Fetal growth restriction (FGR) is the failure of a fetus to reach its pre-determined genetic growth potential during development. FGR is associated both with poor outcome in the neonatal period, and the onset of major adult diseases such as diabetes, hypertension and obesity. Therefore understanding what causes restricted fetal growth is important both for improving neonatal health, and for the minimization of major worldwide healthcare burdens. Described here are two mutant mouse lines, each with a distinct mutation in the Mediator complex gene Med31. These mutations result in reduced fetal growth, allowing for the investigation of the role of Med31 in the proper control of growth during development. The first mutant mouse line (Med31 Null) carries a C/T point mutation in exon 4 of Med31. Homozygous mutant embryos display reduced growth during development, characterized by their reduced size and smaller forelimbs compared to their heterozygous littermate controls. The second mutant mouse line (Med31 Y57C) carries a T/C point mutation in exon 3 of Med31. Similarly, homozygous mutant embryos display reduced fetal growth with reductions in forelimb length compared to their heterozygous littermate controls. In both mutant lines whole embryo growth and endochondral ossification within the limbs is perturbed. This is due to defects in cellular proliferation and the misexpression of the cell cycle genes Ccnb1 and Mtor within the mutant embryos. Additionally, the Med31 Null line is embryonic lethal by E18.5 and displays morphological defects of the placenta compared to heterozygous littermate controls. These morphological differences are suggestive of defects in the function of the placenta, and are proposed as the cause of embryonic lethality. In support of this the Med31 Y57C line is viable with no defects in placental development. New roles for Med31 in embryonic growth, cellular proliferation and placental development are identified. Moreover the two mutant lines constitue an allelic series of Med31, and the two mutations provide insights into the various ways Med31 is able to regulate transcription during development.

L'activation des cellules B est connue pour s’accompagner de remodelages des gènes d’immunoglobulines qui permettent la maturation d'affinité des régions variables d'Ig par hypermutation somatique SHM et la commutation de classe CSR. Ces deux processus sont sous le contrôle de la région régulatrice 3’ (3’RR) du locus IgH. Pendant la CSR, le locus IgH subit des changements tridimensionnels mettant les régions switch ciblés par AID à proximité de la région 3’RR afin de faciliter la recombinaison. La sous-unité MED1 du complexe Médiateur favorise cette interaction à longue distance avec la 3’RR mais elle intervient aussi dans la transcription germinale qui précède la CSR afin de faciliter l’activité d’AID. Comme récemment démontré chez la souris, la région 3’RR peut aussi être ciblée par des recombinaison médiée par AID, mais contrairement à la CSR, ce type de recombinaison qui joint la région Sμ et la 3’RR et qui s’appelle recombinaison suicide du locus IgH ou LSR entraîne une délétion complète de l’ensemble des gènes constants conduisant à la mort des cellules B par la perte de l’expression du BCR. Nous montrons maintenant que la LSR médiée par AID se produit aussi dans les cellules B humaines activées avec les deux régions 3’RR (3’RR1 en aval de Cα1 et 3’RR2 en aval de Cα2) et qui peut toucher l’allèle fonctionnel mais elle peut aussi être biallélique marqué par une quasi-absence de ce type de recombinaison dans les plasmocytes de la moelle mais aussi dans les cellules B mémoires quiescents du sang et qui peut par contre être réinduite à haut niveau lorsque les cellules B mémoires sont réactivées. Toutes nos conditions de stimulation utilisées in-vitro induit la LSR, sans permettre de discerner comment se fait « le choix » entre la CSR et la LSR. Nos résultats montrent par contre que la sous-unité MED1 semble influencer la transcription de la 3’RR et la recombinaison LSR chez la souris. L’inactivation conditionnelle de MED1 influence l’accessibilité transcriptionnelle et donc les recombinaisons sans affecter les marques épigénétiques du locus IgH. Cette étude de MED1 a aussi révélé que l’ensemble des processus stimulés par l’IgH 3’RR sont « Médiateur-dépendants » (SHM, CSR sans distinction de la cis et la trans-CSR, expression augmentée du locus dans les plasmocytes…), comme semble l’être également le processus de choix des segments