El presente trabajo de tesis estudia diferentes mutantes zeste1 y el efecto que esta mutación tiene sobre la regulación de la transcripción en diversos genes. En primer lugar se estudiaron los machos de las cepas M115 y RM115 cuya característica principal, además de ser portadores de la mutación z1, es la inserción de un elemento FB-NOF en el tercer intrón del gen CG32795, que forma con white un par de genes head-to-tail. Para estudiar el efecto de la inserción de FB-NOF en un entorno z1, fue necesario en primer lugar caracterizar el gen CG32795, a nivel de secuencia del mRNA y de predicción de la posible función de la proteína codificada. Se encontraron diversas variantes de splicing, tanto para su extremo 5' como para el 3', parecidas pero diferentes de las variantes predichas anteriormente. Conociendo más en profundidad este gen, nos propusimos estudiar los posibles efectos de la inserción de FB-NOF respecto a la expresión de los genes w y CG32795. Los resultados nos llevaron a un estudio más detallado de la expresión de w en diferentes partes del cuerpo, teniendo en cuenta la especificidad de tejido que la interacción zeste-white presenta. Así, demostramos que el gen w no se ve afectado por la inserción de FB-NOF en su extremo 3' y que las diferencias de expresión observadas son debidas a la duplicación del Zeste Binding Site de w en un entorno z1. Sin embargo, la inserción de FB-NOF en el tercer intrón de CG32795 si modifica la expresión de este gen. La inserción no es sólo responsable de la alteración de la expresión de CG32795 sino también la reordenación que se produce en la cepa RM115 eliminando la copia de w original y dejando la que se duplicó en M115.La segunda parte de esta tesis estudia las hembras mutantes z1. Analizamos la expresión de dos genes con un ZBS (w y dpp) y observamos como se reduce la expresión de dichos genes en las hembras z1. Además, estudiando la expresión del gen CG32795 constatamos que el efecto de la interacción zeste-white es local, sin afectar a CG32795 aunque su extremo 5' se encuentra a tan solo 700bp del extremo 3' del gen w. La reducción de la expresión podría ser debida a alteraciones en la estructura de la cromatina, puesto que los agregados de Zeste en los ZBS son los responsables de reclutar el complejo remodelador de la cromatina BRM, facilitando así la transcripción. Mediante un ensayo de nucleasa micrococal detectamos algunas modificaciones en el posicionamiento de nucleosomas en los ZBS de w y dpp, siendo el posicionamiento en las hembras z1 más estricto. Si el complejo BRM es el responsable de estas diferencias, los patrones de metilación también podrían verse alterados, pues muchos factores asociados a los complejos SWI/SNF se han relacionado con actividades de regulación de la expresión mediante modificación de los mismos. Mediante el ensayo de modificación del DNA por bisulfito sódico estudiamos los patrones de metilación de cinco regiones. Sorprendentemente, el ZBS de w y de dpp, así como las regiones próximas a ellos, se encontraban hipometilados en las hembras z1. El desconocimiento general sobre la metilación en D. melanogaster nos hizo plantear cuales pueden ser las secuencias diana de la metilación en esta especie. Así realizamos un estudio estadístico sobre cuales son las dos bases anteriores y posteriores a las Cs metiladas en nuestras secuencias. Se obtuvieron diferencias en las frecuencias de cada posición y se estableció como secuencia más frecuente para ser metilada ApDp5mCpDpD. Aún así, es una secuencia muy degenerada, y se hacen necesarios estudios más profundos y amplios para confirmarla. / The present PhD thesis studies several zeste1 mutants and this mutation effect over the transcriptional regulation of some genes. The first part is based on the study of the males of M115 and RM115 strains, mainly characterized by the z1 mutation and the insertion of a FB-NOF element in the third intron of the CG32795 gene, known to form a head-to-tail gene pair with white. To study the FB-NOF insertion effect on a z1 background firstly we need to characterize the CG32795, its mRNA sequence and the codified protein predicted structure and function. Several splicing variants were found, not only for its 5' end but also for its 3'end, similar but different from the ones been predicted previously. Once obtained this information we proceeded to study the effects of the FB-NOF insertion over the w and the CG32795 gene expression. The results