From the
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.
Mammalian bHLH
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.
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.
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).
We also
examined the constitutive functional activity of Arnt on a
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,
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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
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.
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, pHP
2 (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).
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.
(
)
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.
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.
-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).
(
)
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.