Dissertationen zum Thema „Convergent transcription“

Genomic DNA is organised in complex spatial arrangements, and a given stretch of DNA may encode more than one gene. In some cases one gene may be entirely contained within a region of the DNA already occupied by another larger gene. The presence of these nested genes, often situated in introns and in the opposite orientation, poses important implications with regards to gene expression, function and regulation. A consequence of the nested gene arrangement is convergent transcription, occurring when two promoters on opposite DNA strands are active. Elucidating the mechanics of multiple interacting proteins on single DNA templates requires single molecule methods such as atomic force microscopy (AFM). AFM can accurately determine the relative positions of enzymes, such as RNA polymerase (RNAP), on individual DNA templates. The central aim of this thesis is to use AFM to study the outcomes of convergent transcription, using linear DNA templates that function as models for nested genes. Fundamental aspects of imaging DNA on mica with AFM were investigated, with a view to optimising sample preparation. The main processes involved with preparing DNA samples, ready for scanning, were examined in turn. Effective binding was achieved by introducing divalent cations into a deposition buffer. Mica ion exchanged with Ni(II) usually gave rise to kinetically-trapped DNA molecules, however short linear fragments (< 800bp) were seen to deviate from the expected behaviour, indicating that ion-exchanged mica is heterogeneous, and contains patches or domains. These findings can be used to more readily control binding of DNA to substrates. The outcomes of varying the relative humidity while imaging biomolecular systems are largely unexplored to date. Various DNA samples were imaged in conditions of varying humidity. In particular when supercoiled plasmids are scanned at very high relative humidity (> 90% RH), localised DNA backbone motions or conformational changes were observed. Humidity controlled AFM will be a useful technique for probing DNA topology without some of the drawbacks of imaging under bulk solution. Initial studies of transcription utilised templates containing two promoter sites and E. Coli RNAP. Two promoter arrangements were studied: a convergent template containing the promoter sites on opposite strands directed towards each other, and a tandem template containing the promoters in the same direction, on the same stand. It was shown that collisions between RNAPs led to similar outcomes in both cases: RNAPs are unable to pass each other and remain stalled against each other. In the convergent case, it was observed that after collision one RNAP could cause another to backtrack along the template. By end-labelling double-stranded (ds) DNA templates with a single-stranded DNA loop, the polarity of the molecules can be established in the AFM. It allowed better discrimination between outcomes of collision events on single DNA molecules and importantly, it enabled a quantitative comparison of the relative frequencies of the outcomes. The most common outcome is a collision between an actively transcribing elongation complex (EC) and a “sitting duck” (SD), which is a stalled RNAP or open promoter complex (OPC). In collisions initiated from OPCs, the most likely outcome, a collision between an EC and an SD occurs ~74% of the time. This causes sizeable back-tracking of the inactive RNAP, on average 59 nm upstream of the promoter. A significant fraction of the collisions (~15%) are between actively transcribing RNAP while the remainder (~11%) are undetermined. End-labelling of dsDNA using nucleic acid structures did not interfere with AFM sample preparation and can be used as a generic approach to studying interactions of multiple proteins on DNA templates at the single molecule level.

With recent advances in sequencing and mapping of genomes, the occurrence of overlapping and nested transcription units is more common than previously thought in both eukaryotes and prokaryotes. The interleaved genome model means that transcriptional interference by collisions between concurrently transcribing RNA polymerases is more likely than ever before. This thesis presents a study of the outcomes of collisions between RNAPs transcribing concurrently from convergent and tandem promoters using AFM to provide a view of single populations seen after collisions. Through the development of an improved DNA end labelling method and incorporation of an inhibitor of RNAP non-specific binding the results of collisions can be viewed with more confidence than previously possible. It was seen that collisions from both convergent and tandem promoters resulted in both RNAPs remaining bound to the template in hard contact. These collisions occurred by two main mechanisms. Either between two active elongation complexes (ECs) or between an elongation complex and an inactive complex referred to as a sitting duck (SD). EC-EC collisions were found to be the most common for convergent promoters while with tandem promoters the distinction between the two is less clear. In the case of EC-SD collisions it is shown that shunting upstream of up to 100 bp by an EC is possible. By utilizing a linear template that is susceptible to supercoiling due to spin locking, it is shown that a region of highly positive supercoiled domain can prevent two convergently transcribing RNAPS coming into hard contact. It is also shown that topology of the DNA plays a role in the distribution of EC-EC and EC-SD collisions that occur for both promoter arrangements. This indicates that topology influences the outcomes of concurrent transcription and provides a mechanism by which RNAPs can sense one another via the DNA template.

