(Received for publication, October 11, 1994; and in revised form, December 19, 1994)
From the
Transcription of the rat adrenergic receptor
(
AR) gene in the liver is controlled by three
promoters that generate three mRNAs. The middle promoter (P2), located
between -432 and -813 base pairs upstream from the
translation start codon and lacking a TATA box, is responsible for
generating the major, 2.7-kilobase mRNA species expressed in many
tissues (Gao, B., and Kunos, G.(1994) J. Biol. Chem. 269, 15762-15767). DNase I footprinting using rat liver
nuclear extracts identified three protected regions in P2: footprint I
(-432 to -452), footprint II(-490 to -540), and
footprint III (-609 to -690). Putative response elements in
footprints I and III were not analyzed except the AP2 binding site in
footprint III, which could be protected by purified AP2 protein.
Footprint II contains four sites corresponding to half of the NF-I
consensus sequence, but DNA mobility shift assays indicate that this
footprint binds two proteins distinct from NF-I: a ubiquitous
CP1-related factor and another novel factor, termed
-Adrenergic
Receptor Transcription Factor (
ARTF), which binds to two separate
sites in this region. The
ARTF is widely distributed, with the
highest amounts found in brain, followed by liver, kidney, lung, and
spleen, but no detectable activity in heart. Deletions of
ARTF
binding sites nearly abolished P2 promoter activity, which suggests
that the
ARTF is essential for the transcription of the
AR gene in most tissues.
-Adrenergic receptors (
AR) (
)play an important role in key components of the
sympathoadrenal response to stress, such as peripheral
vasoconstriction, increased cardiac contractility, and hepatic
glycogenolysis. Both pharmacological and molecular cloning studies have
indicated the existence of multiple subtypes of
AR
with unique tissue distributions (see reviews by Harrison et
al.(1991) and Ford et al.(1994)). One of these subtypes,
the
AR, has been identified in a variety of mammalian
tissues, with the highest amounts being present in the liver. The
expression of the
AR gene in the rat is regulated in
a complex and tissue-specific manner. For example, hypothyroidism
decreases the level of
AR mRNA in the liver, but
increases it in the heart and the lungs (Lazar-Wesley et al.,
1991). The tissue distribution of the
AR message in
the rat generally parallels that of the
AR binding
sites, with the highest levels present in the liver, followed by heart,
cerebral cortex, brain stem, kidney, lung, and spleen (Lomasney et
al., 1991). The rat
AR gene is composed of two
exons and a single large intron of at least 16 kb in length (Gao and
Kunos, 1993), and has three transcripts in the liver of 2.3, 2.7, and
3.3 kb in length (Gao and Kunos, 1994). The 3.3-kb species is
preferentially expressed in liver, whereas the 2.7-kb species is widely
expressed in many tissues (McGehee et al., 1990; McGehee and
Cornett, 1991; Lomasney et al., 1991). The low abundance
2.3-kb species is difficult to detect and has only been reported in our
earlier study in rat liver (Gao and Kunos, 1994), and by Hu et
al.(1993) in hamster DDT
MF-2 cells. We have further
found that these three
AR mRNAs in rat liver are
transcribed from three distinct promoters (Gao and Kunos, 1994). The
proximal promoter (P1), likely involved in generating the low abundance
2.3-kb mRNA species, does not contain homologies with the consensus
sequences of known transcription factors. The middle promoter (P2),
responsible for generating the major 2.7-kb mRNA species, is
G+C-rich, lacks a TATA box, and contains binding sites for several trans-acting factors. The distal promoter (P3), responsible
for generating the 3.3-kb species, contains a putative TATA and CCAAT
box and has in its vicinity recognition sites for liver-specific
transcription factors. In order to further characterize the functional
organization of the major P2 promoter and its associated trans-acting factors, we have employed DNase I footprinting to
identify the sites of DNA-protein interactions, and DNA mobility shift
assays (DMSA) to analyze the nature of the proteins that bind to these
sites.
Figure 4: DNA mobility shift analysis of the specific proteins interacting with the footprint II region. A, DNA sequence of footprint II region between -483 and -543. Lines represent oligos used in DMSA. B-F, DMSA performed with labeled oligos II, IIb, IIa, IIa1, and IIa2, respectively. Lanes 1, labeled oligo alone; lanes 2, 1 ng of labeled oligo was incubated with 10 µg of liver nuclear extract; lanes 3 and up, 1 ng of labeled oligo was incubated with 10 µg of liver nuclear extract and 100 ng of different competitor oligos, as indicated above the lanes. Various amounts of CP1 oligo used are indicated in the panelsE and F.
