©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Constitutive Function of the Basic Helix-Loop-Helix/PAS Factor Arnt
REGULATION OF TARGET PROMOTERS VIA THE E BOX MOTIF (*)

Camilla Antonsson (1), Velmurugesan Arulampalam (2)(§), Murray L. Whitelaw (1) (2), Sven Pettersson (2), Lorenz Poellinger (1)(¶)

From the (1) Departments of Medical Nutrition and (2) Bioscience, Karolinska Institute, Novum, S-141 86 Huddinge, Sweden

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Arnt is a nuclear basic helix-loop-helix (bHLH) transcription factor that, contiguous with the bHLH motif, contains a region of homology (PAS) with the Drosophila factors Per and Sim. Arnt dimerizes in a ligand-dependent manner with the bHLH dioxin receptor, a process that enables the dioxin-(2,3,7,8-tetrachlorodibenzo-p-dioxin)-activated Arnt-dioxin receptor complex to recognize dioxin response elements of target promoters. In the absence of dioxin, Arnt does not bind to this target sequence motif. The constitutive function of Arnt is presently not understood. Here we demonstrate that Arnt constitutively bound the E box motif CACGTG that is also recognized by a number of distinct bHLH factors, including USF and Max. Importantly, amino acids that have been identified to be critical for E box recognition by Max and USF are conserved in Arnt. Consistent with these observations, full-length Arnt, but not an Arnt deletion mutant lacking its potent C-terminal transactivation domain, constitutively activated CACGTG E box-driven reporter genes in vivo. These results indicate a role of Arnt in regulation of a network of target genes that is distinct from that regulated by the Arnt-dioxin receptor complex in dioxin-stimulated cells.


INTRODUCTION

Mammalian bHLH() factors are characterized by a highly conserved DNA binding and dimerization domain composed of a basic (b) region, followed by the helix-loop-helix (HLH) dimerization motif. In the case of the related group of bHLH/Zip proteins, the bHLH motif is contiguous with a second dimerization surface, the leucine zipper (Zip) motif (for a recent review see Littlewood and Evan(1994)). These classes of transcription factors are often involved in regulation of cell type differentiation and proliferation (for reviews see Jan and Jan(1993); Kadesh, 1993; Weintraub, 1993; Dorschkind, 1994). Most bHLH and bHLH/Zip factors bind as dimers to the consensus hexamer motif CANNTG known as the E box, where the central two nucleotides commonly are either GC or CG (reviewed by Littlewood and Evan(1994)). Thus, bHLH factors can be divided into two classes, depending on their E box target sequence: class A proteins recognize CAGCTG, whereas class B proteins recognize CACGTG (Dang et al., 1992). Structure determinations of homodimeric complexes of the DNA binding domains of the class B bHLH/Zip factors Max and USF (Ferré d'Amaré et al., 1993, 1994), and the class A bHLH factors MyoD and E47 (Ellenberger et al., 1994; Ma et al., 1994), bound to their cognate DNA sequences, have demonstrated the basic regions to recognize the E box and the HLH motifs to form an overall conserved parallel four helix bundle.

Arnt is a bHLH factor (Hoffman et al., 1991) that, juxtaposed to the bHLH motif, contains a region (PAS) which is conserved between Arnt, the dioxin receptor, and the Drosophila developmental regulator Sim and the Drosophila circadian rhythm regulatory protein Per (reviewed by Takahashi(1992)). Arnt dimerizes in a ligand-dependent manner with the structurally related bHLH/PAS dioxin receptor, a process which enables both proteins to recognize and regulate xenobiotic or dioxin response elements (XREs) of target promoters (reviewed by Swanson and Bradfield(1993); Hankinson, 1994; Whitlock, 1994). The XRE core motif (TNGCGTG; Lusska et al., 1993) bears little resemblance to consensus E box motifs, and, notably, it is not recognized by either Arnt or the dioxin receptor individually (Dolwick et al., 1993b; Whitelaw et al., 1993; Matsushita et al., 1993; Antonsson et al., 1995). Moreover, the dioxin-activated Arnt-dioxin receptor heterodimer does not bind the CACGTG E box motif from the adenovirus major late promoter (Mason et al., 1994).

