Mechanisms of MEOX1 and MEOX2 Regulation of the Cyclin Dependent Kinase Inhibitors p21CIP1/WAF1 and p16INK4a in Vascular Endothelial Cells

Senescence, the state of permanent cell cycle arrest, has been associated with endothelial cell dysfunction and atherosclerosis. The cyclin dependent kinase inhibitors p21CIP1/WAF1 and p16INK4a govern the G1/S cell cycle checkpoint and are essential for determining whether a cell enters into an arrested state. The homeodomain transcription factor MEOX2 is an important regulator of vascular cell proliferation and is a direct transcriptional activator of both p21CIP1/WAF1 and p16INK4a. MEOX1 and MEOX2 have been shown to be partially functionally redundant during development, suggesting that they regulate similar target genes in vivo. We compared the ability of MEOX1 and MEOX2 to activate p21CIP1/WAF1 and p16INK4a expression and induce endothelial cell cycle arrest. Our results demonstrate for the first time that MEOX1 regulates the MEOX2 target genes p21CIP1/WAF1 and p16INK4a. In addition, increased expression of either of the MEOX homeodomain transcription factors leads to cell cycle arrest and endothelial cell senescence. Furthermore, we show that the mechanism of transcriptional activation of these cyclin dependent kinase inhibitor genes by MEOX1 and MEOX2 is distinct. MEOX1 and MEOX2 activate p16INK4a in a DNA binding dependent manner, whereas they induce p21CIP1/WAF1 in a DNA binding independent manner

NAD(P)H Quinone Oxidoreductase Protects TAp63γ from Proteasomal Degradation and Regulates TAp63γ-Dependent Growth Arrest

BACKGROUND: p63 is a member of the p53 transcription factor family. p63 is expressed from two promoters resulting in proteins with opposite functions: the transcriptionally active TAp63 and the dominant-negative DeltaNp63. Similar to p53, the TAp63 isoforms induce cell cycle arrest and apoptosis. The DeltaNp63 isoforms are dominant-negative variants opposing the activities of p53, TAp63 and TAp73. To avoid unnecessary cell death accompanied by proper response to stress, the expression of the p53 family members must be tightly regulated. NAD(P)H quinone oxidoreductase (NQO1) has recently been shown to interact with and inhibit the degradation of p53. Due to the structural similarities between p53 and p63, we were interested in studying the ability of wild-type and polymorphic, inactive NQO1 to interact with and stabilize p63. We focused on TAp63gamma, as it is the most potent transcription activator and it is expected to have a role in tumor suppression. PRINCIPAL FINDINGS: We show that TAp63gamma can be degraded by the 20S proteasomes. Wild-type but not polymorphic, inactive NQO1 physically interacts with TAp63gamma, stabilizes it and protects it from this degradation. NQO1-mediated TAp63gamma stabilization was especially prominent under stress. Accordingly, we found that downregulation of NQO1 inhibits TAp63gamma-dependant p21 upregulation and TAp63gamma-induced growth arrest stimulated by doxorubicin. CONCLUSIONS/SIGNIFICANCE: Our report is the first to identify this new mechanism demonstrating a physical and functional relationship between NQO1 and the most potent p63 isoform, TAp63gamma. These findings appoint a direct role for NQO1 in the regulation of TAp63gamma expression, especially following stress and may therefore have clinical implications for tumor development and therapy

Characterization of genome-wide p53-binding sites upon stress response

The tumor suppressor p53 is a sequence-specific transcription factor, which regulates the expression of target genes involved in different stress responses. To understand p53's essential transcriptional functions, unbiased analysis of its DNA-binding repertoire is pivotal. In a genome-wide tiling ChIP-on-chip approach, we have identified and characterized 1546 binding sites of p53 upon Actinomycin D treatment. Among those binding sites were known as well as novel p53 target sites, which included regulatory regions of potentially novel transcripts. Using this collection of genome-wide binding sites, a new high-confidence algorithm was developed, p53scan, to identify the p53 consensus-binding motif. Strikingly, this motif was present in the majority of all bound sequences with 83% of all binding sites containing the motif. In the surrounding sequences of the binding sites, several motifs for potential regulatory cobinders were identified. Finally, we show that the majority of the genome-wide p53 target sites can also be bound by overexpressed p63 and p73 in vivo, suggesting that they can possibly play an important role at p53 binding sites. This emphasizes the possible interplay of p53 and its family members in the context of target gene binding. Our study greatly expands the known, experimentally validated p53 binding site repertoire and serves as a valuable knowledgebase for future research

