Heterodimeric Interactions between Chicken Ovalbumin Upstream Promoter-Transcription Factor Family Members ARP1 and Ear2

Plasmids and Constructs

The bait vectors (pBTM116 and pBL1) for the yeast two-hybrid screen and the yeast reporter strains L40 (26) and PL3 (27) were all generous gifts from Drs. R. Losson and P. Chambon (IGBMC, Illkirch, France). Human ARP1 (ARP1, Ref. 28) was a kind gift from Dr. H. Nakshatri, and NR1 was a kind gift from Dr. S. Nakanishi. A fragment corresponding to the hinge and putative ligand binding domain of ARP1 (regions D and E, amino acids 144–414) was amplified by PCR and cloned into both pBTM116 and pBL1 (26), encoding LexA DBD-ARP1 DE and ER DBD-ARP1 DE fusion proteins, respectively. Receptor bait constructs for mouse RXRα (amino acids 132–467) and human RARγ (amino acids 90–454) have been described previously (29). An unrelated bait corresponding to the carboxyl tail (amino acids 835–938) of the NR1 subunit of the N-methyl-D-aspartate receptor (30) was similarly prepared for these studies. All fragments amplified by PCR were verified by sequence analysis.

A mammalian expression vector encoding an ER DBD/ARP1 DE fusion protein was constructed by inserting the EcoRI fragment of pBL1-ARP1 DE into the EcoRI site of pTL1 (31). A mammalian expression vector encoding the DE region of mouse Ear2 (amino acids 122–390) fused to the transcriptional activation domain of GAL4 (GAL4 AD; amino acids 768–881) was prepared by insertion of the corresponding Ear2 fragment into pACT2 (CLONTECH, Palo Alto, CA), yielding pACT2-Ear2 DE. This plasmid was then digested with HindIII, excising the GAL4 AD-Ear2 DE fragment, which was subsequently cloned into the eukaryotic expression vector, pTL1. An amino-terminal truncation mutant of ARP1, mycARP1ΔAB, with a myc epitope tag at the amino terminus was prepared by PCR amplification of an appropriate fragment and insertion into pTL1. Full-length Ear2, fused to an HA epitope tag and the GAL 4 AD, was excised from the library plasmid (pACT2) by digestion with HindIII, and the resulting fragment was ligated into pTL1 creating HA-Ear2. The latter two constructs were prepared for use in DNA binding experiments designed to distinguish between heterodimeric complexes of intermediate electrophoretic mobility and homodimeric complexes composed of either receptor. The ER-responsive CAT reporter, 17-mer-ERE-globin-CAT, has been described previously (32).

GST fusion proteins were prepared for ARP1 DE (GST-ARP1 DE) and Ear2 DE (GST-Ear2 DE) by PCR amplification of the corresponding fragments and subcloning into pGEX-2T (Amersham Pharmacia Biotech, Uppsala, Sweden) that had been previously digested with BamHI and EcoRI. The integrity of all constructs was verified by sequence analysis.

Yeast Two-hybrid Screen

Saccharomyces cerevisiae L40 reporter strain expressing ARP1 (D and E regions) was transformed with a mouse brain cDNA library (CLONTECH) fused to the GAL4 AD on a leucine-selectable vector, pACT2. The screen for ARP1-interacting clones was conducted in synthetic medium lacking tryptophan, leucine, and histidine and supplemented with 20 mM 3-aminotriazole. The plasmids from positive clones containing the brain cDNAs were rescued, shuttled in Escherichia coli, and sequenced using the dideoxynucleotide dye terminator method on an Applied Biosystems, Inc., model 373 or 377 sequencer (Applied Biosystems, Inc., Foster City, CA) at the Central Services Laboratory of the Oregon State University Center for Gene Research and Biotechnology.

β-Galactosidase Assays

Filter assays were utilized to confirm the positive clones. For quantitative purposes the positive clones were grown in synthetic medium to an A₆₀₀ of approximately 2 units, and β-galactosidase activity was assayed as described by Kippert (33), and modified by Avram and Bakalinsky (34). Ligand-dependent assays were carried out by inclusion of 1 μM 9-cis-RA or identical amounts of vehicle in the culture medium.

Protein-Protein Interaction Assays

GST pull-down experiments were conducted as described previously (29, 35).

Electrophoretic Mobility Shift Assays

EMSAs were conducted essentially as described previously (31). ARP1ΔAB and HA-Ear2 were expressed by in vitro transcription/translation in rabbit reticulocyte lysates (Promega). In each case the receptors used for gel retardation assays were translated in parallel in the presence of [³⁵S]methionine for the purpose of quantification. The DR1(G) response element upper strand had the following sequence (canonical half-sites are bold and underlined): 5′-TCGAGGGTAGAGGTCAGAGGTCACTCG-3′; DR2(G) through DR5(G) were synthesized with identical flanking and repeated hexanucleotide sequence but with the following inter-repeat spacers (5′ to 3′): GA (DR2), CGA (DR3), CGAA (DR4), and CCGAA (DR5). The DR(T) series of oligonucleotides were identical to the DR(G) series except that the repeated hexanucleotide had the sequence AGTTCA.

