Rozprawy doktorskie na temat „Nucleoid Organization”

Durant ce projet, nous nous sommes intéressés à une bactérie, D. radiodurans, un coque particulièrement connu pour ces extraordinaires capacités de résistance à différents facteurs de stress. Cependant, à cause de ses capacités de radiorésistance, cette bactérie a surtout été étudiée dans cette optique. Certaines caractéristiques de son cycle cellulaire restent méconnues, notamment (i) sa morphologie au cours de sa division ainsi que (ii) l’organisation et (iii) la ségrégation de son nucléoïde. Ces méconnaissances touchent aussi de façon plus générale toutes les bactéries de types coques, notamment de par la petite taille relative des bactéries qui a été un frein pour leurs études en microscopie photonique.Le but du projet de thèse est donc de mieux comprendre comment les bactéries sont capables d’avoir un nucléoïde très compacté, mais en même temps, dynamique et restant accessible pour les différents mécanismes tels que la réplication de l’ADN, sa transcription ou sa réparation. Dans ce but, nous avons exploré l’organisation en 4D ainsi que la dynamique du nucléoïde de D. radiodurans, en fonction du cycle de vie de la bactérie, de sa phase de croissance. Afin de réaliser ces objectifs, plusieurs stratégies ont été poursuivies : (i) des timelapses en 3D par microscopie confocale (ii) l’étude dynamique du nucléoïde par FRAP et par SptPALM, et (iii) la cartographie des protéines associées au nucléoïde réalisé par microscopie de super-résolution (PALM)
During this PhD work, we have studied on D. radiodurans, a coccus, known for its intriguing outstanding resistance to different stress factors. Studies on D. radiodurans have been mainly focusing on its tremendous radioresistance. 52 years after its discovery, its nucleoid organization/segregation as well as its cell morphology during its cell cycle still remain elusive. Most of our knowledge on the bacteria shape during division and on the nucleoid organization/segregation arises from the study of a small number of “model bacteria”, that are mainly rod-shaped or ovoid. In contrast, little is known on the nucleoid organization/segregation of cocci. Moreover, the small relative size of bacteria and of their nucleoids (<1µm3) has limited their studies by conventional microscopy.Thus, one of the aims of this PhD project is to contribute to a better understanding of the cell morphology and the nucleoid organization/segregation in cocci. For that matter, we explored the 4D organization and the dynamics of D. radiodurans nucleoids, as a function of the cell cycle progression and growth phase. In order to achieve the objective of this PhD, several strategies were undertaken: (i) timelapse 3D stacks by spinning confocal microscopy (ii) dynamics studies with FRAP analysis and SptPALM acquisitions, and (iii) cartographies of nucleoid associated proteins using super-resolution microscopy (PALM)

Mitochondria are key organelles in a variety of cellular functions such as ATP production, calcium homeostasis, cell differentiation, and apoptosis. All these functions are possible thanks to the complicated mitochondrial ultrastructure that results in the compartmentalization of the different biochemical processes. An example of this is the cristae compartment that host the oxidative phosphorylation machinery, existing a direct correlation between the cristae density and the respiratory capacity of mitochondria. Moreover, the mitochondrial ultrastructure is crucial for the mitochondrial function, indeed, thinner cristae favor the assembly of respiratory complexes in supercomplexes improving the respiratory efficiency. Therefore, the ultrastructure has become an indicator of the mitochondrial functional status and the study of specific ultrastructural parameters is currently used to describe a dysfunction. In this thesis, by using a dynamic complexomic analysis to identify new cristae remodeling modulators, we have recognized the protein ATAD3A as a highly interesting candidate to participate in the process. Although previous studies pointed to ATAD3A as a regulator of mitochondrial ultrastructure based on results obtained after silencing, no details about the molecular mechanism have been revealed. In addition, ATAD3A have been also implicated in many other cellular processes like cholesterol transport, apoptosis and nucleoid-mtDNA stability. The main goal of this work was to understand whether and how ATAD3A regulates mitochondrial structure and whether this is somehow correlated with its implication in mtDNA stability. To address these questions, we used a variety of biochemical and proteomic approaches, that in combination with proper genetic models have led us to the data presented in this work. We have identified by dynamic complexomic analysis three different complexes of ATAD3A at 1MDa, 500kDa and 250kDa. The observation that only the 500kDa complex is disrupted during the cristae remodeling, and that OPA1 was comigrating in the same disrupted complex, led us to hypothesize its involvement in the cristae maintenance. Indeed, ATAD3A silencing or overexpression was able to decrease or increase the number of cristae, respectively. Moreover, this effect was accompanied by alterations on the nucleoid and mtDNA copy number. By using specific ATAD3A mutant in the coiled-coil (CCD) or ATPase domain of ATAD3A we demonstrated that the ATPase is required for the cristae maintenance while the CCD seems to be involved in nucleoid organization. Our experiments also excluded the participation of the ATAD3A oligomerization in the cristae maintenance. The analysis of the ATAD3A complexes in presence of the different mutants further supported the involvement of the 500kDa complex in the process of cristae maintenance since the ATAD3A ATPase-mutant, that does not maintain cristae does not assemble on it. On the contrary, the CCD-mutant that induces cristae biogenesis assembles in the 500kDa complex. Our data also support the idea that ATAD3A 500kDa complex act as a scaffold protein that allows OPA1 to assemble together cooperating in the cristae biogenesis process. We have also studied the role of the ATAD3A 1MDa complex, which looking in our complexomic analysis, appeared enriched in mitoribosomal and other proteins suggested to be involved in nucleoid and mtDNA stability, suggesting the involvement in the process. To support this idea, cells lacking the mtDNA do not present the ATAD3A 1MDa complex, and also control treated shortly with EtBr display a significant reduction of the same complex. Since the CCD-mutant is not assembling in the 1MDa complex, we analyzed nucleoids in presence of the ATAD3A wild type or mutants after silencing of the endogenous protein. This results further confirm the involvement of the 1MDa in nucleoid organization since the CCD mutant exhibited a decrease in nucleoid are that was not present with the wild type or ATPase-mutant. Overall our data demonstrate a dual role of ATAD3A in cristae biogenesis and nucleoid-mtDNA stability that depends on the different domains: the coiled-coil domain is required for nucleoid organization and the ATPase domain for cristae biogenesis. Additional studies have been done in this PhD thesis to understand whether and how ATAD3B might be able or not to complement the absence of ATAD3A, or if as previously proposed it acts as a dominant negative for ATAD3A. Our work supports the latter hypothesis, that seems to be mediated by the direct interaction of ATAD3A and ATAD3B in the 1MDa complex. Surprisingly, our data show that ATAD3B can revert the pathological mitochondrial phenotype observed in cells expressing the ATPase mutant, suggesting that the nullifying effect of ATAD3B is not specific for the wild type protein. Further experiments are required to confirm these results.

