Search

BackgroundThe MYC oncoprotein, also known as the master regulator of genes, is a transcription factor that regulates numerous physiological processes, including cell cycle control, apoptosis, protein synthesis and cell adhesion, among others. MYC is overexpressed in approximately 70% of human cancers. Given its pervasive role in cancer biology, MYC down-regulation has become an attractive cancer treatment strategy. MethodsThe CRISPR/Cas9 method was used to produce KO cell models. Western blot was used to analyzed the expressions of MYC and TATA-binding proteinassociated factors 10 (TAF10) in cancer cells (MCF7, A549, HepG2 cells) Cell culture studies were performed to determine the mechanisms by which small molecules (Z363119456, Z363) affects MYC and TAF10 expressions and functions. Mouse studies were carried out to investigate the impact of Z363 regulation on tumor growth. ResultsZ363 activate Thyroid hormone Receptor-interacting Protein 12 (TRIP12), which phosphorylates MYC at Thr58, resulting in MYC ubiquitination and degradation and thereby regulating MYC target genes. Importantly, TRIP12 also induces TAF10 degradation, which reduces MYC protein levels. TRIP12, an E3 ligase, controls MYC levels both directly and indirectly by inhibiting MYC or TAF10 activity. ConclusionsIn summary,these results demonstrate the anti-cancer properties of Z363, a small molecule that is co-regulated by TAF10 and MYC. Funding Agencies|National Natural Science Foundation of China [82000020, 81970008]; Fundamental Research Funds for the Central Universities [2021CDJZYJH-002, 2019CDYGZD009, 2020CDJYGRH-1005]; Natural Science Foundation of Chongqing, China [cstc2020jcyj-msxmX0460]; Chongqing Talents: Exceptional Young Talents Project [cstc2021ycjh-bgzxm0099]; Graduate Research and Innovation Foundation of Chongqing, China [CYB21046]

