Trans regulation in the Ultrabithorax gene of Drosophila: alterations in the promoter enhance transvection

PMCID: PMC556824We report a genetic and molecular study of UbxMX6 and Ubx195rx1, two mutations in the Ultrabithorax (Ubx) locus which appear to have a strong effect on the activity of the homologous Ubx gene. These mutations show the characteristic embryonic and adult phenotypes of Ubx null alleles, and also fail to produce any detectable Ubx product. Yet, genetic and phenotypic analyses involving a large number of trans heterozygous combinations of UbxMX6 and Ubx195rx1 with different classes of Ubx mutations, indicate that they hyperactivate the homologous gene. This effect is induced on wildtype or mutant forms of Ubx, provided that the pairing in the bithorax region is normal, i.e. these mutations have a strong positive effect on transvection. We also show that, unlike all the other known cases of transvection in Ubx, this is not zeste-dependent. Southern analyses indicate that UbxMX6 is a 3.4 kb deletion, and Ubx195rx1 is an approximately 11 kb insertion of foreign DNA, both in the promoter region. We speculate that the region altered in the mutations may have a wildtype function to ensure cis-autonomy of the regulation of Ubx transcription.This work was supported by grants from the DGICYT and the Fundación Ramón Areces.Peer reviewe

Role of PI3Kgamma complexes in vascular smooth muscle cells biology and implications in arterial diseases

Les cellules musculaires lisses vasculaires (CML) sont les constituants majeurs de la partie médiale des artères et sont responsables de leur contractilité (phénotype contractile). Normalement quiescentes, les CML sont capables de réaliser une transition phénotypique qui leur permet d’acquérir des capacités de prolifération et de migration en réponses à différents stress, c’est le phénotype synthétique. Elles sont ainsi capables de quitter la partie médiale de l’artère et envahir la lumière des vaisseaux causants des remodelages de la paroi artérielle participant ainsi au développement de maladies cardiovasculaires. Dans ce contexte, notre équipe a démontré que la Phosphoinositide 3-Kinase gamma (PI3Kg) joue un rôle clé dans la biologie des CML synthétiques. Cette kinase est composée d’une sous-unité catalytique p110g associée à l’une de ces deux sous-unités adaptatrices, p101 ou p84, permettant chacune l’activité enzymatique de la kinase. Cependant l’implication des différentes sous-unités n’a jamais été étudiée dans les CML. Au cours de ma thèse, nous avons cartographié l’expression des différents complexes de la PI3Kg, p110g/p84 et p110g/p101, en fonction du phénotype des CML, contractile ou synthétique. Nous avons pu montrer une expression préférentielle en phénotype contractile du couple p110g/p84, et à l’inverse, le couple p110g/p101 est dominant dans une CML proliférative. Par la suite, grâce à l’utilisation de l’optogénétique, nous avons montré que le recrutement à la membrane du couple p110g/p84 entrainait une contraction des CML. A l’inverse, p110g/p101 participerait plutôt au contrôle de la prolifération. Nous avons ensuite démontré que la contraction des CML était dépendante d’une entrée de calcium dans la cellule. Enfin, nous avons démontré l’implication physiopathologique de ces résultats dans des modèles murins originaux délétés pour les sous-unités régulatrices soumis à un traitement hypertenseur. Ainsi, nos données démontrent clairement que la PI3Kg fonctionne avec deux complexes différents selon le phénotype des CML. L’ensemble des résultats obtenus au cours de ma thèse tendent à montrer le rôle clef de la sous-unité adaptatrice p84 tant dans le maintien du phénotype contractile que dans la contraction calcique des CML.)Vascular smooth muscle cells (VSMC) are the unique component of the medial layer of normal arteries and are responsible for their contractile properties. In healthy conditions, VSMC are mostly quiescent and differentiated, a phenotype called “contractile”, but keep a high potential of dedifferentiation. Indeed, in response to various external cues VSMC can shift from the contractile to a so-called synthetic phenotype where cells are able to migrate away from the medial layer of the artery to proliferate and accumulate in the intimal layer. Consequently, they cause pathological vascular remodelling occurring during pathologies such as atherosclerotic plaques development or after lesions induced by angioplasty in humans. In this context, our team demonstrated that the Phosphoinositide 3-Kinase gamma (PI3Kg) plays a key role in the biology of synthetic VSMC. This kinase is composed of a catalytic subunit p110g associated with one of the two adaptor subunits, p101 or p84, each allowing the enzymatic activity of the kinase. However, the involvement of the different subunits has never been studied in VSMCs. During my thesis, we mapped the expression of the different PI3K complexes, p110g/p84 and p110g/p101, according to the phenotype of the VSMC. We were able to show a preferential expression in contractile phenotype of the couple p110g/p84, and conversely, the couple p110g/p101 is dominant in proliferative cells. Subsequently, using optogenetics, we showed that the recruitment of the p110g/p84 complex to the plasma membrane resulted in contraction of the VSMC in a kinase and calcium dependent manner. By contrast, p110g/p101 would rather participate in the control of cell proliferation. Finally, we demonstrated the pathophysiological relevance of these findings in new mouse models deleted for the regulatory subunits subjected to an hypertensive treatment. Thus, our data clearly demonstrate that PI3K gamma functions with two different complexes depending on the phenotype of the VSMC. All the results obtained during my thesis suggest a key role of the adaptor subunit p84 in the maintenance of the VSMC contractility phenotype and in the calcium-dependent contraction of these cells.

