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ChIP-seq analysis comparing WT, TAp63-/-, and ΔNp63-/- primary murine keratinocytes

ABSTRACT: The p53 family member TP63 encodes two sets of isoforms, TAp63 and ∆Np63 isoforms, which are characterized by different N-termini and have diverse biological functions in epidermal morphogenesis and in cancer. In the skin, where their activities are best characterized, TAp63 prevents premature aging by regulating cellular senescence and genomic stability of stem cells, while ∆Np63 controls terminal differentiation of the basal cells in the epidermis. This functional diversity is surprising given that these isoforms share a high degree of similarity, including an identical DNA binding domain. To understand the mechanisms involved in the transcriptional programs leading to these diverse biological functions, we performed genome-wide analyses using p63-ChIP-seq and RNA-seq of TAp63-/- and ∆Np63-/- compared to wild-type primary mouse epidermal cells. Our data indicate that TAp63 and ∆Np63 recognize significantly different response elements on DNA and can physically and functionally interact with distinct transcription factors for the downstream regulation of their target genes. Our findings unveil previously uncharacterized transcriptomes activated by the p63 isoforms to regulate diverse biological functions in epidermal morphogenesis and homeostasis and cancer.

ORGANISM(S): Mus musculus

PROVIDER: GSE147723 | GEO | 2023/12/06

REPOSITORIES: GEO

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Gene expression profiles induced by knockdown and overexpression of p63 variants in MCF-10A mammary epithelial cell line

Project description:p63 is critical for epithelial development yet little is known about the transcriptional programmes it regulates. The p63 transactivating (TA) isoforms contain an amino-terminal exon that encodes a p53-like transactivation domain, whereas ÎN-isoforms lack this domain but contain the common DNA binding domain (DBD), suggesting that TAp63 and ÎNp63 isoforms may have opposing functions. By characterising transcriptional changes and cellular effects following modulation of p63 expression, we have defined a vital role for p63 in cellular adhesion. Knockdown of p63 expression caused downregulation of cell adhesion-associated genes, cell detachment and anoikis in mammary epithelial cells and keratinocytes. Conversely, overexpression of the TAp63Î³ or ÎNp63Î± isoforms of p63 upregulated cell adhesion molecules, increased cellular adhesion and conferred resistance to anoikis. Total RNA was isolated 48 h after retroviral or adenoviral infection and was subjected to reverse transcription, labelling and hybridization. The knockdown samples were performed in duplicate with shRNA vector control, shRNA targetting all isoforms of p63 (shDBD) and shRNA targetting p63 isoforms containing the transactivating (TA) domains. The overexpression samples were performed in triplicate with vector control, overexpression of the ÎNp63Î± isoform, and overexpression of the TAp63Î³ isoforms.

2010-03-23 | E-GEOD-20286 | biostudies-arrayexpress

DeltaNp63 silencing, DNA methylation shifts and epithelial mesenchymal transition resulted from TAp63 genome editing in squamous cell carcinoma [RRBS]

Project description:TP63 (p63) is strongly expressed in lower-grade carcinomas of head-and-neck, skin, breast, urothelium, etc. to maintain the well-differentiated phenotype. TP63 has two transcription start sites at exon 1 and exon 3’ to produce TAp63 and DeltaNp63 isoforms, respectively. The major protein, DeltaNp63alpha, functions as a core factor to organize super enhancers of genes essential for epidermal/craniofacial differentiation and for self-activation of DeltaNp63. To examine whether very weakly expressed TAp63 has a specific role, we disrupted exon 1 by CRISPR-Cas9 homology directed repair (HDR) in a head and neck squamous cell carcinoma (SCC) line. Surprisingly, TAp63 knockout cells, with either ‘monoallelic HDR and a frameshift deletion on the other allele’ or ‘biallelic HDR’, caused DeltaNp63 silencing. Loss of keratinocyte-specific gene expression, replacement of KRT5 with VIM, and transcriptional suppression of cell-cell and cell-matrix adhesion components indicated core events of epithelial mesenchymal transition. Most of the positively and negatively impacted genes including DeltaNp63 displayed local CpG methylation changes. Furthermore, DeltaNp63 expression was partially rescued by transfection of TAp63alpha, followed by incubation with DNA methyltransferase inhibitor Zebularine. This study suggests that TAp63 is indispensable for DeltaNp63 expression by which keratinocyte-specific epigenome is maintained in SCC.