Figure 1:
Diagrammatic representation of sense (A and B) and antisense (C and D)
DNA fragments used in the footprinting experiments. End labeling of the
fragments was described under ``Materials and Methods.'' The top line with negative numbers represents the rat
AR gene middle promoter (P2) region. Asterisks represent the
P end
label.
The DNase I
footprinting standard reaction was performed according to Galas and
Schmitz(1978), with some modifications. The binding reaction was
performed in a final volume of 100 µl, containing 20 mM Hepes (pH 7.9), 50 mM KCl, 0.1 mM EDTA, 10%
glycerol, 1 mM DTT, and 0.5 mM PMSF. Twenty to 80
µg of nuclear extract or 1 µg of purified AP2 protein (Promega)
were preincubated with 2 µg of poly(dI-dC) (U. S. Biochemical) for
30 min at 0 °C. Then, about 1 ng of labeled fragment (20,000
counts/min) was added, and the incubation was continued for 30 min at
25 °C. The reaction was then diluted 2-fold with a solution of 5
mM CaCl/10 mM MgCl
and
digested with 0.15-0.075 units of RQ1 RNase-free DNase (Promega)
for 1 min at 25 °C. The reaction was stopped by the addition of 180
µl of stop solution (200 mM NaCl, 30 mM EDTA, 1%
SDS, 100 µg/ml yeast tRNA), and DNase I was removed by proteinase
K. The reaction product was extracted by phenol/chlorform, precipitated
with ethanol, and analyzed on a 8% polyacrylamide, 8 M urea
sequencing gel.
To determine the specific nucleotides protected from DNase I digestion, a sequence ladder derived for each fragment by the method of Maxam and Gilbert(1980) was electrophoresed alongside each set of protected and unprotected DNase I-digested sample.
Figure 2:
DNase
I footprint analysis of the rat AR P2 promoter. PanelsA-D illustrate the footprints
(marked by brackets and Roman numerals) obtained on
DNA fragments A-D, respectively (fragments labeled as in Fig. 1). DNase I-hypersensitive sites are marked by filled
arrows. G + A, Maxam-Gilbert G + A sequencing
reaction; NE, rat liver nuclear
extract.
Figure 3:
DNA
sequence of regions of protein-DNA interactions in the rat
AR gene P2 promoter. Footprints are underlined. The filled arrows mark sites of DNase I
hypersensitivity. Consensus DNA binding sites are boxed. Negativenumbers on the right indicate
nucleotide position relative to the translation start codon (+1).
The ellipses above footprint II represent interactions with
CP1 and
ARTF.
In order
to obtain better resolution of multiple binding proteins, we performed
DMSA using subregions of oligo II as radiolabeled probes. Fig. 4C illustrates the results obtained with oligo
IIb. Incubation of the labeled oligo IIb with liver nuclear extract
generated a single shifted band (lane2), effectively
competed by a 100-fold excess of unlabeled oligo IIb (lane3). This complex is also efficiently inhibited by an
excess of oligo IIa (lane 4), suggesting that the factor that
binds oligo IIb also binds oligo IIa. The oligos containing consensus
binding sites for SP1, AP2, CP1, C/EBP, CTF/NF-I (lanes
5-9), and AP1, AP3, BTE, TFIID, GRE, CREB, NF-B (data
not shown), did not compete this complex. A computer-based search of
the Sitedata data base of several thousand sequence-specific response
elements yielded no homologies with sequences in oligos IIb and IIa,
suggesting that the protein binding to these oligos may be a novel
transcription factor. We tentatively name this factor
ARTF, for
-Adrenergic Receptor Transcription Factor. When
P-labeled oligo IIa was used as a probe, the DMSA (Fig. 4D) yielded two DNA-protein complexes (lane
2), which are specific as they are competed away by an excess of
unlabeled oligo IIa (lane 3). The top (major) complex, but not
the bottom (minor) complex, is abolished by an excess of oligo IIb (lane 4), in agreement with the DMSA analysis of oligo IIb.
This suggests that the protein in the top complex is the same as the
one in the complex revealed with oligo IIb, i.e.
ARTF.
The bottom, but not the top, complex is competed away by an excess of a
consensus oligo for the transcription factor CP1 (lane 5).