lead us to a deeper study of the w expression in different body segments, taking into account the zeste-white interaction tissue specificity. Thus, we proved that the w gene expression is not affected by the FB-NOF insertion downstream of its 3' end. The differences observed in the w expression levels were due to the duplication of the w Zeste Binding Site in a z1 background. On the contrary, the FB-NOF insertion not only does modify CG32795 expression, but also mediates the reordenation produced in the RM115 strain being responsible of the w original copy lost and leaving alone the w copy duplicated in the M115 strain.The second part studies the z1 female mutants. We analyzed the expression of two genes possessing a ZBS (w and dpp). The results obtained showed an expression reduction for both genes in the z1 females. Moreover, through the study of the CG32795 gene expression we showed that the zeste-white interaction effect is local, not having any effect on CG32795 expression even though its 5' end is located at only 700bp from the w gene 3' end. Knowing that the Zeste aggregates in the ZBS recruit the chromatin remodelling complex BRM, facilitating thus transcription, we thought that the expression reduction might occur due to chromatin structure alterations. A micrococal nuclease assay was performed and some modifications in nucleosome positioning were found in w and dpp ZBS, being the positioning in the z1 females stricter. Were the BRM complex responsible of those differences, the DNA methylation patterns might also be changed, since many SWI/SNF associated factors have been related to expression regulation duties through DNA methylation patterns modification. A bisulphite modification assay was performed, studying the DNA methylation pattern of five different regions. Surprisingly, w and dpp ZBS and the regions surrounding them were hypomethylated in the z1 females. The little information available about DNA methylation in D. melanogaster encouraged us to study the DNA methylation sites in this specie. Our sequences were statistically analyzed including the two bases before and after the methylated cytosine. The results showed differences in the each nucleotide frequency in each position. The preferently methylated "consensus sequence": ApDp5mCpDpD. However, it is a much degenerated sequence and deeper and broader studies are required to confirm it.

<p><!--[if gte mso 9]><xml> <w:WordDocument> <w:View>Normal</w:View> <w:Zoom>0</w:Zoom> <w:HyphenationZone>21</w:HyphenationZone> <w:PunctuationKerning /> <w:ValidateAgainstSchemas /> <w:SaveIfXMLInvalid>false</w:SaveIfXMLInvalid> <w:IgnoreMixedContent>false</w:IgnoreMixedContent> <w:AlwaysShowPlaceholderText>false</w:AlwaysShowPlaceholderText> <w:Compatibility> <w:BreakWrappedTables /> <w:SnapToGridInCell /> <w:WrapTextWithPunct /> <w:UseAsianBreakRules /> <w:DontGrowAutofit /> </w:Compatibility> <w:BrowserLevel>MicrosoftInternetExplorer4</w:BrowserLevel> </w:WordDocument> </xml><![endif]--><!--[if gte mso 9]><xml> <w:LatentStyles DefLockedState="false" LatentStyleCount="156"> </w:LatentStyles> </xml><![endif]--> <!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; line-height:150%; mso-pagination:widow-orphan; font-size:12.0pt; mso-bidi-font-size:10.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:EN-US;} p.Standardmedluft, li.Standardmedluft, div.Standardmedluft {mso-style-name:"Standard med luft"; margin-top:14.0pt; margin-right:0cm; margin-bottom:0cm; margin-left:0cm; margin-bottom:.0001pt; text-align:justify; line-height:150%; mso-pagination:widow-orphan; font-size:12.0pt; mso-bidi-font-size:10.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:EN-US;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 70.85pt 2.0cm 70.85pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> <!--[if gte mso 10]><mce:style><! /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-ansi-language:#0400; mso-fareast-language:#0400; mso-bidi-language:#0400;} --><!--[endif]--></p><p>Transvection is a widespread phenomenon affecting chromosomal and gene function. There are many examples of epigenetic machineries controlling gene regulation. Nuclear Lamin proteins could have this function. This project shows <em>zeste</em> dependent transvection<strong> </strong>in loss-of-function<strong> </strong><em>lamin</em> mutants in <em>Drosophila melanogaster</em>.