Nombrosos estudis han constatat el valor adaptatiu del ric polimorfisme d’inversions cromosòmiques al drosofíl· lid D. subobscura. No obstant això, fins ara es coneixien molt poc les bases moleculars que hi ha darrere del seu manteniment a les poblacions naturals. En cercar loci candidats, un experiment previ de xoc tèrmic va quantificar els nivells de la proteïna Hsp70 en soques homocariotípiques dels ordenaments OST, O3+4+8 i O3+4. Inesperadament, els individus de l’ordenament càlid O3+4 mostraven nivells incrementats d’aquesta proteïna, en absència d’estrès tèrmic, que no augmentaven després del xoc tèrmic. Malauradament, en el moment en què es va dur a terme l’experiment hi havia moltes incògnites sobre l’organització molecular del locus Hsp70IR a D. subobscura. Els resultats prèviament esmentats, van donar peu al present treball de tesi, els objectius del qual són localitzar el locus Hsp70IR al cariotip i conèixer-ne l’organització genòmica, característiques moleculars i expressió gènica en diversos ordenaments cromosòmics d’interès que inclouen la regió genòmica on es troba la família gènica hsp70: O3+4+16+2, O3+4+8, O3+4 i OST. Gràcies a la seqüència d’un clon de la genoteca d’una línia OST i a còntigs del genoma de D. subobscura, hem pogut dissenyar una sonda a partir de la regió codificant de hsp70 que ens ha permès determinar la localització del locus on es troba aquesta família gènica (Hsp70IR) mitjançant hibridació in situ (ISH). Paral· lelament, hem pogut completar la seqüenciació d’una regió de 9-10 kb al locus Hsp70IR en 12 línies isogèniques per als ordenaments esmentats i a les espècies properes D. madeirensis i D. guanche per aclarir l’evolució d’aquest locus en els darrers 1,8 - 2,8 milions d’anys (Ma). Els resultats de la ISH van mostrar un únic punt d’hibridació a la banda 94A del segment distal (SI) del cromosoma O que coincidia els 4 cariotips estudiats: O3+4+16+2, O3+4+8, O3+4 i OST. Les seqüències corresponents a les 12 línies isogèniques i a D. madeirensis i D. guanche indiquen que en aquestes tres espècies del clúster subobscura, el locus Hsp70IR consta de dues còpies paràloges de 2,5 – 3,0 kb en orientació divergent i separades per una regió espaiadora central no duplicada de 0,5 – 1,4 kb. Les dues còpies mostren un elevat grau de conservació entre els diferents ordenaments i espècies analitzats, mentre que la regió espaiadora central és altament polimòrfica. Entre els aspectes més rellevants de l’anàlisi del polimorfisme, destaquem l’elevada conservació de les regions codificadores (CDSs) i els diferents elements reguladors en cis (CREs) al promotor proximal de tots els gens hsp70 analitzats, que indicarien que aquests són funcionals a totes les línies estudiades, i que la seva regulació podria ser similar. Curiosament, a nivell de seqüència, les regions paràlogues del promotor proximal i el CDS tendeixen a ser significativament més similars dins del mateix ordenament i, en alguns casos, dins la mateixa línia, probablement com a resultat de conversió gènica ectòpica. Per últim, hem dut a terme la quantificació dels nivells basals de mRNA i proteïna en mascles i femelles adults de sis línies isogèniques per a l’ordenament fred OST i sis per a l’ordenament càlid O3+4. La quantificació dels nivells de mRNA indica que els nivells són similars entre els dos ordenaments però en canvi aquests difereixen entre mascles i femelles de l’ordenament càlid O3+4. Així mateix, la quantificació dels nivells de la proteïna Hsp70 suggereix que no hi ha diferències entre sexes ni entre els dos ordenaments, però en canvi observem una interacció significativa entre sexe i ordenament. Aquests resultats, tant per a mRNA com per a proteïna, indiquen que l’expressió de hsp70 podria estar influïda pel sexe.
Numerous studies have confirmed the adaptive value of the rich chromosomal inversion polymorphism in the drosophilid D. subobscura. However, until recently very little was known about the molecular basis behind its maintenance in natural populations. In search of candidate loci, a previous heat shock experiment quantified Hsp70 protein levels in homokaryotypic strains for the OST, O3+4+8 and O3+4 arrangements. Unexpectedly, individuals of the warm climate-associated O3+4 arrangement showed increased levels in absence of thermal stress that did not boost after the heat shock. Unfortunately, by the time this experiment was performed there was very little data available on the molecular organization of the Hsp70IR locus in D. subobscura. The previously mentioned results led to the present thesis work, whose objectives are to locate the Hsp70IR locus in the karyotype and to know the genomic organization, molecular characteristics and gene expression patterns in several representative chromosomal arrangements that comprise the genomic region where the hsp70 gene family is located: O3+4+16+2, O3+4+8, O3+4 and OST. Using the sequence of a clone from an OST line genomic library and contigs from the unassembled genome of D. subobscura, we designed a probe from the hsp70 coding region that enabled us to determine the location of the locus by in situ (ISH) hybridization. Concomitantly, we completed the sequencing of a 9-10 kb region in the Hsp70IR locus in 12 lines isogenic for the aforementioned arrangements and in D. madeirensis and D. guanche to shed light on the evolution of this locus in the last 1.8 - 2.8 million years (myr). ISH results showed a single hybridization site in the 94A band in the distal segment (SI) of the O chromosome coincident in the 4 studied karyotypes: O3+4+16+2, O3+4+8, O3+4 and OST. The sequences corresponding to the 12 isogenic lines and to D. madeirensis and D. guanche indicated that in these three species of the subobscura cluster, the Hsp70IR locus consists of two 2.5 - 3.0 kb long paralogous copies in divergent orientation separated by a 0.5 - 1.4 kb nonduplicated central spacer region. The two copies show a high degree of conservation between the different gene arrangements and species analyzed, while the central spacer region is highly polymorphic. Among the most relevant aspects of polymorphism analyses, we highlight the high degree of conservation in the coding regions (CDSs) and the cis-regulatory elements (CREs) in the proximal promoter of all the analyzed hsp70 genes, which might indicate that these are functional in all studied lines, and that their regulation might be similar. Curiously, at the sequence level, the paralogous 5'-UTR and CDS regions tend to be significantly more similar within the same arrangement and, in some cases, within the same line, probably as a result of ectopic gene conversion. Lastly, we carried out the quantification of basal hsp70 mRNA and protein levels in adult males and females of six lines isogenic for the cold climate-associated OST and six for the warm climate-associated O3+4 arrangements. Basal mRNA quantification results indicate that the two arrangements exhibit similar levels, yet significant differences are observed between males and females of the warm O3+4 arrangement. Regarding the quantification of basal Hsp70 protein levels, these suggest that there are no differences between sexes nor between the two arrangements, but instead we observe a significant interaction between sex and arrangement. Overall, the results for both, mRNA and protein data, indicate that hsp70 expression might be influenced by sex.

