Genome-wide p63-regulated gene expression in differentiating epidermal keratinocytes

The transcription factor p63 is a key regulator in epidermal keratinocyte proliferation and differentiation. However, the role of p63 in gene regulation during these processes is not well understood. To investigate this, we recently generated genome-wide profiles of gene expression, p63 binding sites and active regulatory regions with the H3K27ac histone mark (Kouwenhoven et al., 2015). We showed that only a subset of p63 binding sites are active in keratinocytes, and that differentiation-associated gene expression dynamics correlate with the activity of p63 binding sites rather than with their occurrence per se. Here we describe in detail the generation and processing of the ChIP-seq and RNA-seq datasets used in this study. These data sets are deposited in the Gene Expression Omnibus (GEO) repository under the accession number GSE59827

Cooperation between the transcription factors p63 and IRF6 is essential to prevent cleft palate in mice

Cleft palate is a common congenital disorder that affects up to 1 in 2,500 live human births and results in considerable morbidity to affected individuals and their families. The etiology of cleft palate is complex, with both genetic and environmental factors implicated. Mutations in the transcription factor–encoding genes p63 and interferon regulatory factor 6 (IRF6) have individually been identified as causes of cleft palate; however, a relationship between the key transcription factors p63 and IRF6 has not been determined. Here, we used both mouse models and human primary keratinocytes from patients with cleft palate to demonstrate that IRF6 and p63 interact epistatically during development of the secondary palate. Mice simultaneously carrying a heterozygous deletion of p63 and the Irf6 knockin mutation R84C, which causes cleft palate in humans, displayed ectodermal abnormalities that led to cleft palate. Furthermore, we showed that p63 transactivated IRF6 by binding to an upstream enhancer element; genetic variation within this enhancer element is associated with increased susceptibility to cleft lip. Our findings therefore identify p63 as a key regulatory molecule during palate development and provide a mechanism for the cooperative role of p63 and IRF6 in orofacial development in mice and humans

p63 exerts spatio-temporal control of palatal epithelial cell fate to prevent cleft palate

Contains fulltext : 174706.pdf (publisher's version ) (Open Access

Mutant Ras and inflammation-driven skin tumorigenesis is suppressed via a JNK-iASPP-AP1 axis.

Concurrent mutation of a RAS oncogene and the tumor suppressor p53 is common in tumorigenesis, and inflammation can promote RAS-driven tumorigenesis without the need to mutate p53. Here, we show, using a well-established mutant RAS and an inflammation-driven mouse skin tumor model, that loss of the p53 inhibitor iASPP facilitates tumorigenesis. Specifically, iASPP regulates expression of a subset of p63 and AP1 targets, including genes involved in skin differentiation and inflammation, suggesting that loss of iASPP in keratinocytes supports a tumor-promoting inflammatory microenvironment. Mechanistically, JNK-mediated phosphorylation regulates iASPP function and inhibits iASPP binding with AP1 components, such as JUND, via PXXP/SH3 domain-mediated interaction. Our results uncover a JNK-iASPP-AP1 regulatory axis that is crucial for tissue homeostasis. We show that iASPP is a tumor suppressor and an AP1 coregulator

The contribution of the nonhomologous region of Prs1 to the maintenance of cell wall integrity and cell viability

Item does not contain fulltextThe gene products of the five-membered PRS gene family in Saccharomyces cerevisiae have been shown to exist as three minimal functional entities, Prs1/Prs3, Prs2/Prs5, and Prs4/Prs5, each capable of supporting cell viability. The Prs1/Prs3 heterodimer can be regarded as the most important because its loss causes temperature sensitivity. It has been shown that the GFP signal generated by an integrated GFP-Prs1 construct is lost in the absence of Prs3. In addition to interacting with Prs3, Prs1 also interacts with Slt2, the MAPK of the cell wall integrity (CWI) pathway. Lack of the nonhomologous region (NHR1-1) located centrally in Prs1 abolished the temperature-induced increase in Rlm1 expression. Furthermore, in vitro point mutations generated in PRS1 corresponding to missense mutations associated with human neuropathies or in the divalent cation and/or 5-phosphoribosyl-1(alpha)-pyrophosphate binding sites also display increased Rlm1 expression at 30 degrees C and 37 degrees C and most give rise to caffeine sensitivity. Human PRPS1 cDNA cannot rescue the synthetic lethality of a prs1Delta prs5Delta strain because it lacks sequences corresponding to NHR1-1 of yeast Prs1. The correlation between caffeine sensitivity and increased basal expression of Rlm1 in the altered versions of PRS1 can be extended to their inability to rescue the synthetic lethality of a prs1Delta prs5Delta strain implying that impaired CWI may contribute to the observed loss of viability

