Transient transfections and reporter gene assays in mammalian cells.

Hepa1c1c7 cells, hereafter referred to as Hepa-1 cells, were maintained in alpha minimal essential medium supplemented with 10% fetal calf serum and 100 U of streptomycin and penicillin per ml. Typically, 500 ng each of the CYP1A1 promoter-driven luciferase and pCMV-β-Gal reporter plasmids was cotransfected with either 2 or 5 μg of either pCMX-SRC-1, pCMX-NCoA-2, or pCMX-p/CIP into six-well plates. The final DNA concentration was adjusted to 6 μg per well with empty expression vector, and transfection was achieved with 15 μl of Superfect transfection reagent (Qiagen, Valencia, Calif.) according to the manufacturer's protocol. Three hours after transfection, cells were treated with either 5 × 10⁻⁹ M TCDD or 0.1% dimethyl sulfoxide (DMSO) and incubated for a further 18 h before harvesting. Cells were lysed in 500 μl of 1× harvest buffer (50 mM Tris/MES [morpholineethanesulfonic acid, pH 7.8], 1 mM dithiothreitol, and 0.1% Triton X-100) on ice.

Cell lysates were vortexed briefly, and cellular debris was pelleted by centrifugation. Then 15 μl of lysate was added to 185 μl of harvest buffer, 15 μl of luciferase assay cocktail [750 mM Tris/MES (pH 7.8), 150 mM magnesium acetate, and 40 mM ATP] and mixed with 100 μl of 1 mM luciferin. Luciferase activity was measured in a Monolight 2010 luminometer (Analytical Luminescence Laboratory, Sparks, Md.). β-Galactosidase activity was determined with standard protocols (45), and luciferase activity was normalized to these values.

For reporter gene studies with GAL4-DBD-ARNT fusions, 250 ng of pG5E4T and 250 ng of pCMV-β-galactosidase were cotransfected either alone or with a GAL4-DBD-ARNT construct and increasing amounts of coactivator expression plasmid into HEK 293T cells (hereafter referred to as 293T cells). Cells were harvested 20 h after transfection and analyzed as described above.

In vivo coimmunoprecipitation.

293T cells were transfected with 5 μg of HA-tagged mARNT, 5 μg of mAHR and 5 μg of SRC-1, NCoA-2, or p/CIP. Approximately 20 h after transfection, cells were exposed to 20 nM TCDD or vehicle (0.1% DMSO). Whole-cell extracts were prepared as follows. Confluent plates were lysed in 1× LysS buffer (50 mM Tris [pH 7.8], 300 mM NaC1, 0.5% NP-40, 1 mM EDTA, 10% glycerol, 5 mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, and 10 μg each of leupeptin, antipain, and aprotinin per ml) for 30 min on ice. Cellular debris was pelleted by ultracentrifugation at 60,000 × g in a Beckman L-65 centrifuge with an SW55 Ti rotor for 30 min at 4°C. Lysates were assayed for protein concentration with Bio-Rad protein assay solution, following the manufacturer's protocol.

Affinity-purified antibodies directed against the influenza virus hemagglutinin (HA) tag epitope were purchased from Santa Cruz Biotechnologies Inc. (Santa Cruz, Calif.). Affinity-purified rabbit polyclonal antibodies generated against SRC-1, NcoA-2, and p/CIP have been described previously (51). For each reaction, 1 mg of cell lysate was incubated with 5 μg of affinity-purified anti-HA or the corresponding preimmune immunoglobulin G (IgG) overnight at 4°C. Complexes were then mixed with 50 μl of protein A-Sepharose beads (50% slurry) on a rotating wheel at 4°C for 1 h. The beads were then washed six times with LysS buffer, and proteins were eluted in sodium dodecyl sulfate sample buffer and boiled for 3 min. Complexes were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting was performed with either affinity-purified anti-SRC-1, anti-NCoA-2, or anti-p/CIP antibodies (51). Precipitated complexes were detected with horseradish peroxidase-linked goat anti-rabbit IgG and an enhanced chemiluminesence (ECL) kit (Pierce, Rockford, Ill.).

Green fluorescent protein assays.

