Epigenetic priming of an epithelial enhancer by p63 and CTCF controls expression of a skin-restricted gene XP33.

The transcription factor p63 is a renowned master regulator of gene expression of stratified epithelia. While multiple proteins have been identified as p63 bona fide targets, little is known about non-coding RNAs (ncRNAs) whose transcription is controlled by p63. Here, we describe a skin-specific non-coding RNA XP33 as a novel target of p63. XP33 levels are increased during keratinocyte differentiation in vitro, while its depletion results in decreased expression of late cornified gene LCE2D. By using publicly available multi-omics data, we show that CTCF and p63 establish an epithelial enhancer to prime XP33 transcription in a tissue-restricted manner. XP33 promoter and enhancer form a chromatin loop exclusively in keratinocytes but not in other cell types. Moreover, the XP33 enhancer is occupied by differentiation-specific factors that control XP33 transcription. Altogether, we identify a tissue-specific non-coding RNA whose expression is epigenetically regulated by p63 and CTCF.

Proteasome-mediated degradation of keratins 7, 8, 17 and 18 by mutant KLHL24 in a foetal keratinocyte model: Novel insight in congenital skin defects and fragility of epidermolysis bullosa simplex with cardiomyopathy.

Epidermolysis bullosa simplex (EBS) with cardiomyopathy (EBS-KLHL24) is an EBS subtype caused by dominantly inherited, gain-of-function mutations in the gene encoding for the ubiquitin-ligase KLHL24, which addresses specific proteins to proteasomal degradation. EBS-KLHL24 patients are born with extensive denuded skin areas and skin fragility. Whilst skin fragility rapidly ameliorates, atrophy and scarring develop over time, accompanied by life-threatening cardiomyopathy. To date, pathogenetic mechanisms underlying such a unique disease phenotype are not fully characterized. The basal keratin 14 (K14) has been indicated as a KLHL24 substrate in keratinocytes. However, EBS-KLHL24 pathobiology cannot be determined by the mutation-enhanced disruption of K14 alone, as K14 is similarly expressed in foetal and postnatal epidermis and its protein levels are preserved both in vivo and in vitro disease models. In this study, we focused on foetal keratins as additional KLHL24 substrates. We showed that K7, K8, K17 and K18 protein levels are markedly reduced via proteasome degradation in normal foetal keratinocytes transduced with the mutant KLHL24 protein (ΔN28-KLHL24) as compared to control cells expressing the wild-type form. In addition, heat stress led to keratin network defects and decreased resilience in ΔN28-KLHL24 cells. The KLHL24-mediated degradation of foetal keratins could contribute to congenital skin defects in EBS-KLHL24. Furthermore, we observed that primary keratinocytes from EBS-KLHL24 patients undergo accelerated clonal conversion with reduced colony forming efficiency (CFE) and early replicative senescence. Finally, our findings pointed out a reduced CFE in ΔN28-KLHL24-transduced foetal keratinocytes as compared to controls, suggesting that mutant KLHL24 contributes to patients' keratinocyte clonogenicity impairment.

TAp63 regulates bone remodeling by modulating the expression of TNFRSF11B/Osteoprotegerin.

ABBREVIATIONS: MSC, mesenchymal stem cells; OPG, osteoprotegerin; RUNX2, Run-trelated transcription factor 2.

The Enigma of Interspecific Plasmodesmata: Insight From Parasitic Plants.

