This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, D.-H.
Right arrow Articles by Lee, Y. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, D.-H.
Right arrow Articles by Lee, Y. C.

 Previous Article  |  Next Article 

Molecular and Cellular Biology, February 2008, p. 913-925, Vol. 28, No. 3
0270-7306/08/$08.00+0     doi:10.1128/MCB.01140-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Functional Conservation of the Glutamine-Rich Domains of Yeast Gal11 and Human SRC-1 in the Transactivation of Glucocorticoid Receptor Tau 1 in Saccharomyces cerevisiae{triangledown}

Dae-Hwan Kim, Gwang Sik Kim, Chul Ho Yun, and Young Chul Lee*

Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, South Korea

Received 27 June 2007/ Returned for modification 1 August 2007/ Accepted 14 November 2007


arrow
ABSTRACT
 
The yeast Gal11 protein, a component of the Mediator complex, is required for the transcriptional activation of many class II genes as a physiological target of various activator proteins in vivo. In this study, we identified the yeast (Saccharomyces cerevisiae) Mediator complex as a novel coactivator of the transcriptional activity of the glucocorticoid receptor (GR) tau 1 ({tau}1), the major transcriptional activation domain of the GR. GR {tau}1 directly interacted with the Mediator complex in vivo and in vitro in a Gal11 module-dependent manner, and the Gal11p subunit interacted directly with GR {tau}1. Specific amino acid residues within the glutamine-rich (Qr) domain of Gal11p (residues 116 to 277) were essential for its interaction with GR {tau}1 and GR {tau}1 transactivity in yeast, as demonstrated by mutational analysis of the Gal11 Qr domain, which is highly conserved among human steroid receptor coactivator (SRC) proteins. A Gal11p variant, mini-Gal11p, comprised of the Mediator association and Qr domains of Gal11p or chimeric mini-Gal11p containing the Qr domain of SRC-1 could potentiate the GR {tau}1 transactivity in a gal11{Delta} yeast strain. These results suggest that there is functional conservation between Qr domains of yeast Gal11p and mammalian SRC proteins as direct targets of activator proteins in yeast.


arrow
INTRODUCTION
 
The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily and a ligand-dependent transcription factor, whose major ligand is glucocorticoid (35). The binding of glucocorticoid to the GR causes the GR to translocate into the nucleus and bind to a specific DNA element, termed the GR-response element (GRE), which is present upstream of glucocorticoid-regulated gene promoters. Upon binding to the GRE, GR regulates the temporal and spatial expression of target genes by recruiting a wide spectrum of coregulator proteins that activate or repress transcription (4, 35). Previous studies have shown that the activation function 1 (AF-1) region of the GR is essential for full transcriptional activity of the GR (15, 49, 53). A specific region of the human GR (residues 77 to 262) within the AF-1 region, termed tau 1 (GR {tau}1), shows constitutive transactivity both in the yeast Saccharomyces cerevisiae and in vitro (11, 53). Molecular dissection of the GR {tau}1 domain identified a {tau}1 core ({tau}1c; residues 187 to 244 of human GR) as a major functional domain required for GR transactivity. Several GR mutants have been isolated in an attempt to identify the molecular determinants essential for its transactivity. Of these, an alanine substitution mutant of helical domain 1 of human GR {tau}1c (H1ala) or a D196Y substitution mutation completely abolishes or enhances, respectively, the transcriptional activity of GR {tau}1 (1).

Transcriptional activation by ligand-bound nuclear receptors requires a wide range of coactivator proteins that are involved in chromatin remodeling and modification, as well as the recruitment of basal transcription factors to promoters (29). The steroid receptor coactivator (SRC) family of proteins, also known as the p160 family, contains three homologous members that serve as transcriptional coactivators for nuclear receptors and other transcription factors; SRC-1 (NCoA-1), SRC-2 (GRIP1/TIF2/NCoA-2), and SRC-3 (p/CIP/RAC3/ACTR/AIB1/TRAM-1) (54). In the presence of cognate ligand, the conserved LXXLL (L, Leu; X, any amino acid) motifs located in central regions of SRC proteins directly interact with the ligand-binding domain of nuclear receptor to mediate ligand-dependent AF-2 functions of nuclear receptors. These interactions result in the recruitment of various secondary coactivator molecules (e.g., CBP/p300, histone acetyltransferases, and histone methyltransferases) to enhancer/promoter regions, which facilitates chromatin remodeling, assembly of the RNA polymerase II (Pol II) machinery, and subsequent transcription of target genes. In particular, AF-1 regions of some steroid receptors can also directly associate with a specific domain or domains of SRC proteins to mediate ligand-independent AF-1 functions of these nuclear receptors (54). For example, SRC-1 was shown to be recruited to AF-1 region of the androgen receptor (AR) via a conserved, glutamine-rich (Qr) region of SRC proteins (41).

Many effector molecules of GR {tau}1 that function as transcriptional coactivators in yeast and directly interact with the GR AF-1 region have been identified. They include the SAGA and the Ada-independent NuA4 histone acetyltransferase complexes (19, 51) and the SWI/SNF chromatin remodeling complex (52). These coactivator complexes have been shown to activate GR {tau}1-dependent transcription of chromatin templates in vitro through a direct interaction with GR {tau}1 (51). The H1ala and D196Y mutations of GR {tau}1 most likely alter the transcriptional activity of the GR by affecting the binding affinity of the GR for its coactivator proteins (51, 52). It has also been shown that GR {tau}1-mediated transcription of naked DNA templates is activated by yeast cell extracts deficient in histone acetyltransferase activity. These results strongly suggest that there are as-yet-unidentified coactivator proteins whose activities are independent of chromatin remodeling that are also involved mediating the transactivity of GR {tau}1 in yeast.

The Mediator complex was first purified from the yeast Saccharomyces cerevisiae, where it was shown to be required for diverse aspects of transcriptional regulation, such as activation, repression, and the stimulation of basal transcription (24, 25). Mediator homologues or related complexes were subsequently isolated from several metazoan species as cofactors that were essential for the regulation of transcription by a number of activator proteins (10, 33, 34). Biochemical studies have identified 21 proteins that are bona fide subunits of the core yeast Mediator complex (7). Molecular and genetic studies have revealed that some Mediator subunits are specifically required in the regulation of a subset of genes, whereas others are necessary for general transcription by Pol II (18, 48). Consistent with these observations, biochemical studies have demonstrated that functionally related Mediator subunits physically associate to form two stable subcomplexes, the Rgr1/Med14 and Srb4/Med17 subcomplexes (30). The Rgr1 subcomplex was subsequently shown to contain several functional modules, including Gal11 and Med9/10 modules, which introduced the concept of the modular structure of Mediator complexes (23). Based on electron crystal image data, the modular structure of the Mediator complex consists of three distinct domains—the head, middle, and tail domains—and this structure is conserved among the eukaryotic Mediator complexes (2). The head domain appears to correspond to the Srb4 subcomplex (Med6, -8, -11, -17, -18, -19, -20, and -22), whereas the tail and middle domains represent Rgr1 plus the Gal11 module (Med2, Hrs1/Med3, Gal11/Med15, and Sin4/Med16) and the Med9/10 module (Med1, -4, -5, -7, -9, -10, and -21), respectively (8). Recently, it was reported that the reconstituted head domain makes direct contacts with the Pol II/transcription factor IIF (TFIIF) complex in vitro (47), which is consistent with the proposed function of Srb4 subcomplex in the regulation of general Pol II activity (31). In contrast, Mediator devoid of the Gal11 module is functionally defective in supporting activated but not basal transcription in vitro (31). Moreover, in vitro binding assays have identified the Gal11 module as the activator-binding target, and this has subsequently been confirmed in vivo by genetic studies (31, 40). These results strongly suggest that the Gal11 module of the Rgr1 subcomplex is required to mediate the interaction of the Mediator complex with activators, resulting in the recruitment of a Mediator/Pol II complex (Pol II holoenzyme) to inducible promoters.

Gal11 protein was first identified as a cofactor required for full activation of galactose-inducible genes (46). Although it is not essential for cell viability, gal11 mutants exhibit pleiotropic phenotypes, such as defects in sucrose utilization, sporulation, {alpha}-pheromone production, and so on (12, 13, 38, 50). These results strongly suggest that Gal11p has both positive and negative roles in the transcriptional regulation of Pol II in yeast. In vitro and in vivo analyses have shown that Gal11p enhances basal transcription via a direct interaction with general transcription factors, such as TFIIE and TFIIH, suggesting that Gal11p plays a role in the stimulation of basal transcription by the Mediator (44, 45). In addition to this, Gal11p is also required for transcriptional activation by many activators in vitro and in vivo (16, 31). Yeast Gal11p has been shown to directly interact with various yeast activator proteins, such as Gcn4, Gal4, and Msn2, as well as artificial nonacidic activator peptide (22, 27, 31, 32, 40). These interactions occurred in the context of a functional transcription complex (43) and were required for full activation of target genes in vivo (32, 40, 55). All of these observations strongly suggest that yeast Gal11p is the direct, physiological target of many yeast activators.