In order to study the interaction between ARNT and SRC-1 in the intact cell, mouse ARNT-EGFP or deletion mutant forms of ARNT-EGFP (0.5 μg) were cotransfected into Hepa1 c4 mutant cells (33) (hereafter referred to as c4 cells) with 4 μg of either pcDNA3-LUC, HA-tag-SRC-1, SRC-1, or pcDNA3 (Invitrogen, Carlsbad, Calif.) expression plasmid and GenePorter2 (Gene Therapy Systems, San Diego, Calif.) according to the manufacturer's protocol. Similarly, in order to study the interaction between AHR and SRC-1, transfections were performed in an identical fashion but in wild-type Hepa1 cells. Twenty hours after transfection, cells were fixed in ice-cold methanol for 15 min. After fixing, cells were washed three times with phosphate-buffered saline (PBS) and incubated with anti-HA IgG for 1 h at room temperature, washed again three times with PBS, and incubated with rhodamine-conjugated anti-rabbit IgG (Molecular Probes, Eugene, Oreg.) for a further 45 min, washed three more times in PBS and once in water, and mounted on glass slides with standard protocols. Cells were visualized by fluorescent microscopy with an AX-70 (Olympus, Melville, N.Y.) upright microscope and photographed with Ektachrome ASA 400 film (Eastman Kodak, Rochester, N.Y.).

Single-cell microinjection assay.

Hepa1 cells were seeded on poly-l-lysine-treated glass coverslips at subconfluent density, and microinjections were performed as previously described (51) with minor modifications. Briefly, to allow for ligand solubility, injected cells were maintained in serumless Dulbecco's modified Eagle's medium supplemented with 3.7% bovine serum albumin. Immediately after injection, cells were treated with either 5 × 10⁻⁹ M TCDD or 0.1% DMSO and incubated for a further 18 h.

Chromatin immunoprecipitation assay.

Hepa-1 cells were treated with 10⁻⁸ M TCDD for 35 to 45 min. Cross-linking was achieved by adding formaldehyde to a final concentration of 1% at room temperature for 10 min. Cells were washed twice with ice-cold PBS and collected in 1 ml of ice-cold PBS. Cells were pelleted at 700 × g at 4°C and resuspended in 0.3 ml of cell lysis buffer (50 mM Tris-HCl [pH 8.1], 10 mM EDTA, 1% SDS, Roche complete protease inhibitor cocktail) and incubated on ice for 10 min. Cell lysates were sonicated to yield DNA fragments ranging in size from 200 to 900 bp. Samples were centrifuged for 10 min at 4°C. Supernatants were diluted 10-fold to a final solution containing 15 mM Tris-HCl (pH 8.1), 1% Triton X-100, 1 mM EDTA, 150 mM NaCl, 0.1% SDS, and complete protease inhibitor cocktail. The solutions were precleared with 50 μl of 50% slurry protein A-Sepharose containing 2.5 μg of sheared salmon sperm DNA for 2 h at 4°C.

Immunoprecipitation was performed overnight at 4°C with antibodies against the various coactivators; 50 μl of protein A-Sepharose containing 2.5 μg of salmon sperm DNA and 2 mg of bovine serum albumin per ml was then added to the solution and incubated for an additional 1 h. The beads were pelleted and washed in the following buffers: buffer A (20 mM Tris-HCl [pH 8.1], 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), buffer B (20 mM Tris-HCl [pH 8.1], 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), and LiCl-detergent buffer (10 mM Tris-HCl [pH 8.1], 0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA). Finally, they were washed twice with TE (Tris-EDTA) buffer. Immunocomplexes were extracted with 1% SDS-0.1 M NaHCO₃ from the beads.

Cross-linking was reversed by heating the eluted material at 65°C overnight. The eluates were then digested with proteinase K at 45°C for 1 h. The solutions were extracted with phenol-chloroform-isoamyl alcohol. DNA was purified by ethanol precipitation and amplified by PCR over the murine CYP1A1 enhancer region (−1141 to −784 from the transcription initiation start site). Primer sequences were 5′-CTATCTCTTAAACCCCACCCCAA-3′ and 5′-CTAAGTATGGTGGAGGAAAGGGTG-3′.

In vitro GST pulldown assays.