Parasitic plants live in intimate physical connection with other plants serving as their hosts. These host plants provide the inorganic and organic compounds that the parasites need for their propagation. The uptake of the macromolecular compounds happens through symplasmic connections in the form of plasmodesmata. In contrast to regular plasmodesmata, which connect genetically identical cells of an individual plant, the plasmodesmata that connect the cells of host and parasite join separate individuals belonging to different species and are therefore termed "interspecific". The existence of such interspecific plasmodesmata was deduced either indirectly using molecular approaches or observed directly by ultrastructural analyses. Most of this evidence concerns shoot parasitic Cuscuta species and root parasitic Orobanchaceae, which can both infect a large range of phylogenetically distant hosts. The existence of an interspecific chimeric symplast is both striking and unique and, with exceptions being observed in closely related grafted plants, exist only in these parasitic relationships. Considering the recent technical advances and upcoming tools for analyzing parasitic plants, interspecific plasmodesmata in parasite/host connections are a promising system for studying secondary plasmodesmata. For open questions like how their formation is induced, how their positioning is controlled and if they are initiated by one or both bordering cells simultaneously, the parasite/host interface with two adjacent distinguishable genetic systems provides valuable advantages. We summarize here what is known about interspecific plasmodesmata between parasitic plants and their hosts and discuss the potential of the intriguing parasite/host system for deepening our insight into plasmodesmatal structure, function, and development.

The p63 C-terminus is essential for murine oocyte integrity.

The transcription factor p63 mediates distinct cellular responses, primarily regulating epithelial and oocyte biology. In addition to the two amino terminal isoforms, TAp63 and ΔNp63, the 3'-end of p63 mRNA undergoes tissue-specific alternative splicing that leads to several isoforms, including p63α, p63ß and p63γ. To investigate in vivo how the different isoforms fulfil distinct functions at the cellular and developmental levels, we developed a mouse model replacing the p63α with p63ß by deletion of exon 13 in the Trp63 gene. Here, we report that whereas in two organs physiologically expressing p63α, such as thymus and skin, no abnormalities are detected, total infertility is evident in heterozygous female mice. A sharp reduction in the number of primary oocytes during the first week after birth occurs as a consequence of the enhanced expression of the pro-apoptotic transcriptional targets Puma and Noxa by the tetrameric, constitutively active, TAp63ß isoform. Hence, these mice show a condition of ovary dysfunction, resembling human primary ovary insufficiency. Our results show that the p63 C-terminus is essential in TAp63α-expressing primary oocytes to control cell death in vivo, expanding the current understanding of human primary ovarian insufficiency.

A highly efficient protocol for transforming Cuscuta reflexa based on artificially induced infection sites.

The parasitic plant genus Cuscuta is notoriously difficult to transform and to propagate or regenerate in vitro. With it being a substantial threat to many agroecosystems, techniques allowing functional analysis of gene products involved in host interaction and infection mechanisms are, however, in high demand. We set out to explore whether Agrobacterium-mediated transformation of different plant parts can provide efficient alternatives to the currently scarce and inefficient protocols for transgene expression in Cuscuta. We used fluorescent protein genes on the T-DNA as markers for transformation efficiency and transformation stability. As a result, we present a novel highly efficient transformation protocol for Cuscuta reflexa cells that exploits the propensity of the infection organ to take up and express transgenes with the T-DNA. Both, Agrobacterium rhizogenes and Agrobacterium tumefaciens carrying binary transformation vectors with reporter fluorochromes yielded high numbers of transformation events. An overwhelming majority of transformed cells were observed in the cell layer below the adhesive disk's epidermis, suggesting that these cells are particularly susceptible to infection. Cotransformation of these cells happens frequently when Agrobacterium strains carrying different constructs are applied together. Explants containing transformed tissue expressed the fluorescent markers in in vitro culture for several weeks, offering a future possibility for development of transformed cells into callus. These results are discussed with respect to the future potential of this technique and with respect to the special characteristics of the infection organ that may explain its competence to take up the foreign DNA.

Identification of Long Noncoding RNA by In Situ Hybridization Approaches.

In situ hybridization (ISH) and fluorescence in situ hybridization (FISH) techniques enable us to detect the expression of a specific RNA in fixed cells or tissue sections. Here, we describe in detail two procedures adjusted to reveal specifically lncRNAs in normal human keratinocytes and in skin tissue samples. Examples of the results obtained by the two different approaches are also shown.

ZNF750 represses breast cancer invasion via epigenetic control of prometastatic genes.