Cell Culture and Transfections

Human embryonic kidney (HEK) 293 cells (ATCC CRL 1573) were cultured in minimum essential medium with Earle's salts (Sigma) supplemented with 10% fetal bovine serum (Summit, Boulder, CO) and 4 mM glutamine (Life Technologies, Inc.). Cells were grown to 50–60% confluence and transiently transfected using the calcium phosphate method. Each 100-mm plate was cotransfected with 1 μg of expression vector encoding the ER DBD or ER DBD/ARP1 DE, 1 μg of GAL4 AD, or GAL4 AD/Ear2 DE, 2 μg of the 17-mer-ERE-Globin-CAT reporter construct, and 10 ng of pCMV-SPORT-βgal (Life Technologies, Inc.) encoding β-galactosidase which was used to normalize for transfection efficiency. Cells were harvested 48 h after transfection, and extracts were prepared using standard techniques. β-Galactosidase activity in cellular extracts was quantified using a colorimetric assay, and CAT activity was determined by thin layer chromatography.

Reverse Transcription-Polymerase Chain Reaction

First strand cDNA was synthesized using 1 μg of RNA (Ambion, Austin, TX) and 100 ng of random hexamer primers (Promega) in a buffer containing 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10 mM dithiothreitol, 100 μM concentrations of each dNTP, and 200 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) in a total volume of 25 μl, and the reactions were carried out for 30 min at 37 °C. One μl of each reverse transcription reaction was subjected to PCR amplification using ARP1-, Ear2-, and 36B4 (36)-specific primers. ARP1 and Ear2 amplifications were carried out for 25 cycles, whereas amplification of 36B4 was conducted for 19 cycles. Amplification of 36B4 was used as a control for the quantity of cDNA present in each sample as well in normalization of gel loading and Southern blotting. Note that, in general, one cycle consisted of the following steps: 94 °C × 30 s, 50 °C for 45 s, 72 °C for 60 s; however, the annealing temperature was varied for each set of primers to optimize amplification. Amplification products were separated on a 2% agarose gel and then transferred to ZetaProbe membranes (Bio-Rad). The blots were probed with specific, end-labeled oligonucleotides corresponding to internal sequence in the PCR products. RT-PCR analyses of ARP1 and Ear2 expression in F9 and P19 embryonal carcinoma cells treated in monolayers with either vehicle or trans-retinoic acid for 24 h were carried out as described above.

Ear2 Interacts with ARP1 in a Yeast Two-hybrid System

A fragment encoding the ARP1 hinge region and putative ligand binding domain was used as a bait in a yeast two-hybrid screen to identify proteins expressed in brain that interact with ARP1, which is highly expressed in brain (Refs. 18, 24, 37, and this report). A protein found to interact with ARP1 was Ear2, another member of steroid hormone receptor superfamily, which is closely related to COUP-TF subfamily, that includes COUP-TFI and ARP1 (Fig. 1).

The Ear2 clone isolated in our screen contains the entire open reading frame identified by Jonk and co-workers (18). In addition, the present clone contains 183 base pairs upstream of the putative initiator methionine previously described (18). However, as there is no in frame stop codon(s) in this upstream sequence, it is possible that the actual Ear2 protein is extended in the amino terminus beyond the previously identified initiator methionine (18).

To evaluate the specificity of ARP1-Ear2 interaction, we examined interactions of Ear2 with RAR, RXR, and an unrelated bait, the carboxyl terminus of the NR1 subunit of the N-methyl-D-aspartate receptor (30). The interaction of Ear2 with ARP1 was specific inasmuch as Ear2 did not interact with RXRα or RARα in the presence or absence of 9-cis-retinoic acid (Fig. 2). Similarly, Ear2 did not interact with the carboxyl terminus of NR1 (Fig. 2).