In the present study, we show that the yeast Mediator complex can potentate the transcriptional activity of GR {tau}1. GR {tau}1 interacted directly with purified yeast Mediator in a Gal11 module-dependent fashion, and this was necessary for GR {tau}1c transactivation in yeast. We found that specific amino acids in the Qr domain of Gal11p (residues 116 to 277) are critical for its physical and functional interaction with GR {tau}1. Consistent with these, mini-Gal11p, which was comprised of the GR-interacting domain and Mediator-association domain of Gal11p, potentiated the transactivity of GR {tau}1. We also showed that the Gal11 Qr domain is highly conserved among mammalian SRC proteins and functionally interchangeable with SRC-1 Qr domain for the transactivation by GR {tau}1 in yeast.


arrow
MATERIALS AND METHODS
 
Yeast strains and plasmids. The yeast strains used in this study are listed in Table 1. Strain EGY48 was generated according to the manufacturer's protocol (Clontech). Strains derived from EGY48 or YPH500 were generated by specific gene disruption according to standard procedures. To construct the gene disruption plasmids, the flanking regions of MED2 (nucleotides –353 to –12 and +1251 to +1501, where the first nucleotide of the starting ATG codon is represented by +1), GAL11 (–300 to +253 and +1851 to +2450), HRS1 (–284 to –11 and +1140 to +1411), SIN4 (–347 to –12 and +2831 to +3076) and SWI3 (–315 to –16 and +2088 to +2438) were amplified from yeast genomic DNA by PCR using Vent polymerase (New England Biolabs). Each pair of 5' upstream and 3' downstream regions was cloned into the BamHI and HindIII sites of pBluescript II SK (Stratagene) by three-piece ligation. TRP1 was then inserted between the cloned 5' and 3' regions by EcoRI restriction digestion and ligation. DNA fragments consisting of TRP1 and the flanking regions of each target gene were obtained by BamHI/HindIII digestion and transformed into an appropriate strain to create the corresponding deletion strains.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Yeast strains used in this study

The cDNAs for hGR {tau}1 (residues 77 to 262) and its mutant derivatives (H1ala, D196Y) in pGAL4(1-100) and pGEX4T were kindly provided by Annika Wallberg (Karolinska Institute, Sweden) (52). For in vitro translation, hGR {tau}1 and its derivatives were excised from pGAL4(1-100) and subcloned into pcDNA3. Wild-type and mutant {tau}1c fragments of hGR (residues 187 to 244) were amplified by PCR from their corresponding pGEX-{tau}1 templates and subcloned into pCL313 to assay transactivation activity (17). To construct the episomal 8XlexAo plasmid for chromatin immunoprecipitation (ChIP) analysis, a DNA fragment corresponding to the 8XlexAo region was amplified by PCR from pSH18-34 (Invitrogen) and subcloned into pRS316 using XhoI restriction digestion and ligation.

The glutathione S-transferase (GST) fusion proteins of Gal11p and Gal11p derivatives were described previously (40). The amino acid substitution mutants of the Gal11 Qr domain (residues 116 to 277), Am (R145A and Q147A), and Bm (Q198 and V199A), were constructed by PCR-based site-directed mutagenesis with overlap extension, and subcloned into pGEX for bacterial expression. For the expression of the mini-Gal11p derivatives M-WT, M-Am, M-Bm, and M-ABm in yeast, wild-type and mutant Gal11p Qr domains (Qr-Am, Qr-Bm, and Qr-ABm) were amplified by PCR using the corresponding pGEX derivatives as templates and subcloned into the BamHI and PstI sites of pRS315, which carried the Mediator association domain of Gal11p (residues 799 to 1081) located between the promoter and terminator regions of GAL11 (pRS315-GAL11MAD). To make expression vectors for F-WT, F-Am, and F-Bm, the internal region of Gal11p (residues 278 to 798) was inserted into the PstI site of mini-Gal11p derivatives (pRS315 M-WT, M-Am, and M-Bm) using the in vivo gap repair system. Then, the N-terminal region of Gal11p (residues 1 to 115) was amplified by PCR and subcloned into the BamHI sites of the clones, resulting in the construction of full-length Gal11 derivatives. The DNA inserts were confirmed by sequencing analysis after clone isolation. To generate the GST fusion protein of the Qr domain of human SRC-1 (residues 1020 to 1185), the corresponding gene sequence was amplified by PCR from a cDNA library and subcloned into pGEX4T-1. Mutated variants of the human SRC-1 Qr domain [Am (R1053A, Q1055A), Bm (Q1105A, M1106A), and the ABm double mutant] were also generated by PCR-based site-directed mutagenesis and subcloned into pGEX4T-1 for GST pull-down assays. Chimeric mini-Gal11p derivatives (SQ-WT and SQ-ABm) were constructed by subcloning the corresponding SRC-1 Qr domain fragments into the BamHI and PstI sites of pRS315-GAL11MAD. For the expression of the LexA fusion protein of the AF-1 region of mouse AR (residues 1 to 556) in yeast, the corresponding gene sequence was isolated from a mammalian expression plasmid and subcloned into pCL313. Full-length clones for GAL11, SIN4, HRS1, and MED2 were subcloned with appropriate restriction enzyme sites into pcDNA3 for in vitro translation.

Assays for GR {tau}1c transactivity in yeast. pCL313 derivatives and a pLGZ-2XlexA reporter plasmid (52) were transformed into wild-type and mutant yeast strains by a lithium acetate method. Several colonies from each transformation were patched on minimal medium plates containing 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) to verify the homogeneity of their transactivation phenotype. Three or more transformants were grown in minimal medium to an optical density at 600 nm (OD600) of 0.2 and then treated for 4 to 6 h with 0.5 mM CuSO4 to induce the expression of LexA fusion proteins. Whole-cell extracts were prepared and assayed for β-galactosidase activity as described previously (40). To monitor chromosomal expression of the LEU2 reporter gene, a spot assay was performed using EGY48-derived yeast strains expressing LexA-GR {tau}1c. Cells at the same concentration were spotted in 10-fold serial dilutions onto duplicate minimal medium plates lacking or containing leucine (as a positive control for spotting) and then incubated for 2 to 3 days.

Western blot analysis. Yeast protein extracts were prepared by growing cells to mid-log phase in the appropriate medium, followed by glass bead disruption in lysis buffer (150 mM Tris-OAc [pH 7.9], 50 mM KOAc, 5 mM MgSO4, 1 mM EDTA, 10% glycerol). Unless otherwise noted, manipulation of all proteins was carried out at 4°C and in the presence of the following protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 5 µg/ml of leupeptin, 5 µg/ml pepstatin). The protein concentrations of the whole-cell extracts were normalized using the Bradford protein assay, according to the manufacturer's instructions (Bio-Rad). Purified proteins or immune complexes were separated on either 10% or 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels and then transferred to a Hybond-ECL enhanced chemiluminescence nitrocellulose membrane (Amersham). Membranes were incubated with a monoclonal antibody for anti-LexA (sc-7544; Santa Cruz Biotech), a polyclonal antibody to anti-GST (sc-459; Santa Cruz Biotech), or polyclonal antibodies to Mediator subunits. The antisera against Mediator subunits Gal11, Rgr1, Hrs1, and Srb5 were generated in rats using bacterially expressed recombinant polypeptides as antigens, as described previously (30). The antisera against Med1 were kindly provided by Stefan Björklund (Umea University, Sweden). Immunoreactive proteins were visualized using an Amersham ECL kit, according to the manufacturer's instructions.

Protein preparations and GST pull-down assay. pGEX4T derivatives were introduced into Escherichia coli strain BL21 or DH5{alpha}, and the resultant transformants were grown to an OD600 of 0.4. Overexpression of GST alone or GST proteins was induced by incubation for 6 h at 30°C in the presence of 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and proteins were purified using glutathione-agarose beads (Promega), according to the manufacturer's instructions. All buffers used for the purification of GST fusion proteins and GST pull-down assays contained protease inhibitors, as described above. Purified GST fusion proteins were stored at –80°C until use. The Pol II holoenzyme fractions or whole-cell extracts were prepared from wild-type, rgr1{Delta}2, hrs1{Delta}, gal11{Delta}, srb5{Delta}, and med1{Delta} mutant cells, as described previously (31). Radiolabeled proteins were synthesized by in vitro-translation using pcDNA3-based expression constructs and the TNT transcription-coupled translation system (Promega). Radiolabeled proteins or Pol II holoenzyme fractions were added to equivalent amounts of GST or GST fusion protein (2 to 4 µg) bound to glutathione-agarose beads that had been preequilibrated with buffer A (150 mM Tris-HCl [pH 7.9], 5% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 1x protease inhibitor, 0.01% NP-40, 150 to 200 mM KCl) in a final volume of 250 µl. Following incubation, the beads were washed three times in equilibration buffer and bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography.

RT and quantitative PCR. Yeast total RNA was isolated from wild-type yeast cells bearing the pRS316-8xlexAop plasmid and overexpressing LexA-GR {tau}1c. Total RNA was treated with RNase-free DNase to remove residual genomic DNA. The reverse transcription (RT) reaction was carried out using the thermoscript RT kit (Invitrogen) in a 50-µl volume of reaction mixture containing 5 µg of total RNA and random hexaoligonuclotide primers. The synthesized cDNA was then subjected to PCR by using three primer sets that were specific for URA3 (U-F, 5'-CCTAGTCCTGTTGCTGCCA-3'; U-R, 5'-ATAATGCCTTTAGCGGCTT-3') and the two flanking regions of the lexAo region (A-F, 5'-TCACTGCCCGCTTTCCAGT-3'; A-R, 5'-CCGCCTTTGAGTGAG CTGA-3'; B-F, 5'-CACACCCGCCGCGCTTAAT-3'; B-R, 5'-ATCGCCTTGCAGCACATCC-3') to determine the levels of transcription from these regions within the pRS316-8xlexAop plasmid. As a positive control, genomic DNA was prepared from an identical strain and subjected to genomic PCR using the same primer sets.

