Autophagic cell death exists.

The term autophagic cell death (ACD) initially referred to cell death with greatly enhanced autophagy, but is increasingly used to imply a death-mediating role of autophagy, as shown by a protective effect of autophagy inhibition. In addition, many authors require that autophagic cell death must not involve apoptosis or necrosis. Adopting these new and restrictive criteria, and emphasizing their own failure to protect human osteosarcoma cells by autophagy inhibition, the authors of a recent Editor's Corner article in this journal argued for the extreme rarity or nonexistence of autophagic cell death. We here maintain that, even with the more stringent recent criteria, autophagic cell death exists in several situations, some of which were ignored by the Editor's Corner authors. We reject their additional criterion that the autophagy in ACD must be the agent of ultimate cell dismantlement. And we argue that rapidly dividing mammalian cells such as cancer cells are not the most likely situation for finding pure ACD.

CHO cell engineering to prevent polypeptide aggregation and improve therapeutic protein secretion.

The ability to efficiently produce recombinant proteins in a secreted form is highly desirable and cultured mammalian cells such as CHO cells have become the preferred host as they secrete proteins with human-like post-translational modifications. However, attempts to express high levels of particular proteins in CHO cells may consistently result in low yields, even for non-engineered proteins such as immunoglobulins. In this study, we identified the responsible faulty step at the stage of translational arrest, translocation and early processing for such a "difficult-to-express" immunoglobulin, resulting in improper cleavage of the light chain and its precipitation in an insoluble cellular fraction unable to contribute to immunoglobulin assembly. We further show that proper processing and secretion were restored by over-expressing human signal receptor protein SRP14 and other components of the secretion pathway. This allowed the expression of the difficult-to-express protein to high yields, and it also increased the production of an easy-to-express protein. Our results demonstrate that components of the secretory and processing pathways can be limiting, and that engineering of the secretory pathway may be used to improve the secretion efficiency of therapeutic proteins from CHO cells.

Creating a list of low-value health care activities in Swiss primary care.

Trisomy 21 is the most frequent genetic cause of cognitive impairment. To assess the perturbations of gene expression in trisomy 21, and to eliminate the noise of genomic variability, we studied the transcriptome of fetal fibroblasts from a pair of monozygotic twins discordant for trisomy 21. Here we show that the differential expression between the twins is organized in domains along all chromosomes that are either upregulated or downregulated. These gene expression dysregulation domains (GEDDs) can be defined by the expression level of their gene content, and are well conserved in induced pluripotent stem cells derived from the twins' fibroblasts. Comparison of the transcriptome of the Ts65Dn mouse model of Down's syndrome and normal littermate mouse fibroblasts also showed GEDDs along the mouse chromosomes that were syntenic in human. The GEDDs correlate with the lamina-associated (LADs) and replication domains of mammalian cells. The overall position of LADs was not altered in trisomic cells; however, the H3K4me3 profile of the trisomic fibroblasts was modified and accurately followed the GEDD pattern. These results indicate that the nuclear compartments of trisomic cells undergo modifications of the chromatin environment influencing the overall transcriptome, and that GEDDs may therefore contribute to some trisomy 21 phenotypes.

Analyse des modifications de la chromatine impliquées dans l'effet barrière de CTF1 aux télomères humains

