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Le contrôle de l’expression des gènes est un processus crucial pour le développement, la reproduction ou les mécanismes d’acclimatation aux stress environnementaux et met en jeu des voies de régulation post-transcriptionnelles agissant sur les ARN messagers (ARNm). Ces molécules portent des modifications chimiques dont l’une des plus abondantes est la N6-méthyladénosine ou m6A. Cette modification permet notamment d’attirer des protéines spécifiques qualifiées de « lecteurs » qui, chez les mammifères, agissent principalement pour favoriser la dégradation et/ou la traduction des ARNm. Mes travaux de thèse ont eu pour objectif de caractériser les fonctions d’un de ces lecteurs, nommé ECT2, chez la plante modèle Arabidopsis thaliana. Dans un premier temps, sa fonction de liaison aux ARNm méthylés ainsi que son rôle dans le développement de la plante ont été démontrés. Au niveau moléculaire, une approche de protéomique a permis d’identifier de nombreux partenaires d’ECT2 dont la majorité est impliquée dans le métabolisme des ARNm parmi lesquels des facteurs inhibiteurs de traduction. Les résultats d’une analyse de translatomique permettent de proposer un modèle où ECT2 jouerait un rôle de répresseur de la traduction d’ARNm en coopération avec ses partenaires LARP1 et DCP5, deux facteurs évolutivement conservés qui agissent dans le contrôle de la traduction des ARNm. Enfin, j’ai également découvert que la protéine ECT2 est dynamiquement modifiée via des phosphorylations en réponse à un stress thermique, ce qui semble notamment affecter sa capacité à reconnaitre les résidus m6A. Ces travaux suggèrent pour la première fois que l’activité d’un lecteur peut être régulée par des phosphorylations en réponse à des variations environnementales. / Control of gene expression is a crucial process for development, reproduction or acclimation to environmental stresses and involves post-transcriptional regulatory pathways acting on messenger RNAs (mRNAs). These molecules carry chemical modifications of which N6-methyladenosine (m6A) is one of the most abundant. This modification allows notably the recruitment of specific proteins qualified as “readers” which, in mammals, mostly act to promote decay and/or translation of mRNAs. My thesis aimed to characterize the functions of one of these readers, named ECT2, in the model plant Arabidopsis thaliana. First, its binding function to methylated mRNAs and its role in plant development was demonstrated. At the molecular level, a proteomic approach identified numerous ECT2’s protein partners, mainly involved in mRNA metabolism including translation inhibition factors. Results obtained from a translatome analysis suggest a model where ECT2 could play a repressive role on the translation of methylated mRNAs cooperatively with its partners LARP1 and DCP5, two evolutionarily conserved factors acting in translational control of mRNAs. Finally, I also discovered that ECT2 is dynamically modified with phosphorylations in response to heat stress affecting especially its ability to recognize m6A residues. These works suggests for the first time that the activity of an m6A reader could be regulated by phosphorylations in response to environmental changes.

Loss of hearing affects more than 10 % of the population, and one newborn in a thousand is born with defects of the inner ear. Transcriptional factors involved in the development of inner ear are important in our understanding of the causes of inner ear defects. ISLET1 is one of these factors. ISLET1 expression is detected in the sensory and neuronal cells of the inner ear. It participates in otocyst formation, and the specification and differentiation of cells of cochlea and vestibular system. The functional role of ISLET1 during inner ear development was investigated. Its role was studied by using Pax2-Isl1 transgenic mice that overexpress Islet1 under the control of the Pax2 promoter. Two transgenic lines were generated, Pax2-Isl1/300 and Pax2- Isl1/52. Two copies of the Pax2-Isl1 transgene were inserted to Pax2-Isl1/300 genome and one copy was inserted to the Pax2-Isl1/52 genome. Defects in sense of hearing were detected in both lines and circling behavior, a defect of balance, was detected in the Pax2-Isl1/300 transgenic mice. We observed high postnatal lethality in heterozygote transgenic mice. Pax2-Isl1/52 homozygote mutation is lethal at embryonic day 10 (E10,5). Pax2-Isl1/300 homozygote letality couldn't be detected because of the inability to breed heterozygote mutated mice of this line....