The skeletal muscle fibre is a syncitium where each myonucleus regulates the gene products in a finite volume of cytoplasm i.e., the myonuclear domain (MND). A novel image analysis algorithm applied to confocal images, analyzing MND size and myonuclear spatial distribution in 3-dimensions in single skeletal muscle fibres has been used in this project. The goal was to explore the modulation of myonuclei count and MND size in response to muscle adaptation processes. The effects of ageing, gender, hormones, muscle hypertrophy and body size were investigated on MND size. A strong linear relationship was found between MND size and body size in the muscle fibres from mammals representing a 100,000-fold difference in body size. Independent of species, MND size was highly dependent on MyHC isoform type and mitochondrial contents of skeletal muscle fibres. In hypertrophic mice, a significant effect of MND size on specific force and myosin content was observed. This effect was muscle fibre type-specific and shows that the bigger MNDs in fast-twitch EDL muscle fibres are optimally tuned for force production while smaller MNDs in slow-twitch soleus muscle fibres have a much more dynamic range of hypertrophy without functional compromise. This indicates a critical volume individual myonuclei can support efficiently for a proportional gain in muscle fibre force and size. In human muscle fibres, spatial organization of myonuclei was affected by both ageing and MyHC isoform expression. In fibres expressing type I MyHC isoform, an increased MND size variability and myonuclear aggregates were observed in old age although average MND size was unchanged. In contrast, in type IIa fibres, the average MND size was smaller reflecting smaller size of muscle fibres. Those changes may influence the transcriptional activity per myonucleus and/or local cooperatively of myonuclei in a gender and muscle fibre-type specific manner. Finally, hormone replacement therapy was shown to negate menopause-related functional impairment in skeletal muscle fibres. The positive effect on force was due to quantitative effect in fibres expressing fast myosin isoform while the effect was both quantitative and qualitative in fibres expressing slow myosin isoform. The effect on MND size was fibre type dependent and was achieved by significantly reducing domain size in slow- but not the fast-twitch muscle fibres. Together, our data suggest that modulation of myonuclei count and MND size is a mechanism contributing to remodelling of skeletal muscle in muscle adaptation process. These findings should be considered when developing therapeutic approaches towards restoring muscle mass and strength in muscle wasting conditions.

