Receptor Accessory Factor Research Articles

Flanking Sequence Composition Differentially Affects the Binding and Functional Characteristics of Glucocorticoid Receptor Homo- and Heterodimers

The core binding sites for a multitude of transcription factors have been identified and characterized, but these sequences cannot fully account for the nuances of cell-specific and gene-specific control of gene transcription. Many factors may contribute to the precise responsiveness of a gene to a particular transcriptional regulatory protein, including the nucleotides in the proximity of the core binding site for that protein. Here, we examine two flanking sequences bordering a site in the gamma-fibrinogen gene regulatory region that binds a heterodimer of the Xenopus glucocorticoid receptor accessory factor (XGRAF) and the glucocorticoid receptor (GR). Mutation of the upstream flank results in a decrease in the level of XGRAF binding but little change in hormone induction. However, alteration of the downstream flank adjacent to the GR binding site causes a decrease in levels of both GR monomer binding and hormone induction. Conversion of the XGRAF-GR binding site to a full glucocorticoid response element (GRE) alters the role of the flanking sequences. A full GRE in this position requires the wild-type upstream flank to bind GR homodimer and induce transcription to maximal levels. In contrast, mutation of the downstream flank is not detrimental to either the binding or the function of the GR dimer. Thus, flanking sequence composition and dimer partner combine to influence GR function, underscoring the complexities involved in the identification of authentic transcription factor response elements.

Heterodimerization between the glucocorticoid receptor and the unrelated DNA-binding protein, Xenopus glucocorticoid receptor accessory factor.

The adrenal steroid hormones, glucocorticoids, control many physiological responses to trauma, including elevated synthesis of fibrinogen, a major blood-clotting protein. Glucocorticoid regulation of the gamma-fibrinogen subunit gene in Xenopus laevis is mediated by a binding site for Xenopus glucocorticoid receptor accessory factor (XGRAF) and a contiguous glucocorticoid response element (GRE) half-site. Here, we characterize the protein:DNA complex formed by a cooperative interaction between XGRAF, GR, and the DNA. We demonstrate that the complex contains XGRAF by competition in a gel shift assay. The presence of GR is established by two criteria: 1) size dependence of the XGRAF:GR:DNA complex on the size of the GR component and 2) interference with complex formation by GR antibody. Cooperative binding of XGRAF and GR to the DNA was quantitated, showing that GR favors binding to XGRAF:DNA compared with free DNA by a factor of 30. The cooperative interaction between XGRAF and GR can occur on nicked DNA but is disrupted when 1 bp is inserted between the XGRAF binding site and half-GRE. Significantly, this loss of physical association in vitro correlates with loss of XGRAF amplification of GR activity in transiently transfected primary Xenopus hepatocytes. The simplest explanation for cooperativity between XGRAF and GR is formation of a DNA-bound heterodimer of these two proteins. This mechanism represents a new mode of transcriptional regulation in which GR and a nonreceptor protein form a heterodimer, with both partners contacting their specific DNA sites simultaneously.

Heterodimerization between the Glucocorticoid Receptor and the Unrelated DNA-Binding Protein, Xenopus Glucocorticoid Receptor Accessory Factor

Heterodimerization between the Glucocorticoid Receptor and the Unrelated DNA-Binding Protein, Xenopus Glucocorticoid Receptor Accessory Factor

The binding site for Xenopus glucocorticoid receptor accessory factor and a single adjacent half-GRE form an independent glucocorticoid response unit.

In Xenopus laevis, transcription of the gamma-fibrinogen subunit gene is activated by glucocorticoids. Hormone induction is regulated by three glucocorticoid response element (GRE) half-sites and an additional DNA sequence which binds a novel hepatocyte nuclear protein, Xenopus glucocorticoid receptor accessory factor (XGRAF). The XGRAF binding site (GAGTTAA) is located directly upstream of the most distal half-GRE. The proximity of the binding sites for XGRAF and the glucocorticoid receptor (GR) led to the hypothesis that these two sites form a glucocorticoid response unit (GRU). By transfecting DNA into primary hepatocytes, we showed that this GRU confers hormone responsiveness in the absence of other half-GREs. The XGRAF binding site enhances function of the half-GRE without itself responding to glucocorticoids. The GRU retains efficacy in other locations relative to the gamma-fibrinogen gene promoter, further increases transcription when present in multiple copies, and activates a heterologous promoter. Despite the contiguity of the XGRAF binding site and half-GRE, the two sites can be occupied simultaneously in vitro. The binding characteristics correlate with function since mutations that disrupt concurrent XGRAF and GR binding also impair transcription. This novel GRU represents a new regulatory mechanism that may be applicable to other glucocorticoid responsive genes that lack a full GRE.