Subcellular fractionation and immunohistochemical studies suggest that the dioxin receptor, in analogy to certain steroid hormone receptors, resides in the cytoplasm of untreated cells and translocates into the nucleus upon exposure to ligand. In contrast, Arnt appears to be a constitutively nuclear protein (Pollenz et al., 1994; Hord and Perdew, 1994). The function of Arnt in dioxin-non-stimulated cells remains unclear. In the present report we demonstrate that Arnt constitutively interacted in vitro with the E box motif CACGTG that is present in, for instance, the adenovirus major late promoter and in certain regulatory regions of immunoglobulin heavy-chain enhancers. In agreement with these observations, full-length Arnt, but not an Arnt deletion mutant lacking its potent C-terminal transactivation domain, constitutively activated CACGTG E box-driven reporter genes in vivo.


MATERIALS AND METHODS

Recombinant Plasmids

Plasmids pCMVArnt and pCMVArnt603 have been described earlier (Mason et al., 1994; Whitelaw et al., 1994). The reporter gene pML-EB-T81-Luc was constructed by subcloning an oligonucleotide spanning the E box motif of the adenovirus major late promoter (from positions -72 to -46; Sawadogo and Roeder, 1985) into HindIII-SacI-digested pT81-Luc (Nordeen, 1988). The 3xµE3/-34SV -globin reporter has been described previously (Grant et al., 1992). pLTR-USF was a kind gift from Dr. K. Meyer (CRC Welcome Research Institute, Cambridge, United Kingdom) and the Pax5 expression vector (phBSAP-1s; Adams et al., 1992) was generously provided by Dr. M. Busslinger (Institute for Molecular Pathology, Vienna, Austria).

Cells and Transient Transfection Experiments

CHO cells were grown in Ham's F-12 medium supplemented with 10% fetal calf serum, whereas COS cells were grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum. In addition, all media were supplemented with 100 units of penicillin and 100 µg of streptomycin (Life Technologies, Inc.)/ml. CHO cells (60-mm dishes) were transiently transfected with 2 µg of reporter plasmid and 1.5 µg of expression vector in 30 µl of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (Boehringer Mannheim) according to the manufacturer's instructions. Luciferase assays were performed as described (Sambrook et al., 1989) using luciferin (Promega) as substrate. COS cells growing in log phase were transfected using calcium phosphate co-precipitation (Graham and van der Eb, 1973) with 25 µg of the reporter plasmid p3xµE3/-34SV -globin and indicated amounts of the different expression vectors. For each transfection, 5 µg of a reference plasmid, pHP2 (Weston, 1988), containing the human 2-globin gene driven by the SV40 enhancer, was included to serve as an internal control. Total cytoplasmic RNA was prepared from transfected cells and correctly initiated globin transcripts were assayed by ribonuclease protection as described previously (Pettersson et al., 1990).

Recombinant Vaccinia Virus Expression of Arnt and the Dioxin Receptor

Vaccinia virus expression vectors containing the mouse dioxin receptor and human Arnt were constructed by subcloning cDNA from pDR/ATG/BS (McGuire et al., 1994) and pGEM/Arnt (Whitelaw et al., 1993) into vectors pgpt--6 and pgpt-ATA-18 (Simmen et al., 1991), respectively. Infection of 3-cm dishes of RK13 cells (70% confluent) with wild type vaccinia virus, followed by transfections of recombinant vectors (2 µg), were performed according to established protocols (Ausubel et al., 1994). After 60-h incubation at 37 °C, the cells were lysed by two freeze/thaw cycles and sonication. Lysates (20% of total) were added to 9-cm dishes of confluent RK13 cells. After 90 min at 37 °C, the medium was replaced with 10 ml minimal essential medium containing mycophenolic acid (25 µl), xanthine (250 µl), and hypoxanthine (15 µl) from 10 mg/ml stock solutions. After 72 h the cells were lysed as before, and 10% of the lysate was used to infect fresh 9-cm dishes of RK13 cells in selection media. Selected plaques were isolated, lysed, and used to infect fresh cell cultures, with three plaque purifications repeated to ensure isolation of recombinant virus. For expression of dioxin receptor and Arnt proteins, RK13 cells were infected for 2 h with recombinant lysates, followed by incubation in fresh media for 15 h. Cells were then washed in phosphate-buffered saline, and hypotonic cytosolic extracts obtained (Whitelaw et al., 1993).

