Novel in vivo targets of ΔNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach

<p>Abstract</p> <p>Background</p> <p>p63 is a transcription factor that plays an important role in skin epidermal development and differentiation. The p63 gene encodes for two major protein isoforms, those containing an amino-terminal trans-activation domain (TAp63) and those lacking this domain (ΔNp63). Both the TA and ΔN transcripts are also alternatively spliced at the 3' end producing proteins with unique C-termini that are designated as α, β and γ isoforms. Recent research has suggested that ΔNp63 is the predominant isoform expressed and active in keratinocytes.</p> <p>Results</p> <p>To better elucidate the biological role of p63 in regulating gene expression in keratinocytes we performed chromatin immunoprecipitation (ChIP) experiments with ΔNp63-specific antibodies. We included an additional step in the ChIP procedure to enrich for ΔNp63 targets by screening the library of immunoprecipitated DNA for its ability to bind recombinant GST-ΔNp63. Cloning of ΔNp63-ChIP-derived DNA fragments identified more than 60 potential ΔNp63 target loci that were located close to or embedded within known or predicted genes. Identity of these target genes suggests that they may participate in a myriad of cellular processes including transcriptional regulation, signaling and metabolism. Here we confirm the binding of ΔNp63 to several of these genomic loci both by EMSA and replicate ChIP assays. Finally we show that the expression of many of these target genes is altered when ΔNp63 levels in keratinocytes are reduced by siRNA, further confirming that these are bona fide targets.</p> <p>Conclusion</p> <p>This unbiased genomic approach has allowed us to uncover functional targets of ΔNp63 and serves as the initial step in further analysis of the transcriptional regulatory mechanisms that are governed by p63 in keratinocytes.</p

Regulation of Mat Responses by a Differentiation MAPK Pathway in Saccharomyces cerevisiae

Fungal species exhibit diverse behaviors when presented with extracellular challenges. Pathogenic fungi can undergo cell differentiation and biofilm formation in response to fluctuating nutrient levels, and these responses are required for virulence. In the model fungal eukaryote Saccharomyces cerevisiae, nutrient limitation induces filamentous growth and biofilm/mat formation. Both responses require the same signal transduction (MAPK) pathway and the same cell adhesion molecule (Flo11) but have been studied under different conditions. We found that filamentous growth and mat formation are aspects of a related response that is regulated by the MAPK pathway. Cells in yeast-form mats differentiated into pseudohyphae in response to nutrient limitation. The MAPK pathway regulated mat expansion (in the plane of the XY-axis) and substrate invasion (downward in the plane of the Z-axis), which optimized the mat's response to extracellular nutrient levels. The MAPK pathway also regulated an upward growth pattern (in the plane of the Z-axis) in response to nutrient limitation and changes in surface rigidity. Upward growth allowed for another level of mat responsiveness and resembled a type of colonial chemorepulsion. Together our results show that signaling pathways play critical roles in regulating social behaviors in which fungal cells participate. Signaling pathways may regulate similar processes in pathogens, whose highly nuanced responses are required for virulence

An Active Role of the ΔN Isoform of p63 in Regulating Basal Keratin Genes K5 and K14 and Directing Epidermal Cell Fate

BACKGROUND: One major defining characteristic of the basal keratinocytes of the stratified epithelium is the expression of the keratin genes K5 and K14. The temporal and spatial expression of these two genes is usually tightly and coordinately regulated at the transcriptional level. This ensures the obligate pairing of K5 and K14 proteins to generate an intermediate filament (IF) network that is essential for the structure and function of the proliferative keratinocytes. Our previous studies have shown that the basal-keratinocyte restricted transcription factor p63 is a direct regulator of K14 gene. METHODOLOGY/PRINCIPAL FINDINGS: Here we provide evidence that p63, specifically the DeltaN isoform also regulates the expression of the K5 gene by binding to a conserved enhancer within the 5' upstream region. By using specific antibodies against DeltaNp63, we show a concordance in the expression between basal keratins and DeltaNp63 proteins but not the TAp63 isoforms during early embryonic skin development. We demonstrate, that contrary to a previous report, transgenic mice expressing DeltaNp63 in lung epithelium exhibit squamous metaplasia with de novo induction of K5 and K14 as well as transdifferentiation to the epidermal cell lineage. Interestingly, the in vivo epidermal inductive properties of DeltaNp63 do not require the C-terminal SAM domain. Finally, we show that DeltaNp63 alone can restore the expression of the basal keratins and reinitiate the failed epidermal differentiation program in the skin of p63 null animals. SIGNIFICANCE: DeltaNp63 is a critical mediator of keratinocyte stratification program and directly regulates the basal keratin genes

Correlated changes between regulatory cis elements and condition-specific expression in paralogous gene families

Gene duplication is integral to evolution, providing novel opportunities for organisms to diversify in function. One fundamental pathway of functional diversification among initially redundant gene copies, or paralogs, is via alterations in their expression patterns. Although the mechanisms underlying expression divergence are not completely understood, transcription factor binding sites and nucleosome occupancy are known to play a significant role in the process. Previous attempts to detect genomic variations mediating expression divergence in orthologs have had limited success for two primary reasons. First, it is inherently challenging to compare expressions among orthologs due to variable trans-acting effects and second, previous studies have quantified expression divergence in terms of an overall similarity of expression profiles across multiple samples, thereby obscuring condition-specific expression changes. Moreover, the inherently inter-correlated expressions among homologs present statistical challenges, not adequately addressed in many previous studies. Using rigorous statistical tests, here we characterize the relationship between cis element divergence and condition-specific expression divergence among paralogous genes in Saccharomyces cerevisiae. In particular, among all combinations of gene family and TFs analyzed, we found a significant correlation between TF binding and the condition-specific expression patterns in over 20% of the cases. In addition, incorporating nucleosome occupancy reveals several additional correlations. For instance, our results suggest that GAL4 binding plays a major role in the expression divergence of the genes in the sugar transporter family. Our work presents a novel means of investigating the cis regulatory changes potentially mediating expression divergence in paralogous gene families under specific conditions

