Bakers yeast rises to the challenge: reconstitution of mammalian steroid receptor signalling in S. cerevisiae.

Steroid hormones are an important class of signalling molecule, regulating a diverse range of processes in metazoan eukaryotes. The actions of these hormones are mediated by intracellular receptor proteins that act as ligand-activated transcription factors. The ability to reconstitute steroid receptor signalling in the budding yeast, Saccharomyces cerevisiae, provides a genetically tractable model system in which to investigate steroid receptor structure and function. Through targeted disruption and genetic screening, an increasing number of genes have been identified that are likely to have a role in steroid receptor action.

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.

Direct interaction of the tau 1 transactivation domain of the human glucocorticoid receptor with the basal transcriptional machinery.

We have used a yeast (Saccharomyces cerevisiae) cell free transcription system to study protein-protein interactions involving the tau 1 transactivation domain of the human glucocorticoid receptor that are important for transcriptional transactivation by the receptor. Purified tau 1 specifically inhibited transcription from a basal promoter derived from the CYC1 gene and from the adenovirus 2 major late core promoter in a concentration-dependent manner. This inhibition or squelching was correlated with the transactivation activity of tau 1. Recombinant yeast TATA-binding protein (yTFIID), although active in vitro, did not specifically reverse the inhibitory effect of tau 1. In addition, no specific interaction between tau 1 and yTFIID could be shown in vitro by affinity chromatography. Taken together, these results indicate that the tau 1 transactivation domain of the human glucocorticoid receptor interacts directly with the general transcriptional apparatus through some target protein(s) that is distinct from the TATA-binding factor. Furthermore, this assay can be used to identify interacting factors, since after phosphocellulose chromatography of a whole-cell yeast extract, a fraction that contained an activity which selectively counteracted the squelching effect of tau 1 was found.

Involvement of the transcription factor IID protein complex in gene activation by the N-terminal transactivation domain of the glucocorticoid receptor in vitro.

HeLa cell nuclear extracts were used to study the mechanism of activation of RNA polymerase II-mediated transcription by the N-terminal transactivation domain (tau1) of the glucocorticoid receptor in vitro. When fused to the Gal4 DNA-binding domain, the tau1 domain activated transcription approximately 9-fold in HeLa nuclear extracts. Using heat treatment to inactivate transcription factor IID (TFIID) in the extract, it was shown that the addition of purified TFIID complex, but not the TATA-binding protein alone, was sufficient to restore this level of activation. The tau1 domain was shown to interact directly with the TFIID complex. This interaction was markedly reduced by a mutation in the tau1 domain that reduces its activity. Furthermore, the interaction was specific for the TFIID complex, since no interaction was seen with TFIIIB, an analogous protein complex involved in RNA polymerase III transcription. The tau1 domain was further shown to interact with the TATA-binding protein subunit of the TFIID complex. These results suggest a mechanism by which the GR tau1 domain might contribute to gene activation by recruitment of the TFIID complex to target promoters.

High level expression of the major transactivation domain of the human glucocorticoid receptor in yeast cells inhibits endogenous gene expression and cell growth.

A number of alternative mechanisms by which the DNA-bound glucocorticoid receptor transactivates gene expression have been suggested. The fact that the glucocorticoid and other steroid hormone receptors function in yeast suggests that at least one of these mechanisms has been conserved throughout evolution. Here we show that overexpression of one of the glucocorticoid receptor transactivation domains (tau 1) in yeast causes a reduction in expression of a yeast reporter gene, followed by a severe reduction in the growth rate of the yeast cells. This is analogous to the phenomenon of squelching, first described for the GAL4 protein, and suggests that the tau 1 domain of the glucocorticoid receptor functions by contacting limiting transcription factors needed for efficient gene activity. A similar level of squelching was seen after removal of the up-stream activation sequences from the yeast reporter gene, suggesting that the squelching interactions were with transcription factors needed for the activity of a basal promoter.

Molecular mechanisms of androgen receptor-mediated gene regulation: structure-function analysis of the AF-1 domain.

The androgen receptor is a ligand-activated transcription factor that binds DNA response elements as a homodimer. Binding sites for the receptor have been identified both upstream and downstream of the transcription start site. Once bound to DNA, the receptor contacts chromatin remodelling complexes, coactivator proteins and components of the general transcription machinery in order to regulate target gene expression. The main transactivation domain, termed AF1, is located within the structurally distinct amino-terminal domain. This region is structurally flexible but adopts a more folded conformation in the presence of the binding partner TFIIF, and this in turn enhances subsequent protein-protein interactions. Thus, there is likely to be a dynamic interplay between protein-protein interactions and protein folding, involving AF1, that is proposed to lead to the assembly and/or disassembly of receptor-dependent transcription complexes.