It has been well documented that mRNA is associated with the cytoskeleton, and that this relationship is involved in translation and mRNA sorting. The molecular components involved in the attachment of mRNA to the cytoskeleton are only poorly understood. The objective of this thesis was to directly visualize the interaction of mRNA with the cytoskeleton, with sufficient resolution to identify the filament systems and structures involved. This work required the development of novel in situ hybridization methods for use with electron microscopy. This allowed resolution to visualize single mRNA molecules and individual filaments. The development of a silver enhancement methodology for both the light and electron microscopic detection of biotinated oligo-dT probes permitted a synoptic view of the intracellular distribution of poly(A) mRNA. At the light microscope, the distribution of poly(A) mRNA did not resemble the individual distribution patterns of microfilaments, intermediate filaments or microtubules. Ultrastructural examination revealed that poly(A) mRNA was not uniformly distributed along cytoskeletal filaments, but clustered at their intersections. The composition of these mRNA containing structures was investigated by both morphologic and in situ hybridization analysis using antibodies to cytoskeletal proteins. In thin sections, polysomes were observed attached to both microfilaments and intermediate filaments. To permit the simultaneous detection of oligo-dT hybridization and specific cytoskeletal proteins, a double labelling method using colloidal gold conjugated antibodies was developed. The majority of poly(A) mRNA was associated with the actin cytoskeleton, with 72% of the hybridization localized within 5nm of a labelled microfilament. Within the actin cytoskeleton, poly(A) mRNA was localized to intersections of orthogonal networks. Greater than 50% of poly(A) colocalized with the actin crosslinking proteins, filamin and α-actinin, but not vinculin. A significant amount of poly(A) mRNA was found to be associated with intermediate filaments. The double label gold analysis demonstrated that 33% of the hybridization signal localized within 5nm of labelled vimentin filaments. Prior disorganization of the actin cytoskeleton using cytochalasin did not disrupt the association of mRNA with vimentin. These observations are consistent with our morphologic results of polysome-intermediate filament associations, and indicate that microfilaments are not the only filament system to which mRNA is bound. Furthermore, a small amount of hybridization signal (12%) consistently was observed along microtubules, providing an additional cytoskeletal network to distribute mRNA. To further characterize the spatial organization of mRNA within the cytoskeleton, ultrastructural methods were developed to directly visualize individual mRNA molecules. First, oligonucleotide probes chemically modified with a single hapten and directly conjugated primary reagents were used to permit detection of an individual hybridized probe molecule by a single gold particle. Second, biotin and digoxigenin labelled oligonucleotide probes were used to simultaneously visualize the intermolecular and intramolecular relationships of two nucleic acid sequences. Third, reverse transcriptase was used to extend hybridized primers in situ which permitted visualization of the poly(A) sequence concomittant with the conformation of an mRNA molecule. These methods have permitted analysis of how single mRNA molecules may be positioned with respect to each other within the cytoskeleton. The ultrastructural visualization of mRNA within its structural environment has demonstrated heterogeneous interactions with the cytoskeleton. Future work will be needed to further characterize the mechanism of mRNA attachment. The proteins which bridge nucleic acid sequences to specific intersections can be identified. It will be interesting to learn how the identified mRNA-cytoskeletal interactions might be involved in the regulation of both mRNA translation and intracellular location. Lastly, and perhaps the most challenging goal, is to investigate whether the identified mRNA-cytoskeletal interactions are used by the cell to influence its own shape, polarity and architecture.

La recrudescence du paludisme a plasmodium falciparum, et l'apparition de formes chloroquino-resistantes ont montre la necessite d'une connaissance plus approfondie des mecanismes de controle de la multiplication et de la differenciation du parasite. Les proteines g et les proteines kinases ont un role essentiel dans la modulation de l'activite de nombreuses enzymes en reponse a des signaux developpementaux, environnementaux ou metaboliques. La mise en evidence de proteines g au cours de notre etude initiale a montre que des voies de signalisation intracellulaires semblables a celles des eucaryotes superieurs existaient chez plasmodium. Ceci nous a amene a etudier egalement les proteines kinases. Les travaux rapportes dans ce memoire concernent l'identification, par pcr a l'aide d'oligonucleotides degeneres, et la caracterisation moleculaire d'une nouvelle proteine serine/threonine kinase plasmodiale de type snf1 que nous avons denommee pfkin. La sequence complete du gene a ete obtenue a partir de clones genomiques et d'adnc. Pour cloner l'extremite 5' du gene la pcr inverse a ete utilisee. Le gene, present a raison d'une copie par genome localisee sur le chromosome xiii, comprend 2298 pb et code pour une proteine de poids moleculaire estime de 90 845 da dont le domaine catalytique contient 40% de residus identiques avec celui de la proteine kinase snf1 de saccharomyces cerevisiae. L'expression preferentielle de pfkin au stade gametocyte, stade terminal du developpement chez l'homme, a ete determinee par rt-pcr. Les proteines kinases apparentees a snf1 constituent un groupe d'enzymes de regulation dont une fonction est de proteger la cellule des stress environnementaux ou metaboliques. Nous proposons un role de pfkin dans la reponse adaptative du gametocyte lors de sa transmission depuis l'hote vertebre homeotherme jusqu'a l'intestin du vecteur poikilotherme

Trois genes de trna**(tyr) ont ete identifies et sequencies. L'analyse de leur sequence nucleotidique a montre que l'un des genes coderait pour un trna**(tyr) fonctionnel (trna**(tyr-2)) et les deux autres seraient des pseudogenes. Le trna**(tyr-2) derive probablement d'un gene trna**(phe) par des evenements de recombinaison genetique. La sequence nucleotidique de deux genes de trna mitochondriaux codant pour un trna**(ser) et un trna**(glu) respectivement a ete determinee. Les deux genes ont une homologie de 99% avec leurs homologues mitochondriaux d'autres plantes

