Activité dominante négative des protéines p53 mutées

La protéine p53 dispose d’une fonction activatrice de l’expression de nombreux gènes cibles. Le rôle de facteur de transcription joué par la protéine p53 nécessite la formation d’une structure homotétramérique. Les résultats de certaines expérimentations montrent que les monomères p53 mutés ont la capacité de se lier à des monomères p53 sauvages pour constituer des complexes hétérotétramériques. La présence de monomères p53 mutés au sein de ces complexes hétérotétramériques peut avoir pour conséquence immédiate une inactivation des monomères sauvages. Cette capacité de liaison et d’inactivation des p53 mutées à l’égard des p53 sauvages est qualifiée d’« effet dominant négatif ». Plusieurs facteurs enrôlés dans cette activité dominante négative ont été identifiés. La compréhension des fonctions moléculaires complexes qui régissent cette activité constitue un des aspects importants qui permettrait de mieux discerner les mécanismes biologiques en jeu dans la cancérogenèse. Le but de cet article est de mettre en lumière des aspects jusqu’à présent occultés de l’activité dominante négative des protéines p53 mutées. De plus, nous allons souligner comment cette activité contribue à la cancérogenèse induite par les rayons ultraviolets.Tumor suppressor gene inactivation as proposed by the Knudson model implies a sequential inactivation of two alleles of a gene. For example, the first allele is inactivated by a missense mutation, and the second one is inactivated by a deletion or insertion. The alteration of the p53 tumor suppressor gene is far to correspond only to this model. In the great majority of cancers, the mutated allele of p53 coexists with the normal allele. It is well known that the transcriptional activity is one of the most important functions of p53. The p53 protein is active as a tetramer (this complex activates the expression of targeted genes by binding to its consensus DNA sequence called the p53 response element). Experimental evidence shows that wild-type p53 interacts with mutant proteins to form heterotetramers. In association with wild-type proteins, mutant proteins drive the wild-type subunits into a mutant conformation. This association leads to a loss of trans-activating function. The capacity of mutant subunits to form heterotetramers with wild-type subunits and to commit them into a mutant conformation is called « dominant negative effect ». Many p53 mutant proteins possess this dominant negative activity. Recently, several factors, which are implicated in the control of the dominant negative activity of p53 mutants, have been identified. The elucidation of these complex molecular functions, which are implicated in the dominant negative activity of the p53 mutated protein represents an important aspect in the comprehension of the biological mechanisms involved in carcinogenesis

Differential binding of the transcription factors Sp1, AP-1, and NFI to the promoter of the human α5 integrin gene dictates its transcriptional activity

Purpose. Damage to the corneal epithelium results in the massive secretion of fibronectin (FN) shortly after injury and induces the expression of its integrin receptor α5β1. The authors reported previously that FN induces α5 expression in human corneal epithelial cells and rabbit corneal epithelial cells by altering the binding of the transcription factor (TF) Sp1 to a regulatory element from the α5 promoter that it is also flanked by binding sites for the TFs NFI and AP-1. Here, they assessed the function of NFI and AP-1 on α5 gene expression and evaluated the contribution of FN to their overall regulatory influence. Methods. TF binding to the α5 promoter was evaluated in vitro by electrophoretic mobility shift assays and in vivo by ligation-mediated PCR or chromatin immunoprecipitation. TFs expression was monitored by Western blot, whereas their influence was assessed by transfection and RNAi analyses. Results. Coexpression of Sp1, NFI, and AP-1 was demonstrated in all cell types, and each TF was shown to bind efficiently to the α5 promoter. Whereas both AP-1 and Sp1 activated expression directed by the α5 promoter, NFI functioned as a potent repressor of that gene. Interestingly, FN could either promote or repress α5 promoter activity in a cell density–dependent manner by differentially altering the ratio of these TFs. Conclusions. These results suggest that α5 gene expression is likely dictated by subtle alterations in the nuclear ratio of TFs that either repress (NFI) or activate (Sp1 and AP-1) α5 transcription in corneal epithelial cells

Efficiency of Manual Scanning in Recovering Rare Cellular Events Identified by Fluorescence In Situ Hybridization: Simulation of the Detection of Fetal Cells in Maternal Blood