Eukaryotic genomes are compartmentalized in different structural domains that can affect positively or negatively gene expression. These regions of euchromatin and heterochromatin are characterized by distinct histones marks which can facilitate or repress gene transcription. The chromatin environment represents thus one of the main problems to control gene expression in biotechnological applications or gene therapy, since its expression is affected by the chromatin neighboring its locus of insertion. Some chromatin regions like telomeres are composed of constitutive heterochromatin which leads to the telomeric position effect (TPE) that silences genes adjacent to the telomere. TPE is known to spread by the selfrecruitment of the SIR histone deacetylase complex from the telomere in S.cerevisiae, but the histone marks that are associated to telomeric chromatin in mammalian cells remain mostly unknown. The transcription factor CTF1 has shown antisilencing properties in mammalian cells and also a boundary activity against TPE in yeast cells when fused to the yeast Gal4 DNA binding domain. In the work presented here, we describe a dual-reporter system to assess the boundary activity of proteins such as CTF1 at human telomeres. When located between the two reporter genes, CTF1 shields the telomere distal gene from TPE, while the telomereproximal gene remains silenced by telomeric heterochromatin. The boundary activity of CTF1 is shown to act regardless its function of transcriptional activator, by opposition to the transcriptional activator VP16 which activates indifferently both transgenes. Moreover, this study shows that CTF1 boundary activity is linked to its H3 binding function, as expected from a chromatin remodeler. ChIP experiments showed that histone deacetylation is the main histone modification involved in gene silencing at mammalian cell telomeres. Distinctly to yeast cells, the histone deacetylation signal in human cells extented over a short range along the chromosome. CTF1 may help to block this propagation and therefore to restore histones acetylation level on telomere protected locus. Surprisingly, other histone marks such as trimethyl-H3K9 or trimethyl-H4K20 were found on telomere protected locus, while in another clone, unsilencing of telomere distal transgene was associated with recruitment of the histone variant H2A.Z. Thus, I conclude that CTF1 displays a chromatin boundary function which is independent of its transcriptional activity and therefore exhibit features required for use as chromatin insulator in biotechnological applications. RESUME Les génomes eucaryotes sont compartementalisés en domaines structurels qui peuvent affecter positivement ou négativement l'expression des gènes avoisinants. Ces régions dites d'euchromatine ou d'hétérochromatine sont caractérisées par des modifications posttraductionnelles des histones qui peuvent faciliter ou au contraire inhiber la transcription des gènes qui s'y trouvent. Ainsi, isoler un gène de son environnement chromatinien est problème fréquent lorsqu'il s'agit de contrôler son expression dans le cadre d'applications en biotechnologie ou encore en thérapie génique. Certaines régions de chromatine telles que les télomères sont composées d'hétérochromatine constitutive qui mène au silençage des gènes avoisinants. Cet effet de position télomérique (TPE) est connu dans la levure S.cerevisiae comme se propageant par auto-recrutement du complexe de déacétylation d'histone SIR, alors que peu de modifications de chromatine ont pu être associées à ce phénomène dans les cellules de mammifères. Le facteur de transcription CTF1 a montré des propriétés d'anti-silençage dans les cellules de mammifères, ainsi qu'une activité barrière contre le silençage télomérique dans les cellules de levures lorsqu'il est fusionné au domaine de liaison à l'ADN de la protéine de levure Gal4. Dans le travail présenté ci-après est décrit un système à deux gènes rapporteurs permettant de mesurer l'activité barrière de protéines telles que CTF1 aux télomères humains, et les modifications de chromatine qui y sont associées. Lorsque CTF1 est placé entre les deux gènes rapporteurs, le gène distant du télomère est protégé du silençage qui lui est associé, alors que le gène proche du télomère reste soumis à ce silençage induit par l'hétérochromatine télomérique. L'activité barrière de CTF1 est montrée ici comme agissant indépendamment de son activité transcriptionnelle, par opposition à l'activateur transcriptionnel VP16 qui active indifféremment les deux transgènes. En outre, cette étude appuie l'hypothèse stipulant que CTF1 agisse comme remodeleur chromatinien puisqu'elle démontre que son activité barrière est directement dépendante de son activité de liaison avec l'histone H3. De plus, des expériences d'immuno-précipitation de la chromatine démontrent que la déacétylation des histones est le majeur phénomène intervenant dans le silençage télomérique. Par opposition à la levure, ce signal de déacétylation ne se propage dans les cellules humaines que sur une courte distance le long du chromosome. CTF1 agit ainsi en bloquant cette propagation et en restaurant le niveau d'acétylation des histones sur le locus protégé du télomère. De manière surprenante et inattendue, d'autres modifications d'histones telles que 4 les H3K9 et H4K20 triméthylées sont aussi observées à ce locus, tandis le recrutement du variant H2A.Z peut aussi être suffisant à restaurer l'expression du gène distant du télomère. En terme de cette analyse, CTF1 exhibe ainsi une fonction de barrière chromatinienne qui exclue une activité transcriptionnelle non désirée - propriété qui est requise dans l'établissement des isolateurs visant à permettre le contrôle d'un transgène dans le cadre d'applications en biotechnologies.

Conformational changes modulate the activity of human RAD51 protein.