variables au cours des réarrangements VHDJH. / B-cell activation is accompanied by remodeling of immunoglobulin genes resulting in affinity maturation of Ig variable regions by somatic hypermutation (SHM) and class switch recombination (CSR). These two processes are under the control of the 3' regulatory region (3’RR) of the IgH locus. During CSR, the IgH locus undergoes three dimensional changes bringing the AID-targeted switch regions near the 3'RR region to facilitate recombination. The MED1 subunit of the Mediator complex promotes this long-distance interaction with the 3'RR, but it is also implicated in germinal transcription preceding CSR in order to facilitate AID activity. As recently demonstrated in mice, the 3'RR region can also be targeted by AID-mediated recombination, but unlike CSR, this type of recombination joining the Sμ region and 3'RR (called Locus Suicide Recombination or LSR) results in a complete deletion of all the constant genes leading to B-cell death by loss of B Cell Receptor expression. We now show that AID-mediated LSR also occurs in activated human B cells with the two 3'RR (3'RR1downstream of Cα1 and 3'RR2 downstream of Cα2) and affects the functional allele. It can also be bi-allelic marked by the absence of this type of recombination in plasma cells of the bone marrow but also in quiescent blood memory B cells. LSR occurs at high level when the memory B cells are reactivated. All in-vitro stimulations induce LSR, without identifying conditions favoring either CSR and the LSR. Our results also show that the MED1 subunit appears to influence 3’ RR transcription and LSR in mice. Conditional inactivation of MED1 influences transcriptional accessibility and therefore recombination without affecting epigenetic markers of the IgH locus. This study also revealed that all the processes controlled by the 3'RR are "mediator -dependent" (SHM, CSR without distinction between Cis and Trans -CSR, increased expression of the IgH locus in the plasma cells ...), as well as the choice of varia ble segments during VDJH rearrangements

Diss. (sammanfattning) Umeå : Umeå universitet, 2010. / Härtill 2 uppsatser.

Transcriptional regulation of gene expression in eukaryotes has evolved over millions of years. The regulatory pathways of nuclear receptors represent an evolutionarily ancient, but conserved mechanism with associated accessory proteins, many of them forming a functional nexus known as the Mediator complex involved in transcription. Despite the versatility of the pathway, e.g. through the adoption of new regulatory functions in phylogenetically more recent Metazoa, we hypothesise that the intrinsic potential of the NR-Mediator axis to directly translate a stimulus to a biological response is conserved across species, and additional regulation could also be achieved through secondary functions of its essential members. To support the hypothesis, we assessed the ligand-binding capability of retinoic X receptor in Trichoplax adhaerens and provided evidence to support the concept that this capability was already present at the base of metazoan evolution. With regards to the potential secondary functions, we took inspiration from previous research and identified the Mediator subunit 28 (MED28) as the only known member having documented nuclear and cytoplasmic dual roles, and thus possessing the potential to transmit signals from the cellular structural states to the nucleus. Due to the lack of...

Nutrient availability regulates eukaryotic cell growth. This study focuses on two signaling pathways, involved in sensing amino acids and carbon sources, which allow cells to respond appropriately to their presence. The first part of this study shows that Ssy1, a plasma membrane localized sensor in the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing pathway, can detect 19 common L-amino acids with different potencies and affinities based on the physiochemical structure of amino acids. Substituents around alpha carbon are critical for amino acid sensing by Ssy1. Furthermore, a high concentration of cysteine is toxic to cells. Inactivation of SPS signaling confers resistance to cysteine. The second part focuses on the regulation of Hap4, the regulatory subunit of the Hap2/3/4/5 transcriptional factor complex. Many components of the 25-subunit Mediator complex negatively regulate HAP4 expression. Srb8 undergoes post-translational modification in response to changes of the carbon source. Gal11 and Med3 positively regulate HAP4 expression.