ChIP assay. ChIP assays were performed essentially as described previously (26), with minor modifications. In brief, cells were grown in minimal medium to an OD600 of 0.2, and then treated for 4 h with 0.5 mM CuSO4 to induce the expression of LexA fusion proteins. Cells were harvested, washed, and treated with formaldehyde (1% [vol/vol]) to cross-link DNA and proteins. Total cell extracts were prepared by bead beating and sonication, and equal volumes of extract were incubated overnight at 4°C with the indicated antibodies. Protein G- or A-agarose beads (Invitrogen) were added to the samples to isolate specific protein-bound DNA, and then the beads were washed three times using wash buffer. Elution buffer was added and the beads were incubated at 65°C overnight to separate protein-DNA complexes. DNA was purified and then amplified by PCR for 25 cycles to detect the chromosomal lexA operator region and the HIS4 promoter or for 20 cycles to detect the episomal lexA operator region. The following primer sets were used for PCR: T set (5'-ATGGATTCATTAGATCCGT-3' and 5'-AATAGGTCCTTAAATAATC-3') for the chromosomal lexA operator region, N set (5'-TAATACGACTCACTATAGG-3' and 5'-AATTAACCCTCACTAAAGG-3') for the episomal lexA operator region, and H set (5'-GAAGACAACAGTGGTGTGA-3' and 5'-CAACACACATCGGAGGTGA-3') for the TATA region of HIS4. The amplified PCR products were separated by 2% agarose gel electrophoresis and visualized by ethidium bromide staining.

Sulfo-SBED biotin label transfer assays. Sulfo-SBED {sulfosuccinimidyl [2-6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]-ethyl-1,3'-dithiopropionate} biotin label transfer assays were essentially carried out according to the manufacturer's protocol (Pierce). In brief, a recombinant form of GST (300 nM) or GST-GR {tau}1 (500 nM) was incubated with a 5 M excess of sulfo-SBED in the dark to conjugate the biotin label to the GST derivatives. The excess cross-linking reagent was removed from the GST proteins by dialysis overnight at 4°C. The protein conjugated to sulfo-SBED was incubated with purified Mediator in binding buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 10% glycerol, 0.1% Tween 20) for 1 h. The reaction mixture was then exposed to UV light to cross-link GST-GR {tau}1 and Mediator, after which 4x SDS sample buffer was added to the reaction mixture and incubated for 10 min. Dithiothreitol (100 mM) was added to reaction mixtures in order to transfer the biotin label from GST proteins to the interacting partner(s) of Mediator. The mixtures were applied to 12% SDS-PAGE and subjected to Western blot analysis using streptavidin-horseradish peroxidase and anti-Gal11 antibody.


arrow
RESULTS
 
The Gal11 module of Mediator is specifically required for the transactivity of hGR {tau}1 in yeast. Previous reports have demonstrated that yeast whole-cell extracts deficient in histone acetyltransferase activity can activate GR {tau}1-mediated transcription of naked DNA templates (51), which suggests that a novel coactivator protein other than a chromatin remodeling/modifying factor is involved in mediating the transactivity of GR {tau}1 in vitro. To determine the functional requirement of Mediator for GR {tau}1 transcriptional activity in yeast, we conditionally expressed a LexA-DNA binding domain (DBD) fusion of GR {tau}1c (LexA-GR {tau}1c) under the control of the CUP1 promoter, whose activation was previously shown to be independent of Mediator (28, 36). An expression plasmid for LexA-GR {tau}1c was transformed along with the episomal 2XlexAo-LacZ reporter construct into wild-type and Mediator mutant strains. Transformants were grown in minimal medium containing copper to induce the expression of LexA-GR {tau}1c and then subjected to a liquid β-galactosidase assay. We initially tested various coactivator mutant strains that were deficient in representative subunits of the tail (rgr1{Delta}2, a C-terminal truncation of Rgr1), middle (med1{Delta} and med9{Delta}), and head (srb2{Delta} and srb5{Delta}) domains of Mediator along with mutant strains of SWI/SNF (swi2{Delta}) and SAGA (gcn5{Delta}) complexes. The transactivity of GR {tau}1c was significantly reduced in the rgr1{Delta}2 strain to the levels of the swi2{Delta} and gcn5{Delta} mutants but not in the other Mediator mutant strains (Fig. 1A). These results indicated that there is a specific requirement for the tail domain of the Mediator complex in GR {tau}1c transactivation in yeast. It was previously demonstrated that a C-terminal truncation of Rgr1 or deletion of any single member of the Gal11 module (Gal11, Med2, Sin4, and Hrs1) results in the loss of the entire Gal11 module from the Mediator complex (31). Therefore, we examined the transactivity of GR {tau}1c in yeast strains deficient in each of the genes encoding components of the Gal11 module. As shown in Fig. 1A, GR {tau}1c transactivity was significantly reduced in all Gal11 module mutant strains examined, similar to the rgr1{Delta}2 strain, which confirmed that the Gal11 module of the Mediator complex is required for GR {tau}1c transactivity in yeast. The expression levels of LexA-GR {tau}1c in all of the Gal11 module mutant strains were similar to wild type; thus, the reduced activity of GR {tau}1c in the mutant strains was not due to changes in its expression level (Fig. 1A, lower panel). Interestingly, a double-deletion mutant of the Gal11 module and SAGA (gcn5{Delta} sin4{Delta}) exhibited a more severe defect in GR {tau}1 transactivity than either of the single-deletion mutants alone (Fig. 1A). These results indicate that Mediator and chromatin-related complexes have nonredundant roles and can potentiate GR {tau}1 transactivity in yeast via independent pathways, which would explain the residual transactivity of GR {tau}1 in the single-deletion mutants of these coactivator complexes.


Figure 1
View larger version (46K):
[in this window]
[in a new window]

  		FIG. 1. Requirement of the Gal11 module for GR {tau}1c transactivity in yeast. (A) Episomal reporter gene assay for GR {tau}1c transactivity in various coactivator mutant strains. The episomal 2XlexAo-LacZ reporter gene is depicted schematically on top. The reporter gene and an expression plasmid for LexA-GR {tau}1c were introduced into the wild-type (WT) and the indicated mutant yeast strains listed in Table 1. Three or more transformants were grown in minimal medium to an OD600 of 0.2 followed by treatment with 0.5 mM CuSO4 to induce the expression of LexA-GR {tau}1c. Liquid β-galactosidase assays were performed using whole-cell extracts, and the relative transcriptional activities in each mutant strain are shown as a percentage of that of the wild type (100%). Expression levels of LexA-GR {tau}1c in each strain are shown below. (B) Spot assays using the chromosomal LEU2 reporter gene to measure GR{tau}1c transactivity in Mediator mutant strains lacking the Gal11 module. The expression plasmid for LexA-GR{tau}1c was transformed into the indicated yeast strains harboring the chromosomal LEU2 reporter gene (depicted schematically at the top). Cells were spotted in 10-fold serial dilutions of the same starting concentration onto duplicate minimal medium plates in the absence (–) or presence (+) of leucine and then incubated for 2 to 3 days. All plates contained 0.01 mM CuSO4 to induce the expression of LexA-GR {tau}1c.

We also examined GR {tau}1c transactivity in wild-type or Gal11 module mutant strains carrying the chromosomal LEU2 reporter gene under the control of the LEU2 TATA box and six copies of the lexA operator. Consistent with the previous results, GR {tau}1c-dependent LEU2 expression was severely reduced in all of the Gal11 module mutant strains, as assessed by growth on minimal medium plates lacking leucine (Fig. 1B). These results demonstrated that the Gal11 module of the Mediator complex is essential for the transcriptional activity of GR {tau}1 in yeast.

GR {tau}1 directly interacts with Mediator complex in a Gal11 module-dependent manner. We next examined whether the Gal11 module physically interacts with GR {tau}1 and whether this interaction is functionally relevant. To examine the direct interaction between GR {tau}1 and Mediator, we performed a GST pull-down assay using GST fusion proteins of GR {tau}1 and GR {tau}1 mutants (wild type, D196Y, and H1ala), and Pol II holoenzyme fraction containing Mediator. Mediator interacted with GR {tau}1 but not with GST alone (Fig. 2A, lanes 2 and 3). Interestingly, Pol II was not associated with GR {tau}1 under these conditions, which indicated that the binding activity between Mediator and GR {tau}1 is relatively stronger than that between Mediator and Pol II. GR {tau}1 D196Y is a gain-of-function mutant that exhibits enhanced transactivity (two- to threefold) compared to wild-type GR {tau}1, whereas the transactivity of GR {tau}1 H1ala is less than 10% of that of the wild type. Consistent with this, the interaction of GR {tau}1 D196Y with Mediator was two- to threefold stronger than that of wild-type GR {tau}1, and the H1ala mutant exhibited severely reduced binding to Mediator (Fig. 2A, lanes 3 to 5). These results demonstrated that there was a good correlation between the ability of GR {tau}1 mutants to bind to Mediator and their transactivities in yeast, although it has been shown that their transcriptional activities are also affected by altered interactions with other coactivators, such as SWI/SNF and SAGA complexes (51, 52). To determine the role of the Gal11 module in the Mediator-GR {tau}1 interaction, we measured the binding strength of GR {tau}1 for Mediator complexes prepared from Gal11 module mutant strains (rgr1{Delta}2, hrs1{Delta}, and gal11{Delta}) using the GST pull-down assay. GR {tau}1 failed to bind to mutant Mediator complexes lacking the Gal11 module (Fig. 2B and C), which was in good agreement with the reduced GR {tau}1 transactivity in these strains. In contrast, deletion of the middle (med1{Delta}) and head (srb5{Delta}) subunits of Mediator had no effect on their interactions with GR {tau}1 (Fig. 2C). These results indicated that GR {tau}1 directly interacts with the Mediator complex via the Gal11 module.