No description available.

Understanding the mechanisms of transcriptional regulation and the constraints that operate in gene promoter sequences is the key step in understanding the evolutionary conservation of transcriptional regulation. It is well known that regulatory regions with the same expression outputs do not have to share the sequence similarity. The most important elements in regulatory sequences are transcription factor binding sites and their position relocation does not usually influence the expression output. The least complex transcriptional regulation is characteristic for housekeeping genes. For their expression they require only basal core promoter elements (sometimes only CpG islands are sufficient) and general transcription factors, so they can be transcribed easily and immediately whenever they are needed. In this study we focused on transcriptional regulation of invertebrate amphioxus (Branchiostoma floridae) housekeeping genes in vertebrate systems. We prepared a set of constructs with amphioxus regulatory regions for testing their activity in different mammalian cell lines and a set of constructs with the same amphioxus regulatory regions for observing their spatial recognition in developing medaka fish embryo. We found that half of investigated amphioxus regulatory regions are recognized by...

Das metastasierende kolorektale Karzinom stellt eine große Herausforderung in der Krebstherapie dar. Verlässliche und effiziente Biomarker zur Prognose des Krankheitsverlaufes oder der Therapieantwort (Prädiktion) sind rar. Metastasis-associated in colon cancer 1 (MACC1) ist ein prognostischer, prädiktiver und kausaler Biomarker für verschiedene Tumorentitäten. Durch die Induzierung von Zielgenen, wie z.B. MET, beeinflusst es Signalwege wie MEK/ERK und AKT/β-catenin und fördert so Zellproliferation und -motilität sowie Tumorprogression und Metastasierung in vivo. Diese Arbeit sollte neue Strategien erforschen diese Prozesse durch die Inhibition von MACC1 auf Transkriptions- und Signaltransduktionsebene zu unterbinden. Mit zwei verschiedenen Screeningmethoden konnten wir Statine als potente transkriptionelle Inhibitoren von MACC1 als auch phosphotyrosin (pY)-abhängige Interaktionen von MACC1 mit essentiellen Signalmolekülen identifizieren: SHP2, GRB2, SHC1, PLCG1 und STAT5B. Statine verringerten MACC1-spezifische Proliferation und Koloniebildung in vitro als auch Tumor Wachstum und Metastasierung in vivo bei Dosen äquivalent der humanen Standardtherapie zur Blutlipidsenkung. Mutation der pY-Bindungsstellen reduzierte die Aktivität des MACC1-induzierten ERK Signalwegs sowie Zellmigration und -proliferation. Anhand unserer Daten orchestriert MACC1, abhängig von MET und EGFR, neue SHP2/SRC/ERK und PKA/SRC/CREB Signalkaskaden zu einem malignen Phänotyp. Gezielte Intervention restringierte die MACC1-abhängige Koloniebildung, was neue therapeutische Interventionspunkte identifiziert und eine hervorragende Basis für Untersuchungen zur Kombinationstherapie darstellt. Die weitere Erforschung der spatiotemporalen Organisation des MACC1 Signalosoms und assoziierter Signalkaskaden soll das volle therapeutische Potential von MACC1 ausschöpfen. Wir empfehlen zudem Statine in der Krebstherapie bzw. -prävention, besonders bei MACC1-stratifzierten Patienten, anzuwenden. / Metastatic colorectal cancer still represents a major challenge in therapy. Reliable and efficient biomarkers for early prognosis of disease course or treatment response (prediction) remain scarce. Metastasis-associated in colon cancer 1 (MACC1) has been established as prognostic, predictive and causal biomarker for several tumor entities. Its induction of target genes such as MET affects several signaling pathways including MEK/ERK and AKT/β-catenin. Thus, it promotes cellular proliferation and motility as well as tumor progression and metastasis formation in vivo. This study intended to explore new strategies to inhibit these processes by targeting MACC1 on transcriptional and signaling level. By two distinct screening methods, we identified statins as potent MACC1 transcriptional inhibitors as well as phosphotyrosine (pY)-dependent interactions of MACC1 with crucial signaling molecules: SHP2, GRB2, SHC1, PLCG1 and STAT5B. Statins showed MACC1-specific reduction of proliferation and colony formation in vitro as well as restriction of tumor growth and metastasis formation in vivo at doses equivalent to human standard lipid reduction therapy. Mutation of the pY-interaction sites abrogated MACC1-dependent ERK signaling as well as cell migration and proliferation. Our data further suggest that MACC1 governs SHP2/SRC/ERK and PKA/SRC/CREB axes conferring a malignant phenotype in response to MET and EGFR. Targeted intervention restricted MACC1-dependent colony formation which indicates new drug intervention points for MACC1 signaling and provides an excellent baseline for further investigations of combinatorial treatments. Additional research about the spatiotemporal organization of MACC1 signalosome formation and downstream signaling will reveal the entire potential of MACC1 as therapeutic target, whereas statins should already be considered for cancer therapy or prevention, especially in patients stratified for MACC1 expression.