<p>The Mediator (Med) complex comprises about 30 subunits and is a transcriptional co-regulator in eukaryotic systems. The core Mediator complex, consisting of the head, middle and tail modules, functions as a bridge between transcription factors and basal transcription machinery, whereas the CDK8 kinase module can attenuate Mediator’s ability to function as either a co-activator or co-repressor. Many Arabidopsis Mediator subunit has been functionally characterized, which reveals critical roles of Mediator in many aspects of plant growth and development, responses to biotic and abiotic stimuli, and metabolic homeostasis. Traditional genetic and biochemical approaches laid the foundation for our understanding of Mediator function, but recent transcriptomic and metabolomic studies have provided deeper insights into how specific subunits cooperate in the regulation of plant metabolism. In Chapter 1, we highlight recent developments in the investigation of Mediator and plant metabolism, with emphasis on the large-scale biology studies of <i>med</i> mutants.</p> <p>We previously found that MED5, an Arabidopsis Mediator tail subunit, is required for maintaining phenylpropanoid homeostasis. A semi-dominant mutation (<i>reduced epidermal fluorescence 4-3</i>, <i>ref4-3</i>) that causes a single amino acid substitution in MED5b functions as a strong suppressor of the pathway, leading to <a>decreased soluble phenylpropanoid accumulation, reduced lignin content and dwarfism</a>. In contrast, loss of MED5a and MED5b (<i>med5</i>) results in increased levels of phenylpropanoids. In Chapter 2, we present our finding that <i>ref4-3</i> requires CDK8, a Mediator kinase module subunit, to repress plant growth even though the repression of phenylpropanoid metabolism in <i>ref4-3 </i>is CDK8-independent. Transcriptome profiling revealed that salicylic acid (SA) biosynthesis genes are up-regulated in a CDK8-dependent manner in <i>ref4-3,</i> resulting in hyper-accumulation of SA and up-regulation of SA response genes. Both growth repression and hyper-accumulation of SA in <i>ref4-3</i> require CDK8 with intact kinase activity, but these SA phenotypes are not connected with dwarfing. In contrast, mRNA-sequencing (RNA-seq) analysis revealed the up-regulation of a DNA J protein-encoding gene in <i>ref4-3</i>, the elimination of which partially suppresses dwarfing. Together, our study reveals genetic interactions between Mediator tail and kinase module subunits and enhances our understanding of dwarfing in phenylpropanoid pathway mutants.</p> <p>In Chapter 3, we characterize other phenotypes of <i>med5</i> and <i>ref4-3</i>, and find that in addition to the up-regulated phenylpropanoid metabolism, <i>med5</i> show other interesting phenotypes including hypocotyl and petiole elongation as well as accelerated flowering, all of which are known collectively as the shade avoidance syndrome (SAS), suggesting that MED5 antagonize shade avoidance in wild-type plants. In contrast, the constitutive <i>ref4-3 </i>mutant protein inhibits the process, and the stunted growth of <i>ref4-3 </i>mutants is substantially alleviated by the light treatment that triggers SAS. Moreover, <i>ref4-3</i> mimics the loss-of-function <i>med5</i> mutants in maintaining abscisic acid (ABA) levels under both normal and drought growth conditions. The phenotypic characterization of <i>med5</i> mutants extend our understanding of the role of Mediator in SAS and ABA signaling, providing further insight into the physiological and metabolic responses that require MED5.</p> <p>In Chapter 4, we explore the function of MED5 and CDK8 in gene expression regulation by investigating the effect of mutations in Mediator including <i>med5</i>, <i>ref4-3</i>, <i>cdk8-1</i> and <i>ref4-3 cdk8-1</i> on genome-wide Pol II distribution. We find that loss of MED5 results in loss of Pol II occupancy at many target genes. In contrast, many genes show enriched Pol II levels in <i>ref4-3</i>, some of which overlap with those showing reduced Pol II occupancy in <i>med5</i>. In addition, Pol II occupancy is significantly reduced when CDK8 is disrupted in <i>ref4-3</i>. Our results help to narrow down the direct gene targets of MED5 and identify genes that may be closely related to the growth deficiency observed in <i>ref4-3</i> plants, providing a critical foundation to elucidate the molecular function of Mediator in transcription regulation.</p>