[³⁵S]methionine-labeled full-length AHR, ARNT, or mutant ARNT proteins were synthesized with a T7 TNT-Quick in vitro transcription-translation system (Promega) according to the manufacturer's protocol. GST-SRC-1 fusion proteins were expressed in the bacterial cell line BL21. Cells were lysed in 1× LysS buffer (50 mM Tris [pH 7.8], 1.0 M NaCl, 1.0 mM EDTA, 10% glycerol, β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, and 10 μg each of leupeptin, antipain, and aprotinin per ml) for 30 min on ice. Cellular debris was pelleted by ultracentrifugation at 60,000 × g in an SW41 Ti rotor for 1 h at 2°C. Recombinant proteins were bound to glutathione-Sepharose beads by incubation at 4°C for 3 h on a rotating wheel. Beads were subsequently washed with 0.5× LysS buffer (except that the NaCl concentration was lowered to 300 mM).

Small samples of each recombinant GST-SRC-1 fusion protein were eluted off the beads in 5× SDS sample buffer (62.5 mM Tris [pH 6.8], 10% glycerol, 2% SDS, 700 mM β-mercaptoethanol, and 0.00125% bromophenol blue) and subjected to SDS-PAGE. Protein concentrations were determined by comparison to bovine serum albumin standards as assessed by Coomassie staining. For GST pulldowns, equimolar amounts of each GST-SRC-1 fusion protein (approximately 10 μg) were incubated with 4 μl of in vitro-translated ARNT for 2 h at 37°C. Bead volumes for each sample were standardized with blank glutathione-Sepharose beads, and reaction volumes were brought up to 200 μl with 1× PPI buffer (20 mM HEPES [pH 7.9], 300 mM NaCl, 1 mM EDTA, 4 mM MgCl₂, 1 mM dithiothreitol, 10% glycerol, 0.1% NP-40, 0.5 mM phenylmethylsulfonyl fluoride). Beads were washed six times with PPI buffer, and protein complexes were eluted with SDS sample buffer and boiled for 3 min. Proteins were subjected to SDS-PAGE, and gels were dried and subjected to autoradiography.

In vivo coimmunoprecipitation.

The above results indicate that dioxin-dependent activation of the AHRC and the concomitant increase in transcription of CYP1A1 and potentially other dioxin-responsive genes is modulated by the activity of the NCoA/SRC/p160 family of transcriptional coactivators. In order to address whether a physical interaction occurs between ARNT or AHR and these coactivators, whole-cell extracts from 293T cells transiently cotransfected with both HA-tagged mARNT and mAHR, together with either SRC-1, NCoA-2, or p/CIP, and treated with either TCDD or vehicle were used for in vivo coimmunoprecipitation studies.

Affinity-purified polyclonal rabbit antibodies generated against the influenza virus hemagglutinin tag (HA), and therefore capable of immunoprecipitating HA-tagged mARNT complexes, coimmunoprecipitated exogenous SRC-1 and NCoA-2 but not p/CIP (Fig. ​(Fig.2).2). Cotransfection with the mAHR expression plasmid and treatment with 20 nM TCDD did little to alter the ability of the anti-HA antibody to precipitate either SRC-1 or NCoA-2 (lane 4 in Fig. ​Fig.2),2), suggesting that ARNT can dimerize with AHR and bind SRC-1 at the same time. Anti-HA IgG failed to precipitate coactivator in cells transfected with mAHR and coactivator expression plasmid but not HA-tagged mARNT, demonstrating an absolute requirement for the latter protein (data not shown). Affinity-purified antibodies to AHR coimmunoprecipitated all three coactivators from cells grown in either the presence or absence of TCDD.

Open in a separate window

FIG. 2.

Coimmunoprecipitation of the SRC family of coactivators with AHR and ARNT. 293T cells were transfected with 5 μg each of AHR and HA-tagged ARNT expression vector and 5 μg of either the SRC-1, NCoA-2, or p/CIP expression vector. Twenty hours after transfection, cells were treated with either 20 nM TCDD or vehicle. Two hours later, cells were harvested and total cellular protein was extracted. Extracts were incubated with either affinity-purified anti-HA IgG or preimmune control IgG bound to protein A-Sepharose beads, and complexes were fractionated by SDS-PAGE. Complexes were then transferred to nitrocellulose, and membranes were probed with affinity-purified rabbit anti-SRC-1, anti-NCoA-2, or anti-p/CIP. Detection was performed with an ECL kit. The left lane of each panel contained 200 μg of HEK 293T whole-cell lysate (25% of input).

Interaction of ARNT with coactivators in mammalian cells analyzed by stimulation of an ARNT-dependent reporter gene.