Homologous recombination provides a major pathway for the repair of DNA double-strand breaks in mammalian cells. Defects in homologous recombination can lead to high levels of chromosomal translocations or deletions, which may promote cell transformation and cancer development. A key component of this process is RAD51. In comparison to RecA, the bacterial homologue, human RAD51 protein exhibits low-level strand-exchange activity in vitro. This activity can, however, be stimulated by the presence of high salt. Here, we have investigated the mechanistic basis for this stimulation. We show that high ionic strength favours the co-aggregation of RAD51-single-stranded DNA (ssDNA) nucleoprotein filaments with naked duplex DNA, to form a complex in which the search for homologous sequences takes place. High ionic strength allows differential binding of RAD51 to ssDNA and double-stranded DNA (dsDNA), such that ssDNA-RAD51 interactions are unaffected, whereas those between RAD51 and dsDNA are destabilized. Most importantly, high salt induces a conformational change in RAD51, leading to the formation of extended nucleoprotein filaments on ssDNA. These extended filaments mimic the active form of the Escherichia coli RecA-ssDNA filament that exhibits efficient strand-exchange activity.

Fluorodeoxyuridine mediated cell cycle synchronization in S-phase increases the Auger radiation cell killing with 125I-iododeoxyuridine.

Aim: 125I-iododeoxyuridine is a potential Auger radiation therapy agent. Its incorporation in DNA of proliferating cells is enhanced by fluorodeoxyuridine. Here, we evaluated therapeutic activities of 125I-iododeoxyuridine in an optimized fluorodeoxyuridine pre-treatment inducing S-phase synchronization. Methods: After S-phase synchronization by fluorodeoxyuridine, cells were treated with 125I-iododeoxyuridine. Apoptosis analysis and S-phase synchronization were studied by flow cytometry. Cell survival was determined by colony-forming assay. Based on measured growth parameters, the number of decays per cell that induced killing was extrapolated. Results: Treatment experiments showed that 72 to 91% of synchronized cells were killed after 0.8 and 8 kBq/ml 125I-iododeoxyuridine incubation, respectively. In controls, only 8 to 38% of cells were killed by corresponding 125I-iododeoxyuridine activities alone and even increasing the activity to 80 kBq/ml gave only 42 % killing. Duplicated treatment cycles or repeated fluorodeoxyuridine pre-treatment allowed enhancing cell killing to >95 % at 8 kBq/ml 125I-iododeoxyuridine. About 50 and 160 decays per S-phase cells in controls and S-phase synchronization, respectively, were responsible for the observed cell killing at 0.8 kBq/ml radio-iododeoxyuridine. Conclusion: These data show the successful application of fluorodeoxyuridine that provided increased 125I-iododeoxyuridine Auger radiation cell killing efficacy through S-phase synchronization and high DNA incorporation of radio-iododeoxyuridine.

PPAR disruption: Cellular mechanisms and physiological consequences

Endocrine disruption is defined as the perturbation of the endocrine system, which includes disruption of nuclear hormone receptor signalling. Peroxisome proliferator-activated receptors (PPARs) represent a family of nuclear receptors that has not yet been carefully studied with regards to endocrine disruption, despite the fact that PPARs are known to be important targets for xenobiotics. Here we report a first comprehensive approach aimed at defining the mechanistic basis of PPAR disruption focusing on one chemical, the plasticizer monethylhexyl phthalate (MEHP), but using a variety of methodologies and models. We used mammalian cells and a combination of biochemical and live cell imaging techniques to show that MEHP binds to PPAR gamma and selectively regulates interactions with coregulators. Micro-array experiments further showed that this selectivity is translated at the physiological level during adipocyte differentiation. In that context, MEHP functions as a selective PPAR modulator regulating only a subset of PPAR gamma target genes compared to the action of a full agonist. We also explored the action of MEHP on PPARs in an aquatic species, Xenopus laevis, as many xenobiotics are found in aquatic ecosystems. In adult males, micro-array data indicated that MEHP influences liver physiology, possibly through a cross-talk between PPARs and estrogen receptors (ER). In early Xenopus laevis embryos, we showed that PPAR beta/delta exogenous activation by an agonist or by MEHP affects development. Taken together our results widen the concept of endocrine disruption by pinpointing PPARs as key factors in that process.