The poor prognosis of pancreatic cancer (PC) is explained by a late diagnosis and lack of efficient treatments. A better understanding of the molecular mechanisms that govern its initiation, progression and resistance to most chemotherapies is essential to improve the patient care. TRIP12 (Thyroid hormone Receptor Interacting Protein 12) is an HECT (Homologous to the E6-AP Carboxyl Terminus) type E3 ubiquitin ligase. TRIP12 regulates cell cycle and chromosome stability, it is involved in chromatin remodeling, DNA damage response and activation of P53 pathway. TRIP12 mRNA is overexpressed in pancreatic cancer. Furthermore, TRIP12 is involved in PTF1a (Pancreas Transcription Factor 1a) degradation, a transcription factor essential for the development and homeostasis of the pancreatic acinar cells. PTF1a is a tumor suppressor that is lost during pancreatic carcinogenesis suggesting a role of TRIP12 in PC. My first thesis goal was to determine why TRIP12 expression increases during PC and what are the consequences of its increased expression. I also hypothesized that TRIP12 plays a role in the PC initiation. My second thesis goal was to determine how TRIP12 contributes to the acinar cells reprogramming into ductal cells, the first step in the preneoplastic lesions formation. I demonstrated that TRIP12 expression is necessary for pancreatic tumor growth and its expression is variable within tumors and patients derived-cell lines. I showed that TRIP12 expression is heterogeneous in these cell lines and is explained 1) - by different mRNA levels linked to the activity of the TRIP12 promoter and 2) - by a different TRIP12 protein regulation during the cell cycle involving the deubiquitinase USP7 and a defect in TRIP12 protein nuclear export. Pancreatic cell lines with a high level of TRIP12 had a better response to anticancerous agents highlighting the role of TRIP12 in chemosensitivity. Moreover, results obtained from a novel mouse model showed that TRIP12 conditional invalidation in the pancreas during embryogenesis does not alter the pancreas development and function in adulthood. However, TRIP12 is essential for PC initiation. Indeed, TRIP12 loss in acinar cells prevents acino-ductal metaplasia. TRIP12 is also important for cancer progression since its absence reduces tumor development mediated by Kras mutation and loss of P53. My thesis researches demonstrated that TRIP12 is a decisive player in PC and explained the TRIP12 heterogeneity of expression observed in pancreatic tumors. This new knowledge could offer new treatment opportunities for PC where the need is huge. TRIP12 is a candidate protein of high importance which may contribute to innovative therapeutic strategies., Le mauvais pronostic du cancer du pancréas (CP) peut s'expliquer par un diagnostic tardif et le manque de traitements efficaces. Une meilleure compréhension des mécanismes moléculaires qui régissent l'initiation, la progression et la résistance aux chimiothérapies est indispensable pour améliorer la prise en charge des patients. TRIP12 (Thyroid hormone Receptor Interacting Protein 12) est une E3 ubiquitine ligase de la famille HECT (Homologous to the E6-AP Carboxyl Terminus). TRIP12 régule le cycle cellulaire et la stabilité chromosomique, elle intervient dans le remodelage de la chromatine, dans la réponse aux dommages à l'ADN et dans l'activation de la voie P53. L'ARNm de TRIP12 est surexprimé dans le CP. De plus, TRIP12 est impliquée dans la dégradation de PTF1a (Pancreas Transcription Factor 1a), facteur de transcription essentiel du développement et de l'homéostasie des cellules acinaires du pancréas, suppresseur de tumeur dont l'expression est perdue au cours de la carcinogénèse pancréatique. Ces données suggèrent un rôle de TRIP12 dans le CP. Mon premier objectif de thèse était de déterminer pourquoi l'expression de TRIP12 augmente dans le CP et quelles en sont les conséquences. J'ai également émis l'hypothèse que TRIP12 joue un rôle dès l'initiation du CP. Mon second objectif de thèse était de déterminer si et comment TRIP12 contribue à la reprogrammation des cellules acinaires en cellules canalaires, première étape de la formation de lésions prénéoplasiques. J'ai découvert que TRIP12 est nécessaire à la croissance des tumeurs pancréatiques et que son expression est variable dans les tumeurs et les lignées cellulaires dérivées de CP. J'ai démontré que l'hétérogénéité d'expression de TRIP12 dans ces lignées cellulaires s'explique 1)- par des niveaux d'ARNm différents liés à l'activité du promoteur du gène Trip12 et 2)- par une régulation différente de la protéine TRIP12 au cours du cycle cellulaire mettant en jeu la déubiquitinase USP7 et un défaut de l'export nucléaire de TRIP12. Les lignées pancréatiques ayant un taux élevé de TRIP12 répondent mieux aux agents anticancéreux mettant en évidence le rôle de TRIP12 dans la chimiosensibilité des cellules cancéreuses pancréatiques. Par ailleurs, les résultats obtenus à partir d'un nouveau modèle murin montrent que l'invalidation conditionnelle de TRIP12 dans le pancréas au cours de l'embryogenèse n'altère pas le développement et la fonction du pancréas à l'âge adulte. Cependant, TRIP12 est capitale pour l'initiation du CP. En effet, la perte de TRIP12 dans les cellules acinaires empêche la métaplasie acino-canalaire. TRIP12 est aussi importante pour la progression du CP car son absence ralentit le développement des tumeurs engendrées par la mutation du gène Kras et la perte du gène P53. L'ensemble de mes travaux de thèse montre que TRIP12 est un acteur décisif du CP et explique l'hétérogénéité d'expression de TRIP12 observée dans les tumeurs pancréatiques. Ces nouvelles connaissances pourraient offrir de nouvelles options thérapeutiques pour le CP où le besoin est énorme. TRIP12 est une protéine candidate de haute importance qui peut contribuer aux stratégies de thérapie par des médicaments innovants.

