The baroclinic secondary instability of the two-dimensional shear layer

The focus of this study is on the numerical investigation of two-dimensional, isovolume, high Reynolds and Froude numbers, variable-density mixing layers. Lagrangian simulations, of both the temporal and the spatial models, are performed. They reveal the breaking-up of the strained vorticity and density-gradient braids, connecting two neighboring primary structures. The secondary instability arises where the vorticity has been intensified by the baroclinic torque. A simplified model of the braid of the variable-density mixing layer, consisting of a strained vorticity and density-gradient filament, is analyzed. It is concluded that the physical mechanism responsible for the secondary instability is the forcing of the vorticity field by the baroclinic torque, itself sensitive to perturbations. This mechanism suggests a rapid route to turbulence for the variable-density mixing layer

TAp73β and DNp73β activate the expression of the pro-survival caspase-2S

p73, the p53 homologue, exists as a transactivation-domain-proficient TAp73 or deficient deltaN(DN)p73 form. Expectedly, the oncogenic DNp73 that is capable of inactivating both TAp73 and p53 function, is over-expressed in cancers. However, the role of TAp73, which exhibits tumour-suppressive properties in gain or loss of function models, in human cancers where it is hyper-expressed is unclear. We demonstrate here that both TAp73 and DNp73 are able to specifically transactivate the expression of the anti-apoptotic member of the caspase family, caspase-2S. Neither p53 nor TAp63 has this property, and only the p73β form, but not the p73α form, has this competency. Caspase-2 promoter analysis revealed that a non-canonical, 18 bp GC-rich Sp-1-binding site-containing region is essential for p73β-mediated activation. However, mutating the Sp-1-binding site or silencing Sp-1 expression did not affect p73β's transactivation ability. In vitro DNA binding and in vivo chromatin immunoprecipitation assays indicated that p73β is capable of directly binding to this region, and consistently, DNA binding p73 mutant was unable to transactivate caspase-2S. Finally, DNp73β over-expression in neuroblastoma cells led to resistance to cell death, and concomitantly to elevated levels of caspase-2S. Silencing p73 expression in these cells led to reduction of caspase-2S expression and increased cell death. Together, the data identifies caspase-2S as a novel transcriptional target common to both TAp73 and DNp73, and raises the possibility that TAp73 may be over-expressed in cancers to promote survival

A dedicated microarray for in-depth analysis of pre-mRNA splicing events: application to the study of genes involved in the response to targeted anticancer therapies.

International audience: Alternative pre-mRNA splicing (AS) widely expands proteome diversity through the combinatorial assembly of exons. The analysis of AS on a large scale, by using splice-sensitive microarrays, is a highly efficient method to detect the majority of known and predicted alternative transcripts for a given gene. The response to targeted anticancer therapies cannot easily be anticipated without prior knowledge of the expression, by the tumor, of target proteins or genes. To analyze, in depth, transcript structure and levels for genes involved in these responses, including AKT1-3, HER1-4, HIF1A, PIK3CA, PIK3R1-2, VEGFA-D and PIR, we engineered a dedicated gene chip with coverage of an average 185 probes per gene and, especially, exon-exon junction probes. As a proof of concept, we demonstrated the ability of such a chip to detect the effects of over-expressed SRSF2 RNA binding protein on the structure and abundance of mRNA products in H538 lung cancer cells conditionally over-expressing SRSF2. Major splicing changes were observed, including in HER1/EGFR pre-mRNA, which were also seen in human lung cancer samples over-expressing the SRSF2 protein. In addition, we showed that variations in HER1/EGFR pre-mRNA splicing triggered by SRSF2 overexpression in H358 cells resulted in a drop in HER1/EGFR protein level, which correlated with increased sensitivity to gefitinib, an EGFR tyrosine kinase inhibitor. We propose, therefore, that this novel tool could be especially relevant for clinical applications, with the aim to predict the response before treatment

CELF proteins regulate CFTR pre-mRNA splicing: essential role of the divergent domain of ETR-3

Cystic fibrosis is a prominent genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Among the many disease-causing alterations are pre-mRNA splicing defects that can hamper mandatory exon inclusion. CFTR exon 9 splicing depends in part on a polymorphic UG(m)U(n) sequence at the end of intron 8, which can be bound by TDP-43, leading to partial exon 9 skipping. CELF proteins, like CUG-BP1 and ETR-3, can also bind UG repeats and regulate splicing. We show here that ETR-3, but not CUG-BP1, strongly stimulates exon 9 skipping, although both proteins bind efficiently to the same RNA motif as TDP-43 and with higher affinity. We further show that the skipping of this exon may be due to the functional antagonism between U2AF65 and ETR-3 binding onto the polymorphic U or UG stretch, respectively. Importantly, we demonstrate that the divergent domain of ETR-3 is critical for CFTR exon 9 skipping, as shown by deletion and domain-swapping experiments. We propose a model whereby several RNA-binding events account for the complex regulation of CFTR exon 9 inclusion, with strikingly distinct activities of ETR-3 and CUG-BP1, related to the structure of their divergent domain

ETV6-RUNX1 and RUNX1 directly regulate RAG1 expression: one more step in the understanding of childhood B-cell acute lymphoblastic leukemia leukemogenesis.