Role of conserved hydrophobic amino acids in androgen receptor AF-1 function.

The intracellular androgen receptor (AR) is a ligand-activated transcription factor. Upon binding the steroids testosterone or dihydrotestosterone, the activated receptor translocates to the nucleus, binds to specific DNA response elements and interacts with the transcription machinery in order to regulate gene transcription. In the present study, we have described a highly conserved region (amino acids 224-258) within the AR AF-1 domain and have investigated the role of conserved bulky hydrophobic residues in gene regulation. Mutating pairs of residues (I229A/L236A; V240A/V242A; L251A/L254A) reduced transactivation activity by 25-40%. Mutating residues M244, L246 and V248 to alanines had a more dramatic affect on receptor activity, disrupting activity by at least 60%. The latter mutations also disrupted binding to the RNA polymerase-associated protein 74 subunit of the general transcription factor TFIIF. The protein conformation and stability of the mutant polypeptide in vitro was not significantly different from the wild type. None of the mutations tested disrupted binding of the AF-1 domain with the coactivator protein steroid receptor coactivator-1a. Thus we have concluded that conserved hydrophobic residues are important for receptor-dependent gene transcription and that M244, L246 and V248 are part of the binding interface for TFIIF.

In vivo and in vitro protein-DNA interactions at the distal oestrogen response element of the chicken vitellogenin gene: evidence for the same protein binding to this sequence in hen and rooster liver.

The major egg white protein, vitellogenin, is synthesized in a tissue specific and oestradiol dependent manner in the liver of egg-laying hens. In this paper, we describe a detailed study of the protein-DNA interactions at the distal oestrogen response element (ERED) located 600 bp upstream of the start of transcription. In vivo footprinting of hepatocytes from adult hens and roosters with 0.5-0.0005% dimethylsulphate (DMS) revealed, at critical concentrations of DMS, protection of distinct guanosine residues within the ERED and adjacent downstream sequence in both cases. From this, it was concluded that there were proteins present in both tissues binding to this region in vivo. In vitro studies using missing base contact probing and proteolytic clipping band shift assays with hen and rooster liver nuclear extracts identified the ERE binding protein to be the same or very closely related in both tissues. Furthermore, the protein from rooster nuclear extracts bound to the ERE sequence even when the DNA was methylated at CpG dinucleotides, u.v. cross-linking experiments performed with bromodeoxyuridine substituted ERE, revealed that a nuclear protein with Mr of about 75,000-80,000 bound specifically to this sequence. These studies demonstrate that apart from the oestrogen receptor, at least one other protein can interact specifically with the chicken vitellogenin ERE, independently of hormonal expression of the gene.

The nuclear-receptor interacting protein (RIP) 140 binds to the human glucocorticoid receptor and modulates hormone-dependent transactivation.

The glucocorticoid receptor (GR) regulates target gene expression in response to corticosteroid hormones. We have investigated the mechanism of transcriptional activation by the GR by studying the role of the receptor interacting protein RIP140. Both in vivo and in vitro protein-protein interaction assays revealed a ligand-dependent interaction between the GR and RIP140. The ligand binding domain of the GR was sufficient for this interaction, while both the N- and C-terminal regions of RIP140 bound to the receptor. In a yeast transactivation assay RIP140 and SRC-1, a member of the steroid receptor coactivator family of proteins, both enhanced the transactivation activity of a GR protein (GRA-1) in which the potent N-terminal tau1 transactivation domain has been deleted. In contrast, in COS-7 cells increasing amounts of RIP140 significantly inhibited GRdeltatau1 function. In cotransfection studies in COS-7 cells, RIP140 also inhibited receptor activity in presence of both SRC-1 and the coactivator protein CBP together. Thus, in yeast cells a stimulation of receptor activity was observed, while in mammalian cells RIP140 repressed GR function. Taken together, these data suggest that, (1) RIP140 is a target protein for the GR and (2) RIP140 can modulate the transactivation activity of the receptor.

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.

Investigation of steroid receptor function in the budding yeast Saccharomyces cerevisiae.