La sénescence, qui est un mécanisme antitumoral majeur, est définie comme un état d'arrêt irréversible de la prolifération cellulaire en réponse à un stress comme l'activation illégitime d'oncogènes. Les cellules qui entrent en sénescence subissent de profonds changements de leur épigénome. Les ARNs antisens sont suspectés de joue des rôles importants dans le contrôle du destin cellulaire et dans des processus cellulaires variés. Dans la levure, le variant d'histone H2A.Z co-opère avec les machineries du RNAi et de l'hétérochromatine pour réprimer sur les loci de gènes convergents l'apparition d'antisens dus à des défauts de terminaison de transcription de un des deux gènes. Chez les mammifères, l'existence et la régulation de tels transcrits antisens restent inconnues. De façon intéressante, la déplétion du variant d'histones H2A.Z est connue pour induire la sénescence. Nous nous sommes donc demandés si la sénescence est accompagnée de la régulation de tels transcrits antisens sur les gènes convergents, si la régulation par H2A.Z est conservée et si ces transcrits pouvaient avoir un rôle fonctionnel. Dans un modèle de sénescence induite par les oncogènes in vitro, nous avons identifiés par RNA-Seq brin spécifiques plusieurs loci de gènes convergents où des ARN antisens pourraient être générés par des défauts de terminaison de transcription sur le gène convergent. Des analyses en profondeurs sur deux loci ont confirmé que les transcrits antisens sont effectivement générés par un tel mécanisme (appelé "read-through transcriptionnel"). Nous avons appelé ces antisens START RNAs (pour " Senescence Triggered Antisense Read-through Transcripts). Nous avons par la suite montré que ces STARTs répriment l'expression du gène pour lequel ils sont antisens. Finalement, nous avons montré qu'ils sont réprimés par H2A.Z dans les cellules en prolifération. Nous proposons donc un modèle où la progression en sénescence s'accompagne d'une diminution de H2A.Z, ce qui se traduit par l'induction de transcrits antisens régulateurs sur une famille de loci de gènes convergents dus à des défauts de contrôle de la terminaison de la transcription
Cellular senescence represents one of the major fail-safe mechanisms that counteracts tumour development is defined as a state of irreversible cell cycle arrest as a consequence of stress response such as oncogenic challenge. Such cells undergoing Oncogene-induced Senescence (OIS) display profound alternation in their epigenome as their chromatin are largely decorated with prominent drivers of constitutive heterochromatin.Antisense RNA-mediated gene regulation has been attributed to play diverse roles in mediating various cellular processes and cell fates per-se. In yeast, histone variant H2A.Z cooperates with RNAi and heterochromatin machinery to regulate antisense transcription at convergent gene loci which can otherwise generate pervasive read-through transcripts owing to improper transcription termination. In mammals, whether such antisense transcripts (occurring by read-through transcription at convergent gene pairs) exist and how they are regulated remains unknown. Interestingly, the depletion of the human H2A.Z histone variant has been reported to induce cellular senescence. We thus wondered if the regulation of particular antisense transcripts at convergent gene pairs occurs in senescence, if their regulation by H2A.Z is conserved in mammals and, if so, if a functional significance can be attributed to these transcripts. To this end we took advantage of a well-established in-vitro OIS model. Briefly, we analysed genome wide strand specific RNA-seq analysis of cells undergoing Oncogene Induced Senescence. This led us to identify numerous convergent gene loci associated with accumulation of transcripts downstream of the designated transcription termination site in senescent cells, and extending to generate an antisense to the next gene located in the opposite strand of the convergent gene pair. We confirmed the RNA-seq data at two of such convergent loci. An RNAi based approach revealed that at least two of these transcripts are generated by transcriptional read-throughs. Hence we designated such pervasive transcripts as Senescence Triggered Antisense Read-through Transcripts (START). Importantly, we further found that the two STARTs for which we performed in depth studies repress the expression of the gene for which they are antisense. Finally, we demonstrate that the histone variant H2A.Z suppresses the accumulation of STARTs in proliferative cells. Since it also prevents senescence induction, this suggests that expression of START is important for cellular senescence. This has lead us to propose a model that human cells undergoing OIS are associated with loss of H2A.Z that leads to the wide spread accumulation of read-through transcripts owing to impaired termination control

L'épissage alternatif de l'ARN précurseur est un processus co-transcriptionnel qui affecte la grande majorité des gènes humains et contribue à la diversité des protéines. Le dérèglement d'un tel processus est impliqué dans diverses maladies, y compris la tumorigènes. Cependant, les mécanismes qui régulent ces processus restaient à caractériser. Dans cette étude, nous avons montré que les perturbations de l'épissage alternatif étaient corrélées aux dérèglements de la transcription convergente et de la méthylation de l'ADN. Une transcription convergente peut être générée entre des paires de gènes voisins en en orientation opposée, ou entre des amplificateurs intragéniques et leur gène hôte. CENPO et ADCY3 ont été identifiés comme une paire de gènes de transcription convergentes. Nous avons constaté, dans un modèle de progression tumorale du cancer du sein, que le changement d'épissage de l'exon22 variant d'ADCY3 était corrélé à une augmentation de sa transcription qui allait contre celle de CENPO. En utilisant le système de répression ciblée de la transcription CRISPRi, nous avons démontré que l’inhibition de la transcription de CENPO ne pouvait pas inverser l'altération d'épissage alternatif d'ADCY3 dans les cellules cancéreuses (DCIS). Un activateur intragénique actif a été identifié dans l'intron16 du gène CD44, en aval de ses exons alternatifs. En utilisant le système d'activation ciblée de transcription CRISPRa, nous avons montré que l’augmentation de la transcription de CD44 ne pouvait pas modifier l'épissage alternatif de CD44 dans les cellules DCIS. Ces résultats suggèrent que la modification de transcription convergente par des changements d’activité des promoteurs ne permet pas d’altérer l'épissage alternatif de ADCY3 et CD44 dans les cellules DCIS. Cependant, en remplaçant l'amplificateur intragénique par un promoteur inductible, nous avons constaté que l'activation de la transcription intragénique augmentait le niveau d'inclusion de plusieurs exons alternatifs de CD44 dans les cellules HCT116. Ce résultat suggère que la transcription convergente local pourrait avoir un impact direct sur l'épissage alternatif de CD44. De plus, en utilisant le système de méthylation de l'ADN ciblée CRISPR/dCas9-DNMT3b, nous avons démontré que la méthylation de l'ADN au niveau des exons variants pouvait modifier l'épissage alternatif de CD44. Ce travail de thèse a également exploré les limites et la faisabilité de l'étude de l'épissage alternatif avec des outils moléculaires basés sur le système CRISPR
Alternative splicing of precursor RNA is a co-transcriptional process that affects the vast majority of human genes and contributes to protein diversity. Dysregulation of such process is implicated in various diseases, including tumorigenesis. However, the mechanisms regulating these processes were still to be characterized. In this study, we showed that perturbations of alternative splicing correlated with dysregulations of convergent transcription and DNA methylation. Convergent transcription could be generated between pairs of neighboring genes in opposite orientation, or between intragenic enhancers and their host gene. CENPO and ADCY3 was identified as a convergent transcription gene pair. We found, in a tumor progression model of breast cancer, that the splicing change of the ADCY3 variant exon22 correlated with an increase of its transcription that went against that of CENPO. By using targeted transcription repression system CRISPRi, we demonstrated that downregulating the transcription of CENPO could not reverse the alternative splicing alteration of ADCY3 in cancer cells (DCIS). An active intragenic enhancer was identified in the intron16 of CD44, at the downstream of its alternative exons. By using targeted transcription activation system CRISPRa, we showed that upregulating the transcription of CD44 could not alter the alternative splicing of CD44 in DCIS cells. These results suggest that convergent transcription modulation through changes of promoter activity does not alter the alternative splicing of ADCY3 and CD44 in DCIS cells. However, through replacing the intragenic enhancer by an inducible promoter, we found that intragenic transcription activation increased the inclusion level of several alternative exons of CD44 in HCT116 cells. This result suggested that local convergent transcription could have a direct impact on the alternative splicing of CD44. Furthermore, by using targeted DNA methylation system CRISPR/dCas9-DNMT3b, we showed that DNA methylation at variant exons could directly modify CD44 alternative splicing. This thesis work also explored the limitation and feasibility of studying alternative splicing with repurposed CRISPR systems

DNA damage is an ongoing threat to both the ability of the cell to faithfully transmit genetic information to its offspring as well as to its own survival. In order to maintain genomic integrity, eukaryotes have developed a highly conserved mechanism to detect, signal and repair damage in DNA, known as the DNA damage response (DDR). In fission yeast the two DDR pathways converge at the regulation of single transcriptional factor complex (MBF) resulting in opposite directions. We have shown that when the DNA-synthesis checkpoint is activated, Max1 is phosphorylated by Cds1 resulting in the abrogation of its binding to MBF. As a consequence, MBF-dependent transcription is maintained active until cells are able to overcome the replication challenge. In contrast, upon DNA damage, Chk1 the effector kinase of DNA damage checkpoint is activated and blocks the cell cycle progression, inducing DNA repair and repressing the MBF dependent transcription. We have revealed that Cdc10 is the target of the DNA-damage checkpoint and when cells are treated with MMS or are exposed to IR, Chk1 phosphorylates Cdc10 inducing the exit of MBF from chromatin. The consequence is that under these conditions, MBF-dependent transcription is repressed. Thus, Max1 and Cdc10 couple normal cell cycle regulation and the DNA-synthesis and DNA-damage checkpoints into MBF.