Figure 2
View larger version (50K):
[in this window]
[in a new window]

  		FIG. 2. GR {tau}1 directly interacts with the Gal11 module of the Mediator complex. (A) GR {tau}1 interacts directly and functionally with the Mediator complex. Purified Pol II holoenzyme was incubated with the indicated GST fusion proteins (shown at the bottom) of GR {tau}1 wild-type (WT) and the GR {tau}1 helical region I mutants (D196Y and H1ala). Western blot analysis was performed using antibodies directed against the Mediator complex proteins indicated on the right. (B and C) The Gal11 module is required for the interaction of GR {tau}1 with the Mediator. GST-GR {tau}1 was incubated with Pol II holoenzyme fractions (prepared from wild-type, rgr1{Delta}2, and hrs1{Delta} strains) or whole-cell extracts (prepared from wild-type, gal11{Delta}, srb5{Delta}, and med1{Delta} strains), and the binding of Mediator was examined by Western blot analysis using antibodies directed against the proteins indicated on the right.

Mediator recruitment by GR {tau}1c to target promoters occurs in the absence of transcription in vivo. To confirm the direct role of the Gal11 module in transcriptional activation by GR {tau}1 in vivo, we carried out ChIP assays using two different DNA templates harboring LexA binding sites (Fig. 3A). The first contained the chromosomal LEU2 reporter gene, which can be actively transcribed by LexA-GR {tau}1c (TATA-containing, transcribed template), and the other contained only the lexA operator region (TATA-less, nontranscribed template). We confirmed that there were no transcripts from the regions neighboring the LexA binding sites on the nontranscribed template by RT-PCR (Fig. 3B). First, we sought to demonstrate the activator-dependent recruitment of the Mediator complex to the lexA operator region by GR {tau}1c. As shown in Fig. 3C, Mediator (Rgr1 ChIP) was recruited to LexA binding sites of the templates only when LexA-GR {tau}1c expression was induced by adding copper to the medium. Next, we examined the in vivo occupancy of the lexA operator region of the two templates by Mediator and TATA-binding protein (TBP) to determine whether the association of GR {tau}1 with Mediator at promoter regions is transcription-dependent. We also examined the occupancy of the TATA region of the HIS4 gene as a control for normalizing the ChIP signals (Fig. 3A). We first investigated the binding of LexA-GR {tau}1c to the two templates and found that GR {tau}1c associated with the lexA operators of transcribed and nontranscribed templates with similar levels of efficiency but not with the TATA region of HIS4 (Fig. 3D, lanes 5 and 6). Second, we observed that Mediator (Rgr1 ChIP) is recruited by GR {tau}1c the to lexA operators of the transcribed and nontranscribed templates with equal levels of efficiency, which indicated that the recruitment of Mediator by GR {tau}1c onto target promoters occurs in the absence of transcription in vivo (Fig. 3D, lanes 9 and 10). Third, GR {tau}1c was able to recruit Mediator to lexA operator regions in the absence of Swi3p (Fig. 3D, lane 11), which is an essential component of the SWI/SNF chromatin remodeling complex. This result suggested that GR {tau}1c-dependent recruitment of Mediator onto target DNA sequences occurs in the absence of chromatin remodeling. The association of Mediator at LexA-GR {tau}1c-bound regions of transcribed and nontranscribed templates required the Gal11 module of Mediator (Fig. 3D, lane 12), indicating that the direct interaction of GR {tau}1c with Mediator is dependent on the Gal11 module in vivo. As a control, GR {tau}1c-dependent TBP occupancy was observed on the transcribed template in wild-type and swi3{Delta} strains, but not in gal11{Delta} strains, and it was not observed on the nontranscribed template in any of the strains examined (Fig. 3D, lanes 14 to 16). In addition, there were no significant changes in the levels of LexA-GR {tau}1c bound to the lexA operator, in swi3{Delta} or gal11{Delta} strains (Fig. 3D, lanes 6 to 8). Taken together, these results indicated that Mediator interacts directly with GR {tau}1 bound to target DNA elements in vivo and that this interaction is dependent on the Gal11 module but not on transcription per se.


Figure 3
View larger version (44K):
[in this window]
[in a new window]

  		FIG. 3. ChIP assays for Mediator and TBP recruitment to target regulatory regions by GR {tau}1c. (A) Schematic representation of the target DNA regions used in the ChIP assay. Arrows indicate the regions amplified by PCR. The LexA binding regions of the two templates containing (T) or lacking (N) a TATA box and the HIS4 TATA region (H) that was used as an internal control are indicated. (B) Absence of transcription in the regions neighboring the LexA binding sites of the pRS316-8xlexAop plasmid (TATA-less template). RT-PCR was performed to detect transcripts from the indicated regions (A and B) flanking lexAo (200 bp apart, respectively) or the URA3 open reading frame region (U). Genomic PCR was also carried out as a size marker. (C) GR {tau}1c-dependent recruitment of Mediator complex to the lexAo region. Cross-linked chromatin fragments were isolated from the wild-type strain with or without copper induction of LexA-GR {tau}1c and then subjected to immunoprecipitation using anti-LexA antibody. Coimmunoprecpitated DNA, as well as DNA from whole-cell extracts (INPUT), was amplified by PCR using specific primer sets for each target region. (D) ChIP analysis of LexA (GR {tau}1), TBP, and Rgr1 (Mediator) recruitment onto the indicated target regions. ChIP assays were performed for the wild-type (WT) and the indicated mutant strains using antibodies directed against LexA, TBP, and Rgr1 as described in panel C.

Gal11p directly interacts with GR {tau}1. To determine which Mediator subunit(s) interacts directly with GR {tau}1, we analyzed the interaction of GST-GR {tau}1 and individual proteins of the Gal11 module that were synthesized by in vitro translation (Fig. 4A). A significant amount of Gal11p bound specifically to GST-GR {tau}1 but not to GST alone. Sin4p also interacted specifically with GR {tau}1, but this interaction was much weaker than that of Gal11p. This result indicated that Gal11p is the major binding partner of GR {tau}1 in the Gal11 module. Since Gal11 module proteins form a subcomplex independently of the rest of the Mediator complex, we investigated the binding of reconstituted Gal11 modules to GR {tau}1. Gal11, Sin4, Hrs1, and Med2 proteins were synthesized together by in vitro translation and incubated with GST-GR {tau}1. As shown in Fig. 4B, in contrast to the results using individual proteins (Fig. 4A), all of the Gal11 module proteins associated with GR {tau}1 under these conditions, which indicated that reconstructed Gal11 modules can directly interact with GR {tau}1 via Gal11p. Next, we determined the GR {tau}1 target subunit of Mediator in the context of the whole complex. We employed a sulfo-SBED label transfer method to cross-link interacting protein in a protein-protein interaction assay. Briefly, bacterially purified GST or GST-GR {tau}1 was labeled with sulfo-SBED (biotin) and incubated with the purified Mediator complex. The biotin label on GR {tau}1 was then cross-linked and transferred to its target subunit in Mediator by UV irradiation and dithiothreitol treatment, respectively. The transferred biotin label was detected by Western analysis using horseradish peroxidase conjugated to streptavidin. We found that the biotin label of GST-GR {tau}1, but not of GST alone, was exclusively transferred to the Gal11 subunit of Mediator, which was confirmed by performing immunoblot analysis of the same blot with anti-Gal11 antibody (Fig. 4C). This result clearly showed that Gal11p is a direct and exclusive target of GR {tau}1 for the Mediator interaction.


Figure 4
View larger version (65K):
[in this window]
[in a new window]

  		FIG. 4. Identification of Gal11p as the GR {tau}1-interacting subunit of Mediator (A) Gal11p binds directly to GR {tau}1. The indicated Mediator subunits were synthesized individually by in vitro translation and used in a pull-down assay with GST-GR {tau}1. (B) Gal11 module proteins interact with GR{tau}1 as a subcomplex. The indicated Gal11 module proteins were synthesized together by in vitro cotranslation and tested for their ability to interact with GST-GR {tau}1. (C) Identification of Gal11p as the direct target for GR {tau}1 in the context of the Mediator complex. As described in Materials and Methods, sulfo-SBED biotin label transfer assays were performed with the purified Mediator and GST or GST-GR {tau}1 that had been previously conjugated with sulfo-SBED. The reaction mixture was resolved on a 12% SDS-polyacrylamide gel, and GR {tau}1-interacting protein was detected by Western blot analysis using streptavidin-horseradish peroxidase. The same blot was immunoblotted again with the anti-Gal11 ({alpha}-Gal11) antibody to compare the molecular weights of Gal11p and the protein band detected in the horseradish peroxidase reaction. (D) Mapping of the GR {tau}1-interaction domain within Gal11p. In vitro-translated GR {tau}1 was incubated with a set of GST fusion proteins of Gal11p (represented as the Coomassie-stained gel in the bottom panel). (E) Interaction of Gal11 116-255 with GR {tau}1 in vitro. A GST pull-down assay was performed using the indicated in vitro-translated GR {tau}1 derivatives (WT, wild-type) and GST-Gal11 116-255. INPUT indicates 10% of the in vitro-translated proteins used in the assay.