ATP7A is a novel target of retinoic acid receptor β2 in neuroblastoma cells

Increased retinoic acid receptor β (RARβ2) gene expression is a hallmark of cancer cell responsiveness to retinoid anticancer effects. Moreover, low basal or induced RARβ2 expression is a common feature of many human cancers, suggesting that RARβ2 may act as a tumour suppressor gene in the absence of supplemented retinoid. We have previously shown that low RARβ2 expression is a feature of advanced neuroblastoma. Here, we demonstrate that the ABC domain of the RARβ2 protein alone was sufficient for the growth inhibitory effects of RARβ2 on neuroblastoma cells. ATP7A, the copper efflux pump, is a retinoid-responsive gene, was upregulated by ectopic overexpression of RARβ2. The ectopic overexpression of the RARβ2 ABC domain was sufficient to induce ATP7A expression, whereas, RARβ2 siRNA blocked the induction of ATP7A expression in retinoid-treated neuroblastoma cells. Forced downregulation of ATP7A reduced copper efflux and increased viability of retinoid-treated neuroblastoma cells. Copper supplementation enhanced cell growth and reduced retinoid-responsiveness, whereas copper chelation reduced the viability and proliferative capacity. Taken together, our data demonstrates ATP7A expression is regulated by retinoic acid receptor β and it has effects on intracellular copper levels, revealing a link between the anticancer action of retinoids and copper metabolism

The Human Papillomavirus E6 Oncogene Represses a Cell Adhesion Pathway and Disrupts Focal Adhesion through Degradation of TAp63β upon Transformation

Cervical carcinomas result from cellular transformation by the human papillomavirus (HPV) E6 and E7 oncogenes which are constitutively expressed in cancer cells. The E6 oncogene degrades p53 thereby modulating a large set of p53 target genes as shown previously in the cervical carcinoma cell line HeLa. Here we show that the TAp63β isoform of the p63 transcription factor is also a target of E6. The p63 gene plays an essential role in skin homeostasis and is expressed as at least six isoforms. One of these isoforms, ΔNp63α, has been found overexpressed in squamous cell carcinomas and is shown here to be constitutively expressed in Caski cells associated with HPV16. We therefore explored the role of p63 in these cells by performing microarray analyses after repression of endogenous E6/E7 expression. Upon repression of the oncogenes, a large set of p53 target genes was found activated together with many p63 target genes related to cell adhesion. However, through siRNA silencing and ectopic expression of various p63 isoforms we demonstrated that TAp63β is involved in activation of this cell adhesion pathway instead of the constitutively expressed ΔNp63α and β. Furthermore, we showed in cotransfection experiments, combined with E6AP siRNA silencing, that E6 induces an accelerated degradation of TAp63β although not through the E6AP ubiquitin ligase used for degradation of p53. Repression of E6 transcription also induces stabilization of endogenous TAp63β in cervical carcinoma cells that lead to an increased concentration of focal adhesions at the cell surface. Consequently, TAp63β is the only p63 isoform suppressed by E6 in cervical carcinoma as demonstrated previously for p53. Down-modulation of focal adhesions through disruption of TAp63β therefore appears as a novel E6-dependent pathway in transformation. These findings identify a major physiological role for TAp63β in anchorage independent growth that might represent a new critical pathway in human carcinogenesis

Multiple Signals Converge on a Differentiation MAPK Pathway

An important emerging question in the area of signal transduction is how information from different pathways becomes integrated into a highly coordinated response. In budding yeast, multiple pathways regulate filamentous growth, a complex differentiation response that occurs under specific environmental conditions. To identify new aspects of filamentous growth regulation, we used a novel screening approach (called secretion profiling) that measures release of the extracellular domain of Msb2p, the signaling mucin which functions at the head of the filamentous growth (FG) MAPK pathway. Secretion profiling of complementary genomic collections showed that many of the pathways that regulate filamentous growth (RAS, RIM101, OPI1, and RTG) were also required for FG pathway activation. This regulation sensitized the FG pathway to multiple stimuli and synchronized it to the global signaling network. Several of the regulators were required for MSB2 expression, which identifies the MSB2 promoter as a target “hub” where multiple signals converge. Accessibility to the MSB2 promoter was further regulated by the histone deacetylase (HDAC) Rpd3p(L), which positively regulated FG pathway activity and filamentous growth. Our findings provide the first glimpse of a global regulatory hierarchy among the pathways that control filamentous growth. Systems-level integration of signaling circuitry is likely to coordinate other regulatory networks that control complex behaviors

Basics of nature-climate typization of housing in Trans-Caucasus and methodology of assessment of climate in architectural design

Available from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio

Novel targets of ΔNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach-5

<p><b>Copyright information:</b></p><p>Taken from "Novel targets of ΔNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach"</p><p>http://www.biomedcentral.com/1471-2199/8/43</p><p>BMC Molecular Biology 2007;8():43-43.</p><p>Published online 23 May 2007</p><p>PMCID:PMC1890296.</p><p></p>omoter demonstrate high levels of activity when co-transfected with an expression plasmid encoding ΔNp63α in PtK2 cells. Luciferase values were determined and normalized against β-galactosidase values. Corrected luciferase values for cells transfected with empty vector pCMV-HA were set at 1. TK; thymidine kinas