Rôle moléculaire de RPAP3 et fonction dans la physiologie de l'intestin

Many substrates of HSP90 are involved in signal transduction pathways and related to tumour progression. Inhibition of HSP90 has anti-tumoral effects. Identification of the R2TP, a new HSP90 co-chaperon, allowed the identification of a new set of HSP90 substrates. HSP90/R2TP is involved in the assembly of snoRNPs, telomerase RNP, the nuclear RNA polymerases and PIKKs, which play key functions in cellular proliferation and tumorigenesis. R2TP is formed of four proteins: RUVBL1, RUVBL2, PIH1D1 and RPAP3, some of which are overexpressed in hepatocellular and colorectal cancer. We thus hypothesize that the co-chaperone R2TP could be involved in colorectal carcinogenesis.To study the role of R2TP in intestinal homeostasis and carcinogenesis, we generated a conditional knock-out murine model for RPAP3. We showed that RPAP3 invalidation in whole organism or only in colon is lethal at embryonic stage. The invalidation of RPAP3 in adult intestine, using an inducible recombinase (RPAP3 fl; Villin>Cre-ERT2), leads to a drastic phenotype as soon as 8 days post-induction, resulting in death after 10 days. This phenotype is reminiscent of proliferative defects.In parallel, we address the possibility of a therapeutic window to target RPAP3 during intestinal tumorigenesis by using heterozygous animals (RPAP3 fl/+; Villin>Cre-ERT2 ) in which tumorignesis is induced (i) either by a chemical treatment : for this, we take advantage of the established AOM /DSS protocol, or, (ii) by a genetic one ( Apc LoxP/+).These ongoing experiments will address the role of R2TP in a tissue with a constant turnover and the relevance of R2TP in tumorigenesis.La protéine chaperon HSP90 a de nombreux substrats par les voies de signalisations. Son inhibition engendre un effet tumoral. L'identification du complexe R2TP,un nouveau cochaperon d'HSP90, a mis en évidence de nouveaux substrats. Le système HSP90/R2TP est impliqué dans l'assemblage de complexes macromoléculaires ( PIKKs, Télomérase RNP, ARN polyméras, snoRNP) jouant un rôle clé dans la prolifération cellulaire et la tumorigénèse. Le R2TP est composé de 4 protéines : RUVBL1, RUVBL2, RPAP3 et PIH1D1 dont certains sont surexprimés dans les cancers hépatocellulaires ou colorectaux. Afin d'étudier le rôle du R2TP dans l'homéostasie intestinale et la carcinogénèse, nous avons généré un modèle d'invalidation de RPAP3 chez la souris. L'invalidation de RPAP3 dans l'organisme entier est létale. Son invalidation à l'âge adulte uniquement dans l'intestin entraine une phénotype sévère en 8jours aboutissant à la mort des individus à 10jours. En parallèle, nous avons étudié le rôle potentiel de RPAP3 dans la tumorigénèse intestinale et colorectale avec deux modèles d'induction , génétique avec l'invalidation du gène APC et chimique avec le protocole AOM/DSS

Molecular role of RPAP3 and function in intestine physiology

La protéine chaperon HSP90 a de nombreux substrats par les voies de signalisations. Son inhibition engendre un effet tumoral. L'identification du complexe R2TP,un nouveau cochaperon d'HSP90, a mis en évidence de nouveaux substrats. Le système HSP90/R2TP est impliqué dans l'assemblage de complexes macromoléculaires ( PIKKs, Télomérase RNP, ARN polyméras, snoRNP) jouant un rôle clé dans la prolifération cellulaire et la tumorigénèse. Le R2TP est composé de 4 protéines : RUVBL1, RUVBL2, RPAP3 et PIH1D1 dont certains sont surexprimés dans les cancers hépatocellulaires ou colorectaux. Afin d'étudier le rôle du R2TP dans l'homéostasie intestinale et la carcinogénèse, nous avons généré un modèle d'invalidation de RPAP3 chez la souris. L'invalidation de RPAP3 dans l'organisme entier est létale. Son invalidation à l'âge adulte uniquement dans l'intestin entraine une phénotype sévère en 8jours aboutissant à la mort des individus à 10jours. En parallèle, nous avons étudié le rôle potentiel de RPAP3 dans la tumorigénèse intestinale et colorectale avec deux modèles d'induction , génétique avec l'invalidation du gène APC et chimique avec le protocole AOM/DSS.Many substrates of HSP90 are involved in signal transduction pathways and related to tumour progression. Inhibition of HSP90 has anti-tumoral effects. Identification of the R2TP, a new HSP90 co-chaperon, allowed the identification of a new set of HSP90 substrates. HSP90/R2TP is involved in the assembly of snoRNPs, telomerase RNP, the nuclear RNA polymerases and PIKKs, which play key functions in cellular proliferation and tumorigenesis. R2TP is formed of four proteins: RUVBL1, RUVBL2, PIH1D1 and RPAP3, some of which are overexpressed in hepatocellular and colorectal cancer. We thus hypothesize that the co-chaperone R2TP could be involved in colorectal carcinogenesis.To study the role of R2TP in intestinal homeostasis and carcinogenesis, we generated a conditional knock-out murine model for RPAP3. We showed that RPAP3 invalidation in whole organism or only in colon is lethal at embryonic stage. The invalidation of RPAP3 in adult intestine, using an inducible recombinase (RPAP3 fl; Villin>Cre-ERT2), leads to a drastic phenotype as soon as 8 days post-induction, resulting in death after 10 days. This phenotype is reminiscent of proliferative defects.In parallel, we address the possibility of a therapeutic window to target RPAP3 during intestinal tumorigenesis by using heterozygous animals (RPAP3 fl/+; Villin>Cre-ERT2 ) in which tumorignesis is induced (i) either by a chemical treatment : for this, we take advantage of the established AOM /DSS protocol, or, (ii) by a genetic one ( Apc LoxP/+).These ongoing experiments will address the role of R2TP in a tissue with a constant turnover and the relevance of R2TP in tumorigenesis