Steroid hormones are small lipophilic molecules that control a wide range of responses in both the developing and adult organism. The actions of these molecules are mediated by soluble receptor proteins that function as hormone-activated transcription factors. The first steroid receptors were expressed in the yeast Saccharomyces cerevisiae over 10 years ago, and to date virtually all the classical steroid receptors, together with a number of non-steroid members of the nuclear receptor superfamily, have been expressed in yeast. The ability to reconstitute steroid receptor signalling in yeast cells by co-expression of the receptor protein and a reporter gene driven by the appropriate hormone response element has presented researchers with a powerful model system for investigating receptor action. In this review, the use of yeast-based steroid receptor transactivation assays to investigate the roles of molecular chaperones, the mechanisms of DNA binding and gene activation, and the functional properties of hormone mimics will be discussed.

Mechanisms of transcription activation by nuclear receptors: studies on the human glucocorticoid receptor tau 1 transactivation domain.

Nuclear receptors are important signalling molecules that directly mediate the effects of hormones, vitamins and xenobiotic compounds at the level of gene expression. Several members of this superfamily of proteins have been implicated in receptor-dependent carcinogenesis. In this review, we summarise how these receptors can function as transcription factors with particular emphasis on the mechanism of transcription activation by the human glucocorticoid receptor tau 1 transactivation domain.

The human androgen receptor AF1 transactivation domain: interactions with transcription factor IIF and molten-globule-like structural characteristics.

The AR (androgen receptor) is a ligand-activated transcription factor and member of the steroid receptor superfamily. The AR responds to the ligands testosterone and dihydrotestosterone and activates multiple downstream genes required in development and reproduction. During the events of transactivation, the AR makes specific protein-protein interactions with several basal transcription factors such as TBP (TATA-box-binding protein) and TFIIF (transcription factor IIF). These interactions occur predominantly within a defined region termed AF1 (activation function-1) located within the highly disordered N-terminal domain of the receptor. Our focus is on the structural aspects of AF1 and how this flexible and disordered domain generates functional interactions with regulators of transcription. Our working hypothesis is that AR-AF1 domain exhibits induced folding when contacted by transcription regulators (such as TFIIF) into a more compact and 'active' conformation, enabling further co-regulator recruitment and ultimately transcription. Structural flexibility and intrinsic disorder of AR-AF1 were studied using predictive algorithms and fluorescence spectroscopy under different experimental conditions and the results revealed this domain retains characteristics indicative of molten-globule or pre-molten-globule-like structures. We hypothesize that this partially folded intermediate state is important for, and enables the AF1 domain to make, multiple protein-protein interactions. The structural aspects of AR-AF1 and interactions with TFIIF are discussed.

Structural dynamics of the human androgen receptor: implications for prostate cancer and neurodegenerative disease.

The AR (androgen receptor) is a ligand-activated transcription factor that mediates the action of the steroids testosterone and dihydrotestosterone. Alterations in the AR gene result in a number of clinical disorders, including: androgen-insensitivity, which leads to disruption of male development; prostate cancer; and a neuromuscular degenerative condition termed spinal bulbar muscular atrophy or Kennedy's disease. The AR gene is X-linked and the protein is coded for by eight exons, giving rise to a C-terminal LBD (ligand-binding domain; exons 4-8), linked by a hinge region (exon 4) to a Zn-finger DBD (DNA-binding domain; exons 2 and 3) and a large structurally distinct NTD (N-terminal domain; exon 1). Identification and characterization of mutations found in prostate cancer and Kennedy's disease patients have revealed the importance of structural dynamics in the mechanisms of action of receptors. Recent results from our laboratory studying genetic changes in the LBD and the structurally flexible NTD will be discussed.

Structural and functional alterations in the androgen receptor in spinal bulbar muscular atrophy.

The androgen receptor is a member of the nuclear receptor superfamily, and regulates gene expression in response to the steroid hormones testosterone and dihydrotestosterone. Mutations in the receptor have been correlated with a diverse range of clinical conditions, including androgen insensitivity, prostate cancer and spinal bulbar muscular atrophy, a neuromuscular degenerative condition. The latter is caused by expansion of a polyglutamine repeat within the N-terminal domain of the receptor. Thus the androgen receptor is one of a growing number of neurodegenerative disease-associated proteins, including huntingtin (Huntington's disease), ataxin-1 (spinocerebellar ataxia, type 1) and ataxin-3 (spinocerebellar ataxia, type 3), which show expansion of CAG triplet repeats. Although widely studied, the functions of huntingtin, ataxin-1 and ataxin-3 remain unknown. The androgen receptor, which has a well-recognized function in gene regulation, provides a unique opportunity to investigate the functional significance of poly(amino acid) repeats in normal and disease states.