Ultraweak photon emission in assessing bone growth factor efficiency using fibroblastic differentiation.

Photons participate in many atomic and molecular interactions and changes. Recent biophysical research has shown the induction of ultraweak photons in biological tissue. It is now established that plants, animal and human cells emit a very weak radiation which can be readily detected with an appropriate photomultiplier system. Although the emission is extremely low in mammalian cells, it can be efficiently induced by ultraviolet light. In our studies, we used the differentiation system of human skin fibroblasts from a patient with Xeroderma Pigmentosum of complementation group A in order to test the growth stimulation efficiency of various bone growth factors at concentrations as low as 5 ng/ml of cell culture medium. In additional experiments, the cells were irradiated with a moderate fluence of ultraviolet A. The different batches of growth factors showed various proliferation of skin fibroblasts in culture which could be correlated with the ultraweak photon emission. The growth factors reduced the acceleration of the fibroblast differentiation induced by mitomycin C by a factor of 10-30%. In view that fibroblasts play an essential role in skin aging and wound healing, the fibroblast differentiation system is a very useful tool in order to elucidate the efficacy of growth factors.

Co- and posttranslational modification of the alpha(1B)-adrenergic receptor: effects on receptor expression and function.

We have characterized the maturation, co- and posttranslational modifications, and functional properties of the alpha(1B)-adrenergic receptor (AR) expressed in different mammalian cells transfected using conventional approaches or the Semliki Forest virus system. We found that the alpha(1B)-AR undergoes N-linked glycosylation as demonstrated by its sensitivity to endoglycosidases and by the effect of tunicamycin on receptor maturation. Pulse-chase labeling experiments in BHK-21 cells demonstrate that the alpha(1B)-AR is synthesized as a 70 kDa core glycosylated precursor that is converted to the 90 kDa mature form of the receptor with a half-time of approximately 2 h. N-Linked glycosylation of the alpha(1B)-AR occurs at four asparagines on the N-terminus of the receptor. Mutations of the N-linked glycosylation sites did not have a significant effect on receptor function or expression. Surprisingly, receptor mutants lacking N-linked glycosylation migrated as heterogeneous bands in SDS-PAGE. Our findings demonstrate that N-linked glycosylation and phosphorylation, but not palmitoylation or O-linked glycosylation, contribute to the structural heterogeneity of the alpha(1B)-AR as it is observed in SDS-PAGE. The modifications found are similar in the different mammalian expression systems explored. Our findings indicate that the Semliki Forest virus system can provide large amounts of functional and fully glycosylated alpha(1B)-AR protein suitable for biochemical and structural studies. The results of this study contribute to elucidate the basic steps involved in the processing of G protein-coupled receptors as well as to optimize strategies for their overexpression.

Transcriptional regulatory patterns of the myelin basic protein and malic enzyme genes by the thyroid hormone receptors alpha1 and beta1.

While there is evidence that the two ubiquitously expressed thyroid hormone (T3) receptors, TRalpha1 and TRbeta1, have distinct functional specificities, the mechanism by which they discriminate potential target genes remains largely unexplained. In this study, we demonstrate that the thyroid hormone response elements (TRE) from the malic enzyme and myelin basic protein genes (METRE and MBPTRE) respectively, are not functionally equivalent. The METRE, which is a direct repeat motif with a 4-base pair gap between the two half-site hexamers binds thyroid hormone receptor as a heterodimer with 9-cis-retinoic acid receptor (RXR) and mediates a high T3-dependent activation in response to TRalpha1 or TRbeta1 in NIH3T3 cells. In contrast, the MBPTRE, which consists of an inverted palindrome formed by two hexamers spaced by 6 base pairs, confers an efficient transactivation by TRbeta1 but a poor transactivation by TRalpha1. While both receptors form heterodimers with RXR on MBPTRE, the poor transactivation by TRalpha1 correlates also with its ability to bind efficiently as a monomer. This monomer, which is only observed with TRalpha1 bound to MBPTRE, interacts neither with N-CoR nor with SRC-1, explaining its functional inefficacy. However, in Xenopus oocytes, in which RXR proteins are not detectable, the transactivation mediated by TRalpha1 and TRbeta1 is equivalent and independent of a RXR supply, raising the question of the identity of the thyroid hormone receptor partner in these cells. Thus, in mammalian cells, the binding characteristics of TRalpha1 to MBPTRE (i.e. high monomer binding efficiency and low transactivation activity) might explain the particular pattern of T3 responsiveness of MBP gene expression during central nervous system development.