Le Médiateur (MED) est un complexe multi-protéique dont le principal rôle est de transmettre à la machinerie transcriptionnelle de base les différents signaux fournis par les facteurs fixés sur des séquences d’ADN spécifique , permettant ainsi une régulation fine de l’expression des gènes. Des mutations dans le MED ou ses partenaires, comme NIPBL, sont à l’origine de diverses maladies telles que des malformations congénitales, des troubles neuro développementaux ou des cancers.A partir de cellules provenant de patients portant différentes mutations dans les sous-unités MED12 ou MED17 du MED ou dans NIPBL, nous avons observé une altération du niveau d’expression de certains gènes qui dépend de la localisation de la mutation et de la nature de leur activation. Ces variations de l’expression des gènes sont la conséquence d’un défaut dans la formation du complexe de transcription et du remodelage de la chromatine (modifications post-traductionnelles des histones). Outre une meilleure appréhension du rôle des sous-unités MED12 et MED17 du MED ainsi que NIPBL, sur la transcription des gènes, ma thèse a permis de mieux comprendre l’étiologie des maladies associées à une mutation dans ces protéines. / Mediator (MED) is a multi-protein complex whose main role is to convey to basal transcriptional machinery the different signals from factors bound at specific DNA sequences , allowing thus a fine regulation of gene expression. Mutations in MED or its partners, like NIPBL, cause various diseases, such as congenital malformations, neurodevelopmental disorders or cancers. Using cells from patients carrying different mutations in the MED subunits, MED12 or MED17, or in NIPBL, we observed an alteration of the expression of studied genes which depend on the position of the mutation and on the nature of the activation. These variations of gene expression are the consequence of a defect in transcription complex formation, as well as in chromatin remodeling(post-translational histones modifications). In addition to better comprehend the role of the MED subunits MED12 and MED17, and of NIPBL on gene transcription, my thesis helped to better understand the ethiology of the disorders associated with mutations in these proteins.

Lors des réponses immunitaires, les lymphocytes B diversifient leur répertoire par l’hypermutation somatique (HMS) et la commutation isotypique (CI). Ces deux mécanismes sont dépendant de l’activité de « activation-induced cytidine deaminase » (AID), une enzyme qui déamine les cytosines de l’ADN en uraciles générant des mésappariements qui sont processés différemment dans le cas de l’HMS et de la CI. Au cours de la CI, le locus de la chaîne lourde des immunoglobulines subit un changement de conformation qui rapproche les promoteurs, les enhancers et les régions de switch afin de permettre la recombinaison des régions de switch. Cependant, les mécanismes moléculaires sous-jacents n’ont pas encore été identifié. Dans le but de comprendre les mécanismes de régulation d’AID, nous avons réalisé un criblage protéomique et identifié CTCF ainsi que les complexes médiateur et cohésine qui constituent des facteurs préalablement impliqués dans les interactions longues distances. Au cours de ce travail de thèse, nous avons montré que le complexe médiateur est requis pour la transcription de la région de switch acceptrice, pour l’interaction de cette dernière avec l’enhancer Eµ et pour le recrutement d’AID au locus des IgH. D’un autre côté, nous avons montré que le complexe cohésine est impliqué dans la réparation des cassures induites par AID et qu’il pourrait être impliqué dans la recombinaison des régions de switch. / During immune responses, B cells diversify their repertoire through somatic hypermutation (SHM) and class switch recombination (CSR). Both of these mechanisms are dependent on the activity of activation-induced cytidine deaminase (AID), an enzyme that deaminates cytosines into uracils generating mismatches that are differentially processed to result in SHM and CSR. During CSR, the Ig heavy chain (IgH) locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers and switch regions are brought into close proximity. Nevertheless, little is known about the underlying mechanisms. To gain insight into the molecular mechanism responsible for AID regulation during CSR, we performed a proteomic screen for AID partners and identified CTCF, cohesin and mediator complexes, which are factors previously implicated in long-range interactions. We showed that during CSR, the mediator complex is required for acceptor switch region transcription, long-range interaction between the enhancer and the acceptor switch region and AID recruitment to the IgH locus whereas the cohesin complex is required for proper AID-induced breaks repair and might favor switch regions synapsis.