De Novo Mutations in the Genome Organizer CTCF Cause Intellectual Disability

Item does not contain fulltextAn increasing number of genes involved in chromatin structure and epigenetic regulation has been implicated in a variety of developmental disorders, often including intellectual disability. By trio exome sequencing and subsequent mutational screening we now identified two de novo frameshift mutations and one de novo missense mutation in CTCF in individuals with intellectual disability, microcephaly, and growth retardation. Furthermore, an individual with a larger deletion including CTCF was identified. CTCF (CCCTC-binding factor) is one of the most important chromatin organizers in vertebrates and is involved in various chromatin regulation processes such as higher order of chromatin organization, enhancer function, and maintenance of three-dimensional chromatin structure. Transcriptome analyses in all three individuals with point mutations revealed deregulation of genes involved in signal transduction and emphasized the role of CTCF in enhancer-driven expression of genes. Our findings indicate that haploinsufficiency of CTCF affects genomic interaction of enhancers and their regulated gene promoters that drive developmental processes and cognition

Development of the palatal shelves in E14.5 <i>Krt5</i>-tTA;pTRE-ΔNp63α bi-transgenic mice.

<p>(<b>A</b>) The horizontal palatal shelves of wild-type embryos have formed a midline epithelial seam which has started to degenerate. (<b>B</b>) In contrast, the palatal shelves of <i>Krt5</i>-tTA;pTRE-ΔNp63α embryos have approximated and adhered via a thickened epithelial midline. (<b>C,D</b>). BrdU immunostaining confirmed that the persistent MES continued to proliferate with no evidence of apoptosis (<b>E, F</b>). (<b>G-J</b>) Immunostaining revealed a persistent double layer of keratin 17-positive periderm cells and an underlying layer of p63-positive basal cells. Scale bars: 50 μm.</p

Genome-wide profiling of p63 DNA-binding sites identifies an element that regulates gene expression during limb development in the 7q21 SHFM1 locus.

Contains fulltext : 88501.pdf (publisher's version ) (Open Access)Heterozygous mutations in p63 are associated with split hand/foot malformations (SHFM), orofacial clefting, and ectodermal abnormalities. Elucidation of the p63 gene network that includes target genes and regulatory elements may reveal new genes for other malformation disorders. We performed genome-wide DNA-binding profiling by chromatin immunoprecipitation (ChIP), followed by deep sequencing (ChIP-seq) in primary human keratinocytes, and identified potential target genes and regulatory elements controlled by p63. We show that p63 binds to an enhancer element in the SHFM1 locus on chromosome 7q and that this element controls expression of DLX6 and possibly DLX5, both of which are important for limb development. A unique micro-deletion including this enhancer element, but not the DLX5/DLX6 genes, was identified in a patient with SHFM. Our study strongly indicates disruption of a non-coding cis-regulatory element located more than 250 kb from the DLX5/DLX6 genes as a novel disease mechanism in SHFM1. These data provide a proof-of-concept that the catalogue of p63 binding sites identified in this study may be of relevance to the studies of SHFM and other congenital malformations that resemble the p63-associated phenotypes

Down-regulation of p63 in the medial edge epithelia allows periderm migration.

<p><i>mKrt17</i>-GFP transgenic mice were used for time-lapse confocal imaging of periderm migration during development of the secondary palate. (<b>A-C</b>) On a wild-type background, GFP-positive periderm cells migrate out of the midline seam (arrowed) to form the epithelial triangles on the oral and nasal surfaces as part of the process whereby mesenchymal continuity across the palate is achieved. (<b>D-F</b>) In contrast, in <i>Tgfb3</i>^-/- embryos, GFP-positive periderm cells fail to migrate out of the midline epithelial seam and the secondary palate remains cleft. (<b>G-I</b>) Reducing p63 dosage in <i>Tgfb3</i>^-/- embryos restores the migratory periderm phenotype allowing palatal fusion and rescuing the cleft palate phenotype (region of midline is arrowed). p: palatal shelves. The images are representative stills taken at the same Z position over a 24-hour culture period. The videos are provided as Supplemental Material, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006828#pgen.1006828.s010" target="_blank">S1</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006828#pgen.1006828.s012" target="_blank">S3 Videos</a>.</p