To begin to understand the physiological significance of the direct interaction of Gal11p with GR {tau}1, we first mapped the GR {tau}1-binding domain of Gal11p using a set of GST fusion proteins containing individual fragments of Gal11p (Fig. 4D). The Gal11 derivatives were incubated individually with 35S-labeled GR {tau}1, and their ability to have a physical interaction with GR {tau}1 was monitored by GST pull-down assay. We identified amino acid residues 116 to 255 of Gal11p as the exclusive GR {tau}1-binding domain (Fig. 4D). Since we had previously observed that Mediator directly interacts with GR {tau}1 (Fig. 2A), we investigated the interaction of GST-Gal11 116-255 with GR {tau}1 D196Y and GR {tau}1 H1ala. The binding of Gal11 116-255 to GR {tau}1 D196Y was two- to threefold stronger than that of wild-type GR {tau}1, and there was negligible binding of Gal11 116-255 to GR {tau}1 H1ala (Fig. 4E). This binding pattern was consistent with the binding of GR {tau}1 to Mediator complex (compare Fig. 2A and 4E) and indicated that amino acids 116 to 255 of Gal11p represent the physiological target for GR {tau}1 binding.

Differential requirements for the Qr domain motifs in the interaction of Gal11 and SRC-1 with GR {tau}1 or AR AF-1. We were interested in identifying the motifs within amino acids 116 to 255 of Gal11p that were important for the interaction of Gal11p with GR {tau}1. We found that amino acids 116 to 255 of Gal11p contain a region that is rich in glutamine, termed the Qr domain, which has similarity to the Qr domain of SRC-1. Interestingly, the Qr domain of SRC-1 has previously been shown to functionally interact with the AF-1 region of AR (5). As shown in Fig. 5A, multiple sequence alignments of the Qr domains of yeast Gal11p and SRC family members revealed a high degree of conservation between yeast Gal11p and SRC proteins. Intriguingly, some of the conserved residues (A-box and B-box in Fig. 5A) were previously shown to be important for the function of SRC-1 as a coactivator of AR AF-1 transactivity in mammalian cells (9, 41). Therefore, we generated two double-substitution mutants of the Gal11 Qr domain, Qr-Am (R145A, Q147A) and Qr-Bm (Q198A, V199A) (Fig. 5A), based on the presence of conserved sequence motifs in Gal11p that corresponded to A-box and B-box motifs, respectively, in the Qr domains of SRC proteins. We first examined the effect of the Am and Bm mutations on the ability of the Gal11 Qr domain to bind to GR {tau}1 in vitro. While Gal11 Qr-Am interacted with GR {tau}1 similarly to the wild type, the Qr-Bm mutant failed to interact with GR {tau}1 (Fig. 5B). To determine whether there was functional similarity between the Qr domains of yeast Gal11 and human SRC-1 proteins, we examined the ability of AR AF-1 to bind directly to the Qr domain or Qr domain mutants of Gal11p. Gal11 Qr-Am bound less well to AR AF-1 than both the wild type and Qr-Bm, which indicated that residues in the A-box are more important than those in the B-box for the interaction of Gal11p with AR AF-1 (Fig. 5B). This result was in contrast to the binding pattern of the Gal11p Qr domain for GR AF-1.


Figure 5
View larger version (44K):
[in this window]
[in a new window]

  		FIG. 5. Differential requirements for Qr domain motifs in the interaction of Gal11 and SRC-1 with GR {tau}1 or AR AF-1. (A) Multiple sequence alignment of the Qr domains of SRC proteins and Gal11p (residues 142 to 216). The conserved A-box and B-box motifs are depicted. Amino acid substitutions of the Qr domain of Gal11p or SRC-1 are indicated by asterisks above the aligned sequences. (B and C) Differential effects of Qr domain mutations on the in vitro interaction of GR {tau}1 or AR AF-1 with Gal11 and SRC-1. The wild type (WT) or mutant derivatives (Am, Bm, and ABm) of Gal11 (B) and SRC-1 (C) Qr domains were incubated with in vitro-synthesized GR {tau}1 or AR AF-1. Bound proteins were separated by SDS-PAGE and visualized by autoradiography. INPUT indicates 10% of the in vitro-translated proteins used in the assay.

Next, we tried to identify the determinants of the interaction between the SRC-1 Qr domain and the AF-1 region of GR or AR. To this end, we constructed mutant derivatives of the SRC-1 Qr domain (residues 1020 to 1185) containing mutations in either the A-box or the B-box [Am (R1053A, Q1055A), Bm (Q1105A, M1106A)] or a double mutant containing substitution mutations in both motifs (ABm) (Fig. 5A) and examined their ability to interact with GR {tau}1 and AR AF-1. Similar to the binding pattern of the Gal11 Qr domain (Fig. 5B), the Bm and ABm mutants, but not the Am mutant, were defective in their interactions with GR {tau}1, whereas only the Am and ABm mutants of the SRC-1 Qr domain were defective in their interaction with AR AF-1 (Fig. 5C). These results demonstrated that there are differential requirements of A-box and B-box residues in the interaction of the Qr domains of Gal11 and SRC-1 with the AF-1 regions of AR and GR.

Functional conservation between the Qr domains of yeast Gal11 and human SRC-1 in the potentiation of GR {tau}1 and AR AF-1 transactivities in yeast. The similar binding patterns of the Qr domain mutants of Gal11 and SRC-1 with the AF-1 regions of GR and AR suggested the functional conservation of these domains for the potentiation of GR and AR transactivities in yeast. To address this possibility, we first examined whether the Am and Bm mutations affected GR {tau}1 transactivity in vivo. We constructed a low-copy plasmid expressing the full-length versions of the Am and Bm mutants from the GAL11 promoter (Fig. 6A) in a gal11{Delta} strain. As shown in Fig. 6B, the wild-type (F-WT) and Am mutant (F-Am), but not the Bm mutant (F-Bm), of Gal11p could potentiate GR {tau}1 transactivity, as suggested by the requirement for the Bm residues in Gal11 and SRC-1 for their in vitro binding to GR {tau}1 (Fig. 5B). Next, we determined whether the interaction between the Gal11 Qr domain and GR {tau}1 was sufficient for GR {tau}1 transactivation in yeast. To this end, we constructed a Gal11p variant, mini-Gal11p, that contained the Gal11 Qr domain fused directly to the Mediator association domain of Gal11p (residues 799 to 1081), which has been shown to be both necessary and sufficient for the incorporation of Gal11p into Mediator complexes (3). Mini-Gal11p could potentiate GR {tau}1 transactivation in a gal11{Delta} strain like the full-length Gal11p did (Fig. 6B), which indicated that the interaction of GR {tau}1 with the Gal11 Qr domain is both necessary and sufficient for Mediator-dependent transactivity of GR {tau}1. We also generated mini-Gal11p proteins that contained the Am or Bm mutations (M-Am and M-Bm) and examined their ability to function as coactivators of GR {tau}1 transactivity in a gal11{Delta} strain. The level of GR {tau}1 transactivity in cells expressing M-Am was approximately 90% that in cells expressing wild-type mini-Gal11p (M-WT). In contrast, Gal11 M-Bm failed to potentiate transcriptional activation by GR {tau}1. These results were consistent with the in vitro binding patterns of the Am and Bm Qr domain mutants (Fig. 5B) and indicated that amino acids Q198 and V199 within the B-box of the Gal11 Qr domain are essential for the GR {tau}1-Gal11 interaction and the coactivator function of Mediator in GR {tau}1 transactivity. Notably, when we tested the abilities of Gal11 derivatives to potentiate AR AF-1 transactivity, all of the mutant versions of full-length Gal11p could mediate AR AF-1 function (Fig. 6C). Moreover, mini-Gal11p was unable to rescue AR AF-1 transactivity in the absence of Gal11p (Fig. 6C). Taken together, these results indicate that the in vitro interaction between the Gal11 Qr domain and AR AF-1 (Fig. 5B) is not sufficient to mediate AR-AF-1 transactivity in yeast. Interestingly, we detected a strong physical interaction between AR AF-1 and another Qr region located in the middle region of Gal11 that is essential for AR AF-1 transactivity in yeast (D. H. Kim and Y. C. Lee, unpublished data).


Figure 6
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 6. Functional conservation of the Qr domains of Gal11 and SRC-1 for the transactivities of GR {tau}1 and AR AF-1 in yeast. (A) Schematics of full-length Gal11 (F-WT), mini-Gal11 (M-WT), and chimeric mini-Gal11 (SQ-WT) proteins along with their mutant derivatives (Am, Bm, and ABm). The asterisks indicate the mutation sites within the Qr domain of Gal11p (gray box) or SRC-1 (dotted box) shown in Fig. 5A. The Mediator association domain (residues 799 to 1081) of Gal11 is represented by MAD (hatched box). A low-copy-number plasmid expressing Gal11p derivatives from the GAL11 promoter was transformed into a gal11{Delta} strain harboring the pLGZ-2xlexA reporter and the expression plasmids for GR {tau}1c (B) or AR AF-1 (C). Liquid β-galactosidase assays were performed to measure GR {tau}1c and AR AF-1 transactivities as described in Fig. 1A. The wild-type strain (GAL11) was used as a positive control.

Finally, we investigated whether the SRC-1 Qr domain could functionally substitute for the Gal11 Qr domain in the potentiation of AR AF-1 and GR {tau}1 transactivities in yeast. We constructed chimeric mini-Gal11 proteins consisting of the wild-type SRC-1 Qr domain (SQ-WT) or its ABm mutant derivative (SQ-ABm) fused directly to the Mediator-association domain of Gal11p (residues 799 to 1081) (Fig. 6A). As shown in Fig. 6B, the reduced GR {tau}1 transactivity in a gal11{Delta} strain was restored by SQ-WT (to approximately 50% that of full-length Gal11p) and this effect was completely abolished by the ABm mutations. Similarly, SQ-WT, but not SQ-ABm, potentiated AR AF-1 transactivity in a gal11{Delta} strain (Fig. 6C).