[ES] Los cilios son estructuras eucariotas complejas conservadas evolutivamente que, proyectando desde la superficie de las células, desempeñan un gran número de funciones biológicas. Los cilios se clasifican tradicionalmente en móviles o sensoriales y en su composición intervienen cientos de proteínas. Este conjunto de genes que codifican para los componentes ciliares se conoce como cilioma. Las mutaciones en el cilioma subyacen a un grupo cada vez mayor de enfermedades multisistémicas altamente pleiotrópicas denominadas globalmente como ciliopatías. Estas enfermedades se caracterizan, entre otros síntomas, por retraso mental, defectos sensoriales y/o trastornos metabólicos. A pesar de que se estima que 1 de cada 1.000 personas está afectada por estas enfermedades, las bases moleculares de las ciliopatías son todavía poco conocidas. El adecuado ensamblaje y funcionalidad del cilio requieren de la expresión estrechamente coordinada de los componentes del cilio; sin embargo, se sabe poco sobre la lógica reguladora que controla la transcripción del cilioma. La mayoría de los genes del cilioma son compartidos tanto por cilios móviles como sensoriales. Los factores de transcripción (FTs) de la familia RFX tienen un papel evolutivamente conservado en la regulación transcripcional del cilioma tanto móvil como sensorial. En los vertebrados, la transcripción del cilioma móvil también está regulada directamente por FoxJ1, un FT de la familia forkhead (FKH). Sin embargo, hasta la fecha, se desconocen los FTs que actúan junto a RFX en la transcripción del cilioma sensorial en cualquier organismo. En este trabajo, hemos identificado a FKH-8, un FT de la familia FKH, como selector terminal del cilioma sensorial de C. elegans. fkh-8 se expresa de forma consistente en las sesenta neuronas sensoriales ciliadas de C. elegans, se une a las regiones reguladoras de los genes del cilioma sensorial, también es necesario para la correcta expresión de los genes del cilioma y actúa de forma sinérgica con el conocido regulador maestro de la ciliogénesis DAF19/RFX. En consecuencia, los mutantes para fkh-8 muestran una amplia gama de defectos de comportamiento en una plétora de paradigmas sensoriales, incluyendo la olfacción, la gustación y la mecano-sensación. Así, hemos identificado, por primera vez, un FT que actúa junto con los FTs de la familia RFX en la regulación directa del cilioma sensorial. Además, nuestros resultados, junto con trabajos anteriores, muestran que los FTs FKH y RFX actúan conjuntamente en la regulación de los cilios tanto móviles como sensoriales, lo que sugiere que esta lógica reguladora podría ser un rasgo evolutivo antiguo anterior a la subespecialización funcional de los cilios. Finalmente, esperamos que los resultados de nuestro trabajo ayuden a entender mejor las bases biológicas de las ciliopatías huérfanas / [CA] Els cilis són estructures eucariotes complexes conservades evolutivament que, projectant des de la superfície de les cèl·lules, exerceixen un gran nombre de funcions biològiques. Els cilis es classifiquen tradicionalment en mòbils o sensorials i en la seua composició intervenen centenars de proteïnes. Aquest conjunt de gens que codifiquen per als components ciliars es coneix com el cilioma. Les mutacions en el cilioma subjauen a un grup cada vegada major de malalties multisistèmiques altament pleiotròpiques denominades globalment com ciliopaties. Aquestes malalties es caracteritzen, entre altres símptomes, per retard mental, defectes sensorials i/o trastorns metabòlics. A pesar que s'estima que 1 de cada 1.000 persones està afectada per aquestes malalties, les bases moleculars de les ciliopaties són encara poc conegudes. L'adequat assemblatge i funcionalitat del cili requereixen de l'expressió estretament coordinada dels components del cili; no obstant això, se sap poc sobre la lògica reguladora que controla la transcripció