<strong> </strong>The <em>zeste</em> locus<strong> </strong>encodes a regulatory gene product affecting the expression of other loci, e.g. <em>white</em>. No transvection effect in loss-of-function <em>lamin </em>mutants has so far been shown. The effect of homozygosity versus heterozygosity of <em>lamin</em> on <em>zeste</em>-dependent transvection at paired <em>white</em> loci was analysed by crossing fruit flies to get homozygous<em> </em><em>z¹</em>; <em>lam</em>^D395 individuals. Whether or not the <em>zeste (z¹</em>) transvection effect on <em>white</em> was affected by <em>lam</em> ^D395 loss-of-function mutation was determined by comparing the eye colour phenotypes of double mutant <em>z¹</em>; <em>lam</em>^D395 females to that of <em>z¹/Y</em>; <em>lam</em>^D395 males, which were used as an internal negative control since they are hemizygous for <em>zeste</em> that is located on the X chromosome. Females homozygous for <em>z¹</em> and <em>lam</em>^D395 displayed the <em>z¹</em>-characteristic yellow eye colour. The conclusion is that <em>zeste</em>-dependent transvection effect at <em>white</em> also occurs in <em>lamin</em> mutants. Future research on transvection is needed in order to understand the exact mechanisms of gene regulation. Even gene therapies for some human diseases can take advantage of <em>trans</em>-acting sequences to correct gene expression.</p><p> </p>

The development of animals and plants needs a higher order of regulation of gene expression to maintain proper cell state. The mechanisms that control what, when and where a gene should (or should not) be expressed are essential for correct organism development. The Polycomb group (PcG) is a family of genes responsible for maintaining gene silencing and Suppressor of zeste 12 (Su(z)12) is one of the core components in the PcG. The gene is highly conserved in organisms ranging from plants to humans, however, the specific function is not well known. The main tasks of this thesis was to investigate the function of Su(z)12 and its expression at different stages of Drosophila development. In polytene chromosomes of larval salivary glands, Su(z)12 binds to about 90 specific euchromatic sites. The binding along the chromosome arms is mostly in interbands, which are the most DNA de-condensed regions. The binding sites of Su(z)12 in polytene chromosomes correlate precisely with those of the Enhancer-of-zeste (E(z)) protein, indicating that Su(z)12 mainly exists within the Polycomb Repressive Complex 2 (PRC2). However, the binding pattern does not overlap well with Histone 3 lysine 27 tri-methylations (H3K27me3), the specific chromatin mark created by PRC2. The Su(z)12 binding to chromatin is dynamically regulated during mitotic and meiotic cell division. The two different Su(z)12 isoforms: Su(z)12-A and Su(z)12-B (resulting from alternative RNA splicing), have very different expression patterns during development. Functional analyses indicate that they also have different functions he Su(z)12-B form is the main mediator of silencing. Furthermore, a neuron specific localization pattern in larval brain and a giant larval phenotype in transgenic lines reveal a potential function of Su(z)12-A in neuron development.  In some aspects the isoforms seem to be able to substitute for each other. The histone methyltransferase activity of PRC2 is due to the E(z) protein. However, Su(z)12 is also necessary for H3K27me3 methylation in vivo, and it is thus a core component of PRC2. Clonal over-expression of Su(z)12 in imaginal wing discs results in an increased H3K27me3 activity, indicating that Su(z)12 is a limiting factor for silencing. When PcG function is lost, target genes normally become de-repressed. The segment polarity gene engrailed, encoding a transcription factor, is a target for PRC2 silencing. However, we found that it was not activated when PRC2 function was deleted. We show that the Ultrabithorax protein, encoded by another PcG target gene, also acts as an inhibitor of engrailed and that de-regulation of this gene causes a continued repression of engrailed. The conclusion is that a gene can have several negative regulators working in parallel and that secondary effects have to be taken into consideration, when analyzing effects of mutants. PcG silencing affects very many cellular processes and a large quantity of knowledge is gathered on the overall mechanisms of PcG regulation. However, little is known about how individual genes are silenced and how cells “remember” their fate through cell generations.