In conclusion, our results suggest that the Qr domains of yeast Gal11 and human SRC-1 proteins might be evolutionally conserved and serve as common targets for certain activators (e.g., AF-1 of steroid receptors) and that they play comparable roles in the mediation of the transcriptional activities of these activator proteins in yeast.


arrow
DISCUSSION
 
In this study, we showed that the yeast Mediator complex is essential for the transcriptional activity of GR {tau}1 in yeast. We demonstrated that promoter-bound GR {tau}1 can recruit the Mediator complex to DNA target regions, via a direct interaction with Gal11p, and that this is independent of transcription per se. We found that specific amino acids (B-box motif) in the Gal11 Qr domain are crucial for transactivation by GR {tau}1 in yeast and Gal11 interaction with GR {tau}1 in vitro. Intriguingly, the Gal11 Qr domain showed a high degree of similarity to mammalian SRC proteins, suggesting that there is functional conservation between Qr domains of yeast Gal11p and mammalian SRC proteins as direct targets of activator proteins in yeast.

Identification of Mediator complex as a novel coactivator that mediates GR {tau}1 transactivity in yeast. In general, most eukaryotic coactivator proteins (or complexes) are directly or indirectly recruited to activator-bound regulatory regions and enhance the transcriptional efficiency of target genes by either antagonizing the repressive effect of nucleosome structure and/or directly interacting with the transcriptional machinery (29). The former group of coactivator proteins includes a wide range of protein complexes with chromatin remodeling and/or histone modification activities, such as the SAGA, NuA4, and SWI/SNF complexes of yeast, whereas the latter group includes yeast TFIID and Mediator complexes, which directly interact with components of the general transcription machinery, such as TBP and Pol II. In the current model of transcriptional activation, the concerted or combined action of the two classes of coactivator proteins results in the relief of nucleosomal repression, followed by the assembly of the Pol II transcriptional machinery to achieve full activation of target genes.

It has been reported that GR {tau}1 directly and functionally interacts with SAGA and Ada-independent NuA4 histone acetyltransferase complexes, as well as the SWI/SNF histone remodeling complex, all of which are essential for GR {tau}1 transactivity in yeast (51, 52). Interestingly, Wallberg et al. showed that yeast cell extracts deficient in histone acetyltransferase activity can also mediate GR {tau}1-dependent transcriptional activation from naked DNA templates in vitro (51). Although the human TFIID complex has been shown to be involved in GR {tau}1 transactivation in vitro (14), this observation strongly suggested that a novel transcriptional coactivator that functions independently of chromatin state is involved in GR {tau}1 transactivation in yeast.

Our study demonstrated that in addition to previously identified chromatin remodeling/modifying factors, yeast Mediator complex functions as a novel coactivator of the AF-1 region of the GR in yeast. This finding fits well in the current model of transcriptional activation, in which two classes of coactivators act cooperatively to maximize transcription efficiency. Our observation that a double-deletion mutant of the Gal11 module protein Sin4 and the Gcn5 subunit of SAGA exhibited a more severe defect in GR {tau}1 transactivity than single-deletion mutants also supports the model (Fig. 1A). This result also indicate that different types of coactivator complexes are required for the transactivity of GR {tau}1 and that Mediator and chromatin-related complexes have nonredundant, specialized roles in transcriptional activation by GR {tau}1 in yeast.

Requirement of Gal11p of the Mediator complex for GR {tau}1 transactivity in yeast. The Gal11 subunit of the Mediator complex is required for transcriptional activation by many activators in vitro and in vivo (16, 31) and functions as a direct, physiological target of various activator proteins, as well as artificial nonacidic activator peptide (22, 27, 31, 32, 40). In the present study, we showed that the mammalian activators GR {tau}1 and AR AF-1 are among the activator proteins that functionally interact with Gal11p via direct binding and that this interaction potentiates their transactivities in yeast. In previous studies, mammalian Med14 (TRAP170/ARC150/DRIP150), the proposed homologue of yeast Rgr1, was identified as a GR AF-1-interacting protein based on a yeast two-hybrid screen (20). GR {tau}1 transactivity was impaired in a strain carrying a C-terminal deletion of Rgr1 (Fig. 1A, rgr1{Delta}2); thus, we first examined the physical interaction between the Rgr1 C-terminal region and GR {tau}1. We found that there was no physical interaction between these two proteins in a GST pull-down assay (data not shown). Also, although the intact Rgr1 existed within the purified Mediator from the strains lacking the Gal11 module, we could not observe the interaction between these mutant Mediators with GR {tau}1 (Fig. 2B and C). Rather, we found that GR {tau}1 directly interacts with the Gal11 module, whose association with the Mediator complex is completely dependent on the C-terminal region of Rgr1p (31). This result clearly demonstrated that the impaired transactivity of GR {tau}1 in an rgr1{Delta}2 strain was due to the lack of the Gal11 module rather than to the lack of the C-terminal region of Rgr1p.

The Gal11p subunit of the Gal11 module interacted directly with GR {tau}1 and was required for the recruitment of Mediator onto promoter-bound GR {tau}1 in vivo, even in the absence of transcription (Fig. 3 and 4). The Sin4 subunit of the Gal11 module also interacted with GR {tau}1. However, this interaction was much weaker than that of Gal11p, and we found no evidence of a functional interaction between Sin4 and GR {tau}1 mutants (data not shown), which ruled out the possibility that Sin4p was the physiological target of GR {tau}1. Based on the specialized role of Gal11p in the function of various activator proteins, several mammalian homologues of yeast Gal11p have been proposed. Bioinformatics-based approaches using data from functional and structural studies have identified human Med23 (hSur2/TRAP150/DRIP130) as a Gal11 orthologue (6, 7). In support of this, Med23, Med24 (TRAP100/DRIP100), and Med16 (TRAP95/DRIP92) form a submodule of the mammalian Mediator complex that is regarded as the functional counterpart of the yeast Gal11 module (21). Other studies have identified human Med15 (PCQAP/ARC1051) as the mammalian homologue of yeast Gal11p, based on its homology to the Gal11 N-terminal region and its function as a coactivator for the acidic activator VP16 (39). However, rigorous experimental data are needed to confirm the identity of these mammalian homologues, as these predictions were based on preliminary data and weak homology between short fragments. We found that the N-terminal KIX homology domain of Gal11p was not essential for GR {tau}1 transactivity in yeast and were unable to detect a physical interaction between mammalian Med15 and Med23 and GR {tau}1 in vitro (data not shown). These results indicate that the mammalian orthologues of Gal11p that have been proposed to date are not the functional counterparts of yeast Gal11p in the potentiation of GR {tau}1 transactivity in yeast.

Functional conservation between the Qr domains of Gal11p and SRC as a direct targets for the AF-1 regions of steroid receptors in yeast. We showed that the Qr domain of Gal11p (residues 116 to 255) was the exclusive target for GR {tau}1 binding and was both necessary and sufficient for GR {tau}1 transactivity in yeast, as shown by the analysis of mini-Gal11p (Fig. 6). To identify a conserved motif(s) within the Gal11 Qr domain, we carried out a sequence alignment of the functional orthologues of yeast Gal11p and other known GR AF-1 coactivators. We first aligned Gal11p 116-255 with the reported mammalian homologues of yeast Gal11p (hMed15 and hMed23) and the GR AF-1-interacting subunit of mammalian Mediator (hMed14) and failed to identify any significant homologies between them. Next, we aligned the Gal11p Qr domain with the Qr regions of ySwi1p (residues 286 to 536) and ySnf5p (residues 28 to 273), which are known to serve as the targets of various activator proteins (37, 42). However, this search revealed again that there were no meaningful similarities between these domains except for certain stretches of conserved Q residues. In contrast, amino acids 116 to 255 of Gal11p showed a high degree of sequence similarity to the Qr domains of SRC proteins (Fig. 5A). The A-box and B-box motifs of SRC-1 Qr domain have been shown to be required for the function of SRC-1 as a coactivator of AR AF-1 transactivity (5). Interestingly, only mutations in the B-box (Qr-Bm) of the Gal11 Qr domain resulted in defects in the physical association of Gal11p with GR {tau}1, whereas double- or triple-alanine substitutions in the A-box (Q143/L144/R145, R145/Q147) did not affect the interaction (Fig. 5B and data not shown). The B-box motif was also important in the interaction of GR {tau}1 with the SRC-1 Qr domain (Fig. 5C), indicating the B-box motif of Gal11p and SRC is the binding interface for GR {tau}1. Additional experiments in a relevant biological system are needed to determine the functional importance of the B-box motif in the stimulation of GR {tau}1 transactivity by SRC-1.

In contrast to GR {tau}1, the interaction of the Qr domains of Gal11 and SRC-1 with AR AF-1 required the A-box motif, suggesting that there are different requirements for the two motifs in the interaction of Gal11p with the AF-1 regions of nuclear receptors. In support of this hypothesis, the AF-1 regions of other nuclear receptors (including estrogen receptor and Nurr1) also showed differential requirements for conserved A- and B-box motifs in their interaction with the Gal11p Qr domain and in the activation of their transactivities in yeast (D. H. Kim, G. S. Kim, and Y. C. Lee, unpublished data). It was shown using a mammalian system that both the A- and B-box motifs of the Qr domain were essential for the stimulation of AR AF-1 transactivity by SRC-1 (9, 41). These previous experiments also showed that the A-box motif is more important than the B-box motif, which is somewhat consistent with our in vitro and in vivo interaction data (Fig. 5 and 6).