Trois genes codant pour l'histone h3 et trois genes codant pour l'histone h4 ont ete isoles dans une banque genomique de mais a l'aide de sondes d'oursin, et leurs sequences nucleotidiques ont ete etablies. Ces genes ne renferment pas d'introns et presentent une utilisation des codons tres biaisee en faveur des bases c et g. Un octanucleotide conserve dans les six genes etudies ainsi que dans les qutres genes d'histones de plantes sequences, a ete mis en evidence 200 a 250 nucleotides en amont de l'atg. Les arn messagers transcrits possedent de longues extremites non traduites en 3' et sont polyadenyles. Les genes h3 et h4 existent en 30 a 40 et 50 a 60 copies respectivement par genome haploide. Ces deux familles multigeniques sont organisees en "sous famille

Deux genes d'histones h3 et deux genes h4 ont ete clones a partir d'une banque genomique d'arabidopsis thaliana, et ont ete sequences. Les 2 genes h3 codent pour une meme histone bien que leurs sequences codante aient 85% d'homologie seulement. L'environnement de ces 2 genes est divergent. A l'exception d'un gene h3, les regions en amont de ces genes contiennent les sequences consensus classiques des genes d'histones. Un octanucleotide conserve "cgcggatc" specifique des genes d'histones de plante est trouve, dans les quatre genes etudies (200 a 250 nucleotides en amont). La reiteration des genes histones h3 et h4 d'arabidopsis a ete estimee a 5 a 7 copies par genome haploide. Il existe un polymorphisme peu important qui se limite uniquement a un gene h3 et deux genes h4. Par comparaison a l'organisation des genes d'histones des cereales, le petit genome d'arabidopsis pouvant etre une image simplifiee de la topologie et de l'evolution des genes d'histones dans les genomes de plantes supercenies

Les genes p1 avec les genes lsp1, lsp2 et p6 appartiennent a un petit groupe de genes qui sont exprimes specifiquement dans le corps gras des larves de troisieme stade de drosophila melanogaster. L'utilisation d'un mutant thermosensible, le mutant ecdl, a montre que la transcription du gene p1 etait inductible pour les ecdysteroides. La structure et organisation du gene ont ete etablies. La technique de transformation de la lignee germinale par l'element p et la technique de fusion des genes ont permis de cartographier les sequences de regulation qui agissent en cis sur l'expression de ce gene dans un fragment de 184 pb s'etendant sur 140 pb en amont du site d'initiation de la transcription

On procede au sequencage du genome complet du virus constitue d'un arn de polarite positive a 6318 nucleotides et contenant 3 cistrons. Parmi les strategies mises en oeuvre pour synthetiser ses proteines, on presente la maturation proteolytique de la proteine 206 k codee par ce virus. Une approche "arn-sens" pour la lutte anti-virale est testee experimentalement: des arn "pseudogenomiques" rentrent en competition avec l'arn du virus et sont capables d'inhiber sa replication in vitro

Le virus 3 grenouille (fv3), famille des iridoviridae, a un genome d'adn double brin de 160 kb hautement methyle, a redondances terminales et circulairement permute. Une arn polymerase ii de la cellule hote est impliquee dans la transcription precoce, mais l'adn viral purifie n'est pas infectieux, il est reactive par des proteines du virion. L'expression des genes est sequentielle. L'ordre des genes est determinee par selection des arns par hybridation aux recombinants suivi de leur traduction in vitro. L'organisation de transcripts est obtenue par cartographie a la nuclease s1. La comparaison des extremites 3'des arns revele la presence d'une sequence repetee inversee. Les sequences regulant l'initiation et la terminaison de la transcription sont voisines, sinon communes

Le papovavirus de hamster (hapv) possede un tropisme restreint in vivo vis a vis des keratinocytes et des lymphocytes. Il se replique dans les tumeurs cutanes qui apparaissent chez des hamsters syriens, et induit egalement des lymphomes chez le hamster. L'organisation genetique du hapv deduite de sa sequence a montre qu'il appartient a la famille des polyomavirus. Le hapv est present dans les lymphomes sous forme de multiples copies libres possedant toujours une deletion localisee dans la meme region du genome. Les signaux de transcription precoce du hapv semblent etre actives par la region precoce de ce virus