Fluorescence in situ hybridization (FISH) and manual scanning is a widely used strategy for retrieving rare cellular events such as fetal cells in maternal blood. In order to determine the efficiency of these techniques in detection of rare cells, slides of XX cells with predefined numbers (1–10) of XY cells were prepared. Following FISH hybridization, the slides were scanned blindly for the presence of XY cells by different observers. The average detection efficiency was 84% (125/148). Evaluation of probe hybridization in the missed events showed that 9% (2/23) were not hybridized, 17% (4/23) were poorly hybridized, while the hybridization was adequate for the remaining 74% (17/23). In conclusion, manual scanning is a relatively efficient method to recover rare cellular events, but about 16% of the events are missed; therefore, the number of fetal cells per unit volume of maternal blood has probably been underestimated when using manual scanning

Formation of stress-specific p53 binding patterns is influenced by chromatin but not by modulation of p53 binding affinity to response elements†

The p53 protein is crucial for adapting programs of gene expression in response to stress. Recently, we revealed that this occurs partly through the formation of stress-specific p53 binding patterns. However, the mechanisms that generate these binding patterns remain largely unknown. It is not established whether the selective binding of p53 is achieved through modulation of its binding affinity to certain response elements (REs) or via a chromatin-dependent mechanism. To shed light on this issue, we used a microsphere assay for protein–DNA binding to measure p53 binding patterns on naked DNA. In parallel, we measured p53 binding patterns within chromatin using chromatin immunoprecipitation and DNase I coupled to ligation-mediated polymerase chain reaction footprinting. Through this experimental approach, we revealed that UVB and Nutlin-3 doses, which lead to different cellular outcomes, induce similar p53 binding patterns on naked DNA. Conversely, the same treatments lead to stress-specific p53 binding patterns on chromatin. We show further that altering chromatin remodeling using an histone acetyltransferase inhibitor reduces p53 binding to REs. Altogether, our results reveal that the formation of p53 binding patterns is not due to the modulation of sequence-specific p53 binding affinity. Rather, we propose that chromatin and chromatin remodeling are required in this process

Disruption of AP1S1, Causing a Novel Neurocutaneous Syndrome, Perturbs Development of the Skin and Spinal Cord

Adaptor protein (AP) complexes regulate clathrin-coated vesicle assembly, protein cargo sorting, and vesicular trafficking between organelles in eukaryotic cells. Because disruption of the various subunits of the AP complexes is embryonic lethal in the majority of cases, characterization of their function in vivo is still lacking. Here, we describe the first mutation in the human AP1S1 gene, encoding the small subunit σ1A of the AP-1 complex. This founder splice mutation, which leads to a premature stop codon, was found in four families with a unique syndrome characterized by mental retardation, enteropathy, deafness, peripheral neuropathy, ichthyosis, and keratodermia (MEDNIK). To validate the pathogenic effect of the mutation, we knocked down Ap1s1 expression in zebrafish using selective antisens morpholino oligonucleotides (AMO). The knockdown phenotype consisted of perturbation in skin formation, reduced pigmentation, and severe motility deficits due to impaired neural network development. Both neural and skin defects were rescued by co-injection of AMO with wild-type (WT) human AP1S1 mRNA, but not by co-injecting the truncated form of AP1S1, consistent with a loss-of-function effect of this mutation. Together, these results confirm AP1S1 as the gene responsible for MEDNIK syndrome and demonstrate a critical role of AP1S1 in development of the skin and spinal cord

Comprendre la mutagenèse somatique grâce à la cartographie des dommages à l’ADN

La théorie de la mutagenèse somatique indique que la transformation maligne des cellules est intimement liée à la formation de dommages à l’ADN et au risque que ces derniers puissent mener à des mutations favorables à la croissance cellulaire anarchique. Elle énonce le lien logique qui associe le cancer à l’exposition à des agents génotoxiques. Les spectres de mutations, notamment dans le gène p53, recensent les mutations dans un type de cancer donné en fonction de leur position nucléotidique. Les positions les plus fréquemment mutées sont différentes selon les cancers et reflètent donc la signature du ou des agents mutagènes qui en sont à l’origine. La technologie LMPCR (ligation-mediated PCR) permet de cartographier au niveau nucléotidique les sites les plus fréquemment endommagés par un agent génotoxique donné. Ainsi, on a la possibilité de chercher une corrélation entre ces sites fréquemment endommagés et les sites fréquemment mutés dans un type de cancer. Cela a été notamment appliqué pour confirmer à l’échelle moléculaire le lien étiologique reliant une exposition aux UVB au cancer de la peau, ainsi que celui qui s’établit entre plusieurs substances de la fumée de tabac et le cancer du poumon