Funder: Société Française de Biochimie et Biologie Moléculaire ; French Research MinistryFunder: Cancéropole Grand Ouest ; Région Bretagne ; Société Française d’HématologieFunder: Ligue Régionale contre le cancer ;ETV6-RUNX1 and RUNX1 directly promote RAG1 expression. ETV6-RUNX1 and RUNX1 preferentially bind to the −1200 bp enhancer of RAG1 and the −80 bp promoter of RAG1 gene respectively, and compete for these bindings. ETV6-RUNX1 and RUNX1 induce an excessive RAG recombinase activity. ETV6-RUNX1 participates directly in two events of the multi-hit ALL leukemogenesis: as an initiating event and as an activator of RAG1 expression

Reduction of RUNX1 transcription factor activity by a CBFA2T3-mimicking peptide: application to B cell precursor acute lymphoblastic leukemia.

Funder: FP7 People: Marie-Curie Actions; doi: http://dx.doi.org/10.13039/100011264; Grant(s): 291851BACKGROUND: B Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) is the most common pediatric cancer. Identifying key players involved in proliferation of BCP-ALL cells is crucial to propose new therapeutic targets. Runt Related Transcription Factor 1 (RUNX1) and Core-Binding Factor Runt Domain Alpha Subunit 2 Translocated To 3 (CBFA2T3, ETO2, MTG16) are master regulators of hematopoiesis and are implicated in leukemia. METHODS: We worked with BCP-ALL mononuclear bone marrow patients' cells and BCP-ALL cell lines, and performed Chromatin Immunoprecipitations followed by Sequencing (ChIP-Seq), co-immunoprecipitations (co-IP), proximity ligation assays (PLA), luciferase reporter assays and mouse xenograft models. RESULTS: We demonstrated that CBFA2T3 transcript levels correlate with RUNX1 expression in the pediatric t(12;21) ETV6-RUNX1 BCP-ALL. By ChIP-Seq in BCP-ALL patients' cells and cell lines, we found that RUNX1 is recruited on its promoter and on an enhancer of CBFA2T3 located - 2 kb upstream CBFA2T3 promoter and that, subsequently, the transcription factor RUNX1 drives both RUNX1 and CBFA2T3 expression. We demonstrated that, mechanistically, RUNX1 and CBFA2T3 can be part of the same complex allowing CBFA2T3 to strongly potentiate the activity of the transcription factor RUNX1. Finally, we characterized a CBFA2T3-mimicking peptide that inhibits the interaction between RUNX1 and CBFA2T3, abrogating the activity of this transcription complex and reducing BCP-ALL lymphoblast proliferation. CONCLUSIONS: Altogether, our findings reveal a novel and important activation loop between the transcription regulator CBFA2T3 and the transcription factor RUNX1 that promotes BCP-ALL proliferation, supporting the development of an innovative therapeutic approach based on the NHR2 subdomain of CBFA2T3 protein

Etude de la regulation de l'expression de genes codant pour des cytochromes P450 dans les cellules d'hepatome de rat

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Les récepteurs nucléaires CAR et PXR contrôlent l’induction des cytochromes P450 par le phénobarbital

Les enzymes du métabolisme des xénobiotiques permettent l’assimilation d’une quasi-infinie variété de molécules synthétiques ou naturelles avec lesquelles l’organisme n’a pas été confronté a priori. Ces enzymes sont synthétisées dans la grande majorité des tissus, mais elles sont plus diversifiées et abondantes dans le foie, où elles sont sujettes à de nombreuses régulations. L’induction est une réponse adaptative rapide permettant d’augmenter transitoirement la capacité cellulaire et tissulaire d’élimination du xénobiotique. A contrario, l’inhibition peut conduire à en augmenter la demi-vie biologique. Une nouvelle ère de la pharmacologie moléculaire s’ouvre actuellement grâce à la mise en évidence récente des récepteurs nucléaires CAR et PXR. Ces récepteurs orphelins sont les médiateurs de l’activation transcriptionnelle de gènes codant pour des enzymes de biotransformation hépatique sous l’effet du phénobarbital, un médicament anti-épileptique aux propriétés inductrices connues de très longue date. Ces récepteurs coordonnent l’assimilation des xéno-biotiques et constituent des maillons essentiels de voies de signalisation spécialisées