Simple Summary Over the past two decades, there have been many published studies reporting high-copy SB transgenic lines in which the transposon allele includes both gene-trap and internal promoter elements to drive tumorigenesis in vivo. Cancer gene discovery from end-stage solid tumors in these studies performed by our labs and others has found few proto-oncogenic insertions. However, the question remains whether these tumors are initiated by SB insertions in proto-oncogenes to promote permissible phenotypes for tumor initiation, which become dispensable for tumor maintenance. Here, our study expands upon these indirect observations to demonstrate that high-copy SB transposon alleles designed with only gene-trap elements that inactivate genes (Onc2.3) can drive tumor initiation, progression, and maintenance to end-stage tumors in the absence of sensitizing mutations. Abstract A central challenge in cancer genomics is the systematic identification of single and cooperating tumor suppressor gene mutations driving cellular transformation and tumor progression in the absence of oncogenic driver mutation(s). Multiple in vitro and in vivo gene inactivation screens have enhanced our understanding of the tumor suppressor gene landscape in various cancers. However, these studies are limited to single or combination gene effects, specific organs, or require sensitizing mutations. In this study, we developed and utilized a Sleeping Beauty transposon mutagenesis system that functions only as a gene trap to exclusively inactivate tumor suppressor genes. Using whole body transposon mobilization in wild type mice, we observed that cumulative gene inactivation can drive tumorigenesis of solid cancers. We provide a quantitative landscape of the tumor suppressor genes inactivated in these cancers and show that, despite the absence of oncogenic drivers, these genes converge on key biological pathways and processes associated with cancer hallmarks.

Traballo Fin de Graorn Veterinaria. Curso 2020-2021 Los trastornos del neurodesarrollo son un grupo de trastornos que se manifiestan en períodos tempranos del desarrollo. Entre ellos se encuentran la discapacidad intelectual y los trastornos del espectro autista, que se solapan con gran frecuencia y en cuya etiología influyen factores genéticos y ambientales. Los análisis realizados para descubrir genes involucrados en la discapacidad intelectual han señalado la implicación de TRIP12, el cual también puede provocar autismo. Para investigar la implicación de TRIP12 en este tipo de trastornos, se realizó un bloqueo transitorio del gen mediante morfolinos en pez cebra, analizando el fenotipo morfológico resultante y la locomoción, y se realizó una validación de genes housekeeping así como del bloqueo transitorio a través de análisis de expresión génica por qPCR. Asimismo, se caracterizaron genotípica y fenotípicamente individuos de una línea mutante en pez cebra de trip12 generada mediante el sistema CRISPR/Cas9. Los resultados del análisis fenotípico de los individuos con bloqueo transitorio del gen revelan diferencias significativas (p < 0,05) en la longitud del cuerpo, el diámetro de la cabeza, la distancia del otolito al ojo y el área del ojo, siendo todas las medidas menores en individuos wild type. En relación al análisis fenotípico de los individuos mutantes, existe un aumento estadísticamente significativo respecto a los individuos wild type en todas las medidas (p < 0,05), a excepción del diámetro de la cabeza, que no presenta diferencias en ningún grupo. En cuanto al análisis de locomoción, se detectaron diferencias significativas (p < 0,05), presentando los individuos morfantes un menor movimiento que los wild type tanto en condiciones de luz como de oscuridad.

The Thyroid hormone Receptor Interacting Protein 12 (TRIP12) protein belongs to the 28-member Homologous to the E6-AP C-Terminus (HECT) E3 ubiquitin ligase family. First described as an interactor of the thyroid hormone receptor, TRIP12’s biological importance was revealed by the embryonic lethality of a murine model bearing an inactivating mutation in the TRIP12 gene. Further studies showed the participation of TRIP12 in the regulation of major biological processes such as cell cycle progression, DNA damage repair, chromatin remodeling, and cell differentiation by an ubiquitination-mediated degradation of key protein substrates. Moreover, alterations of TRIP12 expression have been reported in cancers that can serve as predictive markers of therapeutic response. The TRIP12 gene is also referenced as a causative gene associated to intellectual disorders such as Clark–Baraitser syndrome and is clearly implicated in Autism Spectrum Disorder. The aim of the review is to provide an exhaustive and integrated overview of the different aspects of TRIP12 ranging from its regulation, molecular functions and physio-pathological implications.