Neither complex is competed by oligos containing consensus binding
sites for C/EBP, NF-I (lanes 6 and 7), or AP1, AP2,
AP3, SP1, TFIID, GRE, CREB, or NF-
B (data not shown). Taken
together, these findings can be interpreted to indicate that oligos IIb
and IIa each contain a binding site for the same protein,
ARTF,
while oligo IIa contains an additional protein binding site for a
CP1-related factor.
To further delimit the binding domains of oligo
IIa for ARTF and CP1, we synthesized two overlapping oligos, IIa1
and IIa2, which encompass oligo IIa, and employed them in DMSA with rat
liver nuclear extract. As shown in Fig. 4E,
P-labeled oligo IIa1 binds a complex specifically (lane 2), as it is competed away by unlabeled oligo IIa1 (lane 3). This complex is also abolished by unlabeled oligo
IIb (lane 5) but not by oligo IIa2 (lane 4) or the
consensus oligo for CP1 (lanes 6-9), suggesting that the
factor that binds oligo IIa1 is
ARTF. Fig. 4F shows that oligo IIa2 binds two specific proteins (lane
2), which is abolished by self competition (data not shown). The
major protein in the top complex is the CP1-related factor, as it is
competed away dose-dependently by the consensus oligo for CP1 (lanes 5-8) or by oligo IIa (lane 4), but not
by oligo IIb (lane 3). The factor in the bottom complex is
ARTF, which is competed away by oligo IIb (lane 3), but
not by CP1 (lanes 5-8). In summary, footprint II appears
to contain two binding sites for
ARTF and one for CP1, as
schematically illustrated in Fig. 3.
Figure 5:
ARTF is a ubiquitous factor.
P-Labeled oligos IIb, IIa1, and NF-I (A) or M-CAT (B) were incubated with 10 µg of nuclear extract prepared
from different rat tissues and subjected to DMSA, as described under
``Materials and Methods.''
Figure 6:
The effects of footprint deletions on the
activity of the P2 promoter of the rat AR gene. The left side is the schematic representation of the pCAT
constructs, containing the intact P2 promoter or its variants with
deletions of footprints I, II, III, IIa, or IIb, used in cell
transfection experiments. The right side shows CAT activities
measured in DDT
MF-2 cells, expressed as percent of the
positive control. CAT activities were corrected for transfection
efficiencies, as described (Gao and Kunos, 1994). Means ± S.E.
from three experiments are shown.
The experiments reported in this paper suggest a complex
interaction of specific DNA-binding proteins with the major, middle
promoter (P2) of the rat AR gene. We have identified
three groups of binding sites (footprints I-III) for potential trans-acting factors within the 381-bp segment of the
5`-flanking region, which was found necessary and sufficient for
maintaining maximal transcriptional activity of P2 (Gao and Kunos,
1994).
Footprint I contains a CRE and a GC box, which may play
positive roles in the transcription of the rat AR
gene, since deletion of the region -432 to -460, which
contains footprint I, abolished the P2 promoter activity (see Fig. 6). In some genes with TATA-less promoters containing
multiple GC boxes, binding of Sp1 has been shown to be critical for
transcription initiation (Pugh and Tjian, 1990, 1991). It remains to be
determined whether mutation of the GC box in footprint I rather than
deleting the entire footprint is sufficient to eliminate P2 promoter
activity.
Footprint III contains an AP2 binding site, but the
footprint is much larger than the region protected by the purified AP2
protein, which suggests the binding of some additional factors to this
footprint. Sequence analysis reveals that this region contains putative
binding sites for the liver specific factors HNF-1, HNF-5, and C/EBP (Fig. 3). If these binding sites are functional, they may, in
part, account for the much stronger expression of the 2.7-kb
AR mRNA in the liver, than in other tissues of the
rat. Deletion of footprint III also abolishes P2 promoter activity (Fig. 6), but it is not clear which factor or combination of
factors plays the critical role.
Footprint II contains four binding
sites for half of the NF-I palindrome. However, several lines of
evidence suggest that the major factor that binds to footprint II is
distinct from NF-I. (a) In DMSA, the NF-I consensus oligo did
not compete with oligos II, IIb, and IIa1 (Fig. 4, B-D) and, conversely, oligos IIb and IIa1, which contain
half of the NF-I palindrome, did not compete with the labeled NF-I
oligo (data not shown). (b) The factors binding to footprint
II and NF-I have distinct DNA contact points identified by methylation
interference assays. ()(c) The tissue distribution
pattern of this factor is different from that of NF-I (Fig. 5).
It is interesting to note that the protein BTEB, which is distinct from
NF-I, has been identified as the factor binding to the half-NF-I
consensus sequence TGGC in the regulatory domains of the rat P-450c
gene (Yanagida et al., 1990; Imataka et al., 1992).