<!--[if gte mso 9]><xml> <w:WordDocument> <w:View>Normal</w:View> <w:Zoom>0</w:Zoom> <w:HyphenationZone>21</w:HyphenationZone> <w:PunctuationKerning /> <w:ValidateAgainstSchemas /> <w:SaveIfXMLInvalid>false</w:SaveIfXMLInvalid> <w:IgnoreMixedContent>false</w:IgnoreMixedContent> <w:AlwaysShowPlaceholderText>false</w:AlwaysShowPlaceholderText> <w:Compatibility> <w:BreakWrappedTables /> <w:SnapToGridInCell /> <w:WrapTextWithPunct /> <w:UseAsianBreakRules /> <w:DontGrowAutofit /> </w:Compatibility> <w:BrowserLevel>MicrosoftInternetExplorer4</w:BrowserLevel> </w:WordDocument> </xml><![endif]--><!--[if gte mso 9]><xml> <w:LatentStyles DefLockedState="false" LatentStyleCount="156"> </w:LatentStyles> </xml><![endif]--> <!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; line-height:150%; mso-pagination:widow-orphan; font-size:12.0pt; mso-bidi-font-size:10.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:EN-US;} p.Standardmedluft, li.Standardmedluft, div.Standardmedluft {mso-style-name:"Standard med luft"; margin-top:14.0pt; margin-right:0cm; margin-bottom:0cm; margin-left:0cm; margin-bottom:.0001pt; text-align:justify; line-height:150%; mso-pagination:widow-orphan; font-size:12.0pt; mso-bidi-font-size:10.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:EN-US;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 70.85pt 2.0cm 70.85pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> <!--[if gte mso 10]><mce:style><! /* Style Definitions */ table.MsoNormalTable{mso-style-name:"Table Normal";mso-tstyle-rowband-size:0;mso-tstyle-colband-size:0;mso-style-noshow:yes;mso-style-parent:"";mso-padding-alt:0cm 5.4pt 0cm 5.4pt;mso-para-margin:0cm;mso-para-margin-bottom:.0001pt;mso-pagination:widow-orphan;font-size:10.0pt;font-family:"Times New Roman";mso-ansi-language:#0400;mso-fareast-language:#0400;mso-bidi-language:#0400;} --><!--[endif]--> Transvection is a widespread phenomenon affecting chromosomal and gene function. There are many examples of epigenetic machineries controlling gene regulation. Nuclear Lamin proteins could have this function. This project shows zeste dependent transvection in loss-of-function lamin mutants in Drosophila melanogaster. The zeste locus encodes a regulatory gene product affecting the expression of other loci, e.g. white. No transvection effect in loss-of-function lamin mutants has so far been shown. The effect of homozygosity versus heterozygosity of lamin on zeste-dependent transvection at paired white loci was analysed by crossing fruit flies to get homozygous z1; lamD395 individuals. Whether or not the zeste (z1) transvection effect on white was affected by lam D395 loss-of-function mutation was determined by comparing the eye colour phenotypes of double mutant z1; lamD395 females to that of z1/Y; lamD395 males, which were used as an internal negative control since they are hemizygous for zeste that is located on the X chromosome. Females homozygous for z1 and lamD395 displayed the z1-characteristic yellow eye colour. The conclusion is that zeste-dependent transvection effect at white also occurs in lamin mutants. Future research on transvection is needed in order to understand the exact mechanisms of gene regulation. Even gene therapies for some human diseases can take advantage of trans-acting sequences to correct gene expression.

