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Transcriptional regulation of egr-1 gene in murine cells. Towards the understanding of the role of chromatin.

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Transcriptional regulation of egr-1 gene in murine cells. Towards the understanding of the role of chromatin.

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dc.contributor.advisor López Rodas, Gerardo es_ES
dc.contributor.advisor Rodríguez García, José Luís es_ES
dc.contributor.author Tur Arlandis, Gema es_ES
dc.contributor.other Universitat de València - BIOQUÍMICA I BIOLOGIA MOLECULAR es_ES
dc.date.accessioned 2010-07-07T08:02:13Z
dc.date.available 2010-07-07T08:02:13Z
dc.date.issued 2007 es_ES
dc.date.submitted 2007-06-07 es_ES
dc.identifier.uri http://hdl.handle.net/10550/15014
dc.description.abstract Eukaryotic gene expression is a highly regulated process that has to ensure the right cellular response to any type of stimulus. Nevertheless, chromatin structure, despite the fact of being dinamic, imposes certain contraints to this regulation. To solve this restrictions, cells have organized the recruitment of chromatin modifying enzymes and ATP-dependent chromatin remodelling enzymes. The first class of enzymes are responsible for the covalent modifications of histones which alter the global charge of the histones and also serve to recruit specific co-factors to the chromatin. The second group of enzymes uses the energy from ATP hydrolisis to modify the interactions DNA-histones, therefore increasing the nucleosomal DNA accesibility to the transcriptional machinery. The understanding of the mechanisms that the cell uses to regulate expression of an immediate-early gene such as egr-1, are very important, not only for the basic molecular knowledge but also for the understanding of different pathologies. The coordinated action of the signalling pathways, the union of transcriptional factors and enzymatic complexes that act over the chromatin is crucial for the right interpretation of the genetic code, which is specific for each gene under study. In the case of egr-1, treating MLP29 cells with TPA activates expression after 5 min of treatment, has a peak around 15 and 30 min, and decreases subsequently to be undetectable at 180min after the treatment. This induction takes place through the MAP and p38 kinase cascades which also act phosphorylating the promoter transcriptional factors ELK1 and CREB. Chromatin immunoprecipitation experiments (ChIP), suggest that egr-1 is a gene ready to be expressed, as the RNAPol II and the SRF, ELK1, CREB and SP1 transcriptional factors are already present in the basal state of the gene. During the induction process SP1 leaves the promoter and EGR-1, NAB1 and NAB2 are recruited. In the same manner, ChIP experiments have detected the presence of two HDAC complexes implicated in egr-1 repression (mSIN3A and HDAC3-NcoR) and the presence of two HAT complexes (CBP, with a possible role as maintaining the basal acetylation levels and GCN5 which putative role would favour the induction process). The presence of these enzymes has been correlated with a general increase in the levels of acetylation of histone H3 and H4 by immunoprecipitating against specific histone modifications. Additionally, experiments of Micrococcal nuclease protection have suggested the existence of three positioned nucleosomes in egr-1 promoter and also the movement of the more proximal to the transcription start during the induction process. That nucleosomal mobility has been related to the presence of the ATPase BRM and BRG1 in the promoter of the gene and also with specific increases in the acetylation levels of the three nucleosomes as revealed by mononuclesomal ChIP experiments. Moreover, to study the biological effect of the lack of EGR1, it has been designed a siRNA sequence that is able to decrease egr-1 expression up to a 70%, and which biological activity has been shown by a decrease in nab2 (EGR1 target gene) expression of 40%. Lastly, the interconnection between these results has allowed us to hypothesize a model for the epigenetic regulation of egr-1 gene in murine cells treated with TPA. __________________________________________________________________________________________________ RESUMEN La regulación de la expresión génica en eucariotas se encuentra condicionada por la estructura de la cromatina. Por ello, la comprensión de los mecanismos que utiliza la célula para regular la expresión de un gen inmediato-temprano, como egr-1, es de gran importancia tanto para el conocimiento básico molecular como para la compresión de diferentes patologías. El tratamiento de células MLP29 con TPA activa la expresión del gen egr-1 a los 5 min., alcanza un pico entre los 15 y 30 min. y deja de ser detectable a los 180 min. Dicha inducción, se produce a través de las cascadas de quinasas MEK y p38, que además fosforilan a los factores ELK1 y CREB, presentes en el promotor del gen. Experimentos de inmunoprecipitación de cromatina (ChIP), indican que egr-1 posee la RNAPol II y los factores SRF, ELK1, CREB y SP1 unidos al promotor en el estado basal. Durante la inducción, SP1 se libera y EGR1, NAB1 y NAB2 son reclutados. Asimismo, experimentos de ChIP han detectado la presencia en el promotor de dos complejos HDAC (mSIN3A y HDAC3-NCoR) y de dos complejos HAT (CBP y GCN5). Estos enzimas se han correlacionado con un incremento general en los niveles de acetilación de las histonas H3 y H4 mediante ChIP con anticuerpos específicos. Además, experimentos de protección frente a digestión con nucleasa de micrococo sugieren la existencia de 3 nucleosomas posicionados en el promotor de egr-1 así como el movimiento del más próximo al inicio de la transcripción durante el proceso de inducción. Dicha movilidad nucleosomal se ha correlacionado mediante ChIP con la presencia de las ATPasas BRM y BRG1 en el promotor del gen y con incrementos específicos de acetilación de los tres nucleosomas, estudiados mediante ChIP de mononucleosomas. El efecto biológico de EGR1 se ha estudiado mediante RNA de interferencia capaz de producir una disminución del nivel de expresión de egr-1 de un 70% y la inhibición parcial de genes diana como es el caso de nab2. Finalmente, la interconexión de estos resultados nos ha permitido hipotetizar un posible mecanismo de regulación epigenética del gen egr-1 en células MLP29 inducidas por TPA. en_US
dc.description.abstract es_ES
dc.format.mimetype application/pdf es_ES
dc.language cat-en-es es_ES
dc.rights eng es_ES
dc.rights Copyright information available at source archive es_ES
dc.subject none es_ES
dc.title Transcriptional regulation of egr-1 gene in murine cells. Towards the understanding of the role of chromatin. es_ES
dc.type doctoral thesis es_ES

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