In Vitro DNA Binding Assays

E box binding activity of vaccinia virus expressed Arnt was monitored by gel mobility shift assay as described (Hapgood et al., 1989) using a double-stranded oligonucleotide containing the USF recognition sequence of the adenovirus major late promoter (Gregor et al., 1990). Vaccinia virus-expressed dioxin receptor was activated in the absence or presence of Arnt by incubation for 2 h at 25 °C with 5 nM dioxin (Cambridge Isotope Laboratories), and dioxin receptor-dependent DNA binding activities were monitored by gel mobility shift assay (Hapgood et al., 1989) using a double-stranded XRE oligonucleotide (Cuthill et al., 1991). DNA binding reactions were assembled in the presence of 2 µg of nonspecific poly(dI-dC) in a total volume ranging between 20 and 30 µl, incubated for 30 min at 25 °C, and protein-DNA complexes were resolved on 4% (acrylamide/bisacrylamide ratio of 29:1) native polyacrylamide gels at 30 mA and 25 °C using a Tris/glycine EDTA buffer (Hapgood et al., 1989). In DNA competition experiments a double-stranded oligonucleotide µE5 (5`-AGCTTGAACCTGCAGCTGCAGGTGGGGGAGA-3`) was used a class A E box motif. In indicated DNA binding experiments, polyclonal antibodies against the dioxin receptor (Whitelaw et al., 1993), Arnt (Mason et al., 1994) or USF (Pognonec and Roeder, 1991), or preimmune serum were added to the binding reaction mixtures together with protein extracts and the radiolabeled probe to assess the specificity of protein-DNA complexes. USF was translated in vitro from pdI2 in rabbit reticulocyte lysate (Promega) as described (Gregor et al., 1990).


RESULTS AND DISCUSSION

Alignment of the bHLH Domains of Arnt, Max, and USF

Determination of the three-dimensional structures of Max and USF, two class B bHLH/Zip factors, in complex with DNA, has identified three critical residues from the basic region of these two factors to make specific contacts with the 2-fold symmetric CACGTG target sequence (Ferré d'Amaré et al., 1993, 1994). As schematically indicated in Fig. 1, these residues are His-28, Glu-32, and Arg-36 (in the positions of the Max protein). Strikingly these three amino acids are present in the basic region of Arnt but not in the corresponding domain of the dioxin receptor (Fig. 1). Given the critical role of these amino acids for specificity in class B E box recognition (reviewed by Littlewood and Evan(1994); Wolberger, 1994), it is, therefore, possible that Arnt may have the structural prerequisites for recognition of the CACGTG E box motif.


Figure 1: Alignment of selected bHLH proteins. Comparative analysis of the amino acid sequences of the following transcription factors: human dioxin receptor (DR; Dolwick et al., 1994a; Ema et al., 1994), human Arnt (Hoffman et al., 1991), human Max (Blackwood and Eisenman, 1991; Prendergast et al., 1991), and human USF (Gregor et al., 1990). For each protein the region shown is indicated by the amino acid numbers adjacent to the sequence. Asterisks indicate amino acids in Max and USF that are establishing contact with the E Box target sequence and are conserved in Arnt (boxed).