However, the major factor that binds to footprint II is not competed by
a consensus oligo for the BTE binding sequence, suggesting that it is
not BTEB. Since computer-based sequence analysis of footprint II
revealed no apparent homologies with the consensus binding sites for
any other known transcription factors, we have tentatively named the
major factor binding to footprint II
ARTF.
The results of DNA
mobility shift assays indicated that oligos IIb and IIa1, which cover
adjacent, partially overlapping segments of footprint II, were able to
cross-compete. This suggests that the same factor, most likely
ARTF, binds to both oligos. Examination of the sequence of oligos
IIb and IIa1 reveals some similarities, such as the presence of the
motif GCTGG in both (IIb: -513 to -520; IIa1: -501 to
-505). Furthermore, in DNA mobility shift assays both oligo II
and IIa bound
ARTF strongly and CP1 weakly, whereas oligo IIa2
showed strong CP1 binding. This suggests that
ARTF can interfere
with CP1 binding, probably as a result of adjacent or partially
overlapping binding domains. Oligo IIa2 contains an inverted GCAAT
sequence which has been shown to bind the heat-resistant protein C/EBP
with high affinity (Graves et al., 1986; Johnson et
al., 1987). However, the protein binding of oligo IIa2 is
abolished by heating the nuclear extract at 90 °C for 5 min (data
not shown). Furthermore, binding was not competed by the oligo AlbD,
which binds the C/EBP protein (Lichtsteiner et al., 1987), but
it was competed by the CP1 oligo. These observations suggest that the
protein that binds to oligo IIa2 is a CP1-related protein and not
C/EBP. In summary, the footprint II region of P2 contains two binding
sites for
ARTF and one for CP1, and
ARTF can interfere with
CP1 binding. These complex protein interactions may contribute to the
formation of the multiple DNase hypersensitity sites in footprint II.
The critical role of
ARTF in the transcription of the
AR gene is indicated by the finding that deletion of
either footprint IIa or IIb abolishes the activity of the major, P2
promoter (see Fig. 6). The results of the deletion studies thus
indicate that each of the three footprint domains is essential for P2
promoter activity and they do not have a simple additive effect. This
could suggest that the multiple proteins that bind to these elements
also interact with each other to form the primary transcription
complex, where removal of any one component may have a
``domino'' effect.
The tissue distribution of ARTF
generally fits the relative abundance of the 2.7-kb
AR mRNA species, with the exception of the heart. The
rat heart expresses high levels of
AR binding sites
and of the 2.7-kb
AR mRNA (Lomasney et al.,
1991; McGehee et al., 1992), but little if any
ARTF was
detected (see Fig. 5A). This suggests that
transcription of the
AR gene in the rat heart
involves some alternative mechanisms, such as the binding of
heart-specific transcription factor(s). Indeed, sequence analysis
reveals that the 5`-flanking region of the rat
AR
gene contains several putative consensus sequences for cardiac myocyte
nuclear factors; the region between -736 to -730 contains
one mismatch (CATGGCT) to the M-CAT consensus sequence
(CATNC(C/T)(T/A)), which is involved in the expression of several
cardiac-specific genes (Iannello et al., 1991; Farrance et
al., 1992). The region -763 to -768 (CAGTTG) contains
an E-box (Blackwell and Weintraub, 1990), which has also been
implicated in the control of cardiac-specific gene expression (Thompson et al., 1991; French et al., 1991). The possible role
of these cardiac-specific transcription factors in the expression of
the
AR gene in the heart remains to be determined.
Also, in preliminary experiments we have found that the transcription
start point corresponding to the 2.7-kb
AR mRNA is
located at different sites in heart and liver.
Regardless
of the specific mechanisms involved, tissue-dependent differences in
the transcription of the
AR gene have important
physiological implications; they may provide the molecular basis for
opposite changes in
AR gene expression in heart and
liver under certain conditions, such as hypothyroidism (Lazar-Wesley et al., 1991). Whether there is a direct correlation between
the transcription of the rat
AR gene in different
tissues (except in heart) and the levels of
ARTF awaits further
purification and characterization of this factor.
In summary, we
have identified multiple protein-binding sites and complex DNA-protein
interactions in the major promoter of the rat AR
gene, including the involvement of a novel protein factor in its
transcription. Taken together with our previous demonstration of three
different promoters, the present findings further document the complex
mechanisms involved in controlling the transcription of the
AR gene.