Analysis of the DNA-binding Site for XenopusGlucocorticoid Receptor Accessory Factor: CRITICAL NUCLEOTIDES FOR BINDING SPECIFICITY IN VITROAND FOR AMPLIFICATION OF STEROID-INDUCED FIBRINOGEN GENE TRANSCRIPTION

In addition to the glucocorticoid receptor, DNA-binding proteins called accessory factors play a role in hormone activation of many glucocorticoid-responsive genes. Hormonal regulation of the gamma-fibrinogen subunit gene from the frog Xenopus laevis requires a novel DNA sequence that binds a liver nuclear protein called Xenopus glucocorticoid receptor accessory factor (XGRAF). Here we demonstrate that the recognition site for XGRAF encompasses GAGTTAA at positions -175 to -169 relative to the start site of transcription. This sequence is not closely related to the binding sites for known transcription factors. The two guanosines make close contact with XGRAF, as shown by the methylation interference assay. Single-point mutagenesis of every nucleotide in the 9-base pair region from positions -177 to -169 showed an excellent correlation between ability to bind XGRAF in vitro and ability to amplify hormone-induced transcription from DNA transfected into Xenopus primary hepatocytes. Conversely, XGRAF had little or no effect on basal transcription of the gamma-fibrinogen gene. Maximal hormonal induction also requires three half-glucocorticoid response elements (half-GREs) homologous to the downstream half of the consensus GRE. Interestingly, the XGRAF-binding site is immediately adjacent to the most important half-GRE. This close proximity suggests a new mechanism for activation of a gene lacking a conventional full GRE.

Novel Accessory Factor-Binding Site Required for Glucocorticoid Regulation of the γ-Fibrinogen Subunit Gene fromXenopus laevis

Glucocorticoids induce gene expression by binding to an intracellular receptor that interacts with genomic DNA and stimulates transcription of specific genes. The consensus DNA-binding site for the glucocorticoid receptor, called a glucocorticoid response element (GRE), is GGTACAnnnTGTTCT. In the classical model, binding of the receptor as a dimer to the two halves of the GRE is required for activation of transcription. For some glucocorticoid-regulated genes, additional DNA-binding proteins called accessory factors are necessary for hormonal responsiveness. We have identified a new factor required for glucocorticoid-induced expression of the gamma-fibrinogen subunit gene from the frog Xenopus laevis. Transfection of cloned DNA fragments into primary Xenopus hepatocytes showed that the DNA between 163 and 187 bp upstream of the transcription initiation site is essential for hormonal activation. A single complex forms when this small region of DNA is incubated in vitro with Xenopus liver nuclear proteins. The protein recognition site has been narrowed to AAGAGTTAA, a sequence not previously described as a transcription factor-binding site. We have named the protein(s) bound to this sequence Xenopus glucocorticoid receptor accessory factor (XGRAF). In addition to the XGRAF-binding site, glucocorticoid regulation of the gamma-fibrinogen gene requires at least three nearby GREs, each of which is a poor match to the consensus GRE. The position of the binding site for XGRAF overlaps the putative upstream half of the most important GRE. Models are presented to show possible ways that the novel accessory factor and the glucocorticoid receptor could act through closely juxtaposed sites on the DNA.

Dexamethasone Inhibits Insulin Binding to Insulin-Degrading Enzyme and Cytosolic Insulin-Binding Protein p82

We recently demonstrated that insulin specifically binds to several cytosolic insulin-binding proteins (CIBPs) including insulin-degrading enzyme (IDE) and CIBP p82 in cytosol isolated from H35 rat hepatoma cells. Insulin binding to these CIBPs was regulated by culture conditions, such as serum or insulin. In the present study, we examined the effect of dexamethasone on insulin binding to CIBPs in H35 cells. When the cells were treated with 100 nM dexamethasone for 24 hrs, insulin binding to IDE and CIBP p82 was decreased by about 50% without decreasing the expression level of IDE. Insulin added with the dexamethasone prevented the steroid's effect. Furthermore, dexamethasone directly blocked insulin binding to CIBPs in isolated cytosol. These results suggest that dexamethasone, directly or as a complex with other proteins, binds to IDE and CIBP p82 and changes their ability to bind insulin, possibly by inducing a conformational change or by blocking insulin binding sites. IDE was recently identified as a receptor accessory factor for androgen and glucocorticoid receptors and plays an important role in the regulation of gene transcriptional responses. Combined with previous reports, our findings suggest IDE and other CIBPs such as CIBP p82 may play a role in the cross-talk between insulin and the signal transduction pathways of steroid hormones.