BCL6 is a zinc finger transcriptional repressor, which is highly expressed in germinal centre B-cells, and is essential for germinal centre formation and T-dependent antibody responses. Deregulated BCL6 expression is associated with certain non- Hodgkin’s lymphomas. High expression is observed in breast cancer. Tight lineage and temporal regulation of BCL6 is, therefore, required for normal immunity and abnormal regulation occurs in cancer. Regulatory mechanisms have been analysed in two settings. Firstly, BCL6 is strongly induced by the tyrosine kinase inhibitor, Imatinib, in chronic myeloid leukaemia lymphoid blast crisis cell lines, and this effect was used in order to study the effects of phospho-protein signalling on BCL6 expression and a major finding is that p38 MAPK induced BCL6. Also, p38 is, at least in part, responsible for BCL6 expression in basal conditions in the germinal centre representative Burkitt’s lymphoma cell lines and that qualitatively different CD40 stimuli can either induce or repress BCL6 expression. Luciferase assays showed that p38 acts at a 300bp sequence immediately 5’ of exon 1, and probably also at more distal sequences. Overall it appears that the balance between positive and negative regulatory controls BCL6 expression with inhibitory signalling pathways being predominant in most circumstances. Focusing on BCL6 exon 1, a binding site for the multifunctional regulator CTCF was identified. CTCF interacts in vitro and in vivo with this sequence. Reduced expression of CTCF in germinal centre cells caused a moderate reduction of BCL6 expression. Finally, although no clear differences were observed in the methylation status of the CTCF binding site on exon 1, a significant enrichment of active histone modifications at this site was observed in BCL6 expressing cells, suggesting that CTCF may have a role in the epigenetic regulation of BCL6.

Mon projet de thèse s’inscrit dans le cadre des études visant à comprendre comment les cellules embryonnaires intègrent les différents signaux auxquels elles sont exposées pour s’engager dans une voie de différenciation définie. Il est plus particulièrement centré sur le rôle des protéines Smad dans ces processus et peut se diviser en deux axes de recherche. Le premier a trait au rôle de Mad (Smad1) dans les interactions entre signaux Wnt (Wg) et BMP chez la drosophile. Nous avons pu démontrer que la forme Mad non phosphorylée par le récepteur BMP se lie au complexe transcriptionnel ß-catenin/dTCF et est requise pour le signal Wnt canonique. La phosphorylation de Mad par le récepteur BMP dirige Mad vers la voie BMP, créant la possibilité d’une compétition entre ces deux classes de signaux. Le second axe de recherche concerne le facteur de transcription Smad4 qui est requis pour la transduction des signaux TGF-ß et BMP. J’ai pu identifier trois sites potentiels de phosphorylation par la kinase GSK3 dans la séquence primaire de Smad4. En utilisant de nombreuses techniques de biochimie, j’ai pu montrer que Smad4 est phosphorylé par la kinase Erk, puis par GSK-3 en réponse à un signal FGF. Lorsque Smad4 est doublement phosphorylé, il est reconnu par une E3-ligase, beta-TrCP, ce qui entraine sa polyubiquitination et sa dégradation. La voie Wnt étant capable d’inhiber GSK-3, j’ai pu montrer que Smad4 est stabilisé par des signaux Wnt. Ce mécanisme augmente la sensibilité des cellules aux signaux TGF-beta lorsqu’elles reçoivent également un signal Wnt
During my PhD I studied how cells receive and integrate multiple signals from the extracellular milieu. I focused on Smad proteins and my project can be divided into two parts. My first project was centered on the transcription factor Mad (Smad1) and its requirement for the BMP and Wg pathways. Using a combination of genetic and biochemistry experiments, we showed that Mad is required for Wg signaling both in Tcf reporter gene assays and in vivo in Drosophila. We found that the choice for Mad to transduce Dpp or Wg signals is controlled by C-terminal phosphorylations so that Mad binds to Pangolin and participates in Wg target genes transcription only when not phosphorylated at its C-terminus. This results in a competition between Dpp and Wg controlled by the phosphorylation state of Mad. My second project was focused on the tumor suppressor Smad4. When I first joined the lab, I identified three new potential GSK3 phosphorylation sites in Smad4 primary sequence. I used a home-made phospho-specific antibody to demonstrate that FGF or EGF stimulation trigger Erk-mediated phosphorylation of Smad4 which primes subsequent GSK3 phosphorylations. These phosphorylations regulate a transcription activation domain located in Smad4 linker region and generate a Wnt-regulated phosphodegron recognized by the E3 ligase beta-TrCP. This mechanism provides a means of integrating distinct pathways which would otherwise remain insulated, allowing cells to sense FGF and Wnt inputs and adapt TGF-beta outcome to their context. It provides a molecular explanation of the long-standing mystery of the “competence modifier” effect of Wnt on Nodal signals discovered 20 years ago