El presente trabajo trata sobre dos temas coyunturalmente relacionados: el fenotipo de las cepas M115 y RM115 de Drosophila melanogaster y el elemento transponible FB-NOF.La cepa M115 de D. melanogaster presenta el fenotipo zeste1 ampliado a los machos. Este fenotipo se caracteriza por los ojos de color amarillo claro y, aunque normalmente se presenta en hembras pues es necesario el apareamiento de dos copias del gen white para producirse, puede observarse en machos portadores de duplicaciones de white. Es la conocida interacción zeste-white. M115 es una cepa inestable y se pueden encontrar individuos revertientes de forma regular. Un macho revertiente dio lugar a la cepa RM115. Este par de cepas son fenotípicamente parecidas a las descritas por la Dra. Rasmuson-Lestander, w+UZ y w+UR.Siguiendo los pasos de Rasmuson-Lestander en la caracterización de w+UZ y w+UR, identificamos la inserción de un elemento transponible FB-NOF en nuestras cepas M115 y RM115, pocas kilobases a 3' del gen white. Aunque el punto de inserción es exactamente el mismo que en las cepas de Rasmuson-Lestander, nuestros experimentos de Southern Blot demuestran que se trata de inserciones distintas, lo cual plantea si esa región podría ser un hot spot de integración para FB-NOF. Al llevar el análisis un paso más adelante, encontramos que las cepas de ojos amarillos, M115 y w+UZ, eran portadoras de una duplicación del gen white, que explica el fenotipo ampliado a los machos. La reversión en RM115, por su parte, consiste en la deleción de una de las dos copias del gen. Así pues, debemos descartar las hipótesis de Rasmuson-Lestander que apuntaban a la interferencia de FB-NOF en la interacción zeste-white.El elemento transponible FB-NOF es uno de los menos conocidos de D. melanogaster. Dio origen al grupo de transposones denominados foldback por su estructura modular repetitiva y se desconocen los detalles de su biología. En este trabajo proponemos una forma más sencilla de estructurar el elemento que facilita su estudio, sobretodo a partir de secuencias genómicas. Además, analizamos su distribución en el genoma de D. melanogaster, la relación entre las secuencias que lo integran y la expresión de su secuencia codificante. Los resultados de dicho análisis revelan que FB-NOF funciona como un solo transposón con un sesgo en sus preferencias de inserción y que se mantiene activo en la actualidad. / The subject of the present thesis focuses on two related issues: the phenotype of the mutant strains M115 and RM115 from Drosophila melanogaster and the transposable element FB-NOF.The D. melanogaster strain M115 presents an extended zeste1 phenotype to both males and females. This phenotype confers a pale yellow color in the eyes and, however it is usually seen only in females since it is necessary to have paired copies of the white gene, this phenotype can also be observed in males carrying a white duplication. This is known as the zeste-white interaction. M115 is an unstable strain and one can find revertant flies regularly. A revertant male established the so-called RM115 strain. These two strains are phenotipically similar to those described by Dr. Rasmuson-Lestander, w+UZ and w+UR.Following Rasmuson-Lestander's steps in the characterization of w+UZ and w+UR, we identified the insertion of an FB-NOF transposable element in M115 and RM115, just few kilobases downstream the white gene's coding region. Despite the insertion point is exactly the same in both sets of strains, ours and Rasmuson-Lestander's, our Southern blot analysis demonstrate that the two insertions are different. This suggests we could have found a hot spot for the FB-NOF integration. Taking our analysis a step further, we found that the yellow-eyed strains, M115 and w+UZ, carried a white gene duplication, which is the main cause for the male extended zeste1 phenotype. Reversion of the phenotype consists in deletion of one of the two copies of the gene. Therefore, we must discard Rasmuson-Lestander's hypothesis pointing to an FB-NOF interference in the zeste-white interaction as the main cause of the extended phenotype.The FB-NOF transposable element is one of the less known in D. melanogaster. It established the group called foldback transposons due to its modular and repetitive structure, and its biology still remains unknown. In this thesis, we propose a simplest way to arrange the element's sequence to make its study from genomic sequences easier. Moreover, we analyze its distribution in the D. melanogaster's genome, the relationship between the sequences that compose it and the expression of the coding regions within. The results from this analysis reveal that FB-NOF behaves as a single transposon, with a bias in its insertion preferences and it is still active at the present time.