In order to further investigate a potential interaction between ARNT and the coactivators, we employed a GAL4 UAS-thymidine kinase (TK)-luciferase reporter gene (pG5E4T) driven by a fusion of the GAL4 DBD and mARNT and tested whether the coactivators could stimulate the ARNT-dependent reporter gene activity. Reporter gene activity was enhanced by cotransfection of GAL4-DBD-mARNT and increasing amounts of SRC-1 expression plasmid in 293T cells (Fig. ​(Fig.3A).3A). SRC-1 maximally increased ARNT-dependent transcription approximately 6.5-fold, while empty expression vector had no effect on reporter activity. These observations support the notion that SRC-1 can interact with ARNT. The enhancement of luciferase activity is most likely due to the endogenous activation function of SRC-1 (26, 41). Furthermore, NCoA-2 but not p/CIP was capable of enhancing transcription of pG5E4T by GAL4-DBD-ARNT in 293T cells (Fig. ​(Fig.3B),3B), consistent with the coimmunoprecipitation data. The ability of p/CIP to enhance dioxin-dependent transcription (Fig. ​(Fig.1A)1A) is most likely due to p/CIP's ability to interact with mAHR.

Open in a separate window

FIG. 3.

Interaction between ARNT and SRC-1 in mammalian cells as appraised by reporter gene analysis. (A) SRC-1 modulation of ARNT-dependent activation of a GAL4 UAS-TK-driven luciferase reporter. 293T cells were cotransfected with GAL4-DBD-mARNT with either empty vector or increasing amounts of pCMX-SRC-1. (B) A comparison of the coactivator properties of SRC-1, NCoA-2, and p/CIP in the GAL4-DBD-mARNT-responsive luciferase assay.

Fluorescent visualization and colocalization of ARNT/SRC-1 and AHR/SRC-1 complexes.

Coimmunoprecipitation assays are subject to a potential artifactual association of proteins occurring after cellular disruption. Colocalization experiments in mammalian cells in vivo do not suffer from this potential artifact. Such experiments were therefore undertaken in order to further substantiate the interaction of ARNT and AHR with SRC-1. We cotransfected a cDNA plasmid encoding a mARNT-enhanced green fluorescent fusion protein together with either pcDNA3, luciferase expression vector (pCMV-Luc), or pCMX-SRC-1 into the ARNT-deficient Hepa-1 mutant c4 cell line to study how overexpression of SRC-1 might affect the subcellular distribution of mARNT. The c4 cell line was chosen to avoid competition for expressed SRC-1 protein by endogenous ARNT that might potentially confound the interpretation of results. Experiments were performed with equivalent amounts of vector DNA, with pcDNA3 plasmid as a supplement. Representative results of these experiments are presented in Fig. ​Fig.4A.4A.

Open in a separate window

Open in a separate window

FIG. 4.

Intranuclear distribution of ARNT is altered by overexpression of SRC-1. (A) Hepa-1 mutant c4 cells were transiently transfected with pEGFP (0.5 μg) or mARNT-EGFP (0.5 μg) together with 4 μg of either pcDNA3, pLUC-DNA3, or pCMX-SRC-1 and observed for EGFP fluorescence. (B) Hepa-1 mutant c4 cells were transfected with mARNT-EGFP (0.5 μg) and either HA-tagged SRC-1 (4 μg) or untagged SRC-1 (4 μg) expression vector. Fixed cells were incubated with rabbit anti-HA IgG and rhodamine-conjugated goat anti-rabbit IgG. (C) Hepa-1 cells were transiently transfected with the indicated expression vectors, untreated or treated with TCDD for 18 h, as indicated, and observed for EGFP fluorescence.

When pEGFP-C2, encoding green fluorescent protein, was transfected into asynchronous c4 cells, green fluorescent protein distributed throughout the cell, although predominantly in the cytosol. When mARNT fused to enhanced green fluorescent protein was transfected into the c4 cell line, the mARNT-EGFP fusion moiety distributed to the nuclear compartment and appeared to distribute in a homogenous fashion throughout the nucleus. Overexpression of firefly luciferase as a negative control did not alter the expression pattern of mARNT-EGFP. However, expression of SRC-1 caused the redistribution of mARNT-EGFP into discrete foci within the nucleus. This was seen in a large proportion but not all of the transfected cells. The nature of these foci is not clear, although they do not appear to be nucleoli.