Mon projet de thèse s’inscrit dans le cadre des études visant à comprendre comment les cellules embryonnaires intègrent les différents signaux auxquels elles sont exposées pour s’engager dans une voie de différenciation définie. Il est plus particulièrement centré sur le rôle des protéines Smad dans ces processus et peut se diviser en deux axes de recherche. Le premier a trait au rôle de Mad (Smad1) dans les interactions entre signaux Wnt (Wg) et BMP chez la drosophile. Nous avons pu démontrer que la forme Mad non phosphorylée par le récepteur BMP se lie au complexe transcriptionnel ß-catenin/dTCF et est requise pour le signal Wnt canonique. La phosphorylation de Mad par le récepteur BMP dirige Mad vers la voie BMP, créant la possibilité d’une compétition entre ces deux classes de signaux. Le second axe de recherche concerne le facteur de transcription Smad4 qui est requis pour la transduction des signaux TGF-ß et BMP. J’ai pu identifier trois sites potentiels de phosphorylation par la kinase GSK3 dans la séquence primaire de Smad4. En utilisant de nombreuses techniques de biochimie, j’ai pu montrer que Smad4 est phosphorylé par la kinase Erk, puis par GSK-3 en réponse à un signal FGF. Lorsque Smad4 est doublement phosphorylé, il est reconnu par une E3-ligase, beta-TrCP, ce qui entraine sa polyubiquitination et sa dégradation. La voie Wnt étant capable d’inhiber GSK-3, j’ai pu montrer que Smad4 est stabilisé par des signaux Wnt. Ce mécanisme augmente la sensibilité des cellules aux signaux TGF-beta lorsqu’elles reçoivent également un signal Wnt
During my PhD I studied how cells receive and integrate multiple signals from the extracellular milieu. I focused on Smad proteins and my project can be divided into two parts. My first project was centered on the transcription factor Mad (Smad1) and its requirement for the BMP and Wg pathways. Using a combination of genetic and biochemistry experiments, we showed that Mad is required for Wg signaling both in Tcf reporter gene assays and in vivo in Drosophila. We found that the choice for Mad to transduce Dpp or Wg signals is controlled by C-terminal phosphorylations so that Mad binds to Pangolin and participates in Wg target genes transcription only when not phosphorylated at its C-terminus. This results in a competition between Dpp and Wg controlled by the phosphorylation state of Mad. My second project was focused on the tumor suppressor Smad4. When I first joined the lab, I identified three new potential GSK3 phosphorylation sites in Smad4 primary sequence. I used a home-made phospho-specific antibody to demonstrate that FGF or EGF stimulation trigger Erk-mediated phosphorylation of Smad4 which primes subsequent GSK3 phosphorylations. These phosphorylations regulate a transcription activation domain located in Smad4 linker region and generate a Wnt-regulated phosphodegron recognized by the E3 ligase beta-TrCP. This mechanism provides a means of integrating distinct pathways which would otherwise remain insulated, allowing cells to sense FGF and Wnt inputs and adapt TGF-beta outcome to their context. It provides a molecular explanation of the long-standing mystery of the “competence modifier” effect of Wnt on Nodal signals discovered 20 years ago

Les organismes s’adaptent aux variations nutritionnelles en modulant l’expression de leurs gènes. Le contrôle transcriptionnel de l’expression génétique implique de nombreux acteurs, dont les corégulateurs. Un dysfonctionnement de ces corégulateurs est connu pour générer un métabolisme cellulaire inadapté et contribuer à la pathogenèse de maladies métaboliques. Nous avons démontré que l’activation pharmacologique in vivo de SIRT1, une désacétylase membre de la famille des sirtuines, protège les souris de l’obésité en augmentant leur dépense énergétique. Au niveau moléculaire, nous avons lié ces effets à une expression accrue de gènes impliqués dans la biogenèse mitochondriale, la phosphorylation oxydative et l’oxydation des acides gras dans les tissus métaboliques. Ceci est dû au fait que SIRT1 entraîne la désacétylation et l’activation consécutive de PGC-1α, un régulateur majeur de l’activité mitochondriale. Nous avons également montré, qu’à l’instar des désacétylases, les acétyltransférases ont aussi un large impact sur l’homéostasie énergétique. En effet, en induisant l’expression de l’acétyltransférase GCN5, SRC-3 facilite l’acétylation de PGC-1α et par conséquent inhibe son activité. De façon intéressante, le niveau d’expression de SIRT1 est inversement corrélé à celui de GCN5 et SRC-3 dans des situations de jeun ou de surnutrition. Il en résulte une régulation coordonnée de l’activité de PGC-1α, à la fois par les désacétylases et les acétyltransférases, nous menant à considérer ces enzymes comme membres d’un réseau de corégulateurs renseignant PGC-1α sur le statut énergétique cellulaire, PGC-1α adaptant ensuite la production énergétique grâce à son rôle sur l’activité mitochondriale
Organisms respond to variations in hormonal, metabolic, and nutritional signaling by altering the expression of their genetic information, allowing metabolic adaptation. Transcriptional control is achieved through an interwoven and redundant molecular circuitry that involves individual transcription factors, the basal transcriptional machinery, and multiprotein coregulator complexes, which fine-tune metabolic homeostasis. Most of the coregulators interact directly with transcription factors and can either repress or enhance their transcriptional activities. Aberrant signaling by coregulators is known to generate abnormalities of cellular metabolism, and hence can contribute to abnormalities of systemic metabolic pathways and to the pathogenesis of several common disorders, such as obesity and type 2 diabetes. We first demonstrated that an in vivo pharmacological activation of SIRT1, a member of the evolutionary conserved family of NAD-dependant deacetylases named sirtuins, protects the mice against the development of obesity and linked pathophysiologies, through an increased energy expenditure. At the molecular level, the in vivo effects of the sirtuinactivating compounds were associated with an induction of the expression of genes involved in mitochondrial biogenesis, oxidative phosphorylation and fatty acid oxidation in metabolic tissues such as the skeletal muscle and the brown adipose tissue and were explained by a the SIRT1-mediated deacetylation, and subsequent activation, of PGC-1α, a master regulator of mitochondrial function. In parallel, through a genetical approach, we demonstrated that not only deacetylases but also acetyltransferases impact in a major fashion on energy homeostasis. In fact, we have shown that, through promoting the expression the PGC-1α acetyltransferase GCN5, SRC-3 facilitates PGC-1α acetylation in muscle and brown adipose tissue and consequently inhibits PGC-1α activity. Interestingly, the expression levels of SIRT1, the major PGC-1α deacetylase, mirror those of SRC-3 and GCN5 in various physiological situations such as fasting and high fat feeding. This results in a convergent regulation of PGC-1α acetylation by both acetyltransferases and deacetylases and lead us to consider theses enzymes as members of coregulator network that informs PGC-1α about the cellular energy status, which then adapts cellular energy production through its commanding role of master regulator of mitochondrial function. It is therefore tempting to speculate that this converging cofactor network, including SRC-3, GCN5 and SIRT1 can be exploited to design new preventive and therapeutic strategies to combat obesity and associated metabolic disorders such as type 2 diabetes

