Estudos funcionais anteriores identificaram um módulo cis-regulador (MCR) de 67 pb (-253/- 187) na região promotora do gene de pufe de DNA BhC4-1 que dirige a expressão do gene repórter na glândula anelar de Drosophila melanogaster. Uma análise bioinformática identificou 67 sequências de D. melanogaster que são similares a sequências contidas no MCR de glândula anelar. Uma das sequências identificadas reside em um fragmento genômico de 657 pb localizado aproximadamente 2500 pb à montante do CG13711, 400 pb à montante do CG12493, em uma região genômica que constitui um dos íntrons do CG32239 (Gef64C). A caracterização preliminar de três linhagens transformadas com a construção 657 pb-lacZ mostrou expressão do gene repórter no sistema traqueal de larvas e prépupas e no tórax de adultos. Baseado padrão de expressão promovido por este MCR, o mesmo foi denominado Traqueia-Tórax (TT). O principal objetivo do presente trabalho constituiu estender a caracterização molecular das linhagens da série TT-lacZ. Inicialmente embriões, larvas de primeiro, segundo e terceiro estádio, prépupas 0 h, 1 h e 2 h, pupas 24 h e adultos com 1, 3 e 5 dias foram investigados quanto ao padrão de expressão do repórter utilizando ensaio histoquímico que detecta atividade de ?-galactosidase. A expressão do gene repórter é inicialmente detectada no sistema traqueal durante o terceiro estádio larval e continua a ser detectada neste tecido em prépupas 0 h, 1 h e 2 h e pupas 24 h. Em adultos, a expressão do gene repórter é verificada nos músculos longitudinais dorsais em adultos de 3 e 5 dias. Uma vez que o MCR TT reside em uma região intergênica e a informação disponível sobre os CGs próximos ainda é escassa, não foi possível inferir qual dos CGs é regulado pelo MCR TT. Neste contexto, o padrão de expressão do RNAm do gene repórter lacZ e do CG13711, CG12493 e CG32239 foi investigado no sistema traqueal de larvas e prépupas e no tórax de adultos de uma das linhagens da série TT-lacZ utilizando RT-qPCR. Os níveis de expressão do RNAm lacZ aumentam cerca de 3 vezes em prépupas 0 horas, quando comparados com os níveis de expressão do RNAm lacZ presentes no sistema traqueal de larvas de terceiro estádio. Um padrão de expressão similar foi observado no caso do CG32239 e do CG13711. Nos tóraxes de adultos de 3 e 5 dias de idade os níveis de expressão do RNAm lacZ aumentam cerca de 37 vezes e 11 vezes, respectivamente, quando comparados aos níveis de expressão iv do RNAm lacZ presentes nos tóraxes de adultos de 1 dia. No tórax de adultos, o único CG que apresenta um padrão de expressão similar ao padrão de expressão de lacZ constitui o CG12493. Em conjunto, nós concluímos que o MCR TT promove um padrão dinâmico de expressão durante o desenvolvimento. Além disso, com base nos resultados de RT-qPCR, nós sugerimos que o MCR TT regula a expressão do RNAm do CG32239 no sistema traqueal durante a transição larva-prépupa e também a expressão do RNAm do CG12493 no tórax de adultos de 3 e 5 dias de idade. Além de estender a caracterização funcional de um novo MCR, nossos resultados também contribuem com novas informações acerca dos padrões de expressão no desenvolvimento de três CGs de D. melanogaster. / Previous functional studies identified in the DNA puff BhC4-1 promoter region a 67 bp (- 253/-187) cis-regulatory module (CRM) that drives reporter gene expression in the ring gland of D. melanogaster. A bioinformatics analysis identified 67 Drosophila melanogaster sequences that are similar to sequences contained in the ring gland CRM. One of the identified sequences resides in a 657 bp genomic fragment located about 2500 bp upstream CG13711, about 400 bp upstream CG12493, in a genomic region that constitutes one of the introns of CG32239 (Gef64C). The preliminary characterization of three transgenic lines transformed with a 657 bp-lacZ construct revealed reporter gene expression in the larval/prepupal tracheal system and in adult thorax. Based on the pattern of expression driven by this CRM we named it Trachea-Thorax (TT). The main goal of this work was to extend the molecular characterization of the lines of the TT-lacZ series. Initially ?