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

Abstract

Nuclear receptors can function as ligand-inducible transregulators in both mammalian and yeast cells, indicating that important features of control of transcription have been conserved throughout evolution. Here, we report the isolation and characterization of a yeast protein that exhibits properties expected for a coactivator/mediator of the ligand-dependent activation function AF-2 present in the ligand-binding domain (LBD, region E) of the retinoid X (RXRalpha) and estrogen (ERalpha) receptors. This protein is identical to Ada3, a component of the yeast Ada coactivator complex. We demonstrate that: (1) the region encompassing residues 347-702 of Ada3 interacts with the LBD of RXRalpha and ERalpha in a ligand-dependent manner in yeast; (2) this interaction corresponds to a direct binding and requires the integrity of the core of the AF-2 activating domain (AF-2 AD) of both RXRalpha and ERalpha; (3) Ada3 as well as Ada2 and Gcn5, two other components of the Ada complex, are required for maximal AF-2 activity in yeast; and (4) Ada3 is able to enhance the AF-2 activity of RXRalpha and ERalpha when overexpressed in yeast and mammalian cells. Taken together, these data indicate that ligand-dependent transactivation by RXRalpha and ERalpha in yeast is mediated at least in part by the Ada complex, in which the Ada3 subunit directly binds to the holoreceptor LBD.

Figure 1

Identification of Ada3 by a two-hybrid screening for yeast proteins that interact with RXRα. (A) Schematic representation of mouse RXRα. Indicated are the various regions of the receptor (denoted A–E) that are conserved among members of the NR family. Transactivation domains (AF-1 and AF-2), DBD, and LBD are indicated. (Filled bar) Core motif of the AF-2 AD. (Numbers) Amino-acid positions. (B) Schematic representation of the LexA protein unfused or fused to the DE region of RXRα. The VP16 AAD-tagged S. cerevisiae genomic DNA library is represented below. The AAD tag also includes codons specifying the nuclear localization signal (NLS) of the yeast ribosomal protein L29. (C) Transcription of the integrated HIS3 and lacZ reporter genes in the reporter strain L40 is driven by a chimeric GAL1 promoter containing four and eight LexA-binding sites (LexAbs), respectively. (D) Growth complementation by interaction of RXRα(DE) with Ada3(309–702). Yeast L40 cells expressing the indicated fusion proteins were plated on medium containing histidine (His) and on His-negative medium +/− 9C-RA and +/− 3-AT as indicated. Plates were incubated at 30°C for 2 days. (++) Wild-type growth; (+) weak gowth; (−) no growth.

Figure 1

Identification of Ada3 by a two-hybrid screening for yeast proteins that interact with RXRα. (A) Schematic representation of mouse RXRα. Indicated are the various regions of the receptor (denoted A–E) that are conserved among members of the NR family. Transactivation domains (AF-1 and AF-2), DBD, and LBD are indicated. (Filled bar) Core motif of the AF-2 AD. (Numbers) Amino-acid positions. (B) Schematic representation of the LexA protein unfused or fused to the DE region of RXRα. The VP16 AAD-tagged S. cerevisiae genomic DNA library is represented below. The AAD tag also includes codons specifying the nuclear localization signal (NLS) of the yeast ribosomal protein L29. (C) Transcription of the integrated HIS3 and lacZ reporter genes in the reporter strain L40 is driven by a chimeric GAL1 promoter containing four and eight LexA-binding sites (LexAbs), respectively. (D) Growth complementation by interaction of RXRα(DE) with Ada3(309–702). Yeast L40 cells expressing the indicated fusion proteins were plated on medium containing histidine (His) and on His-negative medium +/− 9C-RA and +/− 3-AT as indicated. Plates were incubated at 30°C for 2 days. (++) Wild-type growth; (+) weak gowth; (−) no growth.

Figure 2

Two-hybrid interaction between Ada3 and various nuclear receptors. (A) Residues 347–702 of Ada3 are sufficient for mediating a ligand-dependent interaction with the LBD/AF-2 of RXRα. (Left) Schematic representations of the LexA–RXRα fusions. These chimera were expressed in the yeast reporter strain L40 together with either the VP16 AAD or the VP16 AAD fused to the amino-terminal residues 1–346 of Ada3 [AAD–Ada3(N)] or the VP16 AAD fused to the carboxy-terminal residues 347–702 of Ada3 [AAD–Ada3(C)] or unfused Ada3. Transformants were grown in liquid medium in the presence (+) or absence (−) of 500 nm 9C-RA. β-Galactosidase activities are expressed in nmoles of substrate/min/mg. (B) Ada3 interacts with the LBD/AF-2 of ERα and full-length TRα, but not with the LBD/AF-2 of RARα. The indicated LexA and AAD fusions were assayed for interaction in the yeast reporter strain L40 grown in the presence (+) or absence (−) of the cognate ligand (500 nm T-RA for RARα, 500 nm E2 for ERα, 5 μm T3 for TRα). β-Galactosidase activities are expressed as in A. In all panels, the values (±20%) are the average of at least three independent transformants.