The group working with M. Ptashne has demonstrated that the Gal11 region (residues 261 to 351) flanking the Qr domain is both necessary and sufficient for mediating a weak transactivity of AH (for amphipathic helix) activator in yeast (3), suggesting this region of Gal11p as the physiological target of the artificial activator AH. However, their in vitro binding assay also revealed that AH protein does not significantly bind to the region from 261 to 351 of Gal11p (3). These controversial results raised the intriguing possibility that a weak transactivity of AH activator shown in yeast might have resulted from the artifactual interaction between AH and Gal11 residues 261 to 351, which is too weak to be reproduced in an in vitro condition. In support of this assumption, we could observe that this region of Gal11p was not required for the interactions of Gal11p with many natural activator proteins we tested and in the potentiation of their transactivities in yeast (D. H. Kim, G. S. Kim, and Y. C. Lee, unpublished data).

In summary, our results provide evidence that the Qr domains of Gal11p and SRC family proteins have conserved binding sites for the AF-1 regions of some steroid receptors and that they have comparable functions in mediating their transactivities in yeast. Furthermore, based on our unpublished observations showing that the Qr domain of Gal11p is a general target for various activator proteins, we suggest that the Qr domain of yeast Gal11p is functionally equivalent to the Qr domain of mammalian SRC proteins and that it constitutes the direct, physiological target of transcriptional activators.


arrow
ACKNOWLEDGMENTS
 
We are grateful to Young-Joon Kim, Fred Winston, Annika Wallberg, Stefan Björklund, Marian Carlson, and Richard Young for providing yeast strains, plasmids, and antibodies. We thank all of the previous and current members of Y.C.L.'s laboratory for sharing of materials and helpful discussions.

This work was supported by the grant from Korea Research Foundation (2006-005-J03003) to Y.C.L. G.S.K. is supported in part by the Second Stage BK21 program.


arrow
FOOTNOTES
 
* Corresponding author: Mailing address: Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju 500-757, South Korea. Phone: 82-62-530-0909. Fax: 82-62-530-0500. E-mail: yclee{at}jnu.ac.kr Back

