p63(alpha) belongs to the p53-family of proteins and has full-length (TA) as well as truncated ((delta)N) p63(alpha) isoforms. Previous studies have shown that TA and (delta)Np63(alpha) play multiple roles in cancer and development. In cancer, (delta)Np63(alpha)-mediated transcriptional repression promotes oncogenesis while transactivation by TAp63(alpha) is critical during development. Despite their importance, little is known regarding how TA or (delta)Np63(alpha) is regulated and factors influencing the function of p63(alpha) have yet to be identified. Here, I identify Special AT-rich Binding Protein 2 (SATB2) as a protein that forms a complex with and modulates the function of p63(alpha). SATB2 is detected in multiple head and neck squamous cell carcinoma (HNSCC) cell lines that also show overexpression of (delta)Np63(alpha). Histological analysis on tumor specimens revealed that SATB2 is predominantly expressed in advanced-stage HNSCC cancers. SATB2 increases DNA-binding capabilities of (delta)Np63(alpha), augmenting (delta)Np63(alpha) repression of apoptotic gene expression. Knockdown of SATB2 in HNSCC cells sensitizes cancer cells towards chemotherapy- and radiation-induced apoptosis. These results indicate that SATB2 functions as a co-factor and promotes the transrepression function of (delta)Np63(alpha) in HNSCC. In addition to examining the role of SATB2 in HNSCC, I also investigated the effect of SATB2 on the ability of TAp63(alpha) to induce gene expression. In particular, perp has been shown to be a critical downstream target of p63 during development. ChIP analysis revealed that while SATB2 increases TAp63(alpha)-binding to apoptotic gene promoters, SATB2 decreases TAp63(alpha) localization on the perp promoter and inhibits p63(alpha)-mediated perp induction. SATB2 more readily interacts with human disease-associated p63(alpha) mutations that are found in the SAM domain, further inhibiting transcriptional properties of these mutants. Together, my results suggest that SATB2 is an important modulator of p63(alpha) in cancer and development.

p63(alpha) belongs to the p53-family of proteins and has full-length (TA) as well as truncated ((delta)N) p63(alpha) isoforms. Previous studies have shown that TA and (delta)Np63(alpha) play multiple roles in cancer and development. In cancer, (delta)Np63(alpha)-mediated transcriptional repression promotes oncogenesis while transactivation by TAp63(alpha) is critical during development. Despite their importance, little is known regarding how TA or (delta)Np63(alpha) is regulated and factors influencing the function of p63(alpha) have yet to be identified. Here, I identify Special AT-rich Binding Protein 2 (SATB2) as a protein that forms a complex with and modulates the function of p63(alpha). SATB2 is detected in multiple head and neck squamous cell carcinoma (HNSCC) cell lines that also show overexpression of (delta)Np63(alpha). Histological analysis on tumor specimens revealed that SATB2 is predominantly expressed in advanced-stage HNSCC cancers. SATB2 increases DNA-binding capabilities of (delta)Np63(alpha), augmenting (delta)Np63(alpha) repression of apoptotic gene expression. Knockdown of SATB2 in HNSCC cells sensitizes cancer cells towards chemotherapy- and radiation-induced apoptosis. These results indicate that SATB2 functions as a co-factor and promotes the transrepression function of (delta)Np63(alpha) in HNSCC. In addition to examining the role of SATB2 in HNSCC, I also investigated the effect of SATB2 on the ability of TAp63(alpha) to induce gene expression. In particular, perp has been shown to be a critical downstream target of p63 during development. ChIP analysis revealed that while SATB2 increases TAp63(alpha)-binding to apoptotic gene promoters, SATB2 decreases TAp63(alpha) localization on the perp promoter and inhibits p63(alpha)-mediated perp induction. SATB2 more readily interacts with human disease-associated p63(alpha) mutations that are found in the SAM domain, further inhibiting transcriptional properties of these mutants. Together, my results suggest that SATB2 is an important modulator of p63(alpha) in cancer and development.

No description available.

T cell dysfunction has a crucial role in establishing and maintaining viral persistence. We have previously shown a decline in miR-181a, which regulates CD4+ T cell responses via DUSP6 overexpression, in individuals with hepatitis C virus (HCV) infection. Here, we describe accelerated T cell senescence in HCV-infected individuals compared with age-and sex-matched healthy subjects. Mechanistic studies revealed that up-regulation of transcription factor ∆Np63 led to the decline of miR-181a expression, resulting in an overexpression of the antiaging protein Sirt1, in CD4+ T cells from HCV-infected individuals. Either reconstituting miR-181a or silencing ∆Np63 or Sirt1 expression in CD4+ T cells led to accelerated T cell senescence, as evidenced by an increased senescence-associated b-galactosidase (SA-β-gal) expression, shortened telomere length, and decreased EdU incorporation; this suggests that HCV-induced T cell senescence is counterregulated by the ∆Np63-miR-181a-Sirt1 pathway. An increase of IL-2 production was observed in these senescent CD4+ T cells and was driven by a markedly reduced frequency of Foxp3+ regulatory T (Treg) cells and increased number of Foxp3- effector T (Teff) cells upon manipulating the ∆Np63-miR-181a-Sirt1 pathway. In conclusion, these findings provide novel mechanistic insights into how HCV uses cellular senescent pathways to regulate T cell functions, revealing new targets for rejuvenating impaired T cell responses during chronic viral infection.