We combined de novo mutation (DNM) data from 10,927 individuals with developmental delay and autism to identify 253 candidate neurodevelopmental disease genes with an excess of missense and/or likely gene-disruptive (LGD) mutations. Of these genes, 124 reach exome-wide significance (P

Summary Adenine N6 methylation in DNA (6mA) is widespread among bacteria and phage and is detected in mammalian genomes, where its function is largely unexplored. Here we show that 6mA deposition and removal are catalyzed by the Mettl4 methyltransferase and Alkbh4 dioxygenase, respectively, and that 6mA accumulation in genic elements corresponds with transcriptional silencing. Inactivation of murine Mettl4 depletes 6mA and causes sublethality and craniofacial dysmorphism in incross progeny. We identify distinct 6mA sensor domains of prokaryotic origin within the MPND deubiquitinase and ASXL1, a component of the Polycomb repressive deubiquitinase (PR-DUB) complex, both of which act to remove monoubiquitin from histone H2A (H2A-K119Ub), a repressive mark. Deposition of 6mA by Mettl4 triggers the proteolytic destruction of both sensor proteins, preserving genome-wide H2A-K119Ub levels. Expression of the bacterial 6mA methyltransferase Dam, in contrast, fails to destroy either sensor. These findings uncover a native, adversarial 6mA network architecture that preserves Polycomb silencing., Graphical Abstract, Highlights • 6mA deposition and erasure by mammalian Mettl4 and Alkbh4, respectively • Mettl4-deficient mice display craniofacial dysmorphism • 6mA triggers proteolysis of its cognate sensor proteins ASXL1 and MPND • Adversarial 6mA network architecture preserves Polycomb silencing, Kweon et al. reveal the molecular infrastructure of a mammalian DNA N6-methyladenine (6mA) methylation, erasure, and sensing system. Deposition of 6mA by the Mettl4 methyltransferase triggers the proteolytic destruction of its cognate sensor proteins, the histone H2A deubiquitinases ASXL1 and MPND, thus preserving Polycomb gene silencing.

