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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.

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 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.

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.

Background: Genetic experiments have clarified that p63 is a key transcription factor governing the establishment and maintenance of multilayered epithelia. Key to our understanding of p63 strategy is the identification of target genes. We perfomed an RNAi screening in keratinocytes for p63, followed by profiling analysis., Results: C/EBPdelta, member of a family with known roles in differentiation pathways, emerged as a gene repressed by p63. We validated C/EBPdelta as a primary target of DeltaNp63alpha by RT-PCR and ChIP location analysis in HaCaT and primary cells. C/EBPdelta is differentially expressed in stratification of human skin and it is up-regulated upon differentiation of HaCaT and primary keratinocytes. It is bound to and activates the DeltaNp63 promoter. Overexpression of C/EBPdelta leads to alteration in the normal profile of p63 isoforms, with the emergence of DeltaNp63beta and gamma, and of the TA isoforms, with different kinetics. In addition, there are changes in the expression of most p63 targets. Inactivation of C/EBPdelta leads to gene expression modifications, in part due to the concomitant repression of DeltaNp63alpha. Finally, C/EBPdelta is found on the p63 targets in vivo by ChIP analysis, indicating that coregulation is direct., Conclusion: Our data highlight a coherent cross-talk between these two transcription factors in keratinocytes and a large sharing of common transcriptional targets.

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.

We used a bioinformatics approach to identify Basonuclin2, the second member of the Basonuclin zinc-finger family of transcription factors. The mouse Basonuclin2 protein consists of 1049 amino acids and contains three pairs of zinc fingers in the C-terminus that show a high level of amino acid sequence similarity with Basonuclin1. In addition, other characteristic domains of Basonuclin1, such as the serine strip and a nuclear localization signal, are also present in Basonuclin2. We used genomic and in silico database analysis to identify the human and rat homologs of basonuclin2. A search of the mouse genome showed that the basonuclin2 gene maps to chromosome 4 and consists of six exons spanning approximately 300 kb. Northern blot analysis revealed multiple transcripts of basonuclin2 in tissues of the reproductive system (ovary and testis) and also in kidney and skin. We demonstrate that, as expected from sequence conservation, recombinant Basonuclin2 can bind to a sequence in the promoter of a rRNA gene previously characterized as a Basonuclin-binding site. Full-length Basonuclin2 exclusively localizes to the nucleus, indicating that it likely plays an important role in nuclear function, probably in gene regulation. Our study establishes Basonuclin2 as a novel member of the Basonuclin family. Moreover, the structural and functional similarities with Basonuclin1 suggest that Basonuclin2 may play an analogous function in germ cells and skin keratinocytes.