Breast cancer is the second leading cause of cancer-related deaths among women, largely due to the progression of a significant fraction of primary tumours to the metastatic stage. Here, we show that zinc-finger protein 750 (ZNF750) opposes the migration and invasion of breast cancer cells by repressing a prometastatic transcriptional programme, which includes genes involved in focal adhesion and extracellular matrix interactions, such as LAMB3 and CTNNAL1. Mechanistically, ZNF750 recruits the epigenetic modifiers KDM1A and HDAC1 to the promoter regions of LAMB3 and CTNNAL1, influencing histone marks and transactivating these genomic sites. Gene expression analysis in cancer patient datasets indicated that ZNF750 and its targets were negative prognostic factors in breast cancer. Together, our findings shed light on the molecular mechanism by which ZNF750 regulates cell migration and invasion, suggesting a role in breast cancer metastasis.

Transglutaminase 3 Reduces the Severity of Psoriasis in Imiquimod-Treated Mouse Skin.

Four transglutaminase (TG) isoforms have been detected in epidermal keratinocytes: TG1, TG2, TG3, and TG5. Except for TG1 and TG3, their contribution to keratinocyte development and structure remains undefined. In this paper, we focused on the roles of TG2 and TG3 in imiquimod-induced psoriasis in mouse skin. We evaluated the severity of psoriasis markers in the skin of imiquimod-treated TG3 null and TG2 null mice. Our results showed that compromised TG3KO mouse skin was more responsive than WT or TG2KO mouse skin to the action of the pro-inflammatory drug imiquimod.

Long non-coding RNA uc.291 controls epithelial differentiation by interfering with the ACTL6A/BAF complex.

The mechanisms that regulate the switch between epidermal progenitor state and differentiation are not fully understood. Recent findings indicate that the chromatin remodelling BAF complex (Brg1-associated factor complex or SWI/SNF complex) and the transcription factor p63 mutually recruit one another to open chromatin during epidermal differentiation. Here, we identify a long non-coding transcript that includes an ultraconserved element, uc.291, which physically interacts with ACTL6A and modulates chromatin remodelling to allow differentiation. Loss of uc.291 expression, both in primary keratinocytes and in three-dimensional skin equivalents, inhibits differentiation as indicated by epidermal differentiation complex genes down-regulation. ChIP experiments reveal that upon uc.291 depletion, ACTL6A is bound to the differentiation gene promoters and inhibits BAF complex targeting to induce terminal differentiation genes. In the presence of uc.291, the ACTL6A inhibitory effect is released, allowing chromatin changes to promote the expression of differentiation genes. Thus, uc.291 interacts with ACTL6A to modulate chromatin remodelling activity, allowing the transcription of late differentiation genes.

Selective mineral transport barriers at Cuscuta-host infection sites.

The uptake of inorganic nutrients by rootless parasitic plants, which depend on host connections for all nutrient supplies, is largely uncharted. Using X-ray fluorescence spectroscopy (XRF), we analyzed the element composition of macro- and micronutrients at infection sites of the parasitic angiosperm Cuscuta reflexa growing on hosts of the genus Pelargonium. Imaging methods combining XRF with 2-D or 3-D (confocal) microscopy show that most of the measured elements are present at similar concentrations in the parasite compared to the host. However, calcium and strontium levels drop pronouncedly at the host/parasite interface, and manganese appears to accumulate in the host tissue surrounding the interface. Chlorine is present in the haustorium at similar levels as in the host tissue but is decreased in the stem of the parasite. Thus, our observations indicate a restricted uptake of calcium, strontium, manganese and chlorine by the parasite. Xylem-mobile dyes, which can probe for xylem connectivity between host and parasite, provided evidence for an interspecies xylem flow, which in theory would be expected to carry all of the elements indiscriminately. We thus conclude that inorganic nutrient uptake by the parasite Cuscuta is regulated by specific selective barriers whose existence has evaded detection until now.

Biomarkers for vascular ageing in aorta tissues and blood samples.