To counteract the potentially calamitous effects of genomic instability in the form of double-strand breaks (DSBs), cells have evolved with two major mechanisms. First, DNA non¬homologous end joining (NHEJ) which requires no significant homology, and second, homologous recombination (HR) that uses intact sequences on the sister chromatid or homologous chromosome as a template to repair the broken DNA. Although NHEJ repairs DSBs in all stages of cell cycle; it is generally error-prone due to insertions or deletions of few nucleotides at the breakpoint. In contrast, DSBs that are generated during S and G2 phase of the cell are preferentially repaired by HR that utilizes neighboring sister chromatid as a template. A central role in the HR reaction is promoted by the RAD51 recombinase which polymerizes onto single-stranded DNA (ssDNA), catalyzes pairing and strand invasion with homologous DNA molecule. Assembly of RAD51 monomers onto ssDNA is a relatively slow process and is facilitated by several mediator proteins. The tumor suppressor protein BRCA2 is the best-characterized RAD51 mediator in DSB repair by HR. Many reports in the past two decades have established that RAD51 recruitment at break sites also depends on the RAD51 paralogs. Mammalian cells encode five RAD51 paralogs; RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3 which share 20–30% identity at amino acid level with RAD51 and with each other. In addition to their role in HR, RAD51 paralogs have been identified to be involved in DNA damage signaling and replication fork maintenance. In addition, mouse knockout of RAD51 paralogs causes early embryonic lethality. Recent studies show that germline mutations in all five RAD51 paralogs cause various types of cancer including breast and ovarian cancers. Pedigree analyses revealed that similar to BRCA1 and BRCA2, pathological missense mutants of RAD51C were of high penetrance. Historically, defects in the DNA repair pathways have been exploited for cancer chemo-and radiotherapy. In an attempt to develop better cancer therapeutic approaches, the concept of synthetic lethality for cancer therapy has been recently proposed. One such example is the use of PARP1 inhibitors to treat tumors carrying mutations in HR genes, such as BRCA1 and BRCA2. Inhibition of PARP1 compromises single-strand break repair (SSBR) pathway. Upon replication fork collision, the accumulated SSBs are converted to one-ended DSBs, which are efficiently repaired by the HR for cell survival. As a result, HR-deficient tumors with BRCA1-or BRCA2-deficiency exhibit extreme sensitivity to PARP-1 inhibition resulting in cell death. This approach was highly successful in targeting tumors with severe defects in Fanconi anemia (FA)-BRCA proteins which led to PARP inhibitors being tested in clinical trials. However, targeting cancer cells that express hypomorphic mutants of HR proteins is highly challenging since such partially functional mutants require a high dosage of PARP inhibitors for effective sensitization which renders normal cells toxic and can also lead to tumor resistance. The pathological RAD51C mutants that were identified in breast and ovarian cancer patients are hypomorphic with partial repair function. The first part of my Ph.D. thesis is aimed at developing an effective strategy to target cells that express hypomorphic RAD51C mutants. To this end, we used RAD51C deficient CL-V4B hamster cells and expressed the pathological RAD51C mutants associated with breast and ovarian cancers. Cells expressing RAD51C mutants that were severely defective for HR function exhibited high sensitivity to low doses of PARP1 inhibitor (4-ANI). These cells also accumulated in G2/M and displayed chromosomal aberrations. However, RAD51C mutants that were hypomorphic were partially sensitized even at higher concentrations of PARP inhibitor. RAD51C/ CL-V4B cells displayed higher PARP activity compared WT V79B cells. Notably, PARP activity was directly proportional to the sensitivity of RAD51C mutants towards 4-ANI where highly sensitive RAD51C mutants showed higher PARP activity and vice versa. Increased PARP activity was associated with replication stress as confirmed by an increase of PARP activity in cells treated with replication stress inducer, hydroxyurea (HU). Notably, treatment of CL-V4B cells with PARP1 inhibitor (4-ANI) resulted in the accumulation of PARP1 onto the chromatin which eventually led to the formation of DSBs which suggests that PARP1 entrapment triggers replication fork collapse leading to one-ended DSBs in S-phase. To further understand the molecular mechanism of PARP inhibitor-induced toxicity of RAD51C deficient cells, we carried out chromatin fractionation from V79B and CL-V4B cells at varying time points of 4-ANI treatment. Surprisingly, there was an enhanced loading of NHEJ proteins on chromatin in CL-V4B compared to V79B cells. Consistently, an increased error-prone NHEJ was observed in CL-V4B cells which resulted in increased chromosomal aberrations and cell death. Furthermore, inhibition of DNA-PKcs or depletion of KU70 or Ligase IV restored this phenotype. Thus, error-prone NHEJ in collaboration with PARP inhibition sensitizes RAD51C deficient cells. Since ionizing radiation (IR) is known to stimulate NHEJ activity, we hypothesized that irradiation in combination with PARP inhibitor would further sensitize the RAD51C deficient tumors. Strikingly, stimulation of NHEJ by a low dose of IR in the PARP inhibitor-treated RAD51C deficient cells and cells expressing pathological RAD51C mutants induced enhanced toxicity ‘synergistically’. These results demonstrate that cancer cells arising due to hypomorphic mutations in RAD51C can be specifically targeted by a ‘synergistic approach’ and imply that this strategy can be potentially applied to cancers with hypomorphic mutations in other HR pathway genes. In addition to nuclear functions, RAD51 paralogs RAD51C and XRCC3 have been shown to localize to mitochondria and contribute to mitochondrial genome stability. However, the molecular mechanism by which RAD51 and RAD51 paralogs carry out this function is unclear. The second part of my thesis was dedicated to studying whether RAD51C/XRCC3 facilitates mitochondrial DNA replication and the underlying mechanism by which RAD51C/XRCC3 participate in mitochondrial genome maintenance during unperturbed conditions. Using mitochondrial subfractionation we show that RAD51 and RAD51 paralogs (RAD51C and XRCC3) are an integral part of mitochondrial nucleoid and absence of RAD51C/XRCC3 and RAD51 prevents the restoration of mtDNA upon depletion of mtDNA. This suggests that RAD51 and RAD5C/XRCC3 participate in mtDNA replication. To determine whether this function of RAD51C is exclusive to mitochondria we expressed NLS mutant of RAD51C which was defective for nuclear functions. Interestingly, cells expressing RAD51C R366Q were able to efficiently repopulate the depleted mtDNA after EtBr stress similar to that of WT RAD51C expressing cells, suggesting a nuclear independent function of RAD51C in mitochondrial genome maintenance. mtDNA-IP analysis revealed that RAD51 and RAD51C/XRCC3 are recruited to the mtDNA control regions spontaneously along with mitochondrial polymerase POLG. Moreover, RAD51 was found to associate with TWINKLE helicase and this association was required for the recruitment of RAD51 and RAD51C/XRCC3 at the D-loop. As in nucleus, mtDNA replisome also encounters replication stresses like altered dNTP pools, a collision between replication and transcription machinery, rNTP incorporation, oxidative stress which hampers replication fork progression. Using Dideoxycytidine (ddC) as replication stress inducer in mitochondria, we observed nearly 3-4 fold enrichment of RAD51, RAD51C, XRCC3 and POLG at the mtDNA mutation hotspot region D310. Notably, RAD51C/XRCC3 deficient cells exhibited increased lesions in the mitochondrial genome spontaneously, pointing towards the importance of RAD51C/XRCC3 in the prevention of mtDNA lesions. Moreover, RAD51C/XRCC3 deficiency prevented the repair of ddC induced mtDNA lesions. Given that RAD51C/XRCC3 and RAD51 are localized to mtDNA control regions along with POLG and their deficiency affects mtDNA replication we were curious to learn the effect of RAD51C/XRCC3 deficiency on the recruitment of POLG in mtDNA. To test this we performed a mtDNA-IP assay of POLG in RAD51C deficient cells which revealed that deficiency of RAD51C/XRCC3 and RAD51 affected the recruitment of POLG on mtDNA control regions. As a consequence RAD51C/XRCC3 deficient cells exhibit aberrant mitochondrial functions. These findings propose a mechanism for a direct role of RAD51C/XRCC3 in maintaining the mtDNA integrity under replication stress conditions.