Ear2 and ARP1 Interact in Vitro

A qualitative, in vitro protein-protein interaction study was conducted to confirm the ARP1-Ear2 interaction observed in yeast and to investigate the ability of each protein to self-associate. The hinge and putative LDB regions of each protein were fused to glutathione S-transferase (GST), creating GST-ARP1 DE and GST-Ear2 DE, and both proteins were expressed in bacteria for use in standard GST pull-down experiments (29, 35). [³⁵S]Met-labeled Ear2 interacted with GST-ARP1 DE (Fig. 3A, lane 3) confirming the interaction observed in yeast. [³⁵S]Met-labeled Ear2 was also found to interact with itself (GST-Ear2 DE, lane 4) with efficiency nearly identical to that with which it interacted with GST-ARP1 DE (lane 3) suggesting that Ear2 may form heterodimeric and homodimeric complexes with equal facility. Similarly, [³⁵S]Met-labeled ARP1ΔAB interacted with both GST-Ear2 DE and -ARP1 DE fusion proteins (Fig. 3B, lanes 3 and 4, respectively) but not with glutathione charged with GST alone (lane 2). These in vitro findings corroborate results obtained in yeast (Fig. 2) and also suggest that ARP1 and Ear2 participate in heterodimeric interactions in solution with an apparent efficiency comparable to that of the homodimerization involving either receptor.

ARP1 and Ear2 Form Heterodimers on Directly Repeated Response Elements

COUP-TFI, ARP1, and Ear2 form homodimers in solution and bind promiscuously to direct repeats (DR) of the canonical half-site, AGGTCA, with varied inter-repeat spacing. However, all of these receptors exhibit a degree of selectivity for DRs spaced by a single base pair (DR1) (1, 3). In addition, COUP-TFI and ARP1 bind as a heterodimeric complex to DR elements composed of varied spacing (3). Based on our observation that ARP1 and Ear2 form heterodimers in yeast (Fig. 2) and in solution (Fig. 3), we conducted electrophoretic mobility shift assays (EMSAs) to determine if ARP1 and Ear2 bind to directly repeated response elements as a heterodimeric complex. mycARP1ΔAB homodimeric complexes bound in a protein concentration-dependent manner to the DR1 probe (Fig. 4, lanes 1–3; complex C5). HA-Ear2 formed two complexes, C1 and C2, that exhibited differential electrophoretic mobility on this probe (Fig. 4, lanes 4 and 5). The two species of HA-Ear2, which bound to this probe in a protein concentration-dependent manner, most likely arose from correct and internal initiation of HA-Ear2 translation in rabbit reticulocyte lysates (data not shown). At comparable amounts of protein, HA-Ear2 (C1 and C2) clearly bound to the DR1 probe less efficiently than mycARP1ΔAB (C5; compare lanes 1–3 to 4–6 of Fig. 4). However, incubation of a fixed amount of mycARP1ΔAB with increasing amounts of HA-Ear2 resulted in the complete titration of the mycARP1ΔAB homodimeric complex (C5) into mycARP1ΔAB·HA-Ear2 heterodimeric complexes, C3 and C4 (Fig. 4, lanes 7–9). When excessive amounts of HA-Ear2 were used, the appearance of HA-Ear2 homodimeric complexes (C1 and C2) were also evident (Fig. 4, lanes 8–9). Similarly, titration of a fixed amount of HA-Ear2 with increasing amounts of mycARP1ΔAB quantitatively shifted HA-Ear2 homodimeric complexes (C1 and C2) into mycARP1ΔAB·HA-Ear2 heterodimeric complexes (C3 and C4; Fig. 4, lanes 10–12). The homodimeric mycARP1ΔAB complex (C5) was apparent when this protein was used in excess (Fig. 4, lanes 11 and 12). Complexes C1/C2 and C5 were supershifted with anti-HA and anti-myc antibodies, respectively, whereas both antibodies supershifted complexes C3 and C4, confirming the identity of each complex (data not shown).

ARP1·Ear2 Heterodimeric Complexes Bind Directly Repeated Response Elements with Specificity Differing from Homodimers of Either Receptor

We next sought to determine if the capacity of ARP1 and Ear2 to bind directly repeated response elements as homodimeric and/or heterodimeric complexes was a function of the length of the inter-repeat spacer. mycARP1ΔAB homodimers bound to probes with spacing ranging from 1 to 5 base pairs (complex C5; Fig. 5A, lanes 1, 4, 7, 10, and 13) with somewhat varied efficiency (DR2 ≥ DR1 > DR5 > DR3 > DR4). Conversely, HA-Ear2 did not appear to bind any of these probes with the exception of DR1, to which HA-Ear2 homodimers bound weakly at the relatively low amount of HA-Ear2 protein used in these experiments (Fig. 5B, lanes 2, 5, 8, 11, and 14). mycARP1ΔAB·HA-Ear2 heterodimeric complexes (C3 and C4) bound to probes of all spacings with a distinct preference for DR1 and DR2 (Fig. 5A, lanes 3, 6, 9, 12, and 15). Thus, mycARP1ΔAB×HA-Ear2 heterodimeric complexes exhibited a DNA binding specificity (DR1 ≥ DR2 > DR4 > DR5 > DR3) that was clearly distinct from that of HA-Ear2 homodimers (which bound weakly to DR1 only) and subtly distinct from mycARP1ΔAB homodimeric complexes (see above).