del cilioma. La majoria dels gens del cilioma són compartits tant per cilis mòbils com sensorials. Els factors de transcripció (FTs) de la família RFX tenen un paper evolutivament conservat en la regulació transcripcional del cilioma tant mòbil com sensorial. En els vertebrats, la transcripció del cilioma mòbil també està regulada directament per FoxJ1, un FT de la família forkhead (FKH). No obstant això, fins hui, es desconeixen els FTs que actuen al costat de RFX en la transcripció del cilioma sensorial en qualsevol organisme. En aquest treball, hem identificat a FKH-8, un FT de la família FKH, com a selector terminal del cilioma sensorial de C. elegans. fkh-8 s'expressa de manera consistent en les seixanta neurones sensorials ciliades de C. elegans, s'uneix a les regions reguladores dels gens del cilioma sensorial, també és necessari per a la correcta expressió dels gens del cilioma i actua de manera sinèrgica amb el conegut regulador mestre de la ciliogènesi DAF-19/RFX. En conseqüència, els mutants per a fkh-8 mostren una àmplia gamma de defectes de comportament en una plètora de paradigmes sensorials, incloent la olfacció, la gustació i la mecano-sensació. Així, hem identificat, per primera vegada, un FT que actua juntament amb els FTs de la família RFX en la regulació directa del cilioma sensorial. A més, els nostres resultats, juntament amb treballs anteriors, mostren que els FTs FKH i RFX actuen conjuntament en la regulació dels cilis tant mòbils com sensorials, la qual cosa suggereix que aquesta lògica reguladora podria ser un tret evolutiu antic anterior a la subespecialització funcional dels cilis. Finalment, esperem que els resultats del nostre treball ajuden a entendre millor les bases biològiques de les ciliopaties òrfenes. / [EN] Cilia are complex evolutionary conserved eukaryotic structures that, projecting from cell surfaces, perform a variety of biological roles. Cilia are traditionally classified into motile or sensory and hundreds of proteins take part in their composition. This set of genes coding for ciliary components is known as the ciliome. Mutations in the ciliome underlie an ever-growing group of highly pleiotropic multisystemic diseases globally termed as ciliopathies. These diseases are characterized, among other symptoms, by mental retardation, sensory defects and/or metabolic disorders. Despite an estimated 1 in 1,000 people affected by these diseases, the molecular bases of the ciliopathies are still poorly understood. Proper cilium assembly and functionality requires the tightly co-regulated expression of ciliary components; however, little is known about the regulatory logic controlling ciliome transcription. Most ciliome genes are shared between motile and sensory cilia. RFX transcription factors (TFs) have an evolutionarily conserved role in the transcriptional regulation of both motile and sensory ciliome. In vertebrates, transcription of motile ciliome is also directly regulated by FoxJ1, a Forkhead (FKH) TF. However, to date, TFs working together with RFX in the transcription of the sensory ciliome are unknown in any organism. In this work, we have identified FKH-8, a FKH TF, as a terminal selector of the sensory ciliome in C. elegans. fkh-8 is consistently expressed within the sixty ciliated sensory neurons of C. elegans, it binds the regulatory regions of the sensory ciliome genes, it is also required for correct ciliome gene expression and acts synergistically with the known master regulator of the ciliogenesis DAF-19/RFX. Accordingly, fkh-8 mutants display a wide range of behavioural defects in a plethora of sensory mediated paradigms, including olfaction, gustation, and mechano-sensation. Thus, we have identified, for the first time, a TF that acts together with RFX TFs in the direct regulation of the sensory ciliome. Moreover, our results, together with previous work, show that FKH and RFX