Implications des complexes Polycomb et Trithorax au cours du développement précoce chez Ciona intestinalisLes protéines des groupes Polycomb (PcG) et Trithorax (TrxG) ont été initialement découvertes chez Drosophila melanogaster. Ces deux groupes sont classiquement connus pour leurs rôles respectifs de répresseurs et d'activateurs épigénétiques qui contrôlent et maintiennent les états chromatiniens au cours du temps. Ces facteurs régulent de nombreux gènes cibles dont les gènes homéotiques. Au cours de ma thèse, j'ai étudié trois composants de ces deux groupes : Enhancer of zeste (E(z)), appartenant au complexe PRC2 du PcG et responsable du dépôt de la marque de répression génique H3K27me3, Polyhomeotic (Ph), appartenant au complexe PRC1 du PcG et dont le rôle exact reste à déterminer, et Trithorax (Trx), appartenant au complexe TAC1 du TrxG et responsable du dépôt de la marque d'activation génique H3K4me3. Jusqu'à présent, aucune étude n'a abordé la régulation épigénétique via les PcG et TrxG chez l'ascidie solitaire Ciona intestinalis. Cette espèce présente un cluster des gènes Hox désorganisé et ne possède pas la protéine Polycomb (Pc) du PRC1, responsable de la reconnaissance de la marque de répression H3K27me3 déposée par la protéine E(z).Nos travaux montrent que la protéine E(z) est fonctionnelle et conserve son activité méthyltransférase sur le résidu H3K27 chez Ciona intestinalis. Nous avons ensuite observé, par des expériences de knockdown par micro-injection de morpholinos, que les inhibitions protéiques d'E(z), Ph et Trx ont des conséquences dramatiques sur la différenciation et la mise en place des différents tissus au cours du développement larvaire, notamment sur la mise en place de la notochorde puisque celle-ci est totalement absente chez les morphants E(z) et Ph. Les défauts de phénotype du morphant E(z) sont corrélés à la perte du dépôt d'H3K27me3 et nous avons mis en évidence, lors de l'inhibition d'E(z), une dérépression des gènes tissu-spécifiques impliqués dans le développement embryonnaire précoce alors que les gènes tardivement exprimés sont réprimés. De plus, l'expression des gènes Hox n'est pas significativement modifiée au cours du développement embryonnaire lorsque la protéine E(z) est inhibée, à l'exception du gène Hox12 qui est déréprimé, comme attendu.L'ensemble de ces résultats permet d'émettre l'idée innovante selon laquelle les protéines des PcG et TrxG jouent un rôle déterminant dans la régulation de l'expression génique lors de l'embryogénèse de Ciona intestinalis tout en ayant une implication mineure dans la régulation de l'expression des gènes Hox à ce stade du développement. / Implications of Polycomb and Trithorax complexes in the early development of Ciona intestinalisPolycomb and Trithorax group (PcG and TrxG) proteins were discovered originally in Drosophila melanogaster. Both groups are classically known for their roles in the maintenance of silenced and active chromatin states over time, respectively. These factors regulate many target genes including the homeotic genes. During my PhD, I studied three components of these two groups: Enhancer of zest (E(z)), belonging to the PRC2 complex of PcG and responsible for H3K27me3 mark deposit for gene repression, Polyhomeotic (Ph), belonging to the PRC1 complex of PcG whose role remains to be determined, and Trithorax (Trx), belonging to the TAC1 complex of TrxG and responsible for H3K4me3 mark deposit for gene activation. Until now, no study addresses the epigenetic regulation mediated by PcG and TrxG in the solitary ascidian Ciona intestinalis. This specie has a disorganized Hox cluster and in which the Polycomb (Pc) protein of PRC1, responsible for the recognition of the repressive H3K27me3 mark, is absent.Our work shows that the E(z) protein is functional and retains its methyltransferase activity on H3K27 residue in Ciona intestinalis. Then, we demonstrated, by knockdown experiments with morpholino microinjection, that the inhibition of E(z), Ph and Trx has dramatic consequences on differentiation and on the establishment of different tissues during larval development, particularly on the notochord establishment since it is totally absent in E(z) and Ph morphants. E(z) morphant phenotypic defects are correlated with lack of H3K27me3 mark deposit and we highlighted that, during the E(z) inhibition, tissue-specific genes implied in early development are de-repressed while late-expressed genes are down-regulated. In addition among Hox genes, only Hox12 expression is significantly modified and found to be de-repressed in E(z) morphant context, as expected.Altogether, our results present the innovative idea that the PcG and TrxG proteins play a major role in the gene expression regulation during embryogenesis of Ciona intestinalis while having a minor involvement in the regulation of Hox genes expression at this stage of development.