2023-09-29 | GSE234979 | GEO

Late cornified envelope 1C (LCE1C) gene is a transcriptional target of TAp63.

Project description:As a member of the p53/p63/p73 family, p63 is a transcriptional regulator that induces apoptosis, cell cycle arrest, or senescence. TAp63 protein, a p53-like master transcriptional regulator, plays an essential role in epithelial development because p63-null mice are born alive but die shortly after birth due to developmental defects. However, its molecular mechanism remains elusive. We identified these phosphorylation sites during a search for the targets of Cyclin G-associated kinase (GAK) in vitro. LCE1C was drastically upregulated by doxycycline-dependent expression of Myc-TAp63 wild-type (WT) proteins, but not non-phosphorylatable Myc-TAp63-T46A/T281A (AA) proteins.

2018-03-22 | GSE94635 | GEO

DeltaNp63 silencing, DNA methylation shifts and epithelial mesenchymal transition resulted from TAp63 genome editing in squamous cell carcinoma [microarray]

Project description:TP63 (p63) is strongly expressed in lower-grade carcinomas of head-and-neck, skin, breast, urothelium, etc. to maintain the well-differentiated phenotype. TP63 has two transcription start sites at exon 1 and exon 3’ to produce TAp63 and DeltaNp63 isoforms, respectively. The major protein, DeltaNp63alpha, epigenetically activates genes essential for epidermal/craniofacial differentiation, including DeltaNp63 itself. To examine whether weakly expressed TAp63 has a specific role, we disrupted exon 1 by CRISPR-Cas9 homology directed repair in a head and neck squamous cell carcinoma (SCC) line. Surprisingly, TAp63 knockout cells, either by monoallelic GFP cassette insertion paired with a frameshift deletion allele or by biallelic GFP cassette insertion, caused DeltaNp63 silencing. Loss of keratinocyte-specific gene expression, switching of intermediate filaments from KRT(s) to VIM, and suppression of cell-cell and cell-matrix adhesion components indicated core events of epithelial mesenchymal transition. Most of the positively and negatively impacted genes including DeltaNp63 displayed local DNA methylation changes. Furthermore, DeltaNp63 expression was partially rescued by transfection of TAp63alpha followed by incubation with DNA methyltransferase inhibitor Zebularine. TAp63, as a minor part of TP63 gene, may possibly be involved in the auto-activation mechanism of DeltaNp63 by which keratinocyte-specific epigenome is maintained in SCC.

2023-09-29 | GSE234978 | GEO

p63 ChIP-seq analysis comparing WT, TAp63-null, and DNp63-null primary murine keratinocytes

Project description:We performed a ChIP-seq analysis characterizing the p63 binding sites in primary keratinocytes obtained from E18.5 mice of 3 different genotypes (WT, TAp63-/-, and ΔNp63-/-). We aimed to identify the specific genomic regions bound by TAp63 and ΔNp63 in physiological conditions.

2018-03-20 | GSE79448 | GEO

Gene expression profiles induced by knockdown and overexpression of p63 variants in MCF-10A mammary epithelial cell line

2010-03-12 | GSE20286 | GEO

Transcription profiling of human cell lines from Hay Wells Syndrome-Derived TAp63alphaQ540L mutant rveals impaired transcriptional and cell growth regulatory activity

