Tova Almlöf

Differential Recruitment of the Mammalian Mediator Subunit TRAP220 by Estrogen Receptors ERα and ERβ

Estrogen receptors (ERs) associate with distinct transcriptional coactivators to mediate activation of target genes in response to estrogens. Previous work has provided multiple evidence for a critical role of p160 coactivators and associated histone acetyltransferases in estrogen signaling. In contrast, the involvement of the mammalian mediator complex remains to be established. Further, although the two subtypes ERα and ERβ appear to be similar in regard to principles of LXXLL-mediated coactivator binding to the AF-2 activation domain, there are indications that the context-dependent transcriptional activation profiles of the two ERs can be quite distinct. Potentially, this could be attributed to differences with regard to coregulator recruitment. We have here studied the interactions of the nuclear receptor-binding subunit of the mammalian mediator complex, referred to as TRAP220, with ERα and ERβ. In comparison to the p160 coactivator TIF2, we find that TRAP220 displays ERβ preference. Here, we show that this is a feature of the binding specificity of the TRAP220 LXXLL motifs and demonstrate that the ER subtype-specific F-domain influences TRAP220 interaction. Such differences with regard to coactivator recruitment indicate that the relative importance of individual coregulators in estrogen signaling could depend on the dominant ER subtype.

Structure and function of the glucocorticoid receptor

Glucocorticoids cause changes in the expression of target genes via interaction with an intracellular receptor protein, the glucocorticoid receptor. This signal transduction process can be divided into a number of steps, each of which represents a functional facet of the receptor protein. These steps include (i) receptor transformation to an active form resulting from specific interaction with glucocorticoid steroid hormones, (ii) homo-dimerization, (iii) DNA-binding to specific hormone response elements in the genome and (iv) modulation of the expression levels of linked genes. These aspects of glucocorticoid receptor function have been studied using a combination of tertiary structure determination, biochemical assays and a genetic approach using a yeast system to screen for mutant receptors that are altered in function. The results show that contacts involving both the DNA and steroid binding domains are involved in dimerization and high affinity DNA binding. Genetic experiments have illuminated the role of amino acids within the recognition helix of the DNA-binding domain in discriminating between cognate DNA response elements for the glucocorticoid receptor and closely related binding sites for other nuclear receptors. Squelching experiments suggest that the N-terminal transactivation domain of the receptor contacts components of the general transcriptional machinery that appear to be distinct from the TATA binding protein, TFIID, during transactivation of gene expression by the DNA-bound receptor.

Role of the Ada adaptor complex in gene activation by the glucocorticoid receptor.

We have shown that the Ada adaptor complex is important for the gene activation capacity of the glucocorticoid receptor in yeast. The recently isolated human Ada2 protein also increases the potency of the receptor protein in mammalian cells. The Ada pathway is of key significance for the tau1 core transactivation domain (tau1c) of the receptor, which requires Ada for activity in vivo and in vitro. Ada2 can be precipitated from nuclear extracts by a glutathione S-transferase-tau1 fusion protein coupled to agarose beads, and a direct interaction between Ada2 and tau1c can be shown by using purified proteins. This interaction is strongly reduced by a mutation in tau1c that reduces transactivation activity. Mutations affecting the Ada complex do not reverse transcriptional squelching by the tau1 domain, as they do for the VP16 transactivation domain, and thus these powerful acidic activators differ in at least some important aspects of gene activation. Mutations that reduce the activity of the tau1c domain in wild-type yeast strains cause similar reductions in ada mutants that contain little or no Ada activity. Thus, gene activation mechanisms, in addition to the Ada pathway, are involved in the activity of the tau1c domain.

Role of hydrophobic amino acid clusters in the transactivation activity of the human glucocorticoid receptor.

