are occupied by Elf5 in pregnant mouse mammary glands., Results: Sequencing and genomic localization of cis-regulatory regions bound by Elf5 in vivo has identified several potential target genes covering broad functional categories. A subset of these target genes demonstrates higher expression levels in Elf5-null mammary glands suggesting a repressive functional role for this transcription factor. Here we focus on one putative target of Elf5, the Ccnd2 gene that appeared in our screen. We identify a novel Elf5-binding segment upstream of the Ccnd2 gene and demonstrate that Elf5 can transcriptionally repress Ccnd2 by directly binding to the proximal promoter region. Finally, using Elf5-null mammary epithelial cells and mammary glands, we show that loss of Elf5 in vivo leads to up regulation of Ccnd2 and an altered expression pattern in luminal cells., Conclusions: Identification of Elf5-targets is an essential first step in elucidating the transcriptional landscape that is shaped by this important regulator. Our studies offer new toolbox in examining the biological role of Elf5 in mammary gland development and differentiation.

Mammalian epidermis is a stratified squamous epithelium that serves as a protective barrier against external harmful elements. The development of the epidermis is a highly regulated process that begins by the commitment of a single layer of multipotent ectodermal cells to a keratinocyte cell fate. This is followed by stratification and a subsequent elaborate program of differentiation leading to the generation of a multilayered epidermis and patterned cutaneous appendages such as the hair follicles and sebaceous glands. The stratified epidermis occupying the space between skin appendages is referred to as the interfollicular epidermis (IFE) and is the focus of this review. Within the IFE, keratinocytes in the innermost basal layer are mitotically active. Upon specific cues, these cells undergo cell cycle arrest and execute a terminal differentiation program as they progress through spinous, granular, and cornified layers. This program operates continually throughout the life of an organism; dead cells sloughed off from the skin surface are replenished by basal cells moving outward in a highly synchronized fashion. Not surprisingly, at the heart of the control process is a dedicated group of transcription factors that ensure the integrity of the keratinocyte differentiation program by regulating gene expression in a temporally and spatially coordinated manner. Here we review the transcription factors that play important roles in the development and maintenance of IFE as evidenced by biochemical and genetic studies.

The mammalian AP-2 family of transcription factors consists of four members, AP-2 alpha, AP-2 beta, AP-2 gamma and AP-2 delta, which play an important role in regulating gene expression during development and differentiation of multiple organs and tissues. The defining feature of the AP-2 family is a highly conserved carboxy-terminal basic helix-turn-helix domain that is involved in dimerization and sequence-specific DNA-binding. In this report, we use bioinformatics to identify both the mouse and human AP-2 epsilon, a fifth member of the AP-2 family. The predicted mouse and human AP-2 epsilon proteins consist of 442 amino acids and show a high level of sequence similarity with other AP-2 proteins in the DNA-binding and dimerization domain and weak similarity in the N-terminal activation domain. Northern blot analysis reveals that among the adult mouse tissues examined, AP-2 epsilon is highly expressed in skin tissue. The human AP-2 epsilon gene maps to chromosome 1p42, consists of seven exons spanning 23 kb and exhibits a genomic structure similar to other AP-2 family members. Human AP-2 epsilon mRNA is expressed in human skin and keratinocytes grown in culture. Finally, we show that recombinant AP-2 epsilon can bind to AP-2 binding sequences from keratin promoters in electrophoretic mobility shift assays. Our study establishes AP-2 epsilon as a novel member of the AP-2 family, and suggests that it may play an important role in skin biology.