Project description:p63 mutations have been associated with several human hereditary disorders characterized by ectodermal dysplasia such as EEC syndrome, ADULT syndrome and AEC syndrome . The location and functional effects of the mutations that underlie these syndromes reveal a striking genotype-phenotype correlation. Unlike EEC and ADULT that result from missense mutations in the DNA-binding domain of p63, AEC is solely caused by missense mutations in the SAM domain of p63. We report a study on the TAp63a isoform, the first to be expressed during development of the embryonic epithelia, and on its naturally occurring Q540L mutant derived from an AEC patient. To assess the effects of the Q540L mutation, we generated stable cell lines expressing TAp63a wt, DeltaNp63 alpha or the TAp63 alpha-Q540L mutant protein and used them to systematically compare the cell growth regulatory activity of the mutant and wt p63 proteins and to generate, by microarray analysis, a comprehensive profile of differential gene expression. We found that the Q540L substitution impairs the transcriptional activity of TAp63a and causes misregulation of genes involved in the control of cell growth and epidermal differentiation. Experiment Overall Design: Three biological replicates of not induced TAp63 alpha wt, three biological replicates of induced TAp63 alpha wt, three biological replicates of not induced TAp63Q540L alpha mutant and two biological replicates of induced TAp63Q540L alpha mutant

2008-06-16 | E-GEOD-8646 | biostudies-arrayexpress

Analysis of human TAp63 regulated genes in CD4+ T cells

Project description:By comparing the comprehensive gene expression profiles between human Th17 cells overexpressing TAp63 and those with TAp63 knockdown, FOXP3 was identified as one of the downregulated genes by TAp63

2023-04-10 | GSE181427 | GEO

TAp63-regulated microRNAs suppress cutaneous squamous cell carcinoma

Project description:TAp63 is a transcription factor belonging to the p53 family with important tumor suppressive functions. We show that TAp63-/- mice exhibit an increased susceptibility to UVR-induced cutaneous squamous cell carcinoma (cuSCC). These tumors showed global disruption of miRNA and mRNA expression when compared to tumors arising in wild-type mice. A comparison to similarly sequenced human cuSCC tumors identified miR-30c-2* and miR-497 as being significantly underexpressed in cuSCC. Reintroduction of these miRNAs significantly inhibited the growth of cuSCC cell lines and xenografts. Proteomic profiling of cells transfected with either miRNA showed significant downregulation of proteins related to cell cycle progression and mitosis. A cross-platform comparison of the RNAseq and proteomics signatures identified 7 downregulated proteins, which are also frequently overexpressed in both mouse and human cuSCC. Knockdown of AURKA, KIF18B, PKMYT1, and ORC1 in cuSCC cell lines suppressed tumor cell proliferation and induced cell death. Additionally, we found that an investigational, oral, selective inhibitor of AURKA suppressed cuSCC cell growth and induced cell death, and showed anti-tumor effects in vivo. Our data establishes TAp63 as an essential regulator of miRNA expression during skin carcinogenesis and reveals a novel network of miRNAs and mRNAs, which include potential targets for therapeutic intervention.

2020-07-07 | PXD014261 | Pride

FANCD2 Activates Transcription of TAp63 and Suppresses Tumorigenesis

Project description:Fanconi Anemia (FA) is a rare genetic disorder characterized by an increased susceptibility to squamous cell cancers. Fifteen FA genes are known, and the encoded proteins cooperate in a common DNA repair pathway. A critical step is the monoubiquitination of the FANCD2 protein, and cells from most FA patients are deficient in this step. How monoubiquitinated FANCD2 suppresses squamous cell cancers is unknown. Here we show that Fancd2-deficient mice are prone to Ras oncogene-driven skin carcinogenesis, while Usp1-deficient mice, expressing elevated cellular levels of Fancd2-Ub, are resistant to skin tumors. Moreover, Fancd2-Ub activates the transcription of the tumor suppressor TAp63, thereby promoting cellular senescence and blocking skin tumorigenesis. For FA patients, the reduction of FANCD2-Ub and TAp63 protein levels may account for their susceptibility to squamous cell neoplasia. Taken together, Usp1 inhibition may be a useful strategy for upregulating TAp63 and preventing or treating squamous cell cancers in the general non-FA population. Examination of FANCD2 binding after UV treatment in 293T cells

2013-06-27 | E-GEOD-46902 | biostudies-arrayexpress

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