The yeast Ada complex mediates the ligand-dependent activation function AF-2 of retinoid X and estrogen receptors

Abstract

Nuclear receptors (NRs) represent a large family of ligand-inducible transcriptional regulators that control complex developmental and homeostatic events in vertebrates by binding as homodimers or heterodimers to cognate DNA response elements present in target genes. NRs display a modular structure, with five to six distinct regions (denoted A-E/F; see Fig. 1A). The amino-terminal A/B region contains an autonomous activation function (AF-1), the highly conserved region C belongs to the DNA-binding domain (DBD), and the carboxy-terminal E region contains the ligand-binding domain (LBD), a dimerization surface and a ligand-dependent transcriptional activation function, AF-2 (for reviews and refs, see Mangelsdorf et al. 1995;Chambon 1996;Perlman and Evans 1997). The core of the AF-2 activating domain (AF-2 AD core; see Fig. 1A) has been characterized in the carboxy-terminal part of the E region and corresponds to the conserved amphipathic ␣ helix 12 of the LBD. Upon ligand binding, this helix is thought to fold back over the LBD to generate interacting surface(s) for the binding of transcriptional intermediary factors (TIFs, also denoted coactivators or mediators), whose effects ultimately result in the remodeling of the structure of the chromatin template and/or in the stimulation of initiation of RNA synthesis by the general transcription machinery (for reviews and references, see Chambon 1996;Wurtz et al. 1996;Glass et al. 1997).Although the yeast Saccharomyces cerevisiae does not possess endogenous NRs, it has been shown that a number of NRs, including the estrogen receptor (ER, now designated ER␣), the glucocorticoid receptor (GR), the retinoic acid (RA) receptors (RARs and RXRs), and the thyroid hormone receptor (TRs) can function as liganddependent transactivators in yeast (Metzger et al. , 1992Schena and Yamamoto 1988;Hall et al. 1993;Heery et al. 1993; and references therein). As in vertebrates (Chambon 1996 and references therein), the AF-1 and AF-2 of ER␣, RAR␣, and RXR␣ can activate transcription independently and synergistically in yeast White et al. 1988;Pierrat et al. 1992; Heery et al.1993; and our unpublished results

Human ADA3 Binds to Estrogen Receptor (ER) and Functions As a Coactivator for ER-mediated Transactivation

“…Previous genetic studies of mammalian NRs expressed in the yeast have indicated a coactivator role of the evolutionarily conserved ADA complex as a coactivator of NR-mediated gene expression (17,18), a possibility directly supported by our recent demonstration of a coactivator role for hADA3, a component of the mammalian ADA complex, in RXR function (19). Given that these studies relied solely on hADA3 overexpression, and given the existence of a recent study reporting the inability of mouse ADA3 to interact with estrogen receptor (21), we undertook a systematic examination of the mammalian ADA3 interaction with ER and the functional role of this interaction in the context of ectopic as well as endogenous ADA3.…”

“…NRs when these were expressed in yeast (17,18). Furthermore, we have recently shown that hADA3 binds to and functions as a coactivator for RXR␣ (19).…”

“…The core components of this complex include the adapter proteins ADA3 and ADA2, and a histone acetyltransferase GCN5 (general control non-repressed 5) (16). It is noteworthy that hADA3 exists as a component of a yeast ADA-like complex that includes hADA2 and hGCN5, indicating that the functional roles of the ADA complex are evolutionarily conserved (17). Studies of mammalian RXR and glucocorticoid receptor expressed in the yeast have shown a requirement for the components of yeast ADA complex, including the yADA3 gene product, for transcriptional activation (17,18).…”

“…It is noteworthy that hADA3 exists as a component of a yeast ADA-like complex that includes hADA2 and hGCN5, indicating that the functional roles of the ADA complex are evolutionarily conserved (17). Studies of mammalian RXR and glucocorticoid receptor expressed in the yeast have shown a requirement for the components of yeast ADA complex, including the yADA3 gene product, for transcriptional activation (17,18). However, a functional role of hADA3 in NR transactivation in mammalian cells was not known until our recent work (19).…”

Identification of TATA-binding Protein-free TAFII-containing Complex Subunits Suggests a Role in Nucleosome Acetylation and Signal Transduction

“…To overexpress GCN5, the hGCN5-S-containing cDNA was PCR-amplified from the pcDNA3-hsGCN5 plasmid (14) and inserted in the XhoIBamHI sites of the pET15bEpB10 expression vector (18). Doubletagged hPCAF was expressed using baculovirus in Sf9 cells and purified in the presence of 1 M acetyl-CoA first on a Ni 2ϩ column, then by an anti-FLAG immunoprecipitation (Sigma) and peptide elution.…”