L'objet de cette thèse est la convergence structure-fonction dans les systèmes complexes et ses applications aux systèmes vivants et aux systèmes technico-économiques. Après avoir défini la modularité et identifié les difficultés associées à sa définition, cette thèse formalise le concept de convergence structure-fonction dans les systèmes évolutifs et fonctionnels et montre son intérêt pour l'évolutivité et la robustesse de ces systèmes. Ensuite, elle applique ce concept à des problématiques réelles de systèmes évolutifs et fonctionnels en biologie et en économie afin d'illustrer son utilité. Ainsi, dans le cadre de la génomique, cette thèse montre que la longueur des opérons bactériens, qui sont à la fois des modules structurels et fonctionnels, est limitée du fait de contraintes dues à l'interaction des mécanismes de transcription et de réplication. Ensuite, elle fait l'hypothèse que la modularité structurelle des points chauds de recombinaison correspond au moins partiellement à la modularité fonctionnelle des gènes. Ceci permet de développer une nouvelle méthode d'analyse des études d'association génétique basée sur un découpage en régions géniques du génome dans le but de faciliter la compréhension du mécanisme fonctionnel de leur action sur le caractère étudié en analysant directement l'association de gènes ou de groupe de gènes avec ce caractère. Sur le plan structurel, les résultats sont d'une qualité comparable à ceux des méthodes classiques. En revanche, le découpage en régions devra encore être affiné afin d'obtenir une analyse fonctionnelle pleinement utile. Enfin, dans le cadre de la libéralisation du marché européen de l'électricité, la correspondance effective entre structure et fonction de chaque acteur issu de la restructuration fait supposer que le principe de convergence structure-fonction y est bien appliqué. Cependant, des difficultés subsistent avant de parvenir à mettre en place des relations structurelles permettant d'atteindre l'optimum souhaité. Celui-ci inclut des échanges d'énergie à l'origine des contraintes couplantes entre les acteurs. A partir de la théorie de la décomposition par les prix, nous proposons un cadre permettant de définir des tarifs propres à les faciliter, en particulier celles liant producteurs et transporteurs. En conclusion, cette thèse montre (a) la limite à la convergence structure-fonction que constitue la limite de la longueur des opérons bactériens, (b) la faisabilité de l'utilisation d'un découpage basé sur les limites de gènes afin d'analyser des études d'association génétique à grande échelle et (c) l'importance d'améliorer « la grande boucle » des relations entre producteurs et transporteurs d'électricité afin d'assurer l'optimisation conjointe des investissements en capacité de production et de transport. Elle synthétise l'ensemble de ces résultats dans le cadre conceptuel de la convergence structure-fonction qui postule que la modularité structurelle des systèmes évolutifs et fonctionnels tend à se superposer à leur modularité fonctionnelle afin de leur apporter robustesse et évolutivité.

Les récentes études transcriptomiques chez divers organismes ont montré que la transcription des gènes convergents peut produire des ARN messagers (ARNm) chevauchants. Ce phénomène a été analysé dans le contexte de l’interférence par ARN (ARNi) nucléaire, et peu d’information existe quant au destin cytoplasmique des messagers 3’ chevauchants ou leur impact sur l’expression des gènes. Dans ce travail, nous avons abordé les conséquences potentielles de l’interaction entre des paires d’ARNm sens-antisens chez Saccharomyces cerevisiae, un organisme modèle naturellement dépourvu de l’ARNi. Nous avons démontré que les extrémités 3’ complémentaires des ARNm peuvent interagir dans le cytoplasme et moduler la stabilité ainsi que la traduction d’ARNm. Nos résultats sont issus d’une étude détaillée d’une paire d’ARNm convergents, POR1 et OCA2, ensuite généralisée par l’approche de l’ARNi reconstituée chez S. cerevisiae. L’analyse globale a confirmé que dans les cellules sauvages, les paires d’ARNm sens-antisens forment des duplexes d’ARN in vivo et ont un rôle potentiel à moduler l’expression d’ARNm ou de protéines respectifs, dans des différentes conditions de croissance. Nous avons montré que le destin de centaines des messagers convergents est contrôlé par Xrn1, révélant l’importance de cette exoribonucléase 5’-3’ cytoplasmique très conservée dans la régulation post-transcriptionnelle des gènes convergents. Notre travail ouvre donc la perspective de considérer un nouveau mécanisme de l’interaction entre les paires d’ARNm sens-antisens dans le cytoplasme, chez les organismes contenant ou non la voie de l’interférence par ARN
Recent transcriptome analyses have revealed that convergent gene transcription can produce many 3’ overlapping mRNAs in diverse organisms. This phenomenon has been studied in the context of nuclear RNA interference (RNAi) pathway, however little is known about the cytoplasmic fate of 3’ overlapping messengers or their impact on gene expression. In this work, we address the outcomes of interaction between sense-antisense mRNA pairs in Saccharomyces cerevisiae, a model organism naturally devoid of RNAi. We demonstrate that the complementary tails of 3’ overlapping mRNAs can interact in the cytoplasm in a sequence-specific manner and promote post-transcriptional remodeling of mRNA stability and translation. Our findings are based on the detailed analysis of a convergent mRNA pair, POR1 and OCA2, subsequently generalized using the reconstituted RNAi approach in S. cerevisiae. Genome-wide experiments confirm that in wild-type cells, sense-antisense mRNA pairs form RNA duplexes in vivo and thus have potential roles in modulating the respective mRNA or protein levels under different growth conditions. We show that the fate of hundreds of messenger-interacting messengers is controlled by Xrn1, revealing the extent to which this conserved 5’-3’ cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression. In sum, our work opens a perspective to consider an additional, cytoplasmic mechanism of interaction between sense-antisense mRNA pairs, in both RNAi-positive and negative organisms