TRIP12 is an E3 ubiquitin ligase that belongs to the HECT (Homologous to the E6-AP Carboxyl Terminus) family. Several proteins are targeted by TRIP12 polyubiquitination which triggers their proteasomal degradation. Among its targets, several proteins are involved in DNA damage responses, chromatin remodelling and p53 pathway activation. Unpublished results of my team showed an increased expression of TRIP12 in pancreatic cancer and pre-neoplastic lesions. My group revealed that TRIP12 polyubiquitinates and provokes the degradation of the transcription factor PTF1a (Pancreas Transcription Factor 1a) stability. Describing for the first time a post-translational regulation of PTF1a. PTF1a is essential in pancreatic development and homeostasis. It inhibits the proliferation of pancreatic cells and is considered as a tumour suppressor gene and. Even if several TRIP12 targets are involved in cellular processes that are tightly cell cycle regulated, the regulation of TRIP12 expression and its functions during the cell cycle was unknown at the beginning of my thesis. I showed that TRIP12 expression and nuclear localization are regulated throughout the cell cycle. I identified an intrinsically disordered domain within the N-terminal region of TRIP12 that permits its interaction to euchromatin. I demonstrated TRIP12 implication in mitosis entry by controlling DNA replication timing Independently of its catalytic activity. TRIP12 is also required for maintaining a correct mitotic progression and chromosomes stability. My results propose TRIP12 as a new chromatin-associated protein that is essential for cell cycle progression and to preserve genome integrity. In the end, my studies will be fundamental to explain the increased expression of TRIP12 protein observed in pancreatic cancer and its impact in carcinogenesis., TRIP12 est une E3 ubiquitine ligase de la famille HECT (Homologous to the E6-AP Carboxyl Terminus). TRIP12 a plusieurs cibles connues qu'elle adresse au protéasome par polyubiquitination. Parmi ces cibles, plusieurs protéines ont des rôles importants dans la réponse aux dommages à l'ADN, le remodelage de la chromatine et la voie d'activation de p53. Des résultats non publiés de mon équipe d'accueil ont montré une surexpression de la protéine TRIP12 dans le cancer du pancréas et les lésions pré-néoplasiques. Le groupe a mis en évidence que TRIP12 polyubiquitine et provoque la dégradation du facteur de transcription PTF1a (Pancreas Transcription Factor 1a), décrivant pour la première fois un mécanisme de régulation post-traductionnelle de PTF1a. PTF1a est essentiel au développement et à l'homéostasie du pancréas. Il est capable d'inhiber la prolifération de cellules cancéreuses pancréatiques et est considéré comme un gène suppresseur de tumeur. Bien qu'ayant pour substrats des protéines intervenant dans des processus finement régulés au cours du cycle cellulaire et altérés dans les cancers, la régulation de l'expression et les fonctions de TRIP12 au cours du cycle cellulaire n'étaient pas connues au début de mes travaux de thèse. J'ai démontré que l'expression et la localisation nucléaire de TRIP12 varient au cours du cycle cellulaire. J'ai identifié un domaine intrinsèquement désordonné dans la partie N-terminale de TRIP12 qui lui permet d'interagir avec l'euchromatine. J'ai montré que TRIP12 est impliqué dans l'entrée en mitose en contrôlant la durée de la réplication de l'ADN indépendamment de son activité catalytique. TRIP12 est également indispensable pour une bonne progression en mitose et la stabilité chromosomique. Mes résultats proposent TRIP12 comme une nouvelle protéine associée à la chromatine, essentielle à la progression dans le cycle cellulaire et au maintien de l'intégrité du génome. A terme, ce travail de thèse servira de base pour comprendre la surexpression de TRIP12 dans le cancer du pancréas et son impact dans la carcinogénèse.

Background A sophisticated level of coordinated gene expression is necessary for skeletal muscle fibers to obtain their unique functional identities. We have previously shown that the transcription factor Sox6 plays an essential role in coordinating muscle fiber type differentiation by acting as a transcriptional suppressor of slow fiber-specific genes. Currently, mechanisms regulating the activity of Sox6 in skeletal muscle and how these mechanisms affect the fiber phenotype remain unknown. Methods Yeast two-hybrid screening was used to identify binding partners of Sox6 in muscle. Small interfering RNA (siRNA)-mediated knockdown of one of the Sox6 binding proteins, Trip12, was used to determine its effect on Sox6 activity in C2C12 myotubes using quantitative analysis of fiber type-specific gene expression. Results We found that the E3 ligase Trip12, a HECT domain E3 ubiquitin ligase, recognizes and polyubiquitinates Sox6. Inhibiting Trip12 or the 26S proteasome activity resulted in an increase in Sox6 protein levels in C2C12 myotubes. This control of Sox6 activity in muscle cells via Trip12 ubiquitination has significant phenotypic outcomes. Knockdown of Trip12 in C2C12 myotubes led to upregulation of Sox6 protein levels and concurrently to a decrease in slow fiber-specific Myh7 expression coupled with an increased expression in fast fiber-specific Myh4. Therefore, regulation of Sox6 cellular levels by the ubiquitin-proteasome system can induce identity-changing alterations in the expression of fiber type-specific genes in muscle cells. Conclusions Based on our data, we propose that in skeletal muscle, E3 ligases have a significant role in regulating fiber type-specific gene expression, expanding their importance in muscle beyond their well-established role in atrophy.