The centromere is essential for chromosome segregation and genome stability. It is the site of kinetochore assembly and chromosome attachment to the spindle microtubules, and it is important for chromosome movement during mitosis and meiosis. Normal human chromosomes have one centromere, but genome rearrangements that occur with instability, aging, and disease often result in chromosomes with two centromeres, called dicentrics. Nearly seventy-five years ago, Barbara McClintock demonstrated that dicentric chromosomes in plants are associated with instability through mitotic "breakage-fusion-bridge" cycles. However, human dicentrics are unusually stable due to the poorly understood phenomenon of centromere inactivation. Centromere inactivation has been primarily studied in patient-derived dicentrics, limiting the derivation of a molecular pathway. Key centromere and kinetochore proteins are not present at inactive centromeres, but beyond these observations, the process of centromere inactivation is unclear. Epigenetic and sequence-dependent factors are known to contribute to centromere specification, but requirements for centromere assembly, maintenance, and suppression remain obscure. The aims of this research were to (1) determine the mechanism(s) by which de novo dicentric chromosomes are stabilized, (2) ascertain the factors influencing the involvement of specific chromosomes in de novo fusions, and (3) establish the epigenomic, temporal, and mechanistic basis of centromere inactivation. To uncover the mechanistic foundations of these processes, we developed in vitro cell culture systems to study the formation and stabilization of de novo dicentrics. We demonstrate that transient disruption of human telomere structure non-randomly produces dicentric fusions involving acrocentric chromosomes. This finding is notable since the most prevalent rearrangement in humans involves the acrocentrics and is called Robertsonian translocation (ROB). In some cases, centromere inactivation occurs by an apparently epigenetic mechanism. In other dicentrics, the size of the centromeric DNA array is reduced compared to the same array before dicentric formation. Many functional dicentrics persist for months after formation. Our results indicate that dicentric human chromosomes undergo alternative fates after formation across a broad temporal window. During transient telomere disruption, we observed a dramatic change in nucleolar appearance. Nucleolar proteins did not coalesce into condensed structures, but appeared dispersed throughout the nucleus. This surprising alteration in nucleolar organization and nuclear architecture suggests remodeling of the nucleolus and subsequent effects on nucleolar-associated chromosomes, such as the acrocentrics, could contribute to the high incidence of ROB formation. Further studies and development of additional cell culture systems will allow us to evaluate current models of centromere assembly and disassembly and the importance of chromatin organization to centromere function and genome architecture.

Dissertation

Mitochondrion is an endosymbiotic organelle synthesizing ~1% of its proteome, while remaining ~99% of the proteins are encoded by the nuclear genome and translated on the cytosolic ribosome. Therefore active mitochondrial biogenesis requires efficient protein transport destined for the different sub-compartments. Mitochondrion contains specialized translocation machineries in the outer and in the inner membrane known as TOM40 and TIM23-complex respectively. Import of a majority of mitochondrial proteome is mediated by inner membrane presequence translocase (TIM23 complex). However, the structural organization of Tim23-complex and mechanisms of mitochondrial inner membrane protein translocation is still elusive. Therefore, the present thesis addresses above elusive questions. Chapter 2 highlights the functional significance of different segments of Tim23 in regulating the conformational dynamics of the presequence-translocase- Tim23 is the central channel forming subunit of the presequence-translocase which recruits additional components for the assembly of the core complex. However the functional significance of different segments of Tim23 was not understood due to the lack of suitable conditional mutants. Our study has reported many conditional mutants from different segments of Tim23 which are precisely defective in the organization of the core complex and in the recruitment of the import motor component which enhances our understanding of protein translocation across mitochondrial inner membrane. Chapter 3 highlights the functional cooperativity among mtHsp70 paralogs and orthologs using Saccharomyces cerevisiae as a model organism- mtHsp70s are implicated in a broad spectrum of functions inside the mitochondria. In case of lower eukaryotes gene duplication event has given rise to multiple copies of Hsp70s thereby presenting an opportunity of division of function among these paralogs. The mitochondria of yeast Saccharomyces cerevisiae contains three Hsp70s, including Ssc1, Ssq1 and Ssc3 (Ecm10). The Ssc1 is essential for protein translocation and de novo protein folding functions while Ssq1 is needed for the Fe/S cluster biogenesis inside the mitochondria. Although it has been proposed earlier that, Ssc1 and Ssc3 possesses overlapping functions in protein translocation as a part of import motor in the Tim23-complex. However the physiological relevance and experimental evidences in favor above hypothesis was not established clearly. Our study has reported Ssc3 as an ‘atypical chaperone’ which cannot perform the generalized chaperone functions due to the conformational plasticity associated with both the domains of Ssc3 resulting into weaker client protein affinity, altered interaction with cochaperones and dysfunctional allosteric interface. Additionally, we have also highlighted the role of Nucleotide-binding domain in determining the functional specificity among Hsp70 paralogs and orthologs.