In order to verify that the redistribution of mARNT-EGFP was due to the presence of mSRC-1 protein, colocalization experiments were performed. Hepa-1 mutant c4 cells were cotransfected with mARNT-EGFP and either a HA-tagged SRC-1 or untagged SRC-1 expression vector (pCMX-HA-SRC-1 or pCMX-SRC-1). HA was detected with rabbit anti-HA IgG and rhodamine-conjugated goat anti-rabbit IgG. It was found that cells possessing green fluorescent intranuclear foci (indicating the presence of mARNT-EGFP) were also positive for rhodamine fluorescence when they had been cotransfected with the HA-tagged SRC-1 expression vector. Green fluorescence and rhodamine fluorescence (and thus mARNT-EGFP and HA-tagged SRC-1) colocalized to the same foci in these nuclei (Fig. ​(Fig.4B).4B). In cells cotransfected with the untagged SRC-1 cDNA, there was a complete absence of rhodamine fluorescence. These results support the notion that SRC-1 is directly responsible for causing redistribution of mARNT-EGFP into the nuclear bodies.

When AHR-EGFP was cotransfected with the SRC-1 expression plasmid into Hepa-1 cells in the absence of ligand, SRC-1 caused AHR-EGFP to redistribute into discrete foci within the cytosol. (Presumably SRC-1 can be cytosolic, at least when overexpressed [Fig. ​[Fig.4C].)4C].) In the presence of TCDD, AHR-EGFP was almost completely nuclear and was distributed in a homogenous fashion throughout the nuclear compartment. However, as seen with ARNT, AHR redistributed into discrete foci within the nucleus in the presence of TCDD when SRC-1 was overexpressed, but not when luciferase was overexpressed.

These experiments confirm that both ARNT and AHR can interact with SRC-1 in vivo. It should be noted, however, that these and the previously described experiments were all obtained with transfected cDNAs. It is possible that proteins overexpressed in this manner may not behave identically to the corresponding endogenous proteins. The following experiment studied endogenous proteins and does not suffer from this potential artifact.

Requirement of SRC-1/p160 proteins for TCDD-dependent transactivation.

In order to determine the function of SRC-1, NCoA-2, and p/CIP in TCDD-dependent gene activation, we injected Hepa-1 cells with affinity-purified IgGs specific for each coactivator. The reporter gene lacZ was placed under the control of the p36 minimal promoter and four copies of a classic xenobiotic compound-responsive element. Injection of either anti-SRC-1 or anti-p/CIP IgG prevented TCDD from activating the xenobiotic compound-responsive element-dependent transcription unit (Fig. ​(Fig.5A).5A). Since the cocktail injected in each case contained IgG, injected cells could be scored by immunofluorescence after staining with a rhodamine-conjugated secondary antibody (Fig. ​(Fig.5B).5B). Anti-NCoA-2 IgG failed to affect TCDD-dependent transactivation at this reporter (data not shown). This is not surprising, however. The ability of NCoA-2 to interact with several members of the nuclear receptor family of transcription factors is well documented (23, 26, 41, 51, 52), yet this antibody failed to block retinoic acid receptor-mediated activation in a similar assay (51), indicating that this antibody may not be suitable as an inhibitory factor in this assay.

Open in a separate window

FIG. 5.

Role of SRC-1/p160 proteins in AHR/ARNT function as assessed by microinjection of antibodies into the nuclei of single cells. (A) Nuclei of Hepa-1 cells incubated with or without TCDD were microinjected with control rabbit IgG, with or without either anti-SRC-1 or anti-p/CIP affinity-purified IgG and, where indicated, SRC-1, NCoA-2, or p/CIP expression vector. (B) The cells were probed with rhodamine-conjugated goat anti-rabbit IgG to identify injected cells and stained for β-galactosidase activity. Photomicrographs of rhodamine-stained cells and the corresponding light micrographs after 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) staining are shown.

The inhibitory effect of anti-SRC-1 and anti-p/CIP IgG could be reversed by coinjection of the SRC-1 and p/CIP expression vector, respectively. Unlike retinoic acid receptor-responsive reporter gene activity, rescue from the effects of anti-p/CIP IgG did not require coinjection of CBP expression vector (51), suggesting that the AHRC recruits a distinct p/CIP regulatory complex rather than that of the retinoic acid receptor. Coinjection of either the NCoA-2 or p/CIP expression vectors could not restore the transcriptional activity inhibited by anti-SRC-1. This possibly reflects the inability of NCoA-2 and p/CIP to compete for large macromolecular complexes sequestered by the anti-SRC-1 IgG because of lack of specificity for anti-SRC-1 IgG or may indicate a distinct role for each coactivator in ARHC-dependent transcription, but this remains to be determined.