We have performed a mutagenesis analysis of the 58-amino-acid tau1-core peptide, which represents the core transactivation activity of the tau1 transactivation domain from the glucocorticoid receptor. Mutants with altered activity were identified by phenotypic screening in the yeast Saccharomyces cerevisiae. Most mutants with reduced activity had substitutions of hydrophobic amino acids. Most single-substitution mutants with reduced activity were localized near the N terminus of the tau1-core within a segment that has been shown previously to have a propensity for alpha-helix conformation, suggesting that this helical region is of predominant importance. The particular importance of hydrophobic residues within this region was confirmed by comparing the activities of alanine substitutions of the hydrophobic residues in this and two other helical regions. The hydrophobic residues were shown to be important for the transactivation activity of both the isolated tau1-core and the intact glucocorticoid receptor in mammalian cells. Rare mutations in helical regions I and II gave rise to increased transcriptional activation activity. These mutations increase the hydrophobicity of hydrophobic patches on each of these helices, suggesting a relationship between the hydrophobicity of the patches and transactivation activity. However, certain nonhydrophobic residues are also important for activity. Interestingly, helical region I partially matches a consensus motif found in the retinoic acid receptor, VP16, and several other activator proteins.

Expression Level-Dependent Contribution of Glucocorticoid Receptor Domains for Functional Interaction with STAT5

ABSTRACT The action of the glucocorticoid receptor (GR) on β-casein gene transcription serves as a well-studied example of a case where the action of the GR is dependent on the activity of another transcription factor, STAT5. We have investigated the domain-requirement of the GR for this synergistic response in transfection experiments employing GR mutants and CV-1 or COS-7 cells. The results were influenced by the expression levels of the GR constructs. At low expression, STAT5-dependent transactivation by mutants of the GR DNA binding domain or N-terminal transactivation domain was impaired and the antiglucocorticoid RU486 exhibited a weak agonistic activity. When the N-terminal region of the GR was exchanged with the respective domain of the progesterone receptor, STAT5-dependent transactivation was reduced at low and high expression levels. Only at high expression levels did the GR exhibit the properties of a coactivator and enhanced STAT5 activity in the absence of a functional DNA binding domain and of GR binding sites in the proximal region of the β-casein gene promoter. Furthermore, at high GR expression levels RU486 was nearly as efficient as dexamethasone in activating transcription via the STAT5 dependent β-casein gene promoter. The results reconcile the controversial issue regarding the DNA binding-independent action of the GR together with STAT5 and provide evidence that the mode of action of the GR depends not only on the type of the particular promoter at which it acts but also on the concentration of the GR. GR DNA binding function appears to be mandatory for β-casein gene expression in mammary epithelial cells, since the promoter function is completely dependent on the integrity of GR binding sites in the promoter.

RAP250 Is a Coactivator in the Transforming Growth Factor β Signaling Pathway That Interacts with Smad2 and Smad3

RAP250 is a coactivator for nuclear receptors as well as other transcription factors. Recent studies have established RAP250 as an essential coactivator for many important biological processes, but its exact mechanism of action is not fully understood. To identify novel proteins that can associate with RAP250, we used a yeast two-hybrid system to screen cDNA libraries and identified the intracellular mediators of transforming growth factor-β (TGF-β) response Smad2 and Smad3 as direct interacting proteins. We show that the interaction between RAP250 and Smad2/3 is dependent upon the second LXXLL interaction motif in RAP250 and the MH2 domain in Smad2 and Smad3. Mouse embryonic fibroblasts lacking RAP250 have reduced expression of the TGF-β target gene PAI-1 after stimulation by TGF-β when compared with wild type cells. Furthermore, we demonstrate a cross-talk between TGF-β and liver X receptors (LXR) signaling pathways and show that stimulation of cells with TGF-β and LXR agonists have a synergistic effect on the expression of the LXR target gene ABCG1. Our data identify RAP250 as a new coactivator in the TGF-β signaling pathway that binds Smad2 and Smad3. Our data also suggest that the interaction between RAP250, Smad2, and Smad3 constitutes an important bridging mechanism linking LXR and TGF-β signaling pathways.