-galactosidase histochemical assays were performed in embryos, first, second and third instar larvae, 0h, 1h and 2h prepupae, 24 h pupae and 1, 3 and 5 days old adults. Reporter gene expression is initially detected during the third larval instar in the tracheal system and continues to be detected in this tissue at 0 h, 1h and 2 h prepupa and, 24 h pupa. During the adult stage, reporter gene expression is verified in the dorsal longitudinal muscles of 3 and 5 days old adults. Since the TT CRM lies in an intergenic region and the available information about the nearby CGs is still scarce it was not possible to infer which of the CGs is regulated by the TT CRM. In this context, the mRNA pattern of expression of the lacZ reporter gene and of CG13711, CG12493 and CG32239 was investigated in the tracheal system of both larvae and prepupae and in adult thoraxes of one of the transgenic lines of the TT-lacZ series using RTqPCR. The lacZ mRNA expression levels increase about 3 times in 0 h prepupae when compared to the lacZ mRNA expression levels present in the tracheal system of third instar larvae. A similar pattern of expression was observed for both CG32239 and CG13711. In three and five days old adult thoraxes lacZ mRNA expression levels increase about 37 times and 11 times, respectively, when compared to lacZ mRNA expression levels present in one day old thoraxes. In the adult thorax, the only CG that presents a similar pattern of expression constitutes CG12493. Overall, we conclude that the TT CRM drives a dynamic pattern of ii expression throughout development. Additionally, based on RT-qPCR results, we suggest that the TT CRM regulates the expression of CG32239 mRNA in the tracheal system during the larvae to prepupae transition, as well as the expression of CG12493 mRNA in the thorax of 3 and 5 days old adults. Besides extending the functional characterization of a novel CRM our results also contribute new information about the developmental patterns of expression of three Drosophila melanogaster CGs.

La diversité morphologique des muscles squelettiques permet la précision et la coordination des mouvements propres à chaque espèce animale. L'établissement du patron musculaire a lieu au cours du développement embryonnaire durant le processus de myogenèse. Il a été décomposé en quatre étapes chez la drosophile : la spécification de groupes de myoblastes équivalents (groupes promusculaires) à des positions précises du mésoderme, la sélection d'une ou plusieurs cellules progéniteurs à partir de chaque groupe, la division asymétrique des progéniteurs en cellules fondatrices des muscles, et enfin, la fusion d'une cellule fondatrice avec un nombre défini de myoblastes compétents pour la fusion qui forme une myofibre syncytiale. Ce processus aboutit à la mise en place d'un patron stéréotypé de muscles morphologiquement distincts par leur taille, orientation, forme, et sites d'attachement au squelette ; ces caractères définissant l'identité du muscle. Chez la drosophile, chacun des 30 muscles par hémisegment de la larve est constitué d'une seule myofibre. Il a été proposé que l'identité morphologique de cette fibre soit contrôlée par une combinatoire de facteurs de transcription identitaires (FTi) exprimés par la cellule fondatrice. Mon projet de thèse a porté sur le contrôle transcriptionnel de l'identité musculaire, avec comme modèle d'étude, un muscle dorso-latéral de la larve de drosophile, le muscle DA3 dont un FTi est Collier/EBF (Col). La transcription de col est activée dans un groupe promusculaire, puis transitoirement dans les quatre progéniteurs issus de ce groupe, avant d'être maintenue spécifiquement dans la myofibre DA3. Dans des embryons mutants pour col, le DA3 est transformé en muscle plus dorsal, DA2. Les travaux précédents de l'équipe ont montré que la transcription de col dans le lignage DA3 est contrôlée par deux modules cis-régulateurs, EarlyCRM et LateCRM, séparés physiquement sur le chromosome et agissant séquentiellement. Leur chevauchement temporel d'activité restreint au progéniteur DA3 et l'autorégulation directe du LateCRM ont mené à l'hypothèse d'un mécanisme de " passage de témoin " entre ces deux CRM, spécifique au progéniteur DA3. L'objectif de ma thèse était de tester cette hypothèse et de comprendre comment une information temporelle et spatiale intégrée par un CRM est transmise à un autre CRM, pour définir une identité cellulaire, une question fondamentale au-delà du cas d'espèce que constitue le muscle DA3.