TFs act together in the regulation of both motile and sensory cilia, suggesting this regulatory logic could be an ancient trait pre-dating functional sub-specialization of cilia. Finally, we hope our results could help better understand the biological basis of orphan ciliopathies. / This thesis project has been made possible thanks to a pre-doctoral fellowship from the FPI Programme (BES-2015-072799) conferred by the (now extinct) Spanish Ministry of Economy & Competitivity. The following grants also provided a funding frame throughout the whole research process: “Estudio de los mecanismos transcripcionales que regulan la diferenciación de las neuronas monoaminérgicas y su conservación evolutiva.” SAF2014-56877-R “Dissecting the gene regulatory mechanisms that generate serotonergic neurons and their link to mental disorders.” ERC-St 281920 “Programas de regulación transcripcional asociados a enfermedades genéticas.” SAF2017-84790-R “Regulatory rules and evolution of neuronal gene expression.” ERC-Co 101002203 / Brocal Ruiz, R. (2022). Transcriptional Regulatory Logic of Cilium Formation in C. Elegans [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/181667 / TESIS

Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive human malignancies, is thought to be initiated by KRAS activation. Here, we find that transcriptional activation mediated by the GLI family of transcription factors, although dispensable for pancreatic development, is required for KRAS induced pancreatic transformation. Inhibition of GLI using a dominant-negative repressor (Gli3T) inhibits formation of precursor Pancreatic Intraepithelial Neoplasia (PanIN) lesions in mice, and significantly extends survival in a mouse model of PDAC. Further, ectopic activation of the GLI1/2 transcription factors in mouse pancreas accelerates KRAS driven tumor formation and reduces survival, underscoring the importance of GLI transcription factors in pancreatic tumorigenesis. Interestingly, we find that although canonical GLI activity is regulated by the Hedgehog ligands, in the context of PDAC, GLI transcription factors initiate a unique ligand-independent transcriptional program downstream of KRAS, that involves regulation of the RAS, PI3K/AKT, and NF-кB pathways. We identify I-kappa-B kinase epsilon (IKBKE) as a PDAC specific target of GLI, that can also regulate GLI transcriptional activity via positive feedback mechanism involving regulation of GLI subcellular localization. Using human PDAC cells, and an in vivo model of pancreatic neoplasia, we establish IKBKE as a novel regulator pf pancreatic tumorigenesis that acts as an effector of KRAS/GLI, and mediates pancreatic transformation. We show that genetic knockout of Ikbke leads to a dramatic inhibition of initiation and progression of pancreatic intraepithelial viii neoplasia (PanIN) lesions in mice carrying pancreas specific activation of oncogenic Kras. Furthermore, we find that although IKBKE is a known NF-кB activator, it only modestly regulates NF-кB activity in PDAC. Instead, we find that IKBKE strongly promotes AKT phosphorylation in PDAC in vitro and in vivo, and that IKBKE mediates reactivation of AKT post-inhibition of mTOR. We also show that while mTOR inhibition alone does not significantly affect pancreatic tumorigenesis, combined inhibition of IKBKE and mTOR has a synergistic effect leading to significant decrease tumorigenicity of PDAC cells. Together, our findings identify GLI/IKBKE signaling as an important oncogenic effector pathway of KRAS in PDAC that regulates tumorigenicity, cell proliferation, and apoptosis via regulation of AKT and NF-кB signaling. We provide proof of concept for therapeutic targeting of GLI/IKBKE in PDAC, and support the evaluation of IKBKE as a therapeutic target in treatment of pancreatic cancer, and IKBKE inhibition as a strategy to improve efficacy of mTOR inhibitors in the clinic.