Arnt Constitutively Recognizes the CACGTG E Box Motif in Vitro

To examine if Arnt could recognize class B E box motifs, we performed gel mobility shift experiments using as specific probe spanning the USF recognition sequence of the adenovirus major late promoter. Infection of rabbit kidney RK13 cells with recombinant vaccinia virus encoding either Arnt or dioxin receptor produced high levels of expression of these proteins in cytosolic cell extracts, as assessed by immunoblot analysis (Fig. 2). We detected no CACGTG E box binding activity by a control extract from uninfected RK13 cells, as assessed by gel mobility shift analysis (Fig. 3A, lane 2). In contrast, a cytosolic extract containing vaccinia virus-expressed Arnt produced a distinct complex with the E box probe (Fig. 3A, compare lanes 2 and 3). As a reference we incubated the E Box probe with a corresponding extract containing vaccinia virus expressed dioxin receptor. However, this bHLH/PAS factor did not exhibit any detectable class B E box binding activity (compare lanes 3 and 4).


Figure 2: Vaccinia virus expression of dioxin receptor and Arnt. Crude cytosolic protein (50 µg) from uninfected (lane 1) or recombinant vaccinia virus-infected (lane 2) RK13 cells was separated by 7.5% SDS-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membrane. Arnt (A) or dioxin receptor (DR) (B) expression levels were visualized with polyclonal antiserum against Arnt (Mason et al., 1994) or dioxin receptor (Whitelaw et al., 1993).




Figure 3: Arnt constitutively recognizes the CACGTG E box motif. A, E box binding activity by Arnt. Cytosolic extracts from noninfected RK13 cells (lane 2) or vaccinia virus-infected cells expressing Arnt (lane 3) or dioxin receptor (DR, lane 4) were incubated with a P-labeled probe spanning the E box USF recognition motif of the adenovirus major late promoter and analyzed by gel mobility shift assay. B, XRE binding activity by the ligand-activated Arnt-dioxin receptor complex. Crude cytosolic extracts containing vaccinia virus expressed Arnt or dioxin receptor were treated with dioxin and analyzed for XRE binding activity individually (lanes 2 and 3) or following co-incubation (lane 4) with the labeled XRE probe. C, E box binding activity by vaccinia virus-expressed Arnt was analyzed by gel mobility shift analysis in the absence (lane 2) or presence of anti-Arnt (-Arnt; lane 3), preimmune (P.I.S.; lane 4), or anti-USF (-USF; lane 5) serum. D, DNA binding specificity of Arnt. E box binding reactions were assembled with vaccinia virus-expressed Arnt in the absence (lane 2) or presence of an excess of unlabeled E box probe from the adenovirus major late promoter (ML, lane 3) or an oligonucleotide spanning a class A E box motif (µE5; lane 4). E, E box binding reactions were assembled with in vitro translated USF in the absence or presence of specific antiserum as above. The positions of unbound (Free) probe and of Arnt-, USF-, and dioxin receptor-Arnt (DR)-dependent protein-DNA complexes are indicated. Lane 1 in each panel contains unincubated probe.



In control experiments we examined whether vaccinia virus-expressed Arnt and dioxin receptor were functional with regard to XRE binding activity in vitro in the presence of dioxin. As expected (Dolwick et al., 1993b; Whitelaw et al., 1993; Matsushita et al., 1993; Antonsson et al., 1995), neither Arnt nor the dioxin receptor exhibited any specific XRE binding activity individually (Fig. 3B, compare lanes 2 and 3). It was possible to reconstitute XRE binding activity, however, by co-incubation of vaccinia virus expressed Arnt and the dioxin receptor in the presence of dioxin (Fig. 3B, lane 4), demonstrating that the complex of both proteins exhibited bona fide XRE binding properties.

We next used specific antibodies against Arnt (Mason et al., 1994) to establish that the generated E box complex harbored the Arnt protein. As shown in Fig. 3C, addition of the Arnt antibodies resulted in a supershift of the class B E box complex generated by vaccinia virus expressed Arnt (compare lanes 2 and 3). Addition of preimmune serum did not produce this effect (lane 4). Moreover, polyclonal antibodies against the ubiquitous class B E box binding factor USF (Pognonec and Roeder, 1991) did not alter the mobility of the complex nor inhibit its formation (lane 5). These results demonstrated that the generated E box complex contained Arnt and that no USF-dependent class B E box binding activity was detectable under these conditions.