Analyse fonctionnelle de la protéine Enhancer of Zeste, SlEZ2, chez la tomate, Solanum lycopersicumLes protéines Polycomb, initialement découvertes chez la drosophile, ont récemment caractérisées chez les plantes où elles remplissent des fonctions essentielles au cours du développement de la plante. Chez la drosophile, les protéines polycomb (PcG) agissent sous forme de trois complexes multi-protéiques : PRC1, PRC2 et PhoRC. Seulement, deux de ces complexes ont été identifiés chez les plantes : un orthologue fonctionnel du complexe PRC1 (PRC1-like) et PRC2. Le complexe PRC2 maintien la chromatine dans un état condensé et intervient dans le contrôle du développement des fleurs, des graines, des fruits et des feuilles. Chez la tomate Solanum lycopersicum, le complexe PRC2 est composé de trois protéines polycomb : SlEMF2 (EMbryotic Flower), SlFIE (Fertilization Independent Endosperm) and SlE(Z) (Enhancer of Zeste). Les protéines SlE(Z) portent l’activité histone méthyl transférase qui permet la mise en place de la marque répressive H3K27me3. Chez la plante modèle, Arabidopsis thaliana, cette marque joue un rôle essentiel au cours du développement de la plante Afin d’étudier le rôle du complexe PRC2 dans le développement du fruit et de la plante de tomate, et plus particulièrement de la protéine SlE(Z), nous avons identifié trois gènes codant les protéines SlE(Z) : SlEZ1, SlEZ2 et SlEZ3. Au laboratoire, il a récemment été montré que la protéine SlEZ1 intervient au cours du développement floral (How Kit et al., 2010). L’objectif de ce travail est de déterminer la fonction de la protéine SlEZ2 au cours du développement du fruit et de la plante de tomate. Pour cela, nous avons analysé des plantes transgéniques sous exprimant le gène SlEZ2, orthologue au gène CURLY LEAF d’A. thaliana, par stratégie RNAi. Ce travail indique que la protéine SlEZ2 est impliquée dans la croissance de la plante de tomate, ainsi que dans le développement des feuilles, des fleurs et des fruits. Les plantes transgéniques présentent des phénotypes pléiotropes tels que des fleurs et des feuilles modifiées, un fort taux d’avortement des fruits, des fruits de texture et de couleur altérées ainsi qu’une réduction de la taille des plantes. De plus, nous avons identifiés quatre gènes ciblés par la protéine SlEZ2 dont l’expression est dérégulée dans les feuilles. Il s’agit de deux gènes à MADS box, TAG1 et TAGL1, ainsi que de deux gènes KNOX, LeT6 et TKN4. / Functional analysis SlEZ2, a tomato Enhancer of zeste proteinPolycomb proteins, first discovered in Drosophila, have been identified in plants and play essential functions in plant development. In Drosophila, polycomb proteins (PcG) acts as a complex and three have been identified: PRC1, PRC2 and PhoRC. However, only two polycomb complexes have been identified in plants: like-PCR1 and PRC2. The PCR2 complex maintain chromatin in a closed state and control flower, seed, fruit and leaf development.In tomato Solanum lycopersicum, PRC2 is composed by three polycomb proteins SlEMF2 (EMbryotic Flower), SlFIE (Fertilization Independent Endosperm) and SlE(Z) (Enhancer of Zeste)(Enhancer of Zeste). SlE(Z) proteins have a methyltransferase activity that puts in place an repressive epigenetic mark a trimethylation of lysine 27 histone 3. In plant model, Arabidopsis thaliana, this mark plays an essential role in plant development but little is known about PRC2 role in plant and fruit development of tomato. In order to unravel the function of the E(z) protein in the control of tomato fruit and plant development, we have characterized three E(z) encoding genes, namely SlEz1, SlEz2 and SlEZ3. In a recent work, we reported that SlEZ1 protein plays a role in flower development (How Kit at al., 2010). The aim of this present study was to determine the function of the SlEZ2 protein in plant and fruit development. We present our results focusing on RNAi transgenic plants which underexpressed SlEZ2 gene, homologue of Curly Leaf Arabidopsis gene. This analysis indicates that SlEZ2 protein is implicated in tomato plant growth and affects also leaf, flower and fruit development. Phenotypes include abnormal flowers and leafs, fruit development abortion, altered fruit colour and texture and plant of reduced size. Moreover, we characterize four target genes of SlEZ2 genes in leaves which present a deregulated expression : TAG1, TAGL1, LeT6 and TKN4.

No description available.