OBJECTIVES: Functional and quantitative alterations and senescence of circulating and expanded endothelial progenitor cells (EPC), as well as systemic and tissue modifications of angiogenetic and inflammatory molecules, were evaluated for predicting age-related vessel wall remodeling, correlating them to intima media thickness (IMT) in the common carotid artery (CCA), a biomarker of early cardiovascular disease and aortic root dilation. POPULATIONS AND METHODS: A homogenous Caucasian population was included in the study, constituted by 160 healthy subjects (80 old subjects, mean age 72 ±â€¯6.4, range 66-83 years; and 80 younger blood donors, mean age 26.2 ±â€¯3.4, range 21-33 years), and 60 old subjects (mean age 73 ±â€¯1.4 (range 66-83) years) with aortic root dilatation and hypertension, and 60 old people (70 ±â€¯2.8 (age range 66-83)) with sporadic ascending aorta aneurysm (AAA). In addition, 20 control individuals (10 men and 10 women, mean age: 65 ±â€¯8), were also included in the study for evaluating the gene expression's levels, in aorta tissues. Appropriate techniques, practises, protocols, gating strategies and statistical analyses were performed in our evaluations. RESULTS: Interestingly, old people had a significantly reduced functionality and a high grade of senescence (high SA-ß-Gal activity and high levels of TP53, p21 and p16 genes) of EPC expanded than younger subjects. The values of related parameters progressively augmented from the old subjects, in good healthy shape, to subjects with hypertension and aorta dilation, and AAA. Moreover, they significantly impacted the endothelium than the alterations in EPC number. No changes, but rather increased systemic levels of VEGF and SDF-1 were also assessed in old people vs. younger donors. Old people also showed significantly increased systemic levels of inflammatory cytokines, and a reciprocal significant reduction of systemic s-Notch 1 than younger subjects. These parameters, also including the number EPC alterations, resulted to be significantly sustained in old people bearers of an inflammatory combined genotype. Consistent with these data, a reduced expression of Notch-1 gene, accompanied by a sustained expression of inflammatory genes (i.e. TLR4, IL-1ß, IL-6 and IL-17) were detected in aortic tissues from old control people and AAA cases. Finally, we detected the biological effects induced by all the detected alterations on vessel wall age-related remodeling, by evaluating the IMT in the population studied and correlating it to these alterations. The analysis demonstrated that the unique independent risk predictors for vascular ageing are age, the EPC reduced migratory activity and senescence, high grade of expression of genes inducing EPC senescence and chronic tissue and systemic inflammation. CONCLUSIONS: Thus, we propose these parameters, of easy determination in biological samples (i.e. blood and tissue samples) from alive human population, as optimal biomarkers for vascular ageing.

ZNF185 is a p63 target gene critical for epidermal differentiation and squamous cell carcinoma development.

Development and maintenance of healthy stratified epithelia require the coordination of complex transcriptional programmes. The transcription factor p63, a member of the p53 family, plays a crucial role in epithelial development and homeostasis. Analysis of the p63-dependent transcriptome indicated that one important aspect of p63 functions in epithelial development is the regulation of cell-cell and cell-matrix adhesion programmes. However, limited knowledge exists on the relevant cell-cell adhesion molecules involved in physiological epithelial formation. Similarly, limited data are available to understand if deregulation of the cell-cell adhesion programme is important in tumour formation. Here, using the epidermis as an experimental model with the RNA sequencing approach, we identify a novel p63-regulated gene induced during differentiation, ZNF185. ZNF185 is an actin-cytoskeleton-associated Lin-l 1, Isl-1 and Mec-3 (LIM) domain-containing protein, whose function is poorly known. We found that p63 binds to a specific enhancer region, promoting its expression to sustain epithelial differentiation. ZNF185 silencing strongly impaired keratinocyte differentiation according to gene array analysis. ZNF185 is detected at the cell-cell periphery where it physically interacts with E-cadherin, indicating that it is important to maintain epithelial integrity beyond its pro-differentiation role. Interestingly, poorly differentiated, including head and neck, cervical and oesophageal, squamous cell carcinomas display loss of ZNF185 expression. Together, these studies reinforce that p63 is a crucial gene for maintaining epithelial tissue integrity and support the deregulation of the cell-cell adhesion programme,which plays a critical role in carcinoma development.

ΔNp63 promotes IGF1 signalling through IRS1 in squamous cell carcinoma.