CYP1A1 enhancer occupation by the SRC-1/p160 proteins.

We performed chromatin immunoprecipitation assays to test if SRC-1, NCoA-2, and p/CIP could be recruited to the CYP1A1 enhancer in a TCDD-dependent fashion (Fig. ​(Fig.6).6). Formaldehyde-fixed protein-DNA immunocomplexes precipitated with affinity-purified antibodies raised against SRC-1, NCoA-2, or p/CIP were denatured and subjected to PCR with oligonucleotides flanking the CYP1A1 enhancer. In the absence of TCDD, the CYP1A1 enhancer DNA could not be amplified. However, in the presence of TCDD, the CYP1A1 enhancer region could be amplified after immunoprecipitation with all three coactivator antibodies, demonstrating the presence of the coactivators at the enhancer. Regions flanking the CYP1A1 enhancer could not be amplified by PCR, indicating that these proteins were associated with the CYP1A1 enhancer region and not the proximal promoter (data not shown). These data unambiguously demonstrate TCDD-dependent recruitment of the endogenous SRC-1, NCoA-2, and p/CIP coactivator proteins to the CYP1A1 enhancer in vivo. Differences in the intensities of the SRC-1-, NCoA-2-, and p/CIP-specific bands may reflect the avidity of the antibodies for their respective proteins and do not necessarily indicate a greater affinity of one coactivator for the AHRC complex over another.

Open in a separate window

FIG. 6.

Chromatin immunoprecipitation of the CYP1A1 enhancer region. PCR amplification of the CYP1A1 enhancer by affinity-purified antibodies raised against SRC-1, NCoA-2, and p/CIP. Total input DNA for each PCR can be visualized in the first two lanes of each panel. Precipitation (I.P.) reactions performed in the absence and presence of TCDD are shown in lanes 3 and 4 of each panel, respectively. XRE's, xenobiotic compound-responsive elements.

Determination of ARNT and AHR interaction domains within SRC-1.

In order to determine the ARNT and AHR interaction domains within SRC-1, B42 activation domain-SRC-1 and LexA DNA-binding domain (LexA-DBD)-ARNT/AHR fusion constructs were used in the yeast two-hybrid system to test for protein-protein interactions. Attempts to clearly define the ARNT interaction domain within the SRC-1 protein with the yeast two-hybrid β-galactosidase method were hampered by the ability of ARNT's transcriptional activation domain to confer high-level β-galactosidase activity in cells cotransformed with only empty prey vector. Deletion of sequences encoding amino acids 688 to 791 of the mARNT protein (comprising its carboxy-terminal TAD) in the bait vector abolished the high constitutive activity observed with the full-length protein.

We therefore employed a bait construct (ARNTΔQ) encoding amino acids 1 to 687 of the mARNT protein. Constructs were transformed into the yeast strain L40C and tested for their ability to activate the β-galactosidase reporter gene. Among the SRC-1 deletion mutants tested, the one encoding amino acids 763 to 1100 conferred the highest activity on the lacZ gene, as assayed by β-galactosidase activity (250-fold over basal levels) when cotransformed with ARNTΔQ (Fig. ​(Fig.7A).7A). This region encompasses the CBP interaction domain on SRC-1. Deletion of amino acids 763 to 895 significantly decreased activity in this assay, as evidenced by the reduced interaction of SRC-1_896-1200 with ARNTΔQ compared with SRC-1_763-1100. Deletion of amino acids 1034 to 1100 (represented by SRC-1_763-1033) from SRC-1_763-1100 resulted in only a slight decrease in β-galactosidase activity.

Open in a separate window

FIG. 7.

Identification of interaction domains on SRC-1 for ARNT with yeast two-hybrid and GST pulldown assays. (A) SRC-1 deletion mutants were fused to the B42 activation domain in pJG4.5 and transformed into the yeast strain L40C along with ARNTΔQ fused to the LexA DNA-binding domain and assayed for β-galactosidase activity. Results are expressed as increases (n-fold) over mean values obtained for the empty prey vector. The interaction domains previously identified within SRC-1 for the transcriptional coactivator CBP and for several nuclear hormone receptors (10) are indicated. (B) SRC-1 mutants were fused to glutathione S-transferase and expressed in Escherichia coli. Expressed fusion proteins from bacterial cell lysates were put on glutathione-agarose beads. Approximately 10 μg of protein coupled to beads was incubated with in vitro-translated [³⁵S]methionine-labeled ARNT or RIN1, and absorbed proteins were subjected to SDS-PAGE. The two left lanes contained 1 μl of in vitro-translated RIN1 and 1 μl of in vitro-translated mARNT (25% input) for comparison.