<p>In multicellular organisms all cells in one individual have an identical genotype, and yet their bodies consist of many and very different tissues and thus many different cell types. Somehow there must be a difference in how genes are interpreted. So, there must be signals that tell the genes when and where to be active and inactive, respectively. In some instances a specific an expression pattern (active or inactive) is epigenetic; it is established and maintained throughout multiple rounds of cell divisions. In the developing <i>Drosophila</i> embryo, the proper expression pattern of e.g. the homeotic genes <i>Abd-B</i> and <i>Ubx</i> is to be kept active in the posterior part and silenced in the anterior. Properly silenced homeotic genes are crucial for the correct segmentation pattern of the fly and the Polycomb group (Pc-G) proteins are vital for maintaining this type of stable repression.</p><p>As part of this thesis, <i>Suppressor of zeste 12 (Su(z)12)</i> is characterized as a <i>Drosophila</i> Pc-G gene. Mutations in the gene cause widespread misexpression of several homeotic genes in embryos and larvae. Results show that the silencing of the homeotic genes <i>Abd-B</i> and <i>Ubx</i>, probably is mediated via physical binding of SU(Z)12 to Polycomb Response Elements in the BX-C. <i>Su(z)12</i> mutations are strong suppressors of position-effect-variegation and the SU(Z)12 protein binds weakly to the heterochromatic centromeric region. These results indicate that SU(Z)12 has a function in heterochromatin-mediated repression, which is an unusual feature for a Pc-G protein. The structure of the <i>Su(z)12</i> gene was determined and the deduced protein contains a C2-H2 zinc finger domain, several nuclear localization signals, and a region, the VEFS box, with high homology to mammalian and plant homologues. <i>Su(z)12 </i>was originally isolated in a screen for modifiers of the zeste-white interaction and I present results that suggests that this effect is mediated through an interaction between <i>Su(z)12 </i>and <i>zeste</i>. I also show that <i>Su(z)12</i> interact genetically with other Pc-G mutants and that the SU(Z)12 protein binds more than 100 euchromatic bands on polytene chromosomes. I also present results showing that SU(Z)12 is a subunit of two different E(Z)/ESC embryonic silencing complexes, one 1MDa and one 600 kDa complex, where the larger complex also contains PCL and RPD3. </p><p>In conclusion, results presented in this thesis show that the recently identified Pc-G gene, <i>Su(z)12</i>, is of vital importance for correct maintenance of silencing of the developmentally important homeotic genes.</p>

In multicellular organisms all cells in one individual have an identical genotype, and yet their bodies consist of many and very different tissues and thus many different cell types. Somehow there must be a difference in how genes are interpreted. So, there must be signals that tell the genes when and where to be active and inactive, respectively. In some instances a specific an expression pattern (active or inactive) is epigenetic; it is established and maintained throughout multiple rounds of cell divisions. In the developing Drosophila embryo, the proper expression pattern of e.g. the homeotic genes Abd-B and Ubx is to be kept active in the posterior part and silenced in the anterior. Properly silenced homeotic genes are crucial for the correct segmentation pattern of the fly and the Polycomb group (Pc-G) proteins are vital for maintaining this type of stable repression. As part of this thesis, Suppressor of zeste 12 (Su(z)12) is characterized as a Drosophila Pc-G gene. Mutations in the gene cause widespread misexpression of several homeotic genes in embryos and larvae. Results show that the silencing of the homeotic genes Abd-B and Ubx, probably is mediated via physical binding of SU(Z)12 to Polycomb Response Elements in the BX-C. Su(z)12 mutations are strong suppressors of position-effect-variegation and the SU(Z)12 protein binds weakly to the heterochromatic centromeric region. These results indicate that SU(Z)12 has a function in heterochromatin-mediated repression, which is an unusual feature for a Pc-G protein. The structure of the Su(z)12 gene was determined and the deduced protein contains a C2-H2 zinc finger domain, several nuclear localization signals, and a region, the VEFS box, with high homology to mammalian and plant homologues. Su(z)12 was originally isolated in a screen for modifiers of the zeste-white interaction and I present results that suggests that this effect is mediated through an interaction between Su(z)12 and zeste. I also show that Su(z)12 interact genetically with other Pc-G mutants and that the SU(Z)12 protein binds more than 100 euchromatic bands on polytene chromosomes. I also present results showing that SU(Z)12 is a subunit of two different E(Z)/ESC embryonic silencing complexes, one 1MDa and one 600 kDa complex, where the larger complex also contains PCL and RPD3. In conclusion, results presented in this thesis show that the recently identified Pc-G gene, Su(z)12, is of vital importance for correct maintenance of silencing of the developmentally important homeotic genes.