(Received for publication, November 20, 1995; and in revised form, February 14, 1996)
From the
Expression of CYP1A1 gene is regulated in a
substrate-inducible manner through at least two kinds of regulatory DNA
elements in addition to the TATA sequence, XRE (xenobiotic responsive
element), and BTE (basic transcription element), a GC box sequence. The trans-acting factor on the XRE is a heterodimer consisting of
arylhydrocarbon receptor (AhR) and AhR nuclear translocator (Arnt),
while Sp1 acts as a regulatory factor on the BTE. We have investigated
how these factors interact with one another to induce expression of the CYP1A1 gene. Both in vivo transfection assays using Drosophila Schneider line 2 (SL2) cells, which is devoid of
endogenous Sp1, AhR, and Arnt, and in vitro transcription
assays using baculovirus-expressed AhR, Arnt, and Sp1 proteins revealed
that these factors enhanced synergistically expression of the reporter
genes driven by a model CYP1A1 promoter, consisting of four
repeated XRE sequences and a BTE sequence, in agreement with previous
observation (Yanagida, A., Sogawa, K., Yasumoto, K., and
Fujii-Kuriyama, Y.(1990) Mol. Cell. Biol. 10,
1470-1475). We have proved by coimmunoprecipitation assays and
DNase I footprinting that both AhR and Arnt interact with the zinc
finger domain of Sp1 via their basic HLH/PAS domains. When either the
AhRArnt heterodimer or Sp1 was bound to its cognate DNA element,
DNA binding of the second factor was facilitated. Survey of DNA
sequences in the promoter region shows that the XRE and GC box elements
are commonly found in the genes whose expressions are induced by
polycyclic aromatic hydrocarbons, suggesting that the two regulatory
DNA elements and their cognate trans-acting factors constitute
a common mechanism for induction of a group of drug-metabolizing
enzymes.
Transcriptional activation of eukaryotic genes in response to
exogenous or endogenous signals is accomplished by multiple
sequence-specific protein/DNA and protein/protein interactions.
DNA-binding proteins that recognize the appropriate DNA sequences in
the promoter of a gene can interact with other transcription factors to
transmit transcription-enhancing effects on the basic transcription
machinery, commonly including transcription factor IID and RNA
polymerase II. Transcription of the CYP1A1 (cytochrome P-450c) gene,
which encodes a drug-metabolizing enzyme, is induced by polycyclic
aromatic hydrocarbons such as 3-methylcholanthrene and
TCDD()(1, 2) . The analysis of CYP1A1 gene regulation by the DNA transfer experiments in transient
transfection assays revealed that a high level of inducible expression
required at least two regulatory DNA sequences, designated XRE
(xenobiotic responsive element) and BTE (basic transcription element, a
GC box sequence)(3, 4, 5, 6) . The
regulatory factor on the XRE is a heterodimer complex consisting of AhR
(aryl hydrocarbon receptor; (7) and (8) ) and Arnt
(AhR nuclear translocator; (9) )(10, 11, 12) , while Sp1 is a
regulatory factor acting on the BTE. Like steroid hormone receptors,
AhR exists in the cytoplasm of normal cells in association with
hsp90(13, 14, 15, 16) . As soon as
an inducer is taken up in cells, it is recognized and bound by AhR. The
resultant inducer-bound AhR was stimulated to be dissociated from hsp90
and translocate to the nucleus, where it forms a transcriptionally
active complex with Arnt to activate the CYP1A1 gene via
binding to the XRE. In DNA transfer experiments, deletion of either the
XRE or BTE element in a fusion gene consisting of the CYP1A1 promoter and the CAT (chloramphenicol acetyltransferase)
structural gene resulted in a remarkable reduction in CAT
expression(6) . The fusion gene lacking the BTE sequence in the
promoter exhibited only a low inducible expression of CAT activity in
response to the inducer, because of the decreased constitutive
expression, while the gene deleted of the XRE showed only a
constitutive CAT expression even in the presence of the inducer. These
results suggest a cooperative interaction between the factors acting on
the XRE and BTE sequences to enhance the expression of the gene.