Claudin-1 Is a p63 Target Gene with a Crucial Role in Epithelial Development

The epidermis of the skin is a self-renewing, stratified epithelium that functions as the interface between the human body and the outer environment, and acts as a barrier to water loss. Components of intercellular junctions, such as Claudins, are critical to maintain tissue integrity and water retention. p63 is a transcription factor essential for proliferation of stem cells and for stratification in epithelia, mutated in human hereditary syndromes characterized by ectodermal dysplasia. Both p63 and Claudin-1 null mice die within few hours from birth due to dehydration from severe skin abnormalities. These observations suggested the possibility that these two genes might be linked in one regulatory pathway with p63 possibly regulating Claudin-1 expression. Here we show that silencing of ΔNp63 in primary mouse keratinocytes results in a marked down-regulation of Claudin-1 expression (−80%). ΔNp63α binds in vivo to the Claudin-1 promoter and activates both the endogenous Claudin-1 gene and a reporter vector containing a –1.4 Kb promoter fragment of the Claudin-1 gene. Accordingly, Claudin-1 expression was absent in the skin of E15.5 p63 null mice and natural p63 mutant proteins, specifically those found in Ankyloblepharon–Ectodermal dysplasia–Clefting (AEC) patients, were indeed altered in their capacity to regulate Claudin-1 transcription. This correlates with deficient Claudin-1 expression in the epidermis of an AEC patient carrying the I537T p63 mutation. Notably, AEC patients display skin fragility similar to what observed in the epidermis of Claudin-1 and p63 null mice. These findings reinforce the hypothesis that these two genes might be linked in a common regulatory pathway and that Claudin-1 may is an important p63 target gene involved in the pathogenesis of ectodermal dysplasias

Transforming growth factor-β-inducible early response gene 1 is a novel substrate for atypical protein kinase Cs

The protein kinase C (PKC) family of serine/threonine kinases consists of ten different isoforms grouped into three subfamilies, denoted classical, novel and atypical PKCs (aPKCs). The aPKCs, PKCι/λ and PKCζ serve important roles during development and in processes subverted in cancer such as cell and tissue polarity, cell proliferation, differentiation and apoptosis. In an effort to identify novel interaction partners for aPKCs, we performed a yeast two-hybrid screen with the regulatory domain of PKCι/λ as bait and identified the Krüppel-like factors family protein TIEG1 as a putative interaction partner for PKCι/λ. We confirmed the interaction of both aPKCs with TIEG1 in vitro and in cells, and found that both aPKCs phosphorylate the DNA-binding domain of TIEG1 on two critical residues. Interestingly, the aPKC-mediated phosphorylation of TIEG1 affected its DNA-binding activity, subnuclear localization and transactivation potential

Direct comparison of size-dependent versus EpCAM-dependent CTC enrichment at the gene expression and DNA methylation level in head and neck squamous cell carcinoma