The specificity of the Arnt-E box complex was investigated by oligonucleotide competition experiments. Gel mobility shift analysis performed with the adenovirus major late CACGTG E box probe was performed with vaccinia virus expressed Arnt in the absence or presence of an excess of unlabeled probe or an oligonucleotide, µE5, spanning a class A E box motif from the immunoglobulin heavy-chain 3` enhancer that is recognized by, for instance, E2A bHLH factors.() Strikingly, formation of the Arnt-E box complex was inhibited in the presence of an excess of the CACGTG E box motif, but not in the presence of the CAGCTG E box motif (Fig. 3D, compare lanes 2-4), indicating specificity of Arnt for the class B E box recognition sequence. In excellent agreement with the failure of Arnt to recognize XRE probes in direct gel mobility shift assays (Fig. 3B), binding of Arnt to the adenovirus major late class B E box motif was not competed for in the presence of an excess of the XRE sequence motif (data not shown). Taken together, these data strongly suggest that Arnt constitutively and specifically recognizes class B E Box target sequences.

Although it is presently unclear whether vaccinia virus expressed Arnt bound the E box motif as a homodimeric complex or in association with a putative partner factor endogenous to the RK13 cell extract, USF did not appear to be contained within the Arnt-E box complex. Conversely, E box binding activity of in vitro translated USF was not affected by anti-Arnt antibodies (Fig. 3E, compare lanes 2 and 5), whereas anti-USF antibodies inhibited formation of a complex between USF and the E box motif from the adenovirus major late promoter (compare lanes 2 and 3).

Constitutive Transcriptional Activation of E Box-regulated Promoters by Arnt

To investigate a possible constitutive functional activity of Arnt, we constructed an E-box-regulated reporter gene, pML-EB-T81-Luc, containing a single copy of the USF recognition sequence from the adenovirus major late promoter upstream of a minimal (extending from nucleotide -81 relative to the transcription start site) herpes simplex virus thymidine kinase promoter and the luciferase gene (shown schematically in Fig. 4B). Luciferase activity was analyzed following transient co-transfection of this reporter gene together with Arnt expression vectors into CHO cells. The reporter gene construct showed low levels of constitutive activity upon co-transfection with the naked expression vector pCMV4 (Andersson et al., 1989) containing no Arnt cDNA insert (Fig. 4B). In contrast co-transfection with an expression vector encoding full-length Arnt strongly enhanced the activity of the E box-driven reporter gene, whereas the activity of the parental thymidine kinase promoter reporter gene lacking the E Box motif was not affected by expression of Arnt (Fig. 4B).


Figure 4: Constitutive Regulation of Class B E Box-driven Promoters by Arnt. A, Schematic representations of the Arnt proteins. Two hydrophobic repeats (A and B) within the PAS domain and the transactivation domain (TAD) of Arnt are indicated. B, CHO cells were transiently co-transfected with the pML-EB-T81-Luc or pT81-Luc reporter genes and the blank expression vector pCMV4 (pCMV) or expression vectors encoding full length Arnt (Arnt) or the Arnt deletion mutant (Arnt 603), and luciferase activity was determined after 48 h. Values for the Arnt factors were normalized against protein content and the activity observed with the blank expression vector. Results of a representative experiments are shown. C, Detection of Arnt protein by immunoblotting. Arnt proteins transiently expressed in CHO cells were visualized with anti-Arnt antiserum. The control lane (lane 1) contains an extract from cells transfected with the naked pCMV expression vector. D, COS cells were transiently co-transfected with the schematically indicated -globin reporter gene and a reference -globin reporter construct and blank expression vector (lane 1) or increasing concentrations of Arnt or USF expression vectors or an expression vector encoding the transcription factor Pax5. Correctly initiated globin transcripts were monitored by an RNase protection assay.