Accumulating evidence has proved that deregulation of ΔNp63 expression plays an oncogenic role in head and neck squamous cell carcinomas (HNSCCs). Besides p63, the type 1-insulin-like growth factor (IGF) signalling pathway has been implicated in HNSCC development and progression. Most insulin/IGF1 signalling converges intracellularly onto the protein adaptor insulin receptor substrate-1 (IRS-1) that transmits signals from the receptor to downstream effectors, including the PI3K/AKT and the MAPK kinase pathways, which, ultimately, promote proliferation, invasion, and cell survival. Here we report that p63 directly controls IRS1 transcription and cellular abundance and fosters the PI3K/AKT and MAPK downstream signalling pathways. Inactivation of ΔNp63 expression indeed reduces tumour cell responsiveness to IGF1 stimulation, and inhibits the growth potential of HNSCC cells. In addition, a positive correlation was observed between p63 and IRS1 expression in human HNSCC tissue arrays and in publicly available gene expression data. Our findings indicate that aberrant expression of ΔNp63 in HNSSC may act as an oncogenic stimulus by altering the IGF signalling pathway.

ZNF185 is a p53 target gene following DNA damage.

The transcription factor p53 is a key player in the tumour suppressive DNA damage response and a growing number of target genes involved in these pathways has been identified. p53 has been shown to be implicated in controlling cell motility and its mutant form enhances metastasis by loss of cell directionality, but the p53 role in this context has not yet being investigated. Here, we report that ZNF185, an actin cytoskeleton-associated protein from LIM-family of Zn-finger proteins, is induced following DNA-damage. ChIP-seq analysis, chromatin crosslinking immune-precipitation experiments and luciferase assays demonstrate that ZNF185 is a bona fide p53 target gene. Upon genotoxic stress, caused by DNA-damaging drug etoposide and UVB irradiation, ZNF185 expression is up-regulated and in etoposide-treated cells, ZNF185 depletion does not affect cell proliferation and apoptosis, but interferes with actin cytoskeleton remodelling and cell polarization. Bioinformatic analysis of different types of epithelial cancers from both TCGA and GTEx databases showed a significant decrease in ZNF185 mRNA level compared to normal tissues. These findings are confirmed by tissue micro-array IHC staining. Our data highlight the involvement of ZNF185 and cytoskeleton changes in p53-mediated cellular response to genotoxic stress and indicate ZNF185 as potential biomarker for epithelial cancer diagnosis.

Myoblasts rely on TAp63 to control basal mitochondria respiration.

p53, with its family members p63 and p73, have been shown to promote myoblast differentiation by regulation of the function of the retinoblastoma protein and by direct activation of p21Cip/Waf1 and p57Kip2, promoting cell cycle exit. In previous studies, we have demonstrated that the TAp63γ isoform is the only member of the p53 family that accumulates during in vitro myoblasts differentiation, and that its silencing led to delay in myotube fusion. To better dissect the role of TAp63γ in myoblast physiology, we have generated both sh-p63 and Tet-On inducible TAp63γ clones. Gene array analysis of sh-p63 C2C7 clones showed a significant modulation of genes involved in proliferation and cellular metabolism. Indeed, we found that sh-p63 C2C7 myoblasts present a higher proliferation rate and that, conversely, TAp63γ ectopic expression decreases myoblasts proliferation, indicating that TAp63γ specifically contributes to myoblasts proliferation, independently of p53 and p73. In addition, sh-p63 cells have a defect in mitochondria respiration highlighted by a reduction in spare respiratory capacity and a decrease in complex I, IV protein levels. These results demonstrated that, beside contributing to cell cycle exit, TAp63γ participates to myoblasts metabolism control.

Kruppel-like factor 4 regulates keratinocyte senescence.