Functions of the human papillomavirus type 16 E4 protein

1. Abstract Cervical cancer is thought to be the consequence of infection by human papillomaviruses (HPV). In the majority of cases, DNA from HPV type 16 (HPV16) is found in malignant cervical lesions. The initial steps leading to transformation of an infected cell are not clearly understood but in most cases, disruption and integration of the episomal viral DNA must take place. As a consequence, the E2 and E4 genes are usually not expressed whereas the E6 and E7 oncogenes are highly expressed. However, in a normal infection in which the viral DNA is maintained as an episome, all viral genes are expressed. The pattern according to which the viral proteins are made, and therefore the life cycle of the virus, is tightly linked to the differentiation process of the host keratinocyte. The study of the viral oncogenes E6 and E7 has revealed crucial functions in the process of malignant transformation such as degradation of the p53 tumor suppressor protein, deregulation of the Retinoblastoma protein pathway and activation of the telomerase ribonucleoprotein. All these steps are necessary for cancerous lesions to develop. However, the loss of the E2 gene product seems to be necessary for sufficient expression of E6 and E7 in order to achieve such effects. In normal infections, the E4 protein is made abundantly in the later stages of the viral life cycle. Though extensive amounts of work have been carried out to define the function of E4, it still remains unclear. In this study, several approaches have been used to try and determine the functions of E4. First, a cell-penetrating fusion protein was designed and produced in order to circumvent the chronic difficulties of expressing E4 in mammalian cells. Unfortunately, this approach was not successful due to precipitation of the purified fusion protein. Second, the observation that E4 accumulates in cells having modified their adhesion properties led to the hypothesis that E4 might be involved in the differentiation process of keratinocytes. Preliminary results suggest that E4 triggers differentiation. Last, as E4 has been reported to collapse the cytokeratin network of keratinocytes, a direct approach using atomic force microscopy has allowed us to test the potential modification of mechanical properties of cells harboring reorganized cytokeratin networks. If so, a potential role for E4 in viral particle release could be hypothesized. 2. Résumé Il a été établi que le cancer du col de l'utérus se développe essentiellement à la suite d'une infection par le virus du papillome humain (HPV). Dans la majorité des cas analysés, de l'ADN du HPV de type 16 (HPV16) est détecté. Les étapes initiales de la transformation d'une cellule infectée sont mal connues mais il semble qu'une rupture du génome viral, normalement épisomal, suivi d'une intégration dans le génome de la cellule hôte soient des étapes nécessaires dans la plupart des cas. Or il semble qu'il y ait une sélection pour les cas où l'expression des oncogènes viraux E6 et E7 soit favorisée alors que l'expression des gènes E2 et E4 est en général impossible. Par contre, dans une infection dite normale où le génome viral n'est pas rompu, il n'y pas développement de cancer et tous les gènes viraux sont exprimés. L'ordre dans lequel les protéines virales sont produites, et donc le cycle de réplication du virus, est intimement lié au processus de différentiation de la cellule hôte. L'étude des protéines oncogènes E6 et E7 a révélé des fonctions clés dans le processus de transformation des cellules infectées telles que la dégradation du suppresseur de tumeur p53, la dérégulation de la voie de signalisation Rb ainsi que l'activation de la télomérase. Toutes ces activités sont nécessaires au développement de lésions cancéreuses. Toutefois, il semble que l'expression du gène E2 doit être empêchée afin que suffisamment des protéines E6 et E7 soient produites. Lorsque le gène E2 est exprimé, et donc lorsque le génome viral n'est pas rompu, les protéines E6 et E7 n'entraînent pas de telles conséquences. Le gène E4, qui se trouve dans la séquence codante de E2, a aussi besoin d'un génome viral intact pour être exprimé. Dans une infection normale, le gène E4 est exprimé abondamment dans les dernières étapes de la réplication du virus. Bien que de nombreuses études aient été menées afin de déterminer la fonction virale à E4, aucun résultat n'apparaît évident. Dans ce travail, plusieurs approches ont été utilisées afin d'adresser cette question. Premièrement, une protéine de fusion TAT-E4 a été produite et purifiée. Cette protéine, pouvant entrer dans les cellules vivantes par diffusion au travers de la membrane plasmique, aurait permis d'éviter ainsi les problèmes chroniques rencontrés lors de l'expression de E4 dans les cellules mammifères. Malheureusement, cette stratégie n'a pas pu être utilisée à cause de la précipitation de la protéine purifiée. Ensuite, l'observation que E4 s'accumule dans les cellules ayant modifié leurs propriétés d'adhésion a suggéré que E4 pourrait être impliqué dans le procédé de différentiation des kératinocytes. Des résultats préliminaires supportent cette possibilité. Enfin, il a été montré que E4 pouvait induire une réorganisation du réseau des cytokératines. Une approche directe utilisant le microscope à force atomique nous a ainsi permis de tester une potentielle modification des propriétés mécaniques de cellules ayant modifié leur réseau de cytokératines en présence de E4. Si tel est le cas, un rôle dans la libération de particules virales peut être proposé pour E4.