Recently, we and other groups have isolated cDNA clones for these
regulatory factors(7, 8, 9) , and we have now
investigated how these factors interact with each other on their
cognate DNA elements.
In this paper, we describe the several lines
of evidence for physical interaction between the AhRArnt complex
and Sp1 by in vivo DNA transfer experiment, in vitro transcription assay, coimmunoprecipitation methods, and DNase I
footprinting. Experiments introducing a variety of deletional mutations
into these factors demonstrated that the interaction between
AhR
Arnt and Sp1 is accomplished through the basic HLH/PAS domains
of AhR
Arnt and zinc finger domain of Sp1. Survey of the sequence
data base shows that both the XRE and the GC box are present in
promoter regions of a group of drug-metabolizing enzyme genes,
suggesting that the two DNA elements and their cognate DNA binding
factors constitute a common mechanism for the inducible expression of
this class of genes.
Figure 2: Purification of the recombinant AhR, Arnt and Sp1 expressed in the baculovirus system. Silver staining of SDS-polyacrylamide gel (lanes a) and immunoblot analysis (lanes b) show the purified AhR, Arnt and Sp1. Positions of molecular mass markers are shown on the left.
For the in vitro transcription assay and the DNase I footprinting analysis, the eluate was subject to the PD-10 column (Pharmacia) for change of the KCl concentration to 100 mM KCl.
Figure 1:
Synergistic enhancing
effects on CAT expression under control of CYP1A1 promoter in Drosophila SL2 cells by AhRArnt and Sp1. A,
schematic representation of the CAT reporter plasmid driven by the
model CYP1A1 promoter. B, pGEMAct-Sp1, pGEMAct-hAhR,
and pGEMAct-Arnt (3 µg) were transfected into SL2 cells along with
the reporter p53-ICAT (3 µg). The cells were cultured for 40 h and
then lysed as described. The CAT activities in the cell extracts were
measured as described under ``Experimental Procedures.'' The
CAT activity of lane 1 was arbitrarily assigned to 1.0, and
the other CAT activities were normalized on the basis of this
standard.
Figure 3:
Transcriptional activation by
AhRArnt and Sp1 in in vitro transcription assays. A, schematic representation of G-free cassette template driven
by model CYP1A1 promoter. B and C,
transcriptional activities of AhR
Arnt and Sp1 were measured using
Sf9 cell nuclear extracts in vitro. Transcription reactions
(30 µl) contained 800 ng of CYP1A1 gene promoter template (panel B, p53-IC
AT) or control templates (panel C, p53C
AT and p44-IC
AT). Each
reaction was contained 200 ng of pml
404(180) as an
internal control gene. AhR (20 ng)/Arnt (20 ng) and Sp1 (20 ng) were
used for the reactions. For competition assay, XRE or mutated XRE
oligonucleotides (25-fold) were contained in the reaction mixtures (lanes 7 and 8). Transcripts were detected and
quantified by the Imaging analyzer (Fuji BAS-1000). A representative
result of three experiments is shown.
Figure 4:
Association of Sp1 with AhR and Arnt in vitro. Sp1 synthesized in reticulocyte lysates with
[S]Met was incubated with AhR (lanes 2 and 3) or Arnt (lanes 5 and 6) produced
in the baculovirus system. Immunoprecipitates of the binding complex by
anti-AhR or anti-Arnt antibodies were analyzed by SDS-PAGE. One-fourth
of translated products in immunoprecipitation reaction was used as a
control (lane 1). Positions of molecular mass markers are
shown on the left.
To examine functional domains of Sp1 responsible for
association with AhR and Arnt, we tested the interaction of a series of
Sp1 deletion mutants with AhR and Arnt. Fig. 5A illustrates several deletion constructs of Sp1 and a summary of
their interaction with AhR and Arnt. The experimental data are
demonstrated in Fig. 5B. Removal of the zinc finger
motifs (C649 and C
353) abolished association of Sp1 with AhR
and Arnt (Fig. 5, A and B). In contrast,
removal of most of the sequence N-terminal (
AB and Zf) or
C-terminal (C
755) to the zinc finger had essentially no effect on
the binding activity. Only the zinc finger motifs (Zf
D) retained
an adequate capacity for the association with AhR and Arnt. It was
reported that the region immediately C-terminal to the zinc finger
domain, designated the D region(26) , is necessary for
homologous interaction of Sp1, but this region is not necessary for
interaction with AhR or Arnt. Subdivision of the zinc finger domain for
the interaction activity remains to be investigated.