Figure 3

The AF-2 AD core motif is important for interaction between NRs and Ada3. (A) Point mutations in the conserved hydrophobic residues of the AF-2 AD core of RXRα impair interaction with Ada3. The indicated mutants of RXRα were fused to LexA and assayed for interaction with AAD–Ada3(FL) and AAD–Ada3(C) in the yeast reporter strain L40 grown in the presence or absence of 500 nm 9C-RA. β-Galactosidase activities are expressed as in Fig. 2A. (B) Deletion or point mutations in the AF-2 AD core of ERα abolish interaction with Ada3. L40 transformants expressing the indicated LexA and AAD fusions in the presence or absence of 500 nm E2 were treated as described in A. (C) Ada3 binds directly to the LBD of RXRα, but not of RARα, in a ligand- and AF-2-integrity-dependent manner. Purified His-epitope B10-tagged Ada3 was incubated in a batch assay with control GST (lane 3), GST–RXRα(DE) wild type (WT; lanes 4,5), GST–RXRα(DE) mutated in the conserved hydrophobic residues of the AF-2 AD core motif (lanes 6–9), or GST–RARα(DEF) (lanes 10,11) bound to glutathione–Sepharose beads, in the presence or absence of ligands (1 μm 9C-RA for RXRα and 1 μm T-RA for RARα). Bound Ada3 protein was detected by Western blotting with the B10 antibody. (Lane 1) 1/10 the amount of input His–Ada3 fusion (arrow). (D) Ada3 binds directly to the LBD of ERα in a ligand- and AF-2-integrity-dependent manner. Binding assays were done as in C in the presence or absence of 500 nm E2. (E) E2 (500 nm; lane 5), but not OHT (1 μm; lane 6), induces interaction between the ERα LBD and the carboxy-terminal moiety of Ada3.

Figure 4

Effect of deletion or overexpression Ada3 on the AF-2 activity of RXRα and ERα. (A) Schematic representation of the reporter gene and activators used in this study. The ERE–URA3 reporter gene (integrated in the yeast strain PL3) was created by replacement of the poly[d(A-T)] sequences and the UAS_Ura site [from position −216 to −139 with respect to the ATG (+1) of the URA3 gene] by three EREs (ERE3x) (Pierrat et al. 1992). (B,C) The ligand-dependent activation function AF-2 of RXRα and ERα requires Ada3 for full activity in yeast. Wild-type (WT) and Δada3 PL3 strains were transformed with high-copy-number (YEp90) plasmids containing the receptor derivatives illustrated in A. Transformants were grown exponentially during five generations on selective medium containing uracil and ligand [9C-RA for RXRα(DE)–ER.CAS and E2 for ERα(CDEF)] at the concentrations indicated. OMPdecase assays were performed on each cell-free extract. Enzyme activity is expressed in nmole of substrate/min/mg protein. (D) Ada3 is a limiting coactivator for the AF-2 of RXRα and ERα. The indicated receptor derivatives were expressed from a high- (YEp90) or low- (YCp90) copy-number plasmid in the wild-type and Δada3 PL3 strains in the presence (+) or absence (−) of ligand [500 nm 9C-RA for RXRα(DE)–ER.CAS and 500 nm E2 for ERα(CDEF)]. Cells were also transformed with an episomal expression vector (YEp10) containing Ada3 (+ Ada3) or no insert (+ vector). OMPdecase activity is expressed as in B. The values (±20%) are the mean of at least three independent experiments.

Figure 4

Effect of deletion or overexpression Ada3 on the AF-2 activity of RXRα and ERα. (A) Schematic representation of the reporter gene and activators used in this study. The ERE–URA3 reporter gene (integrated in the yeast strain PL3) was created by replacement of the poly[d(A-T)] sequences and the UAS_Ura site [from position −216 to −139 with respect to the ATG (+1) of the URA3 gene] by three EREs (ERE3x) (Pierrat et al. 1992). (B,C) The ligand-dependent activation function AF-2 of RXRα and ERα requires Ada3 for full activity in yeast. Wild-type (WT) and Δada3 PL3 strains were transformed with high-copy-number (YEp90) plasmids containing the receptor derivatives illustrated in A. Transformants were grown exponentially during five generations on selective medium containing uracil and ligand [9C-RA for RXRα(DE)–ER.CAS and E2 for ERα(CDEF)] at the concentrations indicated. OMPdecase assays were performed on each cell-free extract. Enzyme activity is expressed in nmole of substrate/min/mg protein. (D) Ada3 is a limiting coactivator for the AF-2 of RXRα and ERα. The indicated receptor derivatives were expressed from a high- (YEp90) or low- (YCp90) copy-number plasmid in the wild-type and Δada3 PL3 strains in the presence (+) or absence (−) of ligand [500 nm 9C-RA for RXRα(DE)–ER.CAS and 500 nm E2 for ERα(CDEF)]. Cells were also transformed with an episomal expression vector (YEp10) containing Ada3 (+ Ada3) or no insert (+ vector). OMPdecase activity is expressed as in B. The values (±20%) are the mean of at least three independent experiments.