[...] / The morphological diversity of skeletal muscles allows the precision and coordination of movements specific to each animal species. Establishment of a stereotypic pattern of muscles takes places during the process of myogenesis. Studies in Drosophila, an insect model, have identified four steps in this process: the specification of equivalence groups of myoblasts (promuscular clusters) at defined positions within the somatic mesoderm, the selection of progenitor(s) from each group, asymmetric division of each progenitor into post-mitotic muscle founder cells, and finally the fusion of each founder cell with a given number of fusion competent cells to form a syncytial myofiber. This dynamic, integrated process leads to establishing a stereotyped pattern of morphologically distinct muscles which can each be distinguished, based on size, orientation, shape, sites of attachment to the skeleton, all properties defining muscle identity. In the Drosophila larva, each of the about 30 different muscles per hemisegment is made of a single myofiber. It has been proposed that final morphology of a myofiber reflects the combinatorial code of identity Transcription Factors (iTF) expressed by its founder cell, although many questions remain unanswered. My thesis project aimed at better understanding the mechanism of specification of muscle identity, using as model a dorso-lateral muscle of the Drosophila larva, the DA3 muscle whose identity is controlled by the Collier/EBF (Col) iTF. col transcription is activated in one promuscular cluster, transient in the 4 progenitors issued from this cluster and stably maintained in the DA3 myofiber. In col mutant embryos, the DA3 muscle is transformed into a more dorsal, DA2-like muscle. Previous work has shown that col transcription in the DA3 lineage is controlled by two cis-regulatory modules (EarlyCRM and LateCRM), physically distant on the chromosome and acting sequentially. The temporal overlap of EarlyCRM and LateCRM in the DA3 progenitor and direct col autoregulation via the LateCRM led to hypothesize a handover between the two CRM in the DA3 progenitor. One goal of my thesis project was to challenge this hypothesis and understand how positional and temporal information integrated by EarlyCRM could be memorized via LateCRM, in order to specify cell identity, a fundamental question of developmental biology beyond the specific case of the Drosophila DA3 muscle. [...]

Thesis (M.S.)--Miami University, Dept. of Zoology, 2005. / Title from first page of PDF document. Document formatted into pages; contains [1], ix, 75 p. : ill. Includes bibliographical references (p. 69-75).

Estudos funcionais anteriores identificaram um módulo cis-regulador (MCR) de 67 pb (-253/- 187) na região promotora do gene de pufe de DNA BhC4-1 que dirige a expressão do gene repórter na glândula anelar de Drosophila melanogaster. Uma análise bioinformática identificou 67 sequências de D. melanogaster que são similares a sequências contidas no MCR de glândula anelar. Uma das sequências identificadas reside em um fragmento genômico de 657 pb localizado aproximadamente 2500 pb à montante do CG13711, 400 pb à montante do CG12493, em uma região genômica que constitui um dos íntrons do CG32239 (Gef64C). A caracterização preliminar de três linhagens transformadas com a construção 657 pb-lacZ mostrou expressão do gene repórter no sistema traqueal de larvas e prépupas e no tórax de adultos. Baseado padrão de expressão promovido por este MCR, o mesmo foi denominado Traqueia-Tórax (TT). O principal objetivo do presente trabalho constituiu estender a caracterização molecular das linhagens da série TT-lacZ. Inicialmente embriões, larvas de primeiro, segundo e terceiro estádio, prépupas 0 h, 1 h e 2 h, pupas 24 h e adultos com 1, 3 e 5 dias foram investigados quanto ao padrão de expressão do repórter utilizando ensaio histoquímico que detecta atividade de ?