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

thesis
posted on 2024-09-03, 00:36 authored by Tova Almlöf

The glucocorticoid receptor (GR) is a ligand-inducible transcription factor that belongs to the nuclear receptor family. The GR has important functions in cellular differentiation, development and metabolism in higher eukaryotes. After ligand-binding the GR can bind to specific elements present within genes regulated by glucocorticoids to modulate gene expression. The GR stimulates transcriptional activation through its transactivation domains that interact directly and/or indirectly with components of the basal transcription complex at the promoter. We have investigated how the major transactivation domain of the GR, the Tau 1 domain, functions to stimulate transcription. We have delineated a minimal core region of 58 amino acids in the C-terminal of Tau 1 that accounts for most of the activity. Extensive mutagenesis analyses showed that the Tau 1-core activity depends mainly on hydrophobic residues present within the N-terminus of this Tau 1-core, in a region that has been shown previously to have propensity to form alpha-helical conformation in vitro. Rare mutations that increased the hydrophobicity of hydrophobic patches in this helical region, gave rise to increased transactivation activity suggesting a positive correlation between hydrophobicity of the patches and transactivation activity. However, since hydrophobic amino acid substitutions are only tolerated at certain positions and certain 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 transactivation domains.

We have demonstrated that the Ada adaptor complex plays an important role in GR-dependent transcriptional activation. The Tau 1-core interacts with the Ada2 protein directly in vitro and the human homologue of Ada2 enhances GR dependent transcriptional activation in mammalian cells. We have also shown that the CREB binding protein (CBP), that interacts with the ligand binding domain of many nuclear receptors, contacts the Tau 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 found that the binding of these target factors to a panel of Tau 1-core mutant proteins in vitro reflected the transactivation activities of these mutant proteins in vivo. Since the mutations have a similar effect on the interaction with all the target factors tested, we propose that mutations of hydrophobic residues in the Tau 1-core affect a common property of the transactivation domain, such as protein folding. This suggests that the important hydrophobic residues may form a hydrophobic core in a structured transactivation domain.

History

Defence date

1997-11-28

Department

  • Department of Medicine, Huddinge

Publication year

1997

Thesis type

  • Doctoral thesis

ISBN-10

91-628-2675-1

Language

  • eng

Original publication date

1997-11-07

Author name in thesis

Almlöf, Tova

Original department name

Department of Biosciences and Nutrition

Place of publication

Stockholm

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