Androgen and glucocorticoid receptors interact with insulin degrading enzyme.

We recently identified a protein, designated receptor accessory factor (RAF), that interacts directly with and enhances the DNA binding of androgen (AR) and glucocorticoid (GR) receptor peptide fragments. To determine its identity, RAF was purified from HeLa cell extracts by anion-exchange and DNA affinity chromatography. RAF activity co-purified with a 110-kDa protein, the partial amino acid sequence of which shares 97.5% identity with insulin degrading enzyme (IDE), a metalloendoprotease implicated in the intracellular degradation of insulin. The identity of RAF was confirmed in gel shift assays by demonstrating that AR.RAF and GR.RAF DNA complexes shifted to a slower mobility in the presence of an IDE antibody. Purified preparations of RAF had insulin degrading activity, and this activity was inhibited by AR. In addition, the interaction of AR or GR with RAF was competed by insulin and bacitracin, a competitive inhibitor of IDE. The interactions of AR and GR with IDE may have important implications for both insulin- and steroid-mediated signaling.

Sublocalization of the human interferon-gamma receptor accessory factor gene and characterization of accessory factor activity by yeast artificial chromosomal fragmentation.

A chromosomal fragmentation procedure was employed to produce a deletion set of yeast artificial chromosomes (YACs) from a parental YAC, GART D142H8, known to map to human chromosome 21q and to encode the human interferon-gamma receptor (Hu-IFN-gamma R) accessory factor gene as well as the phosphoribosylglycinamide formyltransferase (GART) gene. When expressed in Chinese hamster ovary cells, these deleted YACs retain accessory factor activity, as judged by major histocompatibility complex class I antigen inducibility, until the deletions from the acentric end exceed 390 kilobases (kb). Therefore, the accessory factor (AF-1) gene can be localized to a 150-kb region at the left (centric) end of the parental 540-kb GART YAC. Cells containing functional YACs are also able to induce the ISGF3 gamma and gamma-activated factor (GAF) transcription factors, but were not protected against encephalomyocarditis virus (EMCV) upon treatment with Hu-IFN-gamma. Therefore, the Hu-IFN-gamma R and the AF-1 are sufficient for some, but not all, of the actions of Hu-IFN-gamma. We postulate that an additional accessory factor (AF-2) required for antiviral activity against EMCV is encoded on chromosome 21q.

Receptor accessory factor enhances specific DNA binding of androgen and glucocorticoid receptors

Protein-protein interactions are common among transcriptional activators and may have important consequences for gene regulation. Using the mobility shift assay, we have identified a factor that enhances specific DNA binding of truncated rat androgen (AR) and glucocorticoid (GR) receptors by 25- and 6-fold, respectively, through the formation of heteromeric complexes. This factor, designated receptor accessory factor, or RAF, also potentiates DNA binding of full-length human GR. RAF is temperature and trypsin sensitive and is present in a variety of cultured mammalian cells. By gel filtration RAF has a predicted molecular mass of 130 kDa. RAF enhancement of AR-DNA binding is optimal with androgen response element DNA. RAF appears to interact directly with AR because 1) deoxycholate, which interferes with protein-protein but not protein-DNA interactions, prevents RAF.AR.DNA complex formation, 2) RAF activity is recovered from an androgen response element DNA affinity column only in the presence of AR, and 3) RAF increases the size of an AR.DNA complex by gel filtration. Mutagenesis of truncated AR fragments indicates that a region in the NH2-terminal domain is required for RAF to enhance AR-DNA binding. The interaction of RAF with AR and GR suggests that RAF might influence the ability of these nuclear receptors to activate transcription.