-galactosidase. A expressão do gene repórter é inicialmente detectada no sistema traqueal durante o terceiro estádio larval e continua a ser detectada neste tecido em prépupas 0 h, 1 h e 2 h e pupas 24 h. Em adultos, a expressão do gene repórter é verificada nos músculos longitudinais dorsais em adultos de 3 e 5 dias. Uma vez que o MCR TT reside em uma região intergênica e a informação disponível sobre os CGs próximos ainda é escassa, não foi possível inferir qual dos CGs é regulado pelo MCR TT. Neste contexto, o padrão de expressão do RNAm do gene repórter lacZ e do CG13711, CG12493 e CG32239 foi investigado no sistema traqueal de larvas e prépupas e no tórax de adultos de uma das linhagens da série TT-lacZ utilizando RT-qPCR. Os níveis de expressão do RNAm lacZ aumentam cerca de 3 vezes em prépupas 0 horas, quando comparados com os níveis de expressão do RNAm lacZ presentes no sistema traqueal de larvas de terceiro estádio. Um padrão de expressão similar foi observado no caso do CG32239 e do CG13711. Nos tóraxes de adultos de 3 e 5 dias de idade os níveis de expressão do RNAm lacZ aumentam cerca de 37 vezes e 11 vezes, respectivamente, quando comparados aos níveis de expressão iv do RNAm lacZ presentes nos tóraxes de adultos de 1 dia. No tórax de adultos, o único CG que apresenta um padrão de expressão similar ao padrão de expressão de lacZ constitui o CG12493. Em conjunto, nós concluímos que o MCR TT promove um padrão dinâmico de expressão durante o desenvolvimento. Além disso, com base nos resultados de RT-qPCR, nós sugerimos que o MCR TT regula a expressão do RNAm do CG32239 no sistema traqueal durante a transição larva-prépupa e também a expressão do RNAm do CG12493 no tórax de adultos de 3 e 5 dias de idade. Além de estender a caracterização funcional de um novo MCR, nossos resultados também contribuem com novas informações acerca dos padrões de expressão no desenvolvimento de três CGs de D. melanogaster. / Previous functional studies identified in the DNA puff BhC4-1 promoter region a 67 bp (- 253/-187) cis-regulatory module (CRM) that drives reporter gene expression in the ring gland of D. melanogaster. A bioinformatics analysis identified 67 Drosophila melanogaster sequences that are similar to sequences contained in the ring gland CRM. One of the identified sequences resides in a 657 bp genomic fragment located about 2500 bp upstream CG13711, about 400 bp upstream CG12493, in a genomic region that constitutes one of the introns of CG32239 (Gef64C). The preliminary characterization of three transgenic lines transformed with a 657 bp-lacZ construct revealed reporter gene expression in the larval/prepupal tracheal system and in adult thorax. Based on the pattern of expression driven by this CRM we named it Trachea-Thorax (TT). The main goal of this work was to extend the molecular characterization of the lines of the TT-lacZ series. Initially ?-galactosidase histochemical assays were performed in embryos, first, second and third instar larvae, 0h, 1h and 2h prepupae, 24 h pupae and 1, 3 and 5 days old adults. Reporter gene expression is initially detected during the third larval instar in the tracheal system and continues to be detected in this tissue at 0 h, 1h and 2 h prepupa and, 24 h pupa. During the adult stage, reporter gene expression is verified in the dorsal longitudinal muscles of 3 and 5 days old adults. Since the TT CRM lies in an intergenic region and the available information about the nearby CGs is still scarce it was not possible to infer which of the CGs is regulated by the TT CRM. In this context, the mRNA pattern of expression of the lacZ reporter gene and of CG13711, CG12493 and CG32239 was investigated in the tracheal system of both larvae and prepupae and in adult thoraxes of one of the transgenic lines of the TT-lacZ series using RTqPCR. The lacZ mRNA expression levels increase about 3 times in 0 h prepupae when compared to the lacZ mRNA expression levels present in the tracheal system of third instar larvae. A similar pattern of expression was observed for both CG32239 and CG13711. In three and five days old adult thoraxes lacZ mRNA expression levels increase about 37 times and 11 times, respectively, when compared to lacZ mRNA expression levels present in one day old thoraxes. In the adult thorax, the only CG that presents a similar pattern of expression constitutes CG12493. Overall, we conclude that the TT CRM drives a dynamic pattern of ii expression throughout development. Additionally, based on RT-qPCR results, we suggest that the TT CRM regulates the expression of CG32239 mRNA in the tracheal system during the larvae to prepupae transition, as well as the expression of CG12493 mRNA in the thorax of 3 and 5 days old adults. Besides extending the functional characterization of a novel CRM our results also contribute new information about the developmental patterns of expression of three Drosophila melanogaster CGs.