Les récentes études transcriptomiques chez divers organismes ont montré que la transcription des gènes convergents peut produire des ARN messagers (ARNm) chevauchants. Ce phénomène a été analysé dans le contexte de l’interférence par ARN (ARNi) nucléaire, et peu d’information existe quant au destin cytoplasmique des messagers 3’ chevauchants ou leur impact sur l’expression des gènes. Dans ce travail, nous avons abordé les conséquences potentielles de l’interaction entre des paires d’ARNm sens-antisens chez Saccharomyces cerevisiae, un organisme modèle naturellement dépourvu de l’ARNi. Nous avons démontré que les extrémités 3’ complémentaires des ARNm peuvent interagir dans le cytoplasme et moduler la stabilité ainsi que la traduction d’ARNm. Nos résultats sont issus d’une étude détaillée d’une paire d’ARNm convergents, POR1 et OCA2, ensuite généralisée par l’approche de l’ARNi reconstituée chez S. cerevisiae. L’analyse globale a confirmé que dans les cellules sauvages, les paires d’ARNm sens-antisens forment des duplexes d’ARN in vivo et ont un rôle potentiel à moduler l’expression d’ARNm ou de protéines respectifs, dans des différentes conditions de croissance. Nous avons montré que le destin de centaines des messagers convergents est contrôlé par Xrn1, révélant l’importance de cette exoribonucléase 5’-3’ cytoplasmique très conservée dans la régulation post-transcriptionnelle des gènes convergents. Notre travail ouvre donc la perspective de considérer un nouveau mécanisme de l’interaction entre les paires d’ARNm sens-antisens dans le cytoplasme, chez les organismes contenant ou non la voie de l’interférence par ARN
Recent transcriptome analyses have revealed that convergent gene transcription can produce many 3’ overlapping mRNAs in diverse organisms. This phenomenon has been studied in the context of nuclear RNA interference (RNAi) pathway, however little is known about the cytoplasmic fate of 3’ overlapping messengers or their impact on gene expression. In this work, we address the outcomes of interaction between sense-antisense mRNA pairs in Saccharomyces cerevisiae, a model organism naturally devoid of RNAi. We demonstrate that the complementary tails of 3’ overlapping mRNAs can interact in the cytoplasm in a sequence-specific manner and promote post-transcriptional remodeling of mRNA stability and translation. Our findings are based on the detailed analysis of a convergent mRNA pair, POR1 and OCA2, subsequently generalized using the reconstituted RNAi approach in S. cerevisiae. Genome-wide experiments confirm that in wild-type cells, sense-antisense mRNA pairs form RNA duplexes in vivo and thus have potential roles in modulating the respective mRNA or protein levels under different growth conditions. We show that the fate of hundreds of messenger-interacting messengers is controlled by Xrn1, revealing the extent to which this conserved 5’-3’ cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression. In sum, our work opens a perspective to consider an additional, cytoplasmic mechanism of interaction between sense-antisense mRNA pairs, in both RNAi-positive and negative organisms

Newborns and older adults aged 65 and over have a heightened risk for severe infections, suggesting suboptimal immune responses. These two populations thus represent age-defined windows of vulnerability to infection. Antigen presenting cells (APC) are important in bridging the innate and adaptive immune systems as they express pathogen recognition receptors such as Toll-like receptors (TLR) that detect the presence of pathogens. They have been proposed to be partially responsible for the altered immunity observed at the extreme ends of the age-spectrum. However, the molecular mechanism/s that underlie this APC deficit have not been fully delineated. We chose to apply newly available cutting-edge tools to begin identifying such mechanisms. With the availability of systems biological tools to interrogate APC function, our overarching hypothesis is that age-dependent differences in the transcriptional response of APC results in functional differences in response to immune stimulation and infection. To address this hypothesis, we employed global transcriptional profiling to comprehensively investigate age-dependent differences in mRNA expression in the most important APC subsets from newborns, healthy young adults, and older adults following TLR stimulation or infection. Following TLR7/8 stimulation, neonatal DC displayed altered expression of signaling pathways involved in the response to viral pathogens. Specifically, IRF-dependent MAPK pathway genes were expressed in an age-dependent manner at baseline, while age-dependent differences in the expression of other IRF-dependent responses only occurred following stimulation. We also investigated the transcriptomic responses of monocytes to Listeria monocytogenes (Lm). Monocytes are one of the primary targets of Lm. Monocytes from newborns, young adults, and older adults differentially expressed guanylate binding proteins (GBP) in an age-dependent manner upon infection, along with significantly reduced IFN-β production in susceptible age groups, while signaling downstream of the IFN receptor complex was comparable. This suggests that age-related differences in IFN-β production in response to Lm infection led to reduced induction of GBPs in newborns and older adults compared to young adults. IFN-β production is also known to be IRF dependent. This convergence of age-dependent differences in immunity on IRF-regulated pathways begins to outline a possible molecular epicenter associated with suboptimal immune responses early and late in life.
Medicine, Faculty of
Medicine, Department of
Experimental Medicine, Division of
Graduate

El gen humano 11-HSD2 es un modelo para investigar la contribución de los efectos de los receptores de esteroides en células de cáncer de mama. El análisis del promotor mostró que la región distal está implicada en la mayor parte de la activación dependiente de hormona. En respuesta a hormona, STAT5A se recluta a la región distal y PR a las regiones distal y proximal del promotor. El reclutamiento de PR se debe a dos mecanismos diferentes, la unión directa de PR a la región proximal, y la implicación vía JAK/STAT en el reclutamiento a la región distal. La inducción del gen 11-HSD2 por hormonas disminuye parcialmente por inhibidores de MAPK y PI3K/Akt y totalmente por inhibidores de JAK/STAT. Así, los efectos citoplasmáticos del PR están implicados en la inducción del gen progesterona. La forma activa de la ARN-polimerasa II es reclutada por la inducción con hormonas a la región distal del promotor 11-HSD2 y la región distal tiene respuesta a hormonas por sí misma, indicando que la inducción del gen por hormonas empieza antes del sitio de inicio de transcripción descrito previamente.
The human 11-HSD2 gene is a model to investigate the contribution of steroid hormone receptors effects on a progesterone responsive promoter in breast cancer cells. Deletion analysis of the 11-HSD2 promoter showed that the distal region is involved in most of the hormone-dependent activation. ChIP showed hormone-dependent STAT5A-recruitment to the distal region and PR-recruitment to the distal and proximal promoter regions. Results suggest two different mechanisms of hormone-induced PR-recruitment, since cells stably expressing PR containing a mutated DNA-binding domain have affected hormone-dependent PR-recruitment to proximal promoter, and JAK/STAT pathway inhibition blocks PR-recruitment to distal promoter. Hormone-stimulated 11-HSD2 gene-expression was partially decreased by MAPK and PI3K/AKT pathway inhibitors and totally blocked by JAK/STAT pathways inhibitors, indicating that cytoplasmic PR effects involvement in progestin-induced 11-HSD2 expression. Importantly, upon hormone induction active RNA-polymerase II is recruited from the 11-HSD2 distal promoter region and the distal minimal promoter has hormone-responsiveness by itself, suggesting that progesterone-dependent 11-HSD2 expression starts upstream the previously characterized transcription start site.

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.