Identification of novel HIV restriction factors

To efficiently replicate within mammalian cells, viruses have to manoeuvre through complex host mechanisms, hijacking a network of host proteins to achieve successful propagation. To prevent this invasion, cells have evolved over time to efficiently block the incursing pathogen by direct or indirect targeting. Human immunodeficiency virus (HIV) is a retrovirus of major global public health issue. In the last decade, extensive focus on innate immune proteins has been given, and particularly restriction factors, proteins inhibiting HIV replication by affecting various stages of the viral cycle. Because of the importance of developing new HIV therapies that are associated with reduced side effects and resistances, there is an urge to understand the antiviral response against HIV. Using common features of known restriction factors as a signature to identify new anti-HIV factors, candidates were identified. Particularly multiple members of the apolipoproteins L (APOL) family were found. Cotransfection experiments confirmed very potent inhibitory effects on HIV-1 expression. Further characterization of APOL6, the best candidate, was carried out. APOL6 was not able to inhibit HIV specifically but rather inhibited any gene-encoded DNA that was cotransfected and therefore APOL6 does not classify as a bona fide restriction factor. In addition, we were able to map the activity of APOL6 to the MAD domain and mainly to residue 174. We also found that other members of the family identified in the screen, APOL1 and 3, could have similar mechanism of action as APOL6. Finally, although the complete mechanism of action of APOL6 has yet to be elucidated, it might be blocked during transfections, potentially improving transfection of primary cells. -- Pour se répliquer efficacement dans les cellules de mammifères, les virus doivent manoeuvrer à travers des mécanismes cellulaires complexes et détourner un réseau de protéines de l'hôte. Pour empêcher cette invasion, les gènes de l'hôte ont évolué dans le temps pour cibler efficacement, directement ou indirectement, l'agent pathogène. Le virus de l'immunodéficience humaine (VIH) est un rétrovirus de problème majeur de santé publique mondiale, mais le faible risque de transmission du virus pourrait être expliqué par la présence d'un système antiviral de l'hôte qui, en cas d'échec, conduit à une infection productive. Durant la dernière décennie, il y a eu un intérêt spécial porté sur les protéines immunitaires innées appelé facteurs de restriction présentant des effets inhibiteurs puissants sur la réplication du VIH en affectant différentes étapes du cycle viral. En raison de l'importance de la recherche de nouvelles thérapies anti-VIH associées à des effets secondaires et des résistances réduites comparé aux traitements actuels, il existe un besoin de comprendre la réponse antivirale innée contre le VIH. Basé sur des caractéristiques communes des facteurs de restriction connus, nous avons proposé d'identifier de nouveaux facteurs anti-VIH. Nous avons trouvé une famille de protéines, les apolipoprotéines L (APOL) montrant les effets inhibiteurs très puissants contre l'expression du VIH-1 dans des expériences de co-transfection. Nous avons décidé d'approfondir le rôle de ces protéines dans l'immunité innée et de se concentrer sur le meilleur candidat APOL6. Nous avons en outre établi qu'APOL6 n'a pas d'activité anti-virale spécifique et donc pas classé comme un facteur de bonne foi de restriction. Par ailleurs, APOL6 est capable d'inhiber fortement l'expression de tout Plasmide cotransfecté. En outre, nous avons été en mesure de cartographier l'activité d'APOL6 au domaine MAD et principalement au résidu 174. Nous avons également constaté que d'autres membres de la famille identifiés dans l'étude, APOL1 et 3, pourraient avoir le même mécanisme d'action qu'APOL6. Enfin, bien que le mécanisme d'action complet d'APOL6 reste à être élucidé, il pourrait être d'une importance biotechnologique car il pourrait potentiellement faciliter la transfection de cellules primaires après l'inhibition d'APOL6.