Keratinocyte replicative senescence has an important role in time-related changes of epidermis. Previous studies demonstrated that miRNAs play key roles in inhibiting proliferation and in the acquisition of the keratinocyte senescent phenotype as well as in individual ageing. Kruppel-like factor 4 is a transcription factor with dual functions in keratinocytes, being a stemness factor and a pro-differentiation factor. Interestingly, in skin squamous cell carcinomas KLF4 expression is strongly down-regulated or absent. While KLF4 involvement in senescence and ageing has not been investigated yet. Here, we show that Klf4 protein decreases during keratinocyte replicative senescence and during physiological skin aging, while its mRNA level does not change. We demonstrated that the senescence-associated miR-34a regulates post-transcriptionally Klf4 expression. KLF4 silencing is sufficient to induce a senescent phenotype in primary keratinocytes and ectopic miR-34a over-expression phenocopies this result. Our findings identify a novel regulatory loop between miR-34a and KLF4 during keratinocytes replicative senescence. This regulatory loop, beside aging, may play a role in age-related pathologies.

Metabolic profiling of visceral adipose tissue from obese subjects with or without metabolic syndrome.

Obesity represents one of the most complex public health challenges and has recently reached epidemic proportions. Obesity is also considered to be primarily responsible for the rising prevalence of metabolic syndrome, defined as the coexistence in the same individual of several risk factors for atherosclerosis, including dyslipidemia, hypertension and hyperglycemia, as well as for cancer. Additionally, the presence of three of the five risk factors (abdominal obesity, low high-density lipoprotein cholesterol, high triglycerides, high fasting glucose and high blood pressure) characterizes metabolic syndrome, which has serious clinical consequences. The current study was conducted in order to identify metabolic differences in visceral adipose tissue (VAT) collected from obese (body mass index 43-48) human subjects who were diagnosed with metabolic syndrome, obese individuals who were metabolically healthy and nonobese healthy controls. Extensive gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS/MS) analyses were used to obtain the untargeted VAT metabolomic profiles of 481 metabolites belonging to all biochemical pathways. Our results indicated consistent increases in oxidative stress markers from the pathologically obese samples in addition to subtle markers of elevated glucose levels that may be consistent with metabolic syndrome. In the tissue derived from the pathologically obese subjects, there were significantly elevated levels of plasmalogens, which may be increased in response to oxidative changes in addition to changes in glycerolphosphorylcholine, glycerolphosphorylethanolamine glycerolphosphorylserine, ceramides and sphingolipids. These data could be potentially helpful for recognizing new pathways that underlie the metabolic-vascular complications of obesity and may lead to the development of innovative targeted therapies.

Metabolic pathways regulated by p63.

The transcription factor p63 belongs to the p53-family and is a master regulator of proliferative potential, lineage specification, and differentiation in epithelia during development and tissue homeostasis. In cancer, p63 contribution is isoform-specific, with both oncogenic and tumour suppressive roles attributed, for ΔNp63 and TAp63, respectively. Recently, p53 and TAp73, in line with other tumour suppressor genes, have emerged as important regulators of energy metabolism and metabolic reprogramming in cancer. To date, p63 contributions in controlling energy metabolism have been partially investigated; given the extensive interaction of the p53 family members, these studies have potential implications in tumour cells for metabolic reprogramming. Here, we review the role of p63 isoforms, TAp63 and ΔNp63, in controlling cell metabolism, focusing on their specific metabolic target genes and their physiological/functional context of action.

Ultraconserved long non-coding RNA uc.63 in breast cancer.

Transcribed-ultraconserved regions (T-UCRs) are long non-coding RNAs (lncRNA) encoded by a subset of long ultraconserved stretches in the human genome. Recent studies revealed that the expression of several T-UCRs is altered in cancer and growing evidences underline the importance of T-UCRs in oncogenesis, offering also potential new strategies for diagnosis and prognosis. We found that overexpression of one specific T-UCRs named uc.63 is associated with bad outcome in luminal A subtype of breast cancer patients. uc.63 is localized in the third intron of exportin-1 gene (XPO1) and is transcribed in the same orientation of its host gene. Interestingly, silencing of uc.63 induces apoptosis in vitro. However, silencing of host gene XPO1 does not cause the same effect suggesting that the transcription of uc.63 is independent of XPO1. Our results reveal an important role of uc.63 in promoting breast cancer cells survival and offer the prospect to identify a signature associated with poor prognosis.