"March 2003"
Bibliography: leaves 133-143.
x, 143 leaves : ill. (some col.) ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biomedical Sciences, 2003

The genome of eukaryotes is packaged into the small volume of the nucleus in an organised manner. This structure of DNA and associated proteins is called chromatin. The basic unit of chromatin is the nucleosome; an octomer of core histone proteins and associated DNA. Other proteins such as linker histones can also associate with the DNA or the core histones. The modular structure of chromatin allows for structural variation with functional consequences including activation or repression of transcription. Alterations can include post-translational modifications to histones, remodelling by multi-protein complexes, DNA methylation, and non-allelic variants of the canonical histones. Changes to chromatin structure have an important impact on all DNA processing events. This thesis investigated the histone variant H2A.Z, a variant of the canonical core histone H2A. H2A.Z is highly conserved and essential in a number of species suggesting it has a critical function. Preliminary work using the Xenopus laevis developmental model system had revealed that disruption of H2A.Z function resulted in defective embryo morphology consistent with disrupted gastrulation and mesoderm development (Ridgway et al., 2004a). This led to the following hypothesis: H2A.Z is important to gastrulation and mesodermal development in X laevis because it plays a developmental role. Temporal and spatial expression patterns of H2A.Z mRNA demonstrated in this study are consistent with a role in mesoderm development. Peak H2A.Z mRNA expression levels occur during gastrulation. H2A.Z mRNA is enriched in the marginal zone of the late blastula, involuting tissue in the gastrula and in notochord (a mesodermal tissue) in tailbud embryos. Significantly, maternal H2A.Z mRNA is enriched asymmetrically in dorsal cells of the early blastula before zygotic transcription, indicating that H2A.Z may play a role in determining polarity of the dorsal ventral axis. Two important processes for development were examined in this thesis: cell fate and cell movement. Determination of mRNA levels and localisations for a selection of mesodermal marker genes indicates that cell fate programs progress normally in embryos where H2A.Z function is disrupted. However, the localisation of mesoderm derived cells is perturbed suggesting cell movement is perturbed. Taken together these studies suggest the H2A.Z histone variant has a specific role in regulating cell mobility during early Xenopus laevis development.