The role of MAR elements in DNA recombination

The untargeted integration of foreign DNA into the mammalian cell genome, extensively used in gene therapy and biotechnology, remains an incompletely understood process. It is believed to be based on cellular DNA double strand break (DSB) repair machinery and to involve two major steps: i) the formation of long gene arrays (concatemers), and ii) recombination of the resulting concatemer with the genome. The main DSB repair pathways in eukaryotes include non-homologous end-joining (NHEJ), homologous recombination (HR), and microhomology-mediated end-joining (MMEJ). However, it is still not clear, which of these pathways are responsible for transgene integration. Here, we show that NHEJ is not the primary pathway used by mammalian cells in the transgene integration process, while the components of the HR pathway seem to be important for genomic integration but not concatemerization. Instead, concatemer formation appears to be mediated by a subset of the MMEJ pathway, termed synthesis-dependent MMEJ (SD-MMEJ). This mechanism also seems to be preferentially used for plasmid integration into the genome, as confirmed by the analysis of plasmid-to-genome junction sequences, which were found to display an SD-MMEJ pattern. Therefore, we propose the existence of two distinct SD-MMEJ subpathways, relying on different subsets of enzymes. One of these mechanisms appears to be responsible for concatemerization, while the other mechanism, partially dependent in HR enzymes, seems to mediate recombination with the genome. Previous studies performed by our group suggested that matrix attachment regions (MARs), which are epigenetic regulatory DNA elements that participate in the formation of chromatin boundaries and augment transcription, may mediate increased plasmid integration into the genome of CHO cells by stimulating DNA recombination. In the present work, we demonstrate that MAR-mediated plasmid integration results from the enhanced SD-MMEJ pathway. Analysis of transgene integration loci and junction DNA sequences validated the prevalent use of this pathway by the MAR elements to target plasmid DNA into gene-rich areas of the CHO genome. We propose that this finding should in the future help to engineer cells for improved recombinant protein production. In addition to investigating the process of transgene integration, we designed recombination assays to better characterize the components of the MMEJ and SD-MMEJ pathways. We also used CHO cells expressing cycle-sensitive reporter genes to demonstrate a potential role of HR proteins in the cell cycle regulation.

