Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2004. / Härtill 4 uppsatser.

published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Also available online.

Thèses (M.Sc.)--Université de Sherbrooke (Canada), 1999. / Titre de l'écran-titre (visionné le 20 juin 2006). Publié aussi en version papier.

Un des enjeux majeurs de la biologie moderne est de comprendre les mécanismes complexes régissant l’expression de gènes d’un organisme en développement. Alors que les activateurs (enhancers) ont été abondamment étudiés et analysés, seul un relatif petit nombre de répresseurs (silencers) a été identifié à ce jour et restent jusqu’à présent assez mal compris. Un nombre non négligeable de CRMs jouent par ailleurs un double rôle à la fois d’amplificateurs et d’inactivateurs de transcription en fonction de l’état ou du type cellulaire dans lequel ils se trouvent, rajoutant un niveau supplémentaire de à la régulation génique dans différents types cellulaires et tissus. De façon surprenante, nous avons découvert que tous les éléments ayant une activité de répression transcriptionnelle que nous avons identifiés, s’avèrent aussi avoir une activité d’activation transcriptionnelle dans d’autres contextes cellulaires. Nos résultats remettent donc en question le paradigme de deux catégories distinctes de CRMs et suggèrent que des milliers, ou plus, d’éléments bifonctionnels restent à être découverts chez la Drosophile et potentiellement 104-105 chez l’humain. Le référencement et la caractérisation de ces éléments devraient s’avérer utiles, si ce n’est cruciaux, afin de comprendre la façon par laquelle ces motifs d’expression sont encodés au sein des génomes d'organismes métazoaires et donc éventuellement chez l’Homme. / A major challenge in biology is to understand how complex gene expression patterns in organismal development are encoded in the genome. While transcriptional enhancers have been studied extensively, few transcriptional silencers have been identified and they remain poorly understood. Here we used a novel strategy to screen hundreds of sequences for tissue-specific silencer activity in whole Drosophila embryos. Strikingly, 100% of the tested elements that we found to act as transcriptional silencers were also active enhancers in other cellular contexts. These elements were enriched in highly occupied target (HOT) region overlap (Roy et al., 2010) and specific transcription factor (TF) motif combinations. CRM bifunctionality complicates the understanding of how gene regulation is specified in the genome and how it is read out differently in different cell types. Our results challenge the common practice of treating elements with enhancer activity identified in one cell type as serving exclusively activating roles in the organism and suggest that thousands or more bifunctional CRMs remain to be discovered in Drosophila and perhaps 104-105 in human (Heintzman et al., 2009). Characterization of bifunctional elements should aid in investigations of how precise gene expression patterns are encoded in the genome.

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No description available.

Estrogen receptor (ER) acts as a ligand-activated transcription factor that regulates the expression of genes. The genomic mechanisms of ER action include ligand-induced dimerization of ER which binds estrogen responsive elements (EREs) in the promoters of target genes. There are also nongenomic mechanisms of ER action which are associated with membrane bound or cytosol ER-dependent activation of various protein-kinase cascades which also influence expression of target genes. Egr-1 is an immediate-early gene induced by 17β-estradiol (E2) in the rodent uterus and breast cancer cells. Deletion analysis of the Egr-1 promoter identified a minimal E2-responsive region that contained serum response element (SRE3) which bound Elk-1 and serum response factor (SRF) in gel mobility shift assays. Hormone-responsiveness of Egr-1 in MCF-7 cells was specifically inhibited by PD98059, a MAPKK inhibitor, but not by LY294002, an inhibitor of PI3-K. These results contrasted with the hormone-dependent activation of the SRE in the c-fos promoter, which was inhibited by both PD98059 and LY294002, suggesting that Egr-1, like c-fos, is activated through non-genomic pathways of estrogen action but through activation of different kinases. COUP-TFs are orphan nuclear receptors expressed in a variety of tissues where they regulate biological functions and organogenesis. In this study, we investigated coactivation of ERα by COUP-TF1 in cell lines transiently cotransfected with the pERE3 construct. COUP-TFI coactivated ERα-mediated transactivation, but unlike many other coactivators, COUP-TFI also enhanced transactivation of ERα when cells were cotransfected with the TAF1-ERα mutant or the 19c-ERα mutant. These data indicate that helix 12 of ERα is not required for coactivation by COUP-TFI when AF-1 of ERα is intact. However, when the AF-1 of ERα is deleted, the intact AF-2 function is required for coactivation by COUP-TFI. Analysis of multiple COUP-TFI deletion mutants showed that the DNA-binding domain and C-terminal region of COUP-TFI were important for coactivation of ERα. Point mutations of the DNA-binding domain of COUP-TFI resulted in loss of interactions with ERα, suggesting that the DNA-binding domain of COUP-TFI is important for its coactivation activity facilitating interactions with ERα. These results demonstrate that COUP-TFI coactivated ERα through a non-classical LXXLL-independent pathway.

Estrogen receptor (ER) acts as a ligand-activated transcription factor that regulates the expression of genes. The genomic mechanisms of ER action include ligand-induced dimerization of ER which binds estrogen responsive elements (EREs) in the promoters of target genes. There are also nongenomic mechanisms of ER action which are associated with membrane bound or cytosol ER-dependent activation of various protein-kinase cascades which also influence expression of target genes. Egr-1 is an immediate-early gene induced by 17β-estradiol (E2) in the rodent uterus and breast cancer cells. Deletion analysis of the Egr-1 promoter identified a minimal E2-responsive region that contained serum response element (SRE3) which bound Elk-1 and serum response factor (SRF) in gel mobility shift assays. Hormone-responsiveness of Egr-1 in MCF-7 cells was specifically inhibited by PD98059, a MAPKK inhibitor, but not by LY294002, an inhibitor of PI3-K. These results contrasted with the hormone-dependent activation of the SRE in the c-fos promoter, which was inhibited by both PD98059 and LY294002, suggesting that Egr-1, like c-fos, is activated through non-genomic pathways of estrogen action but through activation of different kinases. COUP-TFs are orphan nuclear receptors expressed in a variety of tissues where they regulate biological functions and organogenesis. In this study, we investigated coactivation of ERα by COUP-TF1 in cell lines transiently cotransfected with the pERE3 construct. COUP-TFI coactivated ERα-mediated transactivation, but unlike many other coactivators, COUP-TFI also enhanced transactivation of ERα when cells were cotransfected with the TAF1-ERα mutant or the 19c-ERα mutant. These data indicate that helix 12 of ERα is not required for coactivation by COUP-TFI when AF-1 of ERα is intact. However, when the AF-1 of ERα is deleted, the intact AF-2 function is required for coactivation by COUP-TFI. Analysis of multiple COUP-TFI deletion mutants showed that the DNA-binding domain and C-terminal region of COUP-TFI were important for coactivation of ERα. Point mutations of the DNA-binding domain of COUP-TFI resulted in loss of interactions with ERα, suggesting that the DNA-binding domain of COUP-TFI is important for its coactivation activity facilitating interactions with ERα. These results demonstrate that COUP-TFI coactivated ERα through a non-classical LXXLL-independent pathway.