Gene regulation by the glucocorticoid receptor : Post-DNA binding mechanisms of transcriptional activation

Detta är en avhandling från Stockholm : Karolinska Institutet, false

Sammanfattning: The glucocorticoid receptor (GR) is a ligand-inducible transcription factor thatbelongs to the nuclear receptor family. The GR has important functions in cellulardifferentiation, development and metabolism in higher eukaryotes. After ligand bindingthe GR can bind to specific elements present within genes regulated by glucocorticoidsto modulate gene expression. The GR stimulates transcriptional activation throughits transactivation domains that interact directly and/or indirectly with componentsof the basal transcription complex at the promoter. We have investigated how themajor transactivation domain of the GR, the Tau 1 domain, functions to stimulatetranscription. We have delineated a minimal core region of 58 arnino acids in theC-terminal of Tau 1 that accounts for most of the activity. Extensive mutagenesisanalyses showed that the Tau 1-core activity depends mainly on hydrophobic residuespresent within the N-terminus of this Tau 1-core, in a region that has been shownpreviously to have propensity to form alpha-helical conformation in vitro. Rare mutationsthat increased the hydrophobicity of hydrophobic patches in this helical region,gave rise to increased transactivation activity suggesting a positive correlationbetween hydrophobicity of the patches and transactivation activity. However, sincehydrophobic amino acid substitutions are only tolerated at certain positions andcertain negatively charged residues within this region also contribute to the activity,it is not merely the hydrophobicity that contributes to Tau 1-core activity. Interestingly,the helical region I partially matches a consensus motif found in several other transactivationdomains. We have demonstrated that the Ada adaptor complex plays an important role in GR-dependenttranscriptional activation. The Tau 1-core interacts with the Ada2 protein directlyin vitro and the human homologue of Ada2 enhances GR dependent transcriptional activationin mammalian cells. We have also shown that the CREB binding protein (CBP), thatinteracts with the ligand binding domain of many nuclear receptors, contacts theTau l-core in vitro. In order to determine why the Tau-1-core mutants displayed altered activities,we investigated whether amino acid substitutions at hydrophobic residues in the Tau1 core influenced its interaction with the target factors Ada2, CBP and TBP. We foundthat the binding of these target factors to a panel of Tau 1-core mutant proteinsin vitro reflected the transactivation activities of these mutant proteins in vivo.Since the mutations have a similar effect on the interaction with all the targetfactors tested, we propose that mutations of hydrophobic residues in the Tau 1-coreaffect a common property of the transactivation domain, such as protein folding.This suggests that the important hydrophobic residues may form a hydrophobic corein a structured transactivation domain. Key words: Gene activation, Glucocorticoid receptor, Nuclear receptor,Transactivation domain, Transcription, Transcription factor, Yeast ISBN 91-628-2675-1

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