During development, multipotent stem cells establish tissue-specific programmes of gene expression that underlie a process of differentiation into specialized cell types. It was shown in the study that changes in the nuclear architecture during terminal keratinocyte differentiation show correlation with the dynamics of the transcriptional and metabolic activity. In particular, terminal differentiation is accompanied by the decrease of nuclear volume, elongation of its shape, reduction of the number and fusion of nucleoli, increase in the number of centromeric clusters and a dramatic decrease of the transcriptional activity. Global changes in the nuclear architecture of epidermal keratinocytes are associated with marked remodelling of the higher-order chromatin structure of the epidermal differentiating complex (EDC). EDC is positioned peripherally in the epidermal nuclei at E11.5 when its genes show low expression levels and relocates towards the nuclear interior at E16.5 when EDC genes are markedly upregulated. P63 transcription factor serving as a master regulator of epidermal development is involved in the control of EDC relocation in epidermal progenitor cells. The epidermis of E16.5 p63KO exhibits significantly more peripheral positioning of the EDC loci, compared to wild-type. The genome organizer Satb1 serving as a direct p63 target controls higher order chromatin folding of the central part of EDC and Satb1 knockout mice show alterations of epidermal development and expression of the EDC encoded genes. Thus, this study shows that the programme of epidermal development and terminal differentiation is regulated by p63 and other factors and include marked remodelling of three-dimensional nuclear organization and positioning of tissue specific gene loci. In addition to the direct involvement of p63 in controlling the expression of tissue-specific genes, p63 via regulation of the chromatin remodelling factors such as Satb1 promotes establishing specific conformation of the EDC locus required for efficient expression of terminal differentiation-associated genes.

During tissues development multipotent progenitor cells establish tissue-specific gene expression programmes, leading to differentiation into specialized cell types. It has been previously shown that the transcription factor p63, a master regulator of skin development, controls the expression of adhesion molecules and essential cytoskeleton components. It has also been shown that p63 plays an important role in establishing distinct three-dimensional conformations in the Epidermal Differentiation Complex (EDC) locus (Fessing et al., 2011). Here we show that in p63-null mice about 32% of keratinocytes showed altered nuclear morphology. Alterations in the nuclear shape were accompanied by decreased expression of nuclear lamins (Lamin A/C and Lamin B1), proteins of the LINC complex (Sun-1, nesprin-2/3) and Plectin. Plectin links components of the nuclear envelope (nesprin-3) with cytoskeleton and ChIP-qPCR assay with adult epidermal keratinocytes showed p63 binding to the consensus binding sequences on Plectin 1c, Sun-1 and Nesprin-3 promoters. As a possible consequence of the altered expression of nuclear lamins and nuclear envelope-associated proteins, changes in heterochromatin distribution as well as decrease of the expression of several polycomb proteins (Ezh2, Ring1B, Cbx4) has been observed in p63-null keratinocytes. Moreover, recent data in our lab have showed that p63 directly regulates Cbx4, a component of the polycomb PRC1 complex. Here we show that mice lacking Cbx4 displayed a skin phenotype, which partially resembles the one observed in p63-null mice with reduced epidermal thickness and keratinocyte proliferation. All together these data demonstrate that p63-regulated gene expression program in epidermal keratinocytes includes not only genes encoding adhesion molecules, cytoskeleton proteins (cytokeratins) and chromatin remodelling factors (Satb1, Brg1), but also polycomb proteins and components of the nuclear envelope, suggesting the existence of a functional link between cytoskeleton, nuclear architecture and three dimensional nuclear organization. Other proteins important for proper epidermal development and stratification, are cytokeratins. Here, we show that keratin genes play an essential role in spatial organization of other lineage-specific genes in keratinocytes during epidermal development. In fact, ablation of keratin type II locus from chromosome 15 in epidermal keratinocytes led to changes in the genomic organization with increased distance between the Loricrin gene located on chromosome 3 as well as between Satb1 gene located on chromosome 17 and keratin type II locus, resulting in a more peripheral localization of these genes in the nucleus. As a possible consequence of their peripheral localization, reduced expression of Loricrin and Satb1 has also been observed in keratins type II-deficient mice. These findings together with recent circularized chromosome conformation capture (4C) data, strongly suggest that keratin 5, Loricrin and Satb1 are part of the same interactome, which is required for the proper expression of these genes and proper epidermal development and epidermal barrier formation. Taken together these data suggest that higher order chromatin remodelling and spatial organization of genes in the nucleus are important for the establishment of lineage-specific differentiation programs in epidermal progenitor cells. These data provide an important background for further analyses of nuclear architecture in the alterations of epidermal differentiation, seen in pathological conditions, such as psoriasis and epithelial skin cancers.