L’enzyme Activation-induced cytidine deaminase (AID) initie la diversification des immunoglobulines (Ig) par l’induction de dommages à l’ADN. Alors que les lésions induites aux gènes des Ig sont cruciales pour l’établissement de réponses immunes hautement spécifiques et adaptées, ce même type de lésions provoquées ailleurs dans le génome contribue à la transformation cellulaire et à l’apparition de cancer. Les mécanismes impliqués dans la protection de l’intégrité génomique des cellules B restent à définir. D’une part, nous avons développé une approche de protéomique locus-unique en couplant une technique d’identification de protéine par biotinylation de proximité avec l’outil d’édition du génome CRISPR/Cas9. Cette technique innovante, dont nous avons fait la preuve de principe pour des loci abondants, pourra être utilisée pour identifier le protéome des différentes cibles génomiques d’AID. D’autre part, nous avons caractérisé le rôle de Parp3, Parp9 et Med1, identifiées comme partenaires d’AID, éclairant ainsi les mécanismes qui contrôlent l’activité d’AID et la réparation des lésions induites par AID lors de la diversification des Ig. / Activation-induced cytidine deaminase (AID) initiates immunoglobulin (Ig) diversification by inducing DNA damage. While on-target lesions are crucial for mounting highly specific and adaptive immune responses, off-target lesions contribute to malignant cell transformation. Despite its implications, the events following AID recruitment that enforce genome integrity in B cells remain poorly defined. It is not understood why multiple non-Ig loci bound by AID are not mutated or why AID-induced DNA lesions may lead to mutations or DNA breaks. To address this question, we developed a single-locus proteomic approach coupling proximity-dependent protein identification and genome editing (CRISPR/Cas9) to identify and compare the proteins recruited at individual genomic loci bound by AID. We performed the proof of principle of this innovative tool by identifying the proteome of abundant genomic loci. On the other hand, we functionally characterized Parp3, Parp9 and Med1, identified as AID partners, revealing novel mechanisms that tightly control AID activity and DNA repair during Ig diversification.

[ES] Las plantas necesitan acomodar su crecimiento a las condiciones ambientales. Con el objetivo de ajustar su desarrollo a las señales externas, usan una serie de mecanismos moleculares. Uno de estos son las rutas de señalización hormonal, que participan en integrar la información externa con programas de desarrollo propios. Una de las hormonas más relevantes en la biología vegetal son las giberelinas (GAs). La señalización por GAs se inicia con la percepción de la hormona a través del receptor GID1, y continúa por la degradación de las reguladoras transcripcionales DELLA. Sin embargo, solo las plantas vasculares tienen un sistema de percepción de GAs completo. Entender la relevancia de la señalización por GAs requiere estudiar cómo se ensambló la ruta y qué funciones atribuidas a las GAs estaban ya codificadas en las proteínas DELLA ancestrales. Aquí mostramos mediante análisis filogenéticos y bioquímicos que las proteínas DELLA emergieron inequívocamente en un ancestro común de las plantas terrestres, y que el reclutamiento de las DELLAs al módulo de percepción de GAs depende de la presencia de un dominio de transactivación conservado que fue co-optado por el receptor GID1 ancestral para actuar como un degrón dependiente de GAs. Este dominio de transactivación parece regular la co-activación transcripcional de genes concretos por las DELLAs en todas las plantas terrestres mediante el reclutamiento de complejos Mediator a través de su subunidad MED15. Por último, nos hemos centrado en entender las funciones de las proteínas DELLA en briófitas, un clado sin señalización por GAs. Hemos descubierto el rol de la DELLA de Marchantia polymorpha como coordinadora entre las respuestas de crecimiento y estrés, sugiriendo que dicha función estaba ya codificada en proteínas DELLA del ancestro común de plantas terrestres y se ha mantenido durante más de 450 millones de años. / [CA] Les plantes necessiten acomodar el