Immune and Smooth Muscle Cells Interactions in Atherosclerosis: How to Target a Breaking Bad Dialogue?

International audienc

Leucine-Rich Alpha-2 Glycoprotein 1 Accumulates in Complicated Atherosclerosis and Promotes Calcification

Atherosclerosis is the primary cause of cardiovascular disease. The development of plaque complications, such as calcification and neo-angiogenesis, strongly impacts plaque stability and is a good predictor of mortality in patients with atherosclerosis. Despite well-known risk factors of plaque complications, such as diabetes mellitus and chronic kidney disease, the mechanisms involved are not fully understood. We and others have identified that the concentration of circulating leucine-rich α-2 glycoprotein 1 (LRG1) was increased in diabetic and chronic kidney disease patients. Using apolipoprotein E knockout mice (ApoE−/−) (fed with Western diet) that developed advanced atherosclerosis and using human carotid endarterectomy, we showed that LRG1 accumulated into an atherosclerotic plaque, preferentially in calcified areas. We then investigated the possible origin of LRG1 and its functions on vascular cells and found that LRG1 expression was specifically enhanced in endothelial cells via inflammatory mediators and not in vascular smooth muscle cells (VSMC). Moreover, we identified that LRG1 was able to induce calcification and SMAD1/5-signaling pathways in VSMC. In conclusion, our results identified for the first time that LRG1 is a direct contributor to vascular calcification and suggest a role of this molecule in the development of plaque complications in patients with atherosclerosis

Genetic analysis of RNA polymerase I allowed isolation of alleles leading to over-production of rRNA transcripts

Trabajo presentado a la OddPols: International Conference on Transcription by RNA Polymerase I, III, IV and V, celebrada en Toulouse (Francia) del 26 al 29 de junio de 2018.Peer Reviewe

Flexibility of the jaw-lobe region in RNA polymerase I influences transcriptional elongation

Resumen del trabajo presentado al III Meeting Red de Excelencia Temática: "RNA Life", celebrado en Salamanca del 24 al 26 de septiembre de 2018.In growing eukaryotic cells, most transcriptional activity involves synthesis of ribosomal RNA (rRNA) by RNA polymerase I (Pol I). Yeast Pol I is composed by 14 subunits with an overall mass of 600 kDa. Deletion of Pol I subunit A49, which plays a role both in transcription initiation and elongation, causes a severe growth defect. Genetic analysis allowed us to isolate a set of mutants on different Pol I subunits that suppress the A49 deletion phenotype. Most suppressor mutants cluster at the Pol I jaw-lobe region, which encompasses the jaw in subunit A190, the lobe in subunit A135, and the N-terminal and liker domains of subunit A12. We use computational analysis of available Pol I structures to propose a role for the jaw-lobe region in the transition between transcription initiation and elongation. Finally, we show that the most efficient suppressor mutant exhibits increased rRNA production both in vivo and in vitro.Spanish Ministry of Science (BFU2017-87397-P), Ramón Areces Foundation.Peer reviewe

Genetic analyses led to the discovery of a super-active mutant of the RNA polymerase I

Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6 and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I. Author summary The nuclear genome of eukaryotic cells is transcribed by three RNA polymerases. RNA polymerase I (Pol I) is a multimeric enzyme specialized in the synthesis of ribosomal RNA. Deregulation of the Pol I function is linked to the etiology of a broad range of human diseases. Understanding the Pol I activity and regulation represents therefore a major challenge. We chose the budding yeast Saccharomyces cerevisiae as a model, because Pol I transcription apparatus is genetically amenable in this organism. Analyses of phenotypic consequences of deletion/truncation of Pol I subunits-coding genes in yeast indeed provided insights into the activity and regulation of the enzyme. Here, we characterized mutations in Pol I that can alleviate the growth defect caused by the absence of Rpa49, one of the subunits composing this multi-protein enzyme. We mapped these mutations on the Pol I structure and found that they all cluster in a well-described structural element, the jaw-lobe module. Combining genetic and biochemical approaches, we showed that Pol I bearing one of these mutations in the Rpa135 subunit is able to produce more ribosomal RNA in vivo and in vitro. We propose that this super-activity is explained by structural rearrangement of the Pol I jaw/lobe interface