We directly compared two different approaches used for Circulating Tumor Cell (CTC) isolation, a size-dependent microfluidic system versus an EpCAM-dependent positive selection for downstream molecular characterization of CTC both at the gene expression and DNA methylation level in Head and Neck Squamous Cell Carcinoma (HNSCC). A size-dependent microfluidic device (Parsortix, ANGLE) and an EpCAM-dependent positive immune-magnetic isolation procedure were applied in parallel, using 10 mL PB from 50 HNSCC patients and 18 healthy donors. Total RNA was isolated from enriched CTCs and RT-qPCR was used to study the expression levels of CK-19, PD-L1, EGFR, TWIST1, CDH2 and B2M (reference gene). Real time methylation specific PCR (MSP) was used to study the methylation status of RASSF1A and MLL3 genes. In identical blood draws, the label-free size-dependent CTC-isolation system was superior in terms of sensitivity when compared to the EpCAM-dependent CTC enrichment, since a significantly higher percentage of identical PB samples was found positive at the gene expression and DNA methylation level, while the specificity was not affected. Our results indicate that future studies focused on the evaluation of clinical utility of CTC molecular characterization in HNSCC should be based on size-dependent enrichment approaches. © 2020, The Author(s)

Screening for TSC1 and TSC2 mutations using NGS in Greek children with tuberous sclerosis syndrome

Tuberous Sclerosis Complex (TSC) is a rare neurocutaneous syndrome inherited by an autosomal dominant manner. The disorder is commonly manifested by the presence of multiple benign tumors located in numerous tissues, including the brain, heart, skin and kidneys. Seizures, autism, developmental and behavioral delay, as well as non-neurological phenotypic findings, are suggestive of TSC. The identification of one pathogenic mutation in either the TSC1 or TSC2 genes is considered to be an independent diagnostic criterion. In our study, seventeen Greek patients, 2yo on average, were analyzed for the presence of pathogenic germline mutations in the aforementioned loci by Next-Generation Sequencing. A TSC1/2 gene panel was designed for the molecular diagnosis of the disease. Patients underwent initial diagnosis based on their clinical symptoms, most frequently involving the presence of skin lesions and/or epilepsy. Only one case was familial. Sixteen different genetic alterations were identified in TSC1 and TSC2 genes in fifteen patients, giving a 88% detection rate by employing NGS technology. Overall, most pathogenic mutations (11/15) identified were located in the TSC2 gene with exon 41 being the most frequent. With respect to genotype-phenotype association, no patient TSC1 (+) developed SEGA or renal cysts. No significant differences were observed between different types of TSC2 mutations and any clinical feature. Sequencing also revealed 18 different SNPs across the TSC1 and 20 across the TSC2 genes. This is the first registry of the genetic profile of TSC patients in Greece using a custom-made gene panel as molecular diagnostic tool. © 2018 European Paediatric Neurology Societ

Screening for TSC1 and TSC2 mutations using NGS in Greek children with tuberous sclerosis syndrome

Tuberous Sclerosis Complex (TSC) is a rare neurocutaneous syndrome inherited by an autosomal dominant manner. The disorder is commonly manifested by the presence of multiple benign tumors located in numerous tissues, including the brain, heart, skin and kidneys. Seizures, autism, developmental and behavioral delay, as well as non-neurological phenotypic findings, are suggestive of TSC. The identification of one pathogenic mutation in either the TSC1 or TSC2 genes is considered to be an independent diagnostic criterion. In our study, seventeen Greek patients, 2yo on average, were analyzed for the presence of pathogenic germline mutations in the aforementioned loci by Next-Generation Sequencing. A TSC1/2 gene panel was designed for the molecular diagnosis of the disease. Patients underwent initial diagnosis based on their clinical symptoms, most frequently involving the presence of skin lesions and/or epilepsy. Only one case was familial. Sixteen different genetic alterations were identified in TSC1 and TSC2 genes in fifteen patients, giving a 88% detection rate by employing NGS technology. Overall, most pathogenic mutations (11/15) identified were located in the TSC2 gene with exon 41 being the most frequent. With respect to genotype-phenotype association, no patient TSC1 (+) developed SEGA or renal cysts. No significant differences were observed between different types of TSC2 mutations and any clinical feature. Sequencing also revealed 18 different SNPs across the TSC1 and 20 across the TSC2 genes. This is the first registry of the genetic profile of TSC patients in Greece using a custom-made gene panel as molecular diagnostic tool. © 2018 European Paediatric Neurology Societ