La régulation des gènes est à l’origine de la diversité cellulaire en permettant aux cellules de se différencier et de se spécialiser. La régulation génique repose largement sur l’existence de séquences d’ADN non codantes dans le génome, appelées "éléments cis-régulateurs", qui vont permettre de recruter de nombreux facteurs de transcription afin de former d’importants complexes (nucléo)protéiques qui vont agir sur le niveau de transcription des gènes. Ce recrutement est notamment contrôlé par des modifications épigénétiques. Le développement des techniques de séquençage et des méthodes d’analyse bioinformatiques permettent d’intégrer de grandes quantités de données pour étudier le fonctionnement des éléments régulateurs. Dans un premier temps, l’intégration de l’ensemble des données ChIP-seq disponibles dans les bases de données nous a permis de créer un catalogue d’éléments régulateurs putatifs chez l’Homme. L’analyse de ce catalogue nous a alors mené à caractériser ces éléments et à mettre en évidence la complexité combinatoire des facteurs de transcription. Dans un deuxième temps, nous avons réalisé une étude basée sur l’analyse des éléments régulateurs impliqués dans la différenciation précoce des lymphocytes T chez la souris. Cette étude a permis de mettre en évidence deux niveaux de complexité impliqués dans la régulation des gènes : le premier est basé sur la combinatoire des facteurs de transcription au sein des éléments régulateurs et le second repose sur la combinatoire des éléments eux-mêmes. Finalement, nous avons développé une nouvelle technique d’analyse quantitative et à haut débit de l’activité régulatrice de régions génomiques chez les mammifères. / Gene regulation is responsible for cell diversity by allowing cell differentiation and specialisation. Gene expression regulation relies mainly on the existence of non-coding DNA sequences in the genome, called "cis-regulatory elements", which recruit numerous transcription factors to form (nucleo)protein complexes which act on the gene transcription level. This recruitment is controlled in particular by epigenetic modifications. The rapid development of sequencing technologies and bioinformatics methods makes possible the integration of large amounts of data to study regulatory elements. First, the integration of ChIP-seq data for all transcription factors available in public databases has allowed us to create an extensive catalogue of putative regulatory elements in the human genome. The overall analysis of this catalogue led us to further characterize these elements and to highlight the high level of combinatorial complexity of transcription factors in the genome. Secondly, we conducted a more specific study based on the analysis of the regulatory elements involved in the early differentiation of T-cells in mice. This study provided an opportunity to highlight two levels of complexity based on regulatory elements and involved in gene regulation: the first rests on the transcription factor combinatorial in regulatory elements and the second is based on the combinatorial of elements themselves within loci. Finally, to validate experimentally the regulatory elements, we have developed a new quantitative and high-throughput technique to assess the regulatory activity of genomic regions in mammals.