We sought to confirm the location of the ARNT interaction domain with an in vitro GST pulldown assay using full-length ARNT together with eight mutant GST-SRC-1 fusion constructs, each encoding a discrete portion of the protein and together encompassing its entire length. The results of these experiments are presented in Fig. ​Fig.7B.7B. All GST-SRC-1 fusion moieties were quantified on SDS-PAGE with bovine serum albumin standards so that equimolar amounts of each fusion were used in this assay. RIN1, a protein of the same approximate size as mARNT and not likely to interact with SRC-1 in vivo could not be pulled down by any GST-SRC-1 fusion protein in this assay. Firefly luciferase was used as another negative control for this assay. None of the SRC-1 mutant proteins pulled down ³⁵S-labeled luciferase (data not shown). GST-SRC-1_763-1100 and GST-SRC-1_763-1033 were equally effective in precipitating in vitro-translated full-length ARNT, confirming that GST-SRC-1_763-1033 contains the minimal ARNT interaction domain.

The interaction between AHR and SRC-1 was also studied by yeast two-hybrid and GST pulldown analyses. Unlike ARNT, full-length AHR was not constitutively active in yeast cells in the absence of the AHR ligand β-NF. The B42-SRC-1 fusion protein, encoding amino acids 896 to 1200 of the SRC-1 protein, was capable of interacting with AHR in yeast cells in a ligand-dependent fashion, although the severalfold activation of the lacZ reporter in yeast cells by SRC-1 was only approximately 4-fold, compared to approximately 250-fold for the ARNT-SRC-1 interaction. This may be due to AHR's TAD, the known interaction site for SRC-1 (29), being at least partially masked in yeast cells. The same region of SRC-1 was capable of interacting with AHR in the GST interaction assay, but in this case the interaction was ligand independent (Fig. ​(Fig.8A8A and B). SRC-1_763-1100, which interacted maximally with ARNT, was completely incapable of recruiting AHR in both assays.

Open in a separate window

Open in a separate window

FIG. 8.

Identification of AHR interaction domains within SRC-1. (A) Yeast two-hybrid analysis of the interaction between AHR and various deletion mutants of SRC-1. The experiments were performed in the presence or absence of 1 mM β-NF, as indicated. (B) GST pulldown assay of [³⁵S]methionine-labeled AHR and GST-SRC-1 deletion mutant fusion proteins.

The results from the yeast liquid culture and GST pulldown assays are congruent and indicate that the ARNT interaction domain is encompassed within amino acids 763 to 1033 of mSRC-1 and that the AHR interaction domain is encompassed within amino acids 896 to 1200. GST control protein and the fragments encoding regions flanking but not overlapping the above ARNT and AHR interaction domains on SRC-1 were ineffective in precipitating ARNT and AHR, respectively. Therefore, our data indicate that the ARNT and AHR interaction domains within SRC-1 may overlap but are distinct. SRC-1_1-360 contains regions of homology with ARNT and AHR's bHLH and PAS domains, which are known dimerization regions for other bHLH-PAS proteins that interact with ARNT, but surprisingly this fragment was incapable of interacting with ARNT or AHR, as determined by GST pulldown and yeast two-hybrid assays. Recently, it has been reported that the transcriptional coactivator CBP/p300 is recruited by ARNT for dioxin-dependent transactivation, and the respective interaction domains have been identified (28). Interestingly, GST-CBP_1892-2441, encoding the region of CBP that interacts with SRC-1, was not capable of competing with in vitro-translated ARNT for binding to GST-SRC-1_763-1100 (data not shown). These data, taken together, suggest that ARNT's interaction with these two coactivators is not mutually exclusive.

This work was supported by Public Health Service grant CA-28868 from the National Cancer Institute. T.V.B. and S.W. were supported in part by training fellowships from the University of California Toxic Substances Research and Teaching Program's Lead Campus Program.

We gratefully acknowledge the help of Irine Prastio and Rose Estrada in the preparation of the manuscript and Hongdiem Nguyen for excellent technical assistance.