{triangledown} Published ahead of print on 10 December 2007. Back


arrow
REFERENCES
 
    1
  1. Almlöf, T., J.-Å. Gustafsson, and A. P. H. Wright. 1997. Role of hydrophobic amino acid clusters in the transactivation activity of the human glucocorticoid receptor. Mol. Cell. Biol. 17:934-945.[Abstract]
    2. 2
  2. Asturias, F. J., Y. W. Jiang, L. C. Myers, C. M. Gustafsson, and R. D. Kornberg. 1999. Conserved structures of mediator and RNA polymerase II holoenzyme. Science 283:985-987.[Abstract/Free Full Text]
    3. 3
  3. Barberis, A., J. Pearlberg, N. Simkovich, S. Farrell, P. Reinagel, C. Bamdad, G. Sigal, and M. Ptashne. 1995. Contact with a component of the polymerase II holoenzyme suffices for gene activation. Cell 81:359-368.[CrossRef][Medline]
    4. 4
  4. Beato, M., P. Herrlich, and G. Schutz. 1995. Steroid-hormone receptors—many actors in search of a plot. Cell 83:851-857.[CrossRef][Medline]
    5. 5
  5. Bevan, C. L., S. Hoare, F. Claessens, D. M. Heery, and M. G. Parker. 1999. The AF1 and AF2 domains of the androgen receptor interact with distinct regions of SRC1. Mol. Cell. Biol. 19:8383-8392.[Abstract/Free Full Text]
    6. 6
  6. Boube, M., L. Joulia, D. L. Cribbs, and H. M. Bourbon. 2002. Evidence for a mediator of RNA polymerase II transcriptional regulation conserved from yeast to man. Cell 110:143-151.[CrossRef][Medline]
    7. 7
  7. Bourbon, H. M., A. Aguilera, A. Z. Ansari, F. J. Asturias, A. J. Berk, S. Bjorklund, T. K. Blackwell, T. Borggrefe, M. Carey, M. Carlson, J. W. Conaway, R. C. Conaway, S. W. Emmons, J. D. Fondell, L. P. Freedman, T. Fukasawa, C. M. Gustafsson, M. Han, X. He, P. K. Herman, A. G. Hinnebusch, S. Holmberg, F. C. Holstege, J. A. Jaehning, Y. J. Kim, L. Kuras, A. Leutz, J. T. Lis, M. Meisterernest, A. M. Naar, K. Nasmyth, J. D. Parvin, M. Ptashne, D. Reinberg, H. Ronne, I. Sadowski, H. Sakurai, M. Sipiczki, P. W. Sternberg, D. J. Stillman, R. Strich, K. Struhl, J. Q. Svejstrup, S. Tuck, F. Winston, R. G. Roeder, and R. D. Kornberg. 2004. A unified nomenclature for protein subunits of mediator complexes linking transcriptional regulators to RNA polymerase II. Mol. Cell 14:553-557.[CrossRef][Medline]
    8. 8
  8. Chadick, J. Z., and F. J. Asturias. 2005. Structure of eukaryotic Mediator complexes. Trends Biochem. Sci. 30:264-271.[CrossRef][Medline]
    9. 9
  9. Christiaens, V., C. L. Bevan, L. Callewaert, A. Haelens, G. Verrijdt, W. Rombauts, and F. Claessens. 2002. Characterization of the two coactivator-interacting surfaces of the androgen receptor and their relative role in transcriptional control. J. Biol. Chem. 277:49230-49237.[Abstract/Free Full Text]
    10. 10
  10. Conaway, R. C., S. Sato, C. Tomomori-Sato, T. Yao, and J. W. Conaway. 2005. The mammalian Mediator complex and its role in transcriptional regulation. Trends Biochem. Sci. 30:250-255.[CrossRef][Medline]
    11. 11
  11. Dahlman-Wright, K., H. Baumann, I. J. McEwan, T. Almlof, A. P. Wright, J. A. Gustafsson, and T. Hard. 1995. Structural characterization of a minimal functional transactivation domain from the human glucocorticoid receptor. Proc. Natl. Acad. Sci. USA 92:1699-1703.[Abstract/Free Full Text]
    12. 12
  12. Fassler, J. S., and F. Winston. 1988. Isolation and analysis of a novel class of suppressor of Ty insertion mutations in Saccharomyces cerevisiae. Genetics 118:203-212.[Abstract/Free Full Text]
    13. 13
  13. Fassler, J. S., and F. Winston. 1989. The Saccharomyces cerevisiae SPT13/GAL11 gene has both positive and negative regulatory roles in transcription. Mol. Cell. Biol. 9:5602-5609.[Abstract/Free Full Text]
    14. 14
  14. Ford, J., I. J. McEwan, A. P. Wright, and J. A. Gustafsson. 1997. Involvement of the transcription factor IID protein complex in gene activation by the N-terminal transactivation domain of the glucocorticoid receptor in vitro. Mol. Endocrinol. 11:1467-1475.[Abstract/Free Full Text]
    15. 15
  15. Freedman, L. P., S. K. Yoshinaga, J. N. Vanderbilt, and K. R. Yamamoto. 1989. In vitro transcription enhancement by purified derivatives of the glucocorticoid receptor. Science 245:298-301.[Abstract/Free Full Text]
    16. 16
  16. Fukasawa, T., M. Fukuma, K. Yano, and H. Sakurai. 2001. A genome-wide analysis of transcriptional effect of Gal11 in Saccharomyces cerevisiae: an application of "mini-array hybridization technique." DNA Res. 8:23-31.[Abstract]
    17. 17
  17. Han, S. J., J. S. Lee, J. S. Kang, and Y. J. Kim. 2001. Med9/Cse2 and Gal11 modules are required for transcriptional repression of distinct group of genes. J. Biol. Chem. 276:37020-37026.[Abstract/Free Full Text]
    18. 18
  18. Han, S. J., Y. C. Lee, B. S. Gim, G.-H. Ryu, S. J. Park, W. S. Lane, and Y.-J. Kim. 1999. Activator-specific requirement of yeast mediator proteins for RNA polymerase II transcriptional activation. Mol. Cell. Biol. 19:979-988.[Abstract/Free Full Text]
    19. 19
  19. Henriksson, A., T. Almlöf, J. Ford, I. J. McEwan, J.-Å. Gustafsson, and A. P. H. Wright. 1997. Role of the Ada adaptor complex in gene activation by the glucocorticoid receptor. Mol. Cell. Biol. 17:3065-3073.[Abstract]
    20. 20
  20. Hittelman, A. B., D. Burakov, J. A. Iniguez-Lluhi, L. P. Freedman, and M. J. Garabedian. 1999. Differential regulation of glucocorticoid receptor transcriptional activation via AF-1-associated proteins. EMBO J. 18:5380-5388.[CrossRef][Medline]
    21. 21
  21. Ito, M., H. J. Okano, R. B. Darnell, and R. G. Roeder. 2002. The TRAP100 component of the TRAP/Mediator complex is essential in broad transcriptional events and development. EMBO J. 21:3464-3475.[CrossRef][Medline]
    22. 22
  22. Jeong, C. J., S. H. Yang, Y. Xie, L. Zhang, S. A. Johnston, and T. Kodadek. 2001. Evidence that Gal11 protein is a target of the Gal4 activation domain in the mediator. Biochemistry 40:9421-9427.[CrossRef][Medline]
    23. 23
  23. Kang, J. S., S. H. Kim, M. S. Hwang, S. J. Han, Y. C. Lee, and Y. J. Kim. 2001. The structural and functional organization of the yeast mediator complex. J. Biol. Chem. 276:42003-42010.[Abstract/Free Full Text]
    24. 24
  24. Kim, Y. J., S. Bjorklund, Y. Li, M. H. Sayre, and R. D. Kornberg. 1994. A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell 77:599-608.[CrossRef][Medline]
    25. 25
  25. Koleske, A. J., and R. A. Young. 1994. An RNA polymerase II holoenzyme responsive to activators. Nature 368:466-469.[CrossRef][Medline]
    26. 26
  26. Kuo, M. H., and C. D. Allis. 1999. In vivo cross-linking and immunoprecipitation for studying dynamic protein:DNA associations in a chromatin environment. Methods 19:425-433.[CrossRef][Medline]
    27. 27
  27. Lallet, S., H. Garreau, C. Garmendia-Torres, D. Szestakowska, E. Boy-Marcotte, S. Quevillon-Cheruel, and M. Jacquet. 2006. Role of Gal11, a component of the RNA polymerase II mediator in stress-induced hyperphosphorylation of Msn2 in Saccharomyces cerevisiae. Mol. Microbiol. 62:438-452.[CrossRef][Medline]
    28. 28
  28. Lee, D., and J. T. Lis. 1998. Transcriptional activation independent of TFIIH kinase and the RNA polymerase II mediator in vivo. Nature 393:389-392.[CrossRef][Medline]
    29. 29
  29. Lee, J. W., Y. C. Lee, S. Y. Na, D. J. Jung, and S. K. Lee. 2001. Transcriptional coregulators of the nuclear receptor superfamily: coactivators and corepressors. Cell. Mol. Life Sci. 58:289-297.[CrossRef][Medline]
    30. 30
  30. Lee, Y. C., and Y. J. Kim. 1998. Requirement for a functional interaction between mediator components Med6 and Srb4 in RNA polymerase II transcription. Mol. Cell. Biol. 18:5364-5370.[Abstract/Free Full Text]
    31. 31
  31. Lee, Y. C., J. M. Park, S. Min, S. J. Han, and Y.-J. Kim. 1999. An activator binding module of yeast RNA polymerase II holoenzyme. Mol. Cell. Biol. 19:2967-2976.[Abstract/Free Full Text]
    32. 32
  32. Lu, Z., A. Z. Ansari, X. Lu, A. Ogirala, and M. Ptashne. 2002. A target essential for the activity of a nonacidic yeast transcriptional activator. Proc. Natl. Acad. Sci. USA 99:8591-8596.[Abstract/Free Full Text]
    33. 33
  33. Malik, S., and R. G. Roeder. 2005. Dynamic regulation of pol II transcription by the mammalian Mediator complex. Trends Biochem. Sci. 30:256-263.[CrossRef][Medline]
    34. 34
  34. Malik, S., and R. G. Roeder. 2000. Transcriptional regulation through Mediator-like coactivators in yeast and metazoan cells. Trends Biochem. Sci. 25:277-283.[CrossRef][Medline]
    35. 35
  35. Mangelsdorf, D. J., C. Thummel, M. Beato, P. Herrlich, G. Schutz, K. Umesono, B. Blumberg, P. Kastner, M. Mark, P. Chambon, and R. M. Evans. 1995. The nuclear receptor superfamily—the second decade. Cell 83:835-839.[CrossRef][Medline]
    36. 36
  36. McNeil, J. B., H. Agah, and D. Bentley. 1998. Activated transcription independent of the RNA polymerase II holoenzyme in budding yeast. Genes Dev. 12:2510-2521.[Abstract/Free Full Text]
    37. 37
  37. Neely, K. E., A. H. Hassan, C. E. Brown, L. Howe, and J. L. Workman. 2002. Transcription activator interactions with multiple SWI/SNF subunits. Mol. Cell. Biol. 22:1615-1625.[Abstract/Free Full Text]
    38. 38
  38. Nishizawa, M., Y. Suzuki, Y. Nogi, K. Matsumoto, and T. Fukasawa. 1990. Yeast Gal11 protein mediates the transcriptional activation signal of two different transacting factors, Gal4 and general regulatory factor I/repressor/activator site binding protein 1/translation upstream factor. Proc. Natl. Acad. Sci. USA 87:5373-5377.[Abstract/Free Full Text]
    39. 39
  39. Novatchkova, M., and F. Eisenhaber. 2004. Linking transcriptional mediators via the GACKIX domain super family. Curr. Biol. 14:R54-R55.[CrossRef][Medline]
    40. 40
  40. Park, J. M., H.-S. Kim, S. J. Han, M.-S. Hwang, Y. C. Lee, and Y.-J. Kim. 2000. In vivo requirement of activator-specific binding targets of mediator. Mol. Cell. Biol. 20:8709-8719.[Abstract/Free Full Text]
    41. 41
  41. Powell, S. M., V. Christiaens, D. Voulgaraki, J. Waxman, F. Claessens, and C. L. Bevan. 2004. Mechanisms of androgen receptor signalling via steroid receptor coactivator-1 in prostate. Endocr. Relat. Cancer 11:117-130.[Abstract]
    42. 42
  42. Prochasson, P., K. E. Neely, A. H. Hassan, B. Li, and J. L. Workman. 2003. Targeting activity is required for SWI/SNF function in vivo and is accomplished through two partially redundant activator-interaction domains. Mol. Cell 12:983-990.[CrossRef][Medline]
    43. 43
  43. Reeves, W. M., and S. Hahn. 2005. Targets of the Gal4 transcription activator in functional transcription complexes. Mol. Cell. Biol. 25:9092-9102.[Abstract/Free Full Text]
    44. 44
  44. Sakurai, H., and T. Fukasawa. 2000. Functional connections between mediator components and general transcription factors of Saccharomyces cerevisiae. J. Biol. Chem. 275:37251-37256.[Abstract/Free Full Text]
    45. 45
  45. Sakurai, H., and T. Fukasawa. 1998. Functional correlation among Gal11, transcription factor (TF) IIE, and TFIIH in Saccharomyces cerevisiae. Gal11 and TFIIE cooperatively enhance TFIIH-mediated phosphorylation of RNA polymerase II carboxyl-terminal domain sequences. J. Biol. Chem. 273:9534-9538.[Abstract/Free Full Text]
    46. 46
  46. Suzuki, Y., Y. Nogi, A. Abe, and T. Fukasawa. 1988. GAL11 protein, an auxiliary transcription activator for genes encoding galactose-metabolizing enzymes in Saccharomyces cerevisiae. Mol. Cell. Biol. 8:4991-4999. (Erratum, 12:4806, 1992.)[Abstract/Free Full Text]
    47. 47
  47. Takagi, Y., G. Calero, H. Komori, J. A. Brown, A. H. Ehrensberger, A. Hudmon, F. Asturias, and R. D. Kornberg. 2006. Head module control of mediator interactions. Mol. Cell 23:355-364.[CrossRef][Medline]
    48. 48
  48. Thompson, C. M., and R. A. Young. 1995. General requirement for RNA polymerase II holoenzymes in vivo. Proc. Natl. Acad. Sci. USA 92:4587-4590.[Abstract/Free Full Text]
    49. 49
  49. Tsai, S. Y., G. Srinivasan, G. F. Allan, E. B. Thompson, B. W. O'Malley, and M. J. Tsai. 1990. Recombinant human glucocorticoid receptor induces transcription of hormone response genes in vitro. J. Biol. Chem. 265:17055-17061.[Abstract/Free Full Text]
    50. 50
  50. Vallier, L. G., and M. Carlson. 1991. New SNF genes, GAL11 and GRR1 affect SUC2 expression in Saccharomyces cerevisiae. Genetics 129:675-684.[Abstract]
    51. 51
  51. Wallberg, A. E., K. E. Neely, J.-Å. Gustafsson, J. L. Workman, A. P. H. Wright, and P. A. Grant. 1999. Histone acetyltransferase complexes can mediate transcriptional activation by the major glucocorticoid receptor activation domain. Mol. Cell. Biol. 19:5952-5959.[Abstract/Free Full Text]
    52. 52
  52. Wallberg, A. E., K. E. Neely, A. H. Hassan, J.-Å. Gustafsson, J. L. Workman, and A. P. H. Wright. 2000. Recruitment of the SWI-SNF chromatin remodeling complex as a mechanism of gene activation by the glucocorticoid receptor {tau}1 activation domain. Mol. Cell. Biol. 20:2004-2013.[Abstract/Free Full Text]
    53. 53
  53. Wright, A. P., I. J. McEwan, K. Dahlman-Wright, and J. A. Gustafsson. 1991. High level expression of the major transactivation domain of the human glucocorticoid receptor in yeast cells inhibits endogenous gene expression and cell growth. Mol. Endocrinol. 5:1366-1372.[Abstract/Free Full Text]
    54. 54
  54. Xu, J., and Q. Li. 2003. Review of the in vivo functions of the p160 steroid receptor coactivator family. Mol. Endocrinol. 17:1681-1692.[Abstract/Free Full Text]
    55. 55
  55. Zhang, F., L. Sumibcay, A. G. Hinnebusch, and M. J. Swanson. 2004. A triad of subunits from the Gal11/tail domain of Srb mediator is an in vivo target of transcriptional activator Gcn4p. Mol. Cell. Biol. 24:6871-6886.[Abstract/Free Full Text]

Molecular and Cellular Biology, February 2008, p. 913-925, Vol. 28, No. 3
0270-7306/08/$08.00+0     doi:10.1128/MCB.01140-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Balamotis, M. A., Pennella, M. A., Stevens, J. L., Wasylyk, B., Belmont, A. S., Berk, A. J. (2009). Complexity in Transcription Control at the Activation Domain-Mediator Interface. Sci Signal 2: ra20-ra20 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, D.-H.
Right arrow Articles by Lee, Y. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, D.-H.
Right arrow Articles by Lee, Y. C.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»