As schematically represented in Fig. 4A, Arnt harbors within its C terminus a potent constitutively active transactivation domain (Jain et al., 1994; Li et al., 1994; Whitelaw et al., 1994). We therefore also examined the activity of the E box-driven luciferase reporter gene construct upon expression of an Arnt deletion mutant, Arnt 603 (Whitelaw et al., 1994), that lacks the C-terminal transactivation function of Arnt. Only very low levels of reporter activity were observed following expression of this truncated Arnt protein in CHO cells (Fig. 4B). Immunoblot analysis of extracts from transfected cells showed that the truncated Arnt mutant was expressed at levels similar to those produced by the full length protein (Fig. 4C, compare lanes 2 and 3). Thus, these experiments demonstrated a constitutive function of Arnt that was mediated via the E Box recognition motif of the adenovirus major late promoter and was dependent of the C-terminal transactivation domain of Arnt.

We also examined the constitutive functional activity of Arnt on a -globin reporter gene, 3xµE3--globin, driven by the endogenous -globin basal promoter and three copies of a class B E box regulatory element of the immunoglobulin heavy-chain 3` enhancer (Grant et al., 1992). This motif is recognized in vitro by USF (data not shown), and, consistent with this observation, transient expression of USF in COS cells resulted in dose-dependent activation of the 3xµE3--globin reporter gene (Fig. 4D, compare lane 1 with lanes 6-8). In an analogous fashion, expression of Arnt produced an activation response that was similar in potency to that generated by USF (Fig. 4D, compare lanes 2-4 with lanes 6-8). This reporter gene showed very low basal levels of expression in COS cells transfected with the parental CMV expression vector (Fig. 4D, lane 1). Moreover, expression of the lymphoid regulatory factor Pax5 (Adams et al., 1992) did not alter reporter gene activity (lane 5).

In conclusion, Arnt constitutively activated promoter constructs regulated by two distinct CACGTG E box motifs, the USF recognition sequence of the adenovirus major late promoter, and the immunoglobulin heavy-chain 3` enhancer µE3 element. These results indicate that Arnt may represent a novel class of E Box regulatory factors and, thus, mediate at least two different regulatory functions: constitutive regulation of E Box-controlled target genes, and, in partnership with the dioxin receptor, regulation of XRE-driven promoters in dioxin-stimulated cells. Importantly, these two functions of Arnt do not appear to overlap, since Arnt constitutively only recognizes the CACGTG motif and fails to bind the XRE sequence motif, and the dioxin-activated Arnt-dioxin receptor complex only exhibits specificity for XRE sequences but not for E box motifs. It remains to be established whether the constitutive regulatory function of Arnt is mediated by a homodimeric complex or whether this task requires novel, as yet unidentified, partner factors. In support of the notion that Arnt can homodimerize, in vitro translated Arnt sedimented in the 4-6 S region of sucrose gradients,() similar to the sedimentation properties of the dioxin-activated 200-kDa Arnt-dioxin receptor heterodimer (Hapgood et al., 1989). Finally, given the critical role of E box binding factors in regulation of cell differentiation and proliferation (see Kadesh (1993), Jan and Jan(1993), Weintraub(1993), and Dorschkind(1994) for reviews), it will be interesting to investigate the role of Arnt in mammalian development.


FOOTNOTES

*
This work was supported by the Swedish Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a fellowship of the Swedish Cancer Society.

To whom correspondence should be addressed: Dept. of Medical Nutrition, Karolinska Institute, Huddinge University Hospital F-60, Novum, S-141 86 Huddinge, Sweden. Tel.: 46-8-779-7124; Fax: 46-8-711-6659.

The abbreviations and trivial names used are: bHLH, basic helix-loop-helix; dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin; XRE, xenobiotic response element; PAS, Per-Arnt-Sim; CHO, Chinese hamster ovary.

M. Skogsberg and S. Pettersson, manuscript in preparation.

I. Pongratz and L. Poellinger, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Robert G. Roeder (Rockefeller University) for generously providing the anti-USF antiserum and pdI2 and Dr. Oliver Hankinson (UCLA) for pBM5-NEO-M1-1. We also thank Dr. Anders Berkenstam (Karolinska Institute) for advice regarding vaccinia virus expression and Dr. Y. Fujii-Kuriyama (Tohoku University, Sendai, Japan) for fruitful discussions.


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