p63, a homologue of the transcription factor p53, is essential for maintaining the proliferative capacity of epithelial stem cells. The p63 gene yields two major isoforms transcribed from dual promoters at its N-terminus and at least three different splice variants at its C-terminus. The TAp63 isoform functions as a p53-like tumor suppressor and transactivates p53 gene targets. In contrast, ΔNp63α predominates in epithelial stem cells and regulates their proliferative potential, whilst also acting as an oncogene by suppressing the function of both p53 and TAp63 in a dominant-negative manner. ΔNp63α is frequently upregulated in human epithelial cancers and has been theorized to play a role in tumorigenesis. Our prior studies found that an aberrantly spliced ΔNp63α isoform heterozygous for a lack of exon 4-coded sequences (hereafter referred to as Δ4) appeared to be specifically expressed in squamous cell carcinomas, suggesting a potential link between this new isoform and a specific type of cancer. Our study aimed to elucidate the involvement of this aberrantly spliced p63 isoform in the initiation and/or development of epithelial cancer. We established a p63+/Δ4 mouse model to obtain a baseline for the effect of one aberrant allele on both embryonic and adult mouse development. We found that p63+/Δ4 mouse embryos developed normally and that over a period of 74 weeks, the p63+/Δ4 mice developed normally and lived lifespans equivalent to their wildtype counterparts, though a small proportion of the p63+/Δ4 colony showed minor issues with inflammation. After our initial observations, we established an experimental mouse model to test the effect of oncogenic stimulation on p63+/Δ4 mice. In the first model, we administered the oral carcinogen 4-nitroquinoline 1-oxide to mirror the histological and molecular changes seen in human oral carcinogenesis. We observed significant dysplasia in the tongue epithelia of p63+/Δ4 mice compared to that of wildtype. With this data, we developed the hypothesis that expression of Δ4 accelerates tumorigenesis in the presence of oncogenic stimulation. To further test this possibility, we generated a second mouse model: a triple transgenic Cre-inducible mouse model under the control of epithelial cell-specific promotor gene, keratin 14 (K14). This cross introduced a mutated Kras gene to the p63+/Δ4 lineage, as well as to control wildtype mice bred with the same Cre elements. Upon administration of tamoxifen, genetic recombination caused expression of both mutant Kras G12D and Δ4 in K14-positive tissues and subsequent malignant transformation. Among these K14-positive tissues were the oral epithelia, which presented with dysplasia more severe than that of control mice expressing either Kras or Δ4 alone and which matched the phenotype-genotype associations observed in our 4NQO experiment. Lastly, we attempted to create an antibody specific to Δ4 that could eliminate the need for our current two-antibody identification strategy. Though we did not succeed, we found value in elucidating the genetic sequence requirements to create the artificial peptide that we used for mouse immunization. We attempted to optimize established hybridoma protocols and developed a Δ4-specific screening process to determine viable candidates for expansion. In conclusion, this study is the first characterization of a hitherto unknown mutant splice variant of a gene vital to stem cell maintenance and epithelial development. It is the first demonstration of this gene mutation’s responsibility for the acceleration of squamous cell carcinoma, which carries implications for potential therapeutic treatments. / 2026-02-12T00:00:00Z

No description available.

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Yes / The maintenance of a proper nuclear architecture and 3D organization of the genes, enhancer elements and transcription machinery plays an essential role in tissue development and regeneration. Here we show that in the developing skin, epidermal progenitor cells of mice lacking p63 transcription factor display alterations in the nuclear shape accompanied by marked decrease in expression of several nuclear envelop-associated components (Lamin B1, Lamin A/C, SUN1, Nesprin-3, Plectin) compared to controls. Furthermore, ChIP-qPCR assay showed enrichment of p63 on Sun1, Syne3 and Plec promoters, suggesting them as p63 targets. Alterations in the nuclei shape and expression of nuclear envelope-associated proteins were accompanied by altered distribution patterns of the repressive histone marks H3K27me3, H3K9me3 and heterochromatin protein 1- alpha in p63-null keratinocytes. These changes were also accompanied by downregulation of the transcriptional activity and relocation of the keratinocyte-specific gene loci away from the sites of active transcription towards the heterochromatin-enriched repressive nuclear compartments in p63-null cells. These data demonstrate functional links between the nuclear envelope organization, chromatin architecture and gene expression in keratinocytes and suggest nuclear envelope-associated genes as important targets mediating p63-regulated gene expression programme in the epidermis.