From the yeast genome completed in 1996 to the 12 Drosophilagenomes published earlier this year; little more than a decade has provided an incredible amount of genomic data. Yet even with this mountain of genetic information the regulatory networks that control gene expression remain relatively undefined. In part, this is due to the enormous amount of non-coding DNA, over 98% of the human genome, which needs to be made sense of. It is also due to the large number of transcription factors, potentially 2,000 such factors in the human genome, which may contribute to any given network directly or indirectly. Certainly, one of the central limitations has been the paucity of transcription factor (TF) specificity data that would aid in the prediction of regulatory targets throughout a genome. The general lack of specificity data has hindered the prediction of regulatory targets for individual TFs as well as groups of factors that function within a common regulatory pathway. A large collection of factor specificities would allow for the combinatorial prediction of regulatory targets that considers all factors actively expressed in a given cell, under a given condition. Herein we describe substantial improvements to a previous bacterial one-hybrid system with increased sensitivity and dynamic range that make it amenable for the high-throughput analysis of sequence-specific TFs. Currently we have characterized 108 (14.3%) of the predicted TFs in Drosophilathat fall into a broad range of DNA-binding domain families, demonstrating the feasibility of characterizing a large number of TFs using this technology. To fully exploit our large database of binding specificities, we have created a GBrowse-based search tool that allows an end-user to examine the overrepresentation of binding sites for any number of individual factors as well as combinations of these factors in up to six Drosophila genomes (veda.cs.uiuc.edu/cgi-bin/gbrowse/gbrowse/Dmel4). We have used this tool to demonstrate that a collection of factor specificities within a common pathway will successfully predict previously validated cis-regulatory modules within a genome. Furthermore, within our database we provide a complete catalog of DNA-binding specificities for all 84 homeodomains in Drosophila. This catalog enabled us to propose and test a detailed set of recognition rules for homeodomains and use this information to predict the specificities of the majority of homeodomains in the human genome.

The central dogma of biology asserts that DNA is transcribed into RNA and RNA is translated into protein. However, this overtly simplistic assertion fails to portray the highly orchestrated and regulated mechanisms of transcription and translation. During the process of transcription, RNA provides the template for translation and protein synthesis as well as the structural and sequence specificity of many RNA and protein-based machines. While only 1-5% of the genome will escape the nucleus to be translated as mRNAs, complex, parallel, highly-conserved mechanisms have evolved to regulate specific mRNAs. Trans-acting factors bind cis-elements in both the 5" and 3" untranslated regions of mRNA to regulate their stability, localization, and translation. While a few salient examples have been elucidated over the last few decades, mRNA translation can be reversibly regulated by the shortening and lengthening of the 3" polyadenylate tail of mRNA. CPEB, an important factor that nucleates a complex of proteins to regulate the polyadenylate tail of mRNA, exemplifies a major paradigm of translational control during oocyte maturation and early development. CPEB function is also conserved in neurons and somatic foreskin fibroblasts where it plays an important role in protein synthesis dependent synaptic plasticity and senescence respectively. Focusing on the function of CPEB and its role in mRNA polyadenylation during human cellular senescence, the following dissertation documents the important finding that CPEB is required for the normal polyadenylation of p53 mRNA necessary for its normal translation and onset of senescence. Cells that lack CPEB have abnormal levels of mitochondria and ROS production, which are demonstrated to arise from the direct result of hypomorphic p53 levels. Finally, in an attempt to recapitulate the model of CPEB complex polyadenylation in human somatic cells, I unexpectedly find that Gld-2, a poly(A) polymerase required for CPEB-mediated polyadenylation in Xenopus laevis oocytes, is not required for p53 polyadenylation, but instead regulates the stability of a microRNA that in turn regulates CPEB mRNA translation. Furthermore, I demonstrate that CPEB requires Gld-4 for the normal polyadenylation and translation of p53 mRNA.