Yeast hormone response element assays detect and characterize GRIP 1 coactivator-dependent activation of transcription by thyroid and retinoid nuclear receptors ( cell biology y thyroid hormone y transcriptional regulation y subtype specificity y nuclear receptor-interacting protein )

The mouse glucocorticoid receptor-interacting protein (GRIP1) is a member of the ERAP160 family of nuclear receptor (NR) coactivators (including SRC-1 and TIF2) which function as bridging proteins between ligandactivated NRs bound to cognate hormone-response elements (HREs) and the transcription initiation apparatus (TIA). Although these coactivators bind to several NRs, studies overexpressing these coactivators with these NRs in mammalian cells have not uniformly observed a corresponding enhancement of ligand-dependent transactivation. Here, we show that GRIP1 interacts in vitro in a ligand-dependent manner with thyroid receptor, retinoic acid receptor, and retinoid X receptor. Additionally, in yeast (Saccharomyces cerevisiae) GRIP1 coactivator protein markedly increased the ability of these full-length class II NRs to transactivate b-galactosidase reporter genes containing cognate HREs. The magnitude of GRIP1 enhancement of liganded NR homodimer was dependent upon NR subtype and HRE configuration. For most HRE configurations, thyroid receptor and retinoic acid receptor homodimers were essentially unresponsive or very weakly active in the absence of GRIP1, but GRIP1 dramatically restored the ligand-dependent function of these NRs. Although GRIP1 exerted no significant effect on NR homodimers in the absence of their cognate ligands, it increased the transactivation of unliganded NR heterodimers. Whether GRIP1 increased ligand-dependent transactivation of a heterodimer to levels greater than that of the cognate homodimer was determined by HRE configuration and copy number. Compared with the limitations of yeast two-hybrid and mammalian coexpression systems, the yeast HRE-assay systems described in this report facilitated both the detection of putative mammalian NR coactivator function and the elucidation of their mechanisms of transactivational enhancement. Thyroid hormone (T3) receptor (TR), retinoic acid receptor (RAR), and retinoid X receptor (RXR) are members of the nuclear receptor (NR) superfamily of transcriptional proteins which regulate complex gene networks and thereby control diverse biological aspects of growth, development, and homeostasis (1–5). TR, RAR, and RXR are activated by their cognate ligands L-triiodothyronine (T3), all-trans-retinoic acid (at-RA), and 9-cis-retinoic acid (9c-RA), respectively (1–3). Additionally, there are multiple subtypes derived from different genes that have been detected for TRs (1, 4), RARs, and RXRs (1–3). These nuclear receptors (NRs) modulate gene expression primarily by binding as homodimers or heterodimers to AGGTCA hexameric core motifs with variations in sequence, spacing, and orientation called hormone-response elements (HREs), which are located in the regulatory region of target genes (1–5). The steroid (class I) subgroup of NRs bind to HREs as homodimers, whereas members of class II subgroup—i.e., TR, RAR, vitamin D receptor (VDR), and peroxisome proliferator activator receptor (PPARs)may bind not only as homodimers but also with high affinity as heterodimers with RXR to specific HREs (3). Consequently, RXR not only functions as a homodimer that is responsive to its cognate ligand, 9c-RA, but also, through the formation of heterodimers with other class II NRs, plays a central role in the regulation of many intracellular receptor signaling pathways (1–3). NRs function as ligand-dependent DNA-binding activator proteins to stimulate efficient transcriptional initiation of RNA polymerase II by either directly or indirectly interacting with components of the transcription initiation apparatus (TIA) assembled at the TATA box (6, 7). TIA consists of RNA polymerase II and general transcription factors, designated as TFIID [a complex of TATA box-binding protein (TBP) plus TBP-associated factors (TAFs)], TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH, which are evolutionarily conserved between yeast and humans. Some DNA-bound transcriptional activator proteins directly interact with general transcription factors, whereas others require additional proteins called coactivators to activate the TIA (6, 7). Although coactivators cannot function directly as transcriptional activators because they lack DNA-binding domains (DBDs) to interact with target genes, they nevertheless can mediate transcriptional activation by linking DNA-bound activator proteins with the TIA (7). Several proteins which function as coactivators are TAFs—i.e., subunits of TFIID, whereas others are not related to TAFs (6, 7). TR and RAR have been shown to interact directly with The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA 0027-8424y97y943697-6$2.00y0 PNAS is available online at http:yywww.pnas. org. Abbreviations; GR, glucocorticoid receptor; GRIP1, glucocorticoid receptor-interacting protein; TR, thyroid receptor; TRE, thyroid response element; T3, thyroid hormone (L-triiodothyronine); RAR, retinoic acid receptor; RARE, retinoic acid response element; at-RA, all-trans-retinoic acid; RXR, retinoid X receptor; RXRE, retinoid X receptor response element; 9c-RA, 9-cis-retinoic acid; AR, androgen receptor; ER, estrogen receptor; DBD, DNA-binding domain; HBD, hormone-binding domain; HRE, hormone-response element; NR, nuclear receptor; GST, glutathione S-transferase; b-gal, b-galactosidase; TIA, transcription initiation apparatus; TBP, TATA box-binding proteins; TAFs, TBP-associated factors. †To whom reprint requests should be addressed at: Mount Sinai Hospital, Endocrine Unit, Room 781, 600 University Avenue, Toronto, ON M5G 1X5, Canada. e-mail: walfish@mshri.on. ca. ie-mail: butt@mail.med.upenn.edu.