seu creixement a les condicions ambientals. Amb l'objectiu d'ajustar el seu desenvolupament als senyals externs, usen una sèrie de mecanismes moleculars. Un d'aquests són les rutes de senyalització hormonal, que participen en integrar la informació externa amb programes de desenvolupament propis. Una de les hormones més rellevants en la biologia vegetal són les giberel·lines (GAs). La senyalització per GAs s'inicia amb la percepció de l'hormona a través del receptor GID1, i continua per la degradació de les reguladores transcripcionals DELLA. No obstant això, només les plantes vasculars tenen un sistema complet de percepció de GAs. Entendre la rellevància de la senyalització per GAs requereix estudiar com es va assemblar la ruta i quines funcions atribuïdes a les GAs estaven ja codificades en les proteïnes DELLA ancestrals. Ací mostrem mitjançant anàlisis filogenètiques i bioquímiques que les proteïnes DELLA van emergir inequívocament en un ancestre comú de les plantes terrestres, i que el reclutament de les DELLAs al mòdul de percepció de GAs depén de la presència d'un domini de transactivació conservat que va ser co-optat pel receptor GID1 ancestral per a actuar com un degró dependent de GAs. Aquest domini de transactivació sembla regular la co-activació transcripcional de gens concrets per les DELLAs en totes les plantes terrestres mitjançant el reclutament de complexos Mediator a través de la seua subunitat MED15. Finalment, ens hem centrat en entendre les funcions de les proteïnes DELLA en briòfites, un clade sense senyalització per GAs. Hem descobert el rol de la DELLA de Marchantia polymorpha com a coordinadora entre les respostes de creixement i estrés, suggerint que aquesta funció estava ja codificada en proteïnes DELLA de l'ancestre comú de plantes terrestres i s'ha mantingut durant més de 450 milions d'anys. / [EN] Plants need to accommodate their growth habits to environmental conditions. For this aim, several mechanisms are used to adjust developmental responses to exogenous signals. Among them, hormonal signalling pathways participate by integrating external information with endogenous programs. One of the most relevant hormones in plant biology are gibberellins (GAs). GA signalling involves perception of the hormone by the GA receptor GID1 and subsequent degradation of the DELLA transcriptional regulators. However, only vascular plants possess a full GA perception system. Understanding the relevance of GA signalling requires elucidating how this pathway was assembled and which of the functions attributed to GAs were encoded in the ancestral DELLA proteins. Here we show by phylogenetic and biochemical analyses that DELLA proteins emerged unequivocally in a land plant common ancestor and that their recruitment into the GA-perception module relies in the presence of a conserved transactivation domain co-opted by an ancestral GID1 receptor to act as a GA-dependent degron. Moreover, this transactivation domain seems to regulate DELLA-dependent transcriptional co-activation of selected target genes by recruitment of Mediator complexes through the MED15 subunit in all land plants. Finally, we have focused on understanding the functions of DELLA proteins in bryophytes, a clade with no GA signalling. We have uncovered the role of Marchantia polymorpha DELLA protein as a coordinator between growth and stress responses, suggesting that this function was already present in the DELLA protein of a land plant common ancestor and has been maintained for over 450 millions of years. / La realización de esta tesis doctoral ha sido posible gracias a una ayuda para contratos predoctorales FPU (FPU15/01756), dos Ayudas para Estancias Breves FPU (EST17/00237, IPS2, París; EST18/00400, WUR, Wageningen), una ayuda EMBO Short-Term (ASTF 8239, WUR, Wageningen), y la financiación MSCA H2020 RISE para desplazamientos en el contexto del proyecto SIGNAT (RISE Action 644435, PUC, Santiago). Así mismo, el grueso del trabajo experimental incluido ha sido financiado por el proyecto HUBFUN del MINECO (BFU2016-80621-P) / Hernández García, J. (2021). Ancestral Functions of DELLA Proteins [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/169370 / TESIS