Tese de mestrado Bioinformática e Biologia Computacional, Universidade de Lisboa, Faculdade de Ciências, 2018
Uma explosão no número de transcritos que não codificam para proteínas tem sido registada nos últimos anos, muito justificada pela utilização de técnicas de alto rendimento de sequenciação. Apesar da sua abundancia, a função de muitos deles ainda permanece desconhecida. Uma das classes de RNAs não codificantes são os transcritos contra-sentido, que são transcritos na cadeia de DNA oposta a dos genes codificadores (PC) aos quais estão associados. Esta proximidade sugere uma possível função reguladora entre ambas as unidades transcricionais. E hoje sabido que um elevado numero de regiões promotoras de genes apresenta transcrição bidirecional, dando origem a transcritos divergente não codificantes de proteínas. A transcrição contra sentido convergente (NATs) é também comum, mas a sua relevância biológica permanece muito menos compreendida. Várias análises efectuadas contemplando todo o genoma indiciam que a expressão dos NATs leva a uma repressão da expressão dos genes PC sobrepostos. Contudo, recentemente foi estudado um destes pares no nosso laboratório – ZEB2 / ZEB2-NAT – o qual apresentou uma dinâmica oposta a generalidade dos pares descritos na literatura, aumentando os níveis de mRNA quando o NAT e expresso. O ZEB2-NAT (transcrito contrasentido) e transcrito a partir do primeiro intrão do gene ZEB2 (gene codificador para proteínas), terminado a montante do início de transcrição deste. Outros pares, como VIM / VIM-AS1 e SPHK1 / LncRNA Khps1 apresentam os seus elementos contra-sentido a serem transcritos igualmente a partir do primeiro intrão, demonstrando uma correlação positiva face a sua expressão, semelhante ao par estudado no nosso laboratório. Estas evidências levaram-me a perguntar quantos pares havia no genoma humano numa disposição semelhante, e se a sua expressão também aumentaria os níveis dos PC sobrepostos. Para responder a esta questão, baseado nas anotações Ensembl, selecionei pares expressos com uma disposição semelhante aos referidos anteriormente, utilizando RNA-seq da fracção da cromatina, em células HeLa. Nesta identificação foram usados limites de 2 e 1 TPMs (Transcripts per Kilobase Milion) , acima dos quais eram considerados expressos os transcritos de PC e contra-sentido, respectivamente. Apos o trimming com o software Cutadapt e o alinhamento com o Hisat2, efectuei a quantificação de reads com o software StringTie, do qual foram obtidos os valores de TPMs. Daqui, um conjunto de 97 pares foram selecionados como tendo ambas as unidades transcricionais expressas. Seguidamente, realizei uma curação manual, na qual apliquei dados de mammalian native elongating transcript sequencing (mNET-seq), os quais revelaram o sinal da pausa da RNA polimerase junto dos promotores dos transcritos contra-sentido, permitindo aceitar ou rejeitar os pares anteriormente identificados de forma automática. Nesta técnica são sequenciados os RNAs que se encontram a ser transcritos pela RNA Polimerase II, havendo uma selecção pelo estado de fosforilação dos diferentes aminoácidos (UnPh, Y1P, S2P, Th4, S5P, S7P) do domínio Carboxi-terminal (CTD) deste complexo proteico. Era desconhecido para os transcritos contra sentido qual o estado de fosforilação que caracterizava a sua transcrição. Por este motivo analisei inicialmente todas os estados. Na analise efetuada não foram detetadas diferenças nos sinais de CTD da RNA polimerase II entre a transcrição de PCATs e PC, sendo igualmente detetado um forte sinal para os anticorpos associados aos estados Y1P e UnPh da RNA polimerase II. Estes elevados níveis foram cruciais para a identificação de PCATs expressos e não expressos. Esta estratégia de busca permitiu a identificação de 65 pares de unidades transcricionais, nos quais ambos os transcritos eram expressos. Aos elementos não codificantes destes pares atribuímos o nome de promoter-proximal convergent antisense transcripts (PCATs). Após a sua identificação realizei uma caracterização da classe PCAT. Esta análise começou por verificar os níveis de poliadenilação destes transcritos, por comparação a outras classes de genes, para os quais esta modificação pós-transcricional esta bem caracterizada. Para tal, utilizei dois conjuntos de dados de RNA-seq da fração nucleoplasmática. Num deles foram selecionados e sequenciados reads pertencentes a transcritos poliadenilados, enquanto para um segundo, reads de transcritos não poliadenilados. Os meus resultados mostram que para a classe PCAT a abundância relativa de reads pertencentes a transcritos poliadenilados é superior à de não poliadenilados. Observação similar a encontrada em genes PC, os quais sabemos que sofrem esta modificação. Os rácios entre reads poadeniladas / não-poliadeniadas são similares entre PCATs e PCs, o que dita uma poliadenilacao eficiente por parte desta nova classe em estudo. Sabendo que um elevado número de transcritos não codificadores de proteína são mantidos no núcleo e degradado neste compartimento, decidi analisar os níveis de degradação dos PCATs na fração da cromatina e do nucleoplasma pelo exossoma nuclear. Para tal, recorri a dados de RNA-seq das frações de cromatina e nucleoplasma de células HeLa com knock-down (KD) do exossoma. Por comparação a células wild type, demonstrei que os PCATs são estáveis enquanto ligados a cromatina, não apresentando diferenças entre os dois grupos. Quando analisados os dados de nucleoplasma foi possível concluir que a abundância dos PCATs quando feito KD do exossoma nuclear era superior. Esta observação indica que este complexo enzimático tem um efeito de degradação sobre a classe em estudo na fração nucleoplasmática, nao observada na cromatina. Desta forma, os dados sugerem uma possível função preferencial dos PCATs na fração da cromatina, comparativamente a do núcleo plasma. De modo a entender possíveis efeitos da presença destes transcritos na cromatina, recorrendo a dados de ChIP-seq do ENCODE project, estudei a modificação de vários aminoácidos das projeções de histones e proteínas associadas ao DNA (CTCF, EZH2, H2A.Z, H3K27ac, H3K27me3, H3K36me3, H3K4me1, H3K4me2, H3K4me3, H3K79me2, H3K9ac, H3K9me3, H4K20me1 e RNA Polymerase II) em regiões onde os PCATs são expressos. Das várias modificações estudadas, as acetilações H3K9ac e H3K27ac demonstraram estar mais presentes quando os PCATs são expressos, em comparação a regiões que não expressam estes transcritos não codificadores para proteínas. Estas marcas estão intimamente ligadas a ativação de genes e a descompactação do DNA. Desta forma, estes resultados apontam para um efeito regulador sofre a cromatina por parte dos PCATs, podendo levar a uma sobre expressão dos genes PC sobrepostos. Por fim, com dados de RNA-seq da fração da cromatina, previamente usados para a identificação dos PCATs, comparei os níveis de mRNAs para PC com PCATs expressos e não expressos. Desta análise conclui que para PC com PCATs anotados, os níveis de mRNAs são mais elevados aquando da sua expressão. Verificamos ainda que os níveis de expressão de genes sem PCATs anotados são semelhantes aos encontrados para aqueles que tem PCATs expressos, sugerindo que certas regiões no genoma necessitam da expressão destes transcritos para terem níveis de expressão semelhantes aos genes convencionais. Deste trabalho surtiu a descoberta de 65 pares de transcritos semelhantes ao par previamente estudado no nosso laboratório - ZEB2 / ZEB2-NAT. Alem disso, os meus resultados apontam que os PCATs identificados têm um poder regulador sobre os PC sobrepostos, idêntico ao ZEB2-NAT. Contudo, o estudo destes pares em laboratório vai ser essencial, não só para confirmar este poder regulador mas também para entender o seu mecanismo de ação. Os resultados obtidos neste trabalho levam-me a propor os PCATs como uma nova classe reguladora de RNA não codificantes.
In recent years, an increasing number of non-coding transcripts have been discovered, powered by the introduction of high-throughput sequencing techniques. Despite their large abundance in the human genome, their function remains largely unknown. Non-coding antisense transcripts are transcribed on the opposite strand of protein-coding genes, which suggests that they have a regulatory function. Most eukaryotic promoter regions are transcribed bi-directionally, generating divergent non-coding RNAs. Antisense transcription convergent to protein-coding genes is also common, but its biological relevance remains unclear. Several genome-wide studies have indicated that the expression of these overlapping non-coding transcripts may inhibit sense genes. However, studies done in our laboratory with the transcriptional pair Zeb2 / Zeb2-NAT have shown that this pair presents an opposite behavior, in which the antisense transcript actually promotes the expression of the sense gene. The non-coding Zeb2-NAT is transcribed from the first intron of the Zeb2 (protein-coding gene) in the opposite direction and ends upstream its Transcription Start Site (TSS). Other similar cases had already been reported. For instance, VIM / VIM-AS1 and SPHK1 / LncRNA Khps1, which are also transcribed from the first intron of protein-coding genes, showing a positive correlation with their sense pair unit, as our case study pair. These results have made me question how frequently in the human genome antisense transcription starting in the first intron enhances sense gene expression. To address this question, I searched for expressed sense (protein-coding) and antisense (non-coding) transcriptional paired units with closely spaced convergent promoters, using RNA-seq datasets from chromatin fractions obtained from HeLa cells and Ensembl annotations. Next, I did a manual curation using datasets obtained by mammalian native elongating transcript sequencing (mNET-seq), which revealed consistent polymerase pausing at the TSS of these antisense transcripts. The mNET-seq data determined whether to accept or reject the identified pairs that resulted from the automated search step. A group of 65 paired transcription units were identified, with both elements (sense and antisense) being expressed. We coined the term promoter-proximal convergent antisense transcripts (PCATs) to refer to the non-coding component of these pairs. Next, I characterized PCATs. Analysis of polyadenylated and non-polyadenylated transcripts in RNAseq datasets from the nucleoplasmic fraction of HeLa cells revealed that an enriched proportion of reads in PCATs regions originates from polyadenylated transcripts, similar to the proportion found for protein-coding genes, suggesting that PCATs are efficiently polyadenylated. To study PCAT stability, I analyzed RNA-seq data from chromatin and nucleoplasm fractions isolated from HeLa cells after knockdown of the nuclear exosome. PCATs associated with chromatin were not altered when the exosome was inhibited, whereas in the nucleoplasm PCATs accumulated after exosome inhibition. Analysis of NET-seq data using antibodies that distinguish different phophorylation-isoforms of the carboxi-terminal domain of RNA polymerase II (RNAP II-CTD) revealed similar patterns for proteincoding and PCATs transcription. In particular, prominent NET-seq peaks were observed at the TSS of both protein-coding genes and PCATs corresponding to CTD unphosphorylated and Y1P isoforms. Analysis of ChIP-seq data from Encode project on HeLa cells further demonstrated higher levels of histone H3 acetylation (H3K9ac and H3K27ac) around the TSS of protein-coding genes when PCATs are expressed. This suggests that PCAT expression is associated with gene activation and chromatin relaxation. Lastly, I compared the levels of protein-coding mRNAs when the corresponding PCATs were either expressed or non-expressed and found a strong positive correlation. I found similar expression levels for protein-coding genes without a PCAT annotated and protein-coding genes with an overlapping expressed PCAT; however, in the absence of PCAT expression, the associated proteincoding genes were expressed at significantly lower levels. Taken together, these results suggest that PCATs represent a novel class of regulatory non-conding RNAs.