Characterization of a MAF1 knockout mouse model

L'ARN polymérase 3 transcrit un petit groupe de gènes fortement exprimés et impliqués dans plusieurs mécanismes moléculaires. Les ARNs de transfert ou ARNt représentent plus ou moins la moitié du transcriptome de l'ARN polymérase 3. Ils sont directement impliqués dans la traduction des protéines en agissant comme transporteurs d'acides aminés qui sont incorporés à la chaîne naissante de polypeptides. Chez des levures cultivées dans un milieu jusqu'à épuisement des nutriments, Maf1 réprime la transcription par l'ARN polymérase 3, favorisant ainsi l'économie énergétique cellulaire. Dans un modèle de cellules de mammifères, MAF1 réprime aussi la transcription de l'ARN polymérase 3 dans des conditions de stress, cependant il n'existe aucune donnée quant à son rôle chez un mammifère vivant. Pendant mon doctorat, j'ai utilisé une souris délétée pour le gène Maf1 afin de connaître les effets de ce gène chez un mammifère. Etonnamment, la souris Maf1-­‐/-­‐ est résistante à l'obésité même si celle-­‐ci est nourrie avec une nourriture riche en matières grasses. Des études moléculaires et de métabolomiques ont montré qu'il existe des cycles futiles de production et dégradation des lipides et des ARNt, ce qui entraîne une augmentation de la dépense énergique et favorise la résistance à l'obésité. En plus de la caractérisation de la souris Maf1-­‐/-­‐, pendant ma thèse j'ai également développé une méthode afin de normaliser les données de ChIP-­‐sequencing. Cette méthode est fondée sur l'utilisation d'un contrôle interne, représenté ici par l'ajout d'une quantité fixe de chromatine provenant d'un organisme différent de celui étudié. La méthode a amélioré considérablement la reproductibilité des valeurs entre réplicas biologiques. Elle a aussi révélé des différences entre échantillons issus de conditions différentes. Une occupation supérieure de l'ARN polymérase 3 sur les gènes Pol 3 chez les souris Maf1 KO entraîne une augmentation du niveau de précurseurs d'ARNt, ayant pour effet probable la saturation de la machinerie de maturation des ARNt. En effet, chez les souris Maf1 KO, le pourcentage d'ARNt modifiés est plus faible que chez les souris type sauvage. Ce déséquilibre entre le niveau de précurseurs et d'ARNt matures entraîne une diminution de la traduction protéique. Ces résultats ont permis d'identifier de nouvelles fonctions pour la protéine MAF1, comme étant une protéine régulatrice à la fois de la transcription mais aussi de la traduction et en étant un cible potentielle au traitement à l'obésité. -- RNA polymerase III (Pol 3) transcribes a small set of highly expressed genes involved in different molecular mechanisms. tRNAs account for almost half of the Pol 3 transcriptome and are involved in translation, bringing a new amino into the nascent polypeptide chain. In yeast, under nutrient deprivation, Maf1 acts for cell energetic economy by repressing Pol 3 transcription. In mammalian cells, MAF1 also represses Pol 3 activity under conditions of serum deprivation or DNA damages but nothing is known about its role in a mammalian organism. During my thesis studies, I used a Maf1 KO mouse model to characterize the effects of Maf1 deletion in a living animal. Surprisingly, the MAF1 KO mouse developed an unexpected phenotype, being resistant to high fat diet-­‐induced obesity and displaying an extended lifespan. Molecular and metabolomics characterizations revealed futile cycles of lipids and tRNAs, which are produced and immediately degraded, which increases energy consumption in the Maf1 KO mouse and probably explains in part the protection to obesity. Additionally to the mouse characterization, I also developed a method to normalize ChIP-­‐seq data, based on the addition of a foreign chromatin to be used as an internal control. The method improved reproducibility between replicates and revealed differences of Pol 3 occupancy between WT and Maf1 KO samples that were not seen without normalization to the internal control. I then established that increased Pol 3 occupancy in the Maf1 KO mouse liver was associated with increased levels of tRNA precursor but not of mature tRNAs, the effective molecules involved in translation. The overproduction of precursor tRNAs associated with the deletion of Maf1 apparently overwhelms the tRNA processing machinery as the Maf1 KO mice have lower levels of fully modified tRNAs. This maturation defect directly impacts on translation efficiency as polysomic fractions and newly synthetized protein levels were reduced in the liver of the Maf1 KO mouse. Altogether, these results indicate new functions for MAF1, a regulator of both transcription and translation as well as a potential target for obesity treatment.

Assaying the regulatory potential of mammalian conserved non-coding sequences in human cells.

BACKGROUND: Conserved non-coding sequences in the human genome are approximately tenfold more abundant than known genes, and have been hypothesized to mark the locations of cis-regulatory elements. However, the global contribution of conserved non-coding sequences to the transcriptional regulation of human genes is currently unknown. Deeply conserved elements shared between humans and teleost fish predominantly flank genes active during morphogenesis and are enriched for positive transcriptional regulatory elements. However, such deeply conserved elements account for <1% of the conserved non-coding sequences in the human genome, which are predominantly mammalian. RESULTS: We explored the regulatory potential of a large sample of these 'common' conserved non-coding sequences using a variety of classic assays, including chromatin remodeling, and enhancer/repressor and promoter activity. When tested across diverse human model cell types, we find that the fraction of experimentally active conserved non-coding sequences within any given cell type is low (approximately 5%), and that this proportion increases only modestly when considered collectively across cell types. CONCLUSIONS: The results suggest that classic assays of cis-regulatory potential are unlikely to expose the functional potential of the substantial majority of mammalian conserved non-coding sequences in the human genome.