Gene regulation through chromatin remodelling by members of the nuclear receptor superfamily.

The intracellular receptors for steroid hormones, thyroid hormones, retinoids and vitamin D(3) are known to bind to specific DNA elements and thus regulate target gene expression. This introductory review and the following papers address some of the mechanisms underlying this action. In particular, the ability of this family of transcription factors to recruit multi-protein complexes that have the capacity to remodel chromatin structure in order to silence or activate target gene expression is discussed.

Interaction of the human androgen receptor transactivation function with the general transcription factor TFIIF.

The human androgen receptor (AR) is a ligand-activated transcription factor that regulates genes important for male sexual differentiation and development. To better understand the role of the receptor as a transcription factor we have studied the mechanism of action of the N-terminal transactivation function. In a protein-protein interaction assay the AR N terminus (amino acids 142-485) selectively bound to the basal transcription factors TFIIF and the TATA-box-binding protein (TBP). Reconstitution of the transactivation activity in vitro revealed that AR142-485 fused to the LexA protein DNA-binding domain was competent to activate a reporter gene in the presence of a competing DNA template lacking LexA binding sites. Furthermore, consistent with direct interaction with basal transcription factors, addition of recombinant TFIIF relieved squelching of basal transcription by AR142-485. Taken together these results suggest that one mechanism of transcriptional activation by the AR involves binding to TFIIF and recruitment of the transcriptional machinery.

Structural studies of mutant glucocorticoid receptor transactivation domains establish a link between transactivation activity in vivo and alpha-helix-forming potential in vitro.

We have previously shown, using circular dichroism spectroscopy, that the tau 1 core peptide has alpha-helix-forming potential in vitro [Dahlman-Wright et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 1699-1703]. The tau 1 core peptide is a 58-amino acid peptide, constituting the core of the transactivation activity of the tau 1 major transactivation domain of the human glucocorticoid receptor [Dahlman-Wright et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 1619-1623]. Further structural studies of the peptide, using NMR spectroscopy, identified three segments with alpha-helical character. In this report we show that reduced protein expression or stability is not responsible for the reduced in vivo transactivation potential of tau 1 core peptides with proline substitutions in proposed alpha-helical regions. Rather, the reduced alpha-helix propensity of the corresponding purified peptides in vitro suggests that alpha-helices are involved in the molecular mechanism of glucocorticoid receptor mediated changes in gene activity.

In vitro regulation of reporter gene transcription by the androgen receptor AF1 domain.

The androgen receptor (AR) is a ligand-activated transcription factor that regulates gene expression in response to the steroids testosterone and dihydrotestosterone. AR-dependent gene expression is likely to play an important role in a number of receptor-associated disorders, such as prostate cancer, spinal bulbar muscular atrophy, male type baldness and hirsutism. The AR contains two transactivation domains, termed AF1 (activation function 1) located in the N-terminus and AF2 (activation function 2) in the C-terminal ligand-binding domain. AF2 exhibits weak transcriptional activity, whereas AF1 is a strong regulator of transcription. Transcriptional regulation by AF1 is thought to be modulated by a number of proteins that interact with this region, and by post-translational modifications. Our focus is on the N-terminal-interacting proteins and their regulation of transcription via interaction with the receptor. To better understand the mechanism of AR-AF1 action, we have reconstituted AR activity in HeLa nuclear extracts using a unique dual reporter gene assay. Multiple LexA-binding sites in the promoter allow transcription to be driven by a recombinant AR-AF1-Lex fusion protein. The findings from initial experiments suggest an increase in transcription initiation and elongation rates by AR-AF1-Lex. The role of protein-protein interactions involving co-activators and basal transcription factors and AR-AF1 activity are discussed.

Mechanism of gene expression by the glucocorticoid receptor: role of protein-protein interactions.

The glucocorticoid receptor belongs to an important class of transcription factors that alter the expression of target genes in response to a specific hormone signal. The glucocorticoid receptor can function at least at three levels: (1) recruitment of the general transcription machinery; (2) modulation of transcription factor action, independent of DNA binding, through direct protein-protein interactions; and (3) modulation of chromatin structure to allow the assembly of other gene regulatory proteins and/or the general transcription machinery on the DNA. This review will focus on the multifaceted nature of protein-protein interactions involving the glucocorticoid receptor and basal transcription factors, coactivators and other transcription factors, occurring at these different levels of regulation.