The RPAP3 C-terminal domain identifies R2TP-like quaternary chaperones

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In Vitro Corticogenesis from Embryonic Stem Cells Recapitulates the In Vivo Epigenetic Control of Imprinted Gene Expression

International audienceIn vitro corticogenesis from embryonic stem cells (ESCs) is an attractive model of cortical development and a promising tool for corticaltherapy. It is unknown to which extent epigenetic mechanisms crucial for cortex development and function, such as parental genomicimprinting, are recapitulated by in vitro corticogenesis. Here, using genome-wide transcriptomic and methylation analyses on hybridmouse tissues and cells, wefind a high concordance of imprinting status between invivo and ESC-derived cortices. Notably, in vitrocorticogenesis strictly reproduced the in vivo parent-of-origin-dependent expression of 41 imprinted genes (IGs), including Mest and Cdkn1c known to control corticogenesis. Parent-of-origin-dependent DNA methylation was also conserved at 14 of 18 imprinted differentially methylated regions. The least concordant imprinted locus was Gpr1-Zdbf2, where the aberrant bi-allelic expression of Zdbf2 and Adam23 was concomitant with a gain of methylation on the maternal allele in vitro. Combined, our data argue for a broad conservation of the epigenetic mechanisms at imprinted loci in cortical cells derived from ESCs. We propose that in vitro corticogenesis helps to define the still poorly understood mechanisms that regulate imprinting in the brain and the roles of IGs in cortical developmen

The HSP90/R2TP assembly chaperone promotes cell proliferation in the intestinal epithelium

International audienceThe R2TP chaperone cooperates with HSP90 to integrate newly synthesized proteins into multi-subunit complexes, yet its role in tissue homeostasis is unknown. Here, we generated conditional, inducible knock-out mice for Rpap3 to inactivate this core component of R2TP in the intestinal epithelium. In adult mice, Rpap3 invalidation caused destruction of the small intestinal epithelium and death within 10 days. Levels of R2TP substrates decreased, with strong effects on mTOR, ATM and ATR. Proliferative stem cells and progenitors deficient for Rpap3 failed to import RNA polymerase II into the nucleus and they induced p53, cell cycle arrest and apoptosis. Post-mitotic, differentiated cells did not display these alterations, suggesting that R2TP clients are preferentially built in actively proliferating cells. In addition, high RPAP3 levels in colorectal tumors from patients correlate with bad prognosis. Here, we show that, in the intestine, the R2TP chaperone plays essential roles in normal and tumoral proliferation

Deep Structural Analysis of RPAP3 and PIH1D1, Two Components of the HSP90 Co-chaperone R2TP Complex

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Loss of cyclin A2 in murine colonic epithelial cells disrupts colon homeostasis by triggering DNA damage and dysplasia and high cyclin A2 expression is a good-prognosis factor in patients with colorectal cancer

Abstract To clarify the function of cyclin A2 in colon homeostasis and colorectal cancer (CRC) we generated mice deficient for cyclin A2 in colonic epithelial cells (CEC). Colons of those mice displayed architectural changes in the mucosa, and signs of inflammation as well as an increased proliferation of CEC associated with the appearance of low- and high-grade dysplasia. The main initial events triggering those alterations in cyclin A2 deficient CEC appear to be abnormal mitoses and DNA damage. Cyclin A2 deletion in CEC promoted the development of dysplasia and adenocarcinomas in the murine colitis-associated cancer model. We next explored the status of cyclin A2 expression in clinical CRC samples at the mRNA and protein level and found higher expression in tumors of stage I and II patients compared to those of stage III and IV. A meta-analysis of 11 transcriptome datasets comprising 2,239 primary CRC tumors displayed different CCNA2 (the mRNA coding for cyclin A2) expression levels among the CRC tumor subtypes with highest in CMS1 and lowest in CMS4. Moreover, high expression of CCNA2 was found to be a good prognosis factor independently from other prognostic factors for the CMS1, CMS3 and CMS4 subtypes