Changes in cis- and trans-regulatory elements are among the prime sources of genetic and phenotypical variation at species level. The introduction of cis- and trans- regulatory variation has played important roles in driving diversity, phenotypical differentiation, and evolution of humans. Therefore, variation that occurs on cis- and trans- regulatory elements becomes imperative to better understanding of human genetic diversity and its evolution. In this research, around 3360 gene regulatory factors (GRF) from the human genome were catalogued. This catalog includes genes that code for proteins that perform gene regulatory activities such DNA-depending transcription, RNA polymerase II transcription cofactor and co-repressor activity, chromatin binding and remodeling, among other 218 regulatory functions. This GRF catalog allowed us to initially explore how some GRF genes have evolved in humans, archaic humans (Neandertal and Denisovan) and non-human primate species. We discussed the likely phenotypical and medical effects that evolutionary changes in GRF genes may have introduced into the human genome; for instance, traits associated to speech and language capabilities, genomic recombination hotspots, diseases, among others. By using genome-wide datasets, we additionally looked for GRFs likely to be candidates for positive selection in three human populations: Utah Residents with Northern and Western Ancestry (CEU), Han Chinese in Beijing (CHB), and Yoruba in Ibadan (YRI). As result, we produced a set of candidates that gathers genes that may have contributed in shaping the phenotypical diversity currently observed in these populations; for instance, by introducing regulatory diversity at population-specific level. We additionally identified six GRF classes enriched for genes located in regions that are likely candidates for positive selection at population specific level. We found that out of the 41 DNA-binding GRF classes classified so far, six groups exhibited enrichment for genes located on regions that may have been under positive selection: C2H2 zinc finger, KRAB-ZNF zinc finger, Homeo domain, Tryptophan cluster, Fork head/winged helix and, and High-mobility HMG domain. We additionally identified three KRAB-ZNF gene clusters, in the chromosomes one, three, and 16, for the Asian population that exhibit regions with extended haplotype homozygosity EHH (larger than 100 kb). This EHH suggests that these regions have undergone positive selection in CHB population. Finally, considering that a representative fraction of the phenotypic diversity observed between humans and its closely related species are likely explained by changes in cis-regulatory elements (CREs), we investigated putative binding sites for the transcription factor GABPa. Using ChIP-Seq data generated from a human cell line (HEK293T), 11,619 putative GABPa CREs were found, Out of which 224 are putative human-specific. To experimentally validate the transcriptional activity of these human-specific CREs, reporter gene essays and knock-down experiments were performed. Our results supported the functionality of these human-specific GABPa CREs and suggest that at least 1,215 genes are primary targets of GABPa. Finally, further analyses depict scenarios that put together transcriptional regulation by GABPa and the evolution of particular human traits; for instance, cognitive abilities, breast morphology, lipids and glucose metabolic pathways, among others.

Les ascidies présentent des propriétés remarquables au sein des métazoaires qui en font un modèle particulièrement intéressant pour étudier le fonctionnement et l’évolution des éléments cis-régulateurs dans un contexte développemental. Ciona intestinalis et Phallusia mammillata, deux espèces d’ascidies qui ont divergé il y a environ 300 millions d’années, combinent une grande conservation de leurs processus développementaux avec une grande divergence de leur séquence génomique. Pour comprendre comment « fabriquer » des embryons similaires avec des génomes divergents, nous avons identifié les éléments cis-régulateurs actifs au cours du développement de Ciona intestinalis et Phallusia mammillata en développant et en appliquant la méthode de ChIP-Seq sur des modifications d’histones sur des jeunes gastrulae. La définition puis la validation fonctionnelle de différentes catégories d'éléments cis-régulateurs nous a permis de révéler quelques propriétés de la cis-régulation au sein de génomes compacts et intensément remaniés. En sus, les données que nous avons produites constituent une resource fonctionnelle unique pour la caractérisation des éléments cis-régulateurs chez les ascidies et l'étude de leur évolution au sein des Chordés. / Ascidians display remarkable features within metazoans making them particularly suited for the study of function and evolution of cis-regulatory elements in the context of embryonic development. Ciona intestinalis and Phallusia mammillata, two ascidian species that diverged about 300M years ago, combine high conservation of their developmental processes with high divergence of their genome sequence. To understand how to “make” similar embryos with divergent genomes, we identified active cis-regulatory elements during Ciona intestinalis and Phallusia mammillata development by developing and applying the ChIP-Seq method on histone modifications in early-gastrula embryos. Definition then functional validation of different categories of cis-regulatory elements led us to reveal some features of cis-regulation within compact and highly dynamic genomes. Together, our data constitute a unique functional resource for characterizing cis-regulatory elements in ascidians and questioning their evolution within the Chordates.

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