Figure 5:
Functional domains of Sp1 responsible for
association with AhR and Arnt. A Schematic representation of
Sp1 and its deletion mutants. CX denotes the C-terminal
deletion from the Xth amino acid of Sp1. S/T and Q indicate serine/threonine and glutamine-rich domain, respectively.
Domains A, B, C, D and +/- were designated by A. J. Coury et al.(26) . B, coimmunoprecipitation of in vitro synthesized Sp1 and its deletion mutants with AhR and
Arnt by anti-AhR (I) and anti-Arnt C34 (II)
antibodies, respectively. The experimental procedures are described in
the figure legend to Fig. 4. Positions of molecular mass markers
are shown on the left.
Figure 6:
Functional domains of AhR responsible for
association with Sp1. A, schematic representation of AhR and
its deletion mutants. B, coimmunoprecipitation of S-labeled AhR and its deletion mutants with Sp1 by
anti-Sp1 antibodies (Santa Cruz). AhR and its deletion mutants in
vitro synthesized in the reticulocyte lysate system with
[
S]Met were incubated with Sp1 produced in
baculovirus system and immunoprecipitated with anti-Sp1 antibodies.
Immunoprecipitates were analyzed by SDS-polyacrylamide gel (lanes
8-14). One-fourth of translated products in
immunoprecipitation reaction were used as controls (lanes
1-7). Positions of molecular mass markers are shown on the left.
Figure 7:
Functional domains of Arnt responsible for
association with Sp1. A Schematic representation of several
domains of Arnt fused with the glutathione S-transferase. B, binding assay of in vitro synthesized Sp1 to
bacterially expressed GST-Arnt fusion proteins. GST-Arnt fusion
proteins purified by the glutathione beads were incubated with S-labeled Sp1. The binding complex on the glutathione
beads were analyzed by a SDS-polyacrylamide gel. One-fourth of
translated products in immunoprecipitation reaction was used as a
control (lane 1).
Figure 8:
DNase I footprinting analyses of Sp1 and
AhRArnt on the CYP1A1 model promoter. A, schematic
representation of a DNA probe used for DNase I footprinting. B, DNase I footprinting analysis by using recombinant AhR,
Arnt, and Sp1. Amounts of factors used for analysis are as follows,
Sp1: 5 ng (lines 3 and 12), 15 ng (lines 4 and 13), and 50 ng (lines 5-8 and 14). AhR or Arnt: 5 ng (lines 6 and 9), 15
ng (lines 7 and 10), and 50 ng (lines 8 and 11-14). Representative DNase I footprinting bands, which
were uniquely changed depending on the simultaneous presence of
AhR
Arnt and Sp1 factors, are indicated by the arrowheads. G+A indicates the markers by the
Maxam-Gilbert method.
In our previous study on gene regulation of CYP1A1 by DNA transfer experiments, the two DNA elements, XRE and BTE,
together with the TATA sequence are necessary for a high level of
inducible expression of the CYP1A1 gene in response to
inducers such as TCDD and 3-MC. Lack of one of the two elements in the
promoter region reduced markedly the expression of the gene, suggesting
a synergistic effect of the two elements on the CYP1A1 gene
expression. In line with this observation, we further demonstrated the
synergistic transactivation by AhRArnt and Sp1 through an in
vivo DNA transfer experiment using SL2 cells and an in vitro transcription assay system. Since SL2 cells is known to be devoid
of Sp1 or its highly related factors and AhR
Arnt, in vivo DNA transfer experiments showed that a high level of inducible CAT
expression in response to the inducer was clearly dependent on the
simultaneous transfection of the expression plasmids of AhR, Arnt and
Sp1 into SL2 cells. On the other hand, transcription of either Sp1
plasmid or AhR and Arnt plasmids alone resulted in only a low level of
the CAT expression. The synergistic enhancing effect on the
transcription between Sp1 and AhR
Arnt was also observed in the in vitro transcription system using Sf9 cell lysates, which
lack Sp1 and AhR
Arnt, although it was not so marked as in the in vivo transient transfection system. It was revealed that an
optimal transcription driven by a model CYP1A1 promoter
requires Sp1, AhR
Arnt, and their cognate recognition sequences.