Mitochondrion is an endosymbiotic organelle synthesizing ~1% of its proteome, while remaining ~99% of the proteins are encoded by the nuclear genome and translated on the cytosolic ribosome. Therefore active mitochondrial biogenesis requires efficient protein transport destined for the different sub-compartments. Mitochondrion contains specialized translocation machineries in the outer and in the inner membrane known as TOM40 and TIM23-complex respectively. Import of a majority of mitochondrial proteome is mediated by inner membrane presequence translocase (TIM23 complex). However, the structural organization of Tim23-complex and mechanisms of mitochondrial inner membrane protein translocation is still elusive. Therefore, the present thesis addresses above elusive questions. Chapter 2 highlights the functional significance of different segments of Tim23 in regulating the conformational dynamics of the presequence-translocase- Tim23 is the central channel forming subunit of the presequence-translocase which recruits additional components for the assembly of the core complex. However the functional significance of different segments of Tim23 was not understood due to the lack of suitable conditional mutants. Our study has reported many conditional mutants from different segments of Tim23 which are precisely defective in the organization of the core complex and in the recruitment of the import motor component which enhances our understanding of protein translocation across mitochondrial inner membrane. Chapter 3 highlights the functional cooperativity among mtHsp70 paralogs and orthologs using Saccharomyces cerevisiae as a model organism- mtHsp70s are implicated in a broad spectrum of functions inside the mitochondria. In case of lower eukaryotes gene duplication event has given rise to multiple copies of Hsp70s thereby presenting an opportunity of division of function among these paralogs. The mitochondria of yeast Saccharomyces cerevisiae contains three Hsp70s, including Ssc1, Ssq1 and Ssc3 (Ecm10). The Ssc1 is essential for protein translocation and de novo protein folding functions while Ssq1 is needed for the Fe/S cluster biogenesis inside the mitochondria. Although it has been proposed earlier that, Ssc1 and Ssc3 possesses overlapping functions in protein translocation as a part of import motor in the Tim23-complex. However the physiological relevance and experimental evidences in favor above hypothesis was not established clearly. Our study has reported Ssc3 as an ‘atypical chaperone’ which cannot perform the generalized chaperone functions due to the conformational plasticity associated with both the domains of Ssc3 resulting into weaker client protein affinity, altered interaction with cochaperones and dysfunctional allosteric interface. Additionally, we have also highlighted the role of Nucleotide-binding domain in determining the functional specificity among Hsp70 paralogs and orthologs.

Indiana University-Purdue University Indianapolis (IUPUI)
My thesis comprises of two individual projects: 1) we have developed a database for operon prediction using high-throughput sequencing datasets for bacterial genomes. 2) Genomics and mechanistic insights of convergent transcription in bacterial genomes. In the first project we developed a database for the prediction of operons for bacterial genomes using RNA-seq datasets, we predicted operons for bacterial genomes. RNA-seq datasets with different condition for each bacterial genome were taken into account and predicted operons using Rockhopper. We took RNA-seq datasets from NCBI with distinct experimental conditions for each bacterial genome into account and analyzed using tool for operon prediction. Currently our database contains 9 bacterial organisms for which we predicted operons. User interface is simple and easy to use, in terms of visualization, downloading and querying of data. In our database user can browse through reference genome, genes present in that genome and operons predicted from different RNA-seq datasets. Further in the second project, we studied the genomic and mechanistic insights of convergent transcription in bacterial genomes. We know that convergent gene pairs with overlapping head-to-head configuration are widely spread across both eukaryotic and prokaryotic genomes. They are believed to contribute to the regulation of genes at both transcriptional and post-transcriptional levels, although factors contributing to their abundance across genomes and mechanistic basis for their prevalence are poorly understood. In this study, we explore the role of various factors contributing to convergent overlapping transcription in bacterial genomes. Our analysis shows that the proportion of convergent overlapping gene pairs (COGPs) in a genome is affected due to endospore formation, bacterial habitat, oxygen requirement, GC content and the temperature range. In particular, we show that bacterial genomes thriving in specialized habitats, such as thermophiles, exhibit a high proportion of COGPs. Our results also conclude that the density distribution of COGPs across the genomes is high for shorter overlaps with increased conservation of distances for decreasing overlaps. Our study further reveals that COGPs frequently contain stop codon overlaps with the middle base position exhibiting mismatches between complementary strands. Further, for the functional analysis using cluster of orthologous groups (COGs) annotations suggested that cell motility, cell metabolism, storage and cell signaling are enriched among COGPs, suggesting their role in processes beyond regulation. Our analysis provides genomic insights into this unappreciated regulatory phenomenon, allowing a refined understanding of their contribution to bacterial phenotypes.