Methylation interference studies have implicated a role for the guanines present in each half-site of a DR1-type response element in the specific interaction of COUP-TFI with DNA (38). To address the potential role of the second guanine (G²) in each AGGTCA half-site in DNA recognition by ARP1·ARP1, ARP1·Ear2, and Ear2·Ear2 complexes, EMSAs were carried out using a series of probes in which G² in each half-site was mutated to thymidine (see Fig. 5B). This G² → T mutation had a dramatic effect on the efficiency of both mycARP1ΔAB homodimer and mycARP1ΔAB·HA-Ear2 heterodimer binding to probes of all spacing (Fig. 5B). However, mycARP1ΔAB homodimers did bind to the “T” series probe with a single base pair spacer, DR1(T) (Fig. 5B, lane 1), and with lower efficiency to DR5(T) (lane 13) and DR3(T) (lane 7). In contrast, mycARP1ΔAB·HA-Ear2 heterodimer binding was not evident on any of the “T” series probes with the possible exception of DR1(T) (Fig. 5B, lane 3). However, HA-Ear2 appeared to inhibit mycARP1ΔAB homodimer binding to DR1(T), DR3(T), and DR5(T) (Fig. 5B, lanes 3, 9, and 15, respectively), the latter of which is identical to the RARE in the RARβ promoter (39). These findings suggest that mycARP1ΔAB and HA-Ear2 may form a heteromeric complex in solution that is not competent for DNA binding to the T series of response elements. Consistent with this hypothesis, mycARP1ΔAB and HA-Ear2 interacted in DNA-binding independent, GST pull-down experiments (Fig. 3, A and B).

ARP1 and Ear2 Interact in Mammalian Cells

Although ARP1 and Ear2 interact in yeast and in vitro, both in solution and on various directly repeated response elements, a physiologically relevant interaction between these two proteins would only occur in cells in which both proteins were expressed. Thus, cotransfection experiments were carried out to determine if the two proteins are capable of interacting in cells using a mammalian two-hybrid system. HEK 293 cells were cotransfected with a plasmid (ER DBD-ARP1 DE) encoding ARP1 DE fused to ER DBD together with a second plasmid (Ear2 DE-GAL4 AD) encoding Ear2 DE fused to GAL4 AD. The previously described 17-mer-ERE-globin-CAT (32) was used as a reporter construct in these studies. ER DBD alone (Fig. 6, lane 4) or when cotransfected with the GAL4 AD (lane 5) failed to activate the reporter construct. Similarly, Ear2 DE-GAL4 AD did not activate the reporter plasmid when cotransfected with the ER DBD (Fig. 6, lane 6). However, cotransfection of ER DBD-ARP1 DE together with Ear2 DE-GAL4 AD resulted in strong induction of the reporter suggesting that Ear2 and ARP1 are capable of interaction in mammalian cells.

Ear2 and ARP1 Expression Patterns Overlap in Several Mouse Tissues

Based on the observation that ARP1 and Ear2 interact in yeast, in solution, on DNA, and in mammalian cells, we next determined if the corresponding genes, Ear2 and Arp1, may exhibit overlapping patterns of expression in several mouse tissues that would facilitate a biologically relevant interaction between the two proteins.

Jonk and co-workers (18) have previously observed, by in situ hybridization, high level Arp1 expression in embryonal brain, lung, and kidney, whereas transcripts derived from the Ear2 gene were found to be present at lower levels and in several tissues in the developing embryo (8.5–14.5 d.p.c.). We have examined Arp1 and Ear2 expression in several tissues of the adult mouse by RT-PCR. The expression patterns of both genes were found to overlap precisely with the highest level of expression in brain, lung, kidney, and in the embryo (10–12 d.p.c.; Fig. 7A). Neither gene appeared to be expressed in thymus, spleen, and testis (Fig. 7A). Although these studies demonstrate that Arp1 and Ear2 genes are expressed in similar tissues, one cannot conclude from these data that the two orphan receptors are expressed in the same cell type(s) that would obviously be required for a physiologically relevant interaction to occur. To address the possibility that Arp1 and Ear2 may be coexpressed in a single cell type, RT-PCR analyses were conducted using RNA derived from two embryonal carcinoma cell lines, F9 and P19. These analyses revealed that Ear2 was constitutively expressed in both P19 and F9 cells and was unaffected by treatment of either cell line with all-trans-retinoic acid (RA; Fig. 7B), consistent with the finding of Jonk and co-workers (18). Arp1 was also found to be constitutively expressed in both cells lines and did not appear to be induced by treatment with RA (Fig. 7C), in contrast to previous studies (18). However, the treatment protocol employed in the current studies involved exposure to RA for only 24 h, whereas Jonk and co-workers (18) observed maximal induction of Arp1 in P19 cells by Northern analysis after a 72-h treatment with RA.