When one of the factors or the DNA elements was missing in this in
vitro system, the transcription activity was markedly reduced,
consistent with a synergistic effect of the XRE and BTE sequences on
the transcription of the CYP1A1 gene in vivo. Survey
of the promoter regions of the genes, whose expressions are known to be
induced in response to aromatic inducers such as TCDD and 3-MC, reveals
that most, if not all, of the genes carry these two regulatory DNA
elements (Fig. 9; (5) and (30) -33).
Although its presence was not reported, there is an apparent GC box
sequence in the promoter region of the glutathione S-transferase gene. These results suggest that the XRE and GC
box sequences and their cognate binding factors constitute a common
mechanism for drug induction of a group of drug-metabolizing enzymes.
Immunoprecipitation assay and GST-fusion protein binding assay
demonstrated that Sp1 and AhR
Arnt physically interact with each
other. The experiments using deletion mutants of various parts of the
three proteins clearly demonstrated that the interaction was mediated
between the zinc finger domain of Sp1 and the HLH/PAS domains of AhR
and Arnt proteins. Recently Sp1 has been reported to interact with
itself or with other transcription factors such as NF-
B, GATA-1,
and YY-1 to synergistically enhance the transcription driven by the
promoter containing the GC box and the corresponding factor binding
sequences(34, 35, 36, 37) .
Interestingly, the interaction of Sp1 with these regulatory factors
including AhR
Arnt is always mediated through its zinc finger
domain. Since there is apparently no common sequence among these
partner factors, it is interesting to investigate whether various parts
of the zinc finger of Sp1 interact with respective partner molecules or
whether a common part of the zinc finger is used for interaction with
these factors in an adaptive manner. Alternatively, it is possible that
apparently different primary sequences from these factors form related
three-dimensional structures, resulting in interaction with a common
part of the zinc finger domain of Sp1. In any event, it is important to
track down the interactive sequence of Sp1 into a small portion of the
molecule for each of the partner proteins. In DNase I footprint
analysis, while separate addition of AhR
Arnt and Sp1 protected
their respective DNA recognition sequences against digestion by DNase
I, the simultaneous presence of the two transcription factors caused
unique alterations in the combination of the two DNase I footprint
patterns (indicated by arrowheads in Fig. 8),
suggesting physical interaction between AhR
Arnt and Sp1 bound on
their DNA elements. In addition, judging from the intensity of the
protected bands, it appeared that the extent of protection by one
factor was increased in the presence of the other. These results
suggest that binding of one of the two factors to its recognition
sequence facilitates the other to bind its cognate DNA element. This
cooperative DNA binding of the two transcription factors could result
in synergistic enhancement of transcription of the CYP1A1 gene.
Figure 9: Schematic representation of the promotor region of drug-inducible genes whose expressions are induced in response to TCDD and 3-MC. References to the structures of the genes are as follows:, CYP1A1(5) , glutathione S-transferase (30) , quinone reductase(31) , aldehyde dehydrogenase-3(32) , and UDP-glucuronosyl transferase(33) .
Although the N-terminal region of Sp1 is known to
interact with TAF110 to enhance basal transcription(38) , AhR
and Arnt have potent transcriptional activation domains in the
C-terminal regions(27, 28) . It remains to be studied
how the physical interaction between AhRArnt and Sp1 leads their
synergistic enhancing effect on transcription of the CYP1A1 gene. In this study, we used a model promoter of the CYP1A1 gene constructed by placing XRE sequences on the -53-bp
segment of the CYP1A1 gene, which carries a GC box sequence on
the 5` end. Therefore, the XRE sequences are brought in such close
proximity to the GC box that the two transcription factors may interact
efficiently with each other. It is generally envisioned that
chromosomal looping may bring together complexes of Sp1 bound on the GC
box sequence and AhR
Arnt on the XREs in the promoter of CYP1A1 gene.