RECEPTOR ACCESSORY FACTOR (RAF) ENHANCES SPECIFIC DNA BINDING OF ANDROGEN AND GLUCOCORTICOID RECEPTORS

The biological responses to steroid hormones are mediated by a family of ligand-inducible intracellular receptors that activate or repress transcription of target genes. Protein-protein interactions arc common among other transcriptional activators and may have important consequences for gene regulation by steroid hormone receptors. Using the mobility shift assay we have identified a factor that enhances specific DNA binding of truncated rat androgen (AR) and glucocorticoid (GR) receptors by 25-fold and 6-fold, respectively, through the formation of heteromeric complexes. This factor, designated receptor accessory factor, or RAF, also potentiates DNA binding of full-length human GR. RAF is temperature and trypsin sensitive and is present in a variety of cultured mammalian cells. By gel filtration RAF has a predicted molecular mass of 130 kDa. RAF enhancement of AR-DNA binding requires androgen response clement DNA. RAF appears to interact directly with AR because deoxycholate, which interferes with protein-protein but not protein-DNA interactions, prevents RAF-AR-DNA complex formation. Furthermore, RAF activity is recovered from an androgen response element DNA affinity column only in the presence of AR. Mutagenesis of truncated AR fragments indicates that a region in the NH2-terminal domain is required for RAF to enhance AR-DNA binding. The interaction of RAF with AR and GR suggests that RAF might influence the ability of these nuclear receptors to activate transcription.

The human gamma interferon receptor accessory factor encoded by chromosome 21 transduces the signal for the induction of 2',5'-oligoadenylate-synthetase, resistance to virus cytopathic effect, and major histocompatibility complex class I antigens.

Mouse fibroblasts, and human-mouse hybrid fibroblasts carrying only human chromosome 21, were transfected with cDNA encoding full-length human gamma interferon (IFN-gamma) receptor or chimeric IFN-gamma receptor (extracellular domain of the human receptor; transmembrane and intracellular domains of mouse origin). These transfected mouse cells were sensitive to human IFN-gamma only when human chromosome 21 was present. These results show that the species-specific accessory protein encoded by human chromosome 21 interacts with the extracellular domain of human IFN-gamma receptor and transduces the IFN-gamma signal not only for up-regulation of mouse major histocompatibility complex class I antigen expression but also for the induction of 2',5'-oligoadenylate-synthetase and resistance to virus cytopathic effect.

Receptor accessory factor enhances specific DNA binding of androgen and glucocorticoid receptors

Receptor accessory factor enhances specific DNA binding of androgen and glucocorticoid receptors

Transcriptional control of vitamin D-regulated proteins.

Vitamin D is a physiological regulator of gene transcription associated with control of a broad spectrum of biological processes that include but is not restricted to growth, differentiation and calcium-mediated homeostatic control. Transcriptional regulation is mediated by sequence-specific interactions of a 1,25(OH)2D3-vitamin D receptor-accessory factor complex with vitamin D responsive elements (VDRE) residing in the promoters of hormone responsive genes. Functioning primarily as a transcription enhancer, activity at the VDRE is controlled by diverse and integrated cellular signalling pathways acting synergistically and/or antagonistically with a series of basal regulatory elements and other hormone regulated sequences that are components of modularly organized vitamin D-responsive gene promoters. Molecular mechanisms that integrate the activities at promoter elements contributing to vitamin D-related transcriptional control include overlapping transcription factor binding domains within regulatory elements and cooperative activities at independent regulatory sequences that determine the level of vitamin D responsiveness.

Stimulation of oligonucleotide binding of estradiol receptor complexes by accessory proteins.

During purification of E2R using oligo(dT)-cellulose chromatography, a receptor accessory factor (RAF) was identified in the cytosol of mouse kidney. This factor stimulates the binding of purified E2R to oligo(dT)-, oligo(dC)-, and oligo(dA)-cellulose as well as to DNA cellulose. It is a heat-stable, trypsin-resistant protein with an apparent molecular weight of between 10 and 30,000 daltons. Although structurally unrelated, similar stimulation of oligonucleotide binding was seen with calf thymus histones and, to a lesser extent, egg white lysozyme. Individual histones, especially H2a, H2B, and H3, also facilitate rebinding of purified E2R to oligo(dT)-cellulose, while H1 is less effective. Furthermore, histones stabilize the holoreceptor during sedimentation at 4 degrees and 12 degrees C. The N- and C-terminal half molecules of H2b were generated by cyanogen bromide-mediated cleavage and the N-terminal half was found to duplicate the effects of the parent molecule, both in binding and holoreceptor stabilization. These data suggest that the in vivo binding of E2R to DNA can be modulated by accessory proteins of cytosol and nuclear origin.