Figure 4

Effect of deletion or overexpression Ada3 on the AF-2 activity of RXRα and ERα. (A) Schematic representation of the reporter gene and activators used in this study. The ERE–URA3 reporter gene (integrated in the yeast strain PL3) was created by replacement of the poly[d(A-T)] sequences and the UAS_Ura site [from position −216 to −139 with respect to the ATG (+1) of the URA3 gene] by three EREs (ERE3x) (Pierrat et al. 1992). (B,C) The ligand-dependent activation function AF-2 of RXRα and ERα requires Ada3 for full activity in yeast. Wild-type (WT) and Δada3 PL3 strains were transformed with high-copy-number (YEp90) plasmids containing the receptor derivatives illustrated in A. Transformants were grown exponentially during five generations on selective medium containing uracil and ligand [9C-RA for RXRα(DE)–ER.CAS and E2 for ERα(CDEF)] at the concentrations indicated. OMPdecase assays were performed on each cell-free extract. Enzyme activity is expressed in nmole of substrate/min/mg protein. (D) Ada3 is a limiting coactivator for the AF-2 of RXRα and ERα. The indicated receptor derivatives were expressed from a high- (YEp90) or low- (YCp90) copy-number plasmid in the wild-type and Δada3 PL3 strains in the presence (+) or absence (−) of ligand [500 nm 9C-RA for RXRα(DE)–ER.CAS and 500 nm E2 for ERα(CDEF)]. Cells were also transformed with an episomal expression vector (YEp10) containing Ada3 (+ Ada3) or no insert (+ vector). OMPdecase activity is expressed as in B. The values (±20%) are the mean of at least three independent experiments.

Figure 5

Yeast Ada3 (yAda3) stimulates the RXRα transcriptional activity in mammalian cells and interacts with hAda2 in a yeast two-hybrid assay. (A) Effect of yeast Ada3 on the RXRα transcriptional activity in transfected Cos-1 cells. DR1-tk/CAT reporter (1 μg), RXRα (100 ng), and pCH110 (expressing β-galactosidase; 1 μg) were transiently cotransfected into Cos-1 cells, together with increasing amounts of yeast Ada3 (1, 2, and 5 μg). Cells were treated with control vehicle (□) or 100 nm 9C-RA (▪). Values for CAT activities (+/− 20%) represent the averages of three independent duplicated transfections after normalization for the internal control β-galactosidase activity of pCH110. (B) Two-hybrid interaction between yeast Ada3 and hAda2. The indicated LexA and AAD fusions were introduced into the yeast reporter strain L40. β-Galactosidase activities are expressed as in Fig. 2A.

Figure 5

Yeast Ada3 (yAda3) stimulates the RXRα transcriptional activity in mammalian cells and interacts with hAda2 in a yeast two-hybrid assay. (A) Effect of yeast Ada3 on the RXRα transcriptional activity in transfected Cos-1 cells. DR1-tk/CAT reporter (1 μg), RXRα (100 ng), and pCH110 (expressing β-galactosidase; 1 μg) were transiently cotransfected into Cos-1 cells, together with increasing amounts of yeast Ada3 (1, 2, and 5 μg). Cells were treated with control vehicle (□) or 100 nm 9C-RA (▪). Values for CAT activities (+/− 20%) represent the averages of three independent duplicated transfections after normalization for the internal control β-galactosidase activity of pCH110. (B) Two-hybrid interaction between yeast Ada3 and hAda2. The indicated LexA and AAD fusions were introduced into the yeast reporter strain L40. β-Galactosidase activities are expressed as in Fig. 2A.

Figure 6

Schematic representation of the known interactions (double-headed arrows) between the ADs of transactivators and the subunits of the yeast Ada complex. Putative interactions between Ada3 and Ada5 and/or Ada1 are indicated by broken lines. Interactions between Ada5 (and possibly Ada1) and TBP, and between the HAT Gcn5 and the histone tails are also shown. It is postulated that transcriptional activation mediated by the Ada complex involves chromatin remodeling and/or recruitment of the general transcription machinery through interactions with GTFs.