(Received for publication, December 3, 1996, and in revised form, February 11, 1997)
From the Department of Molecular and Cell Biology, The University of Texas at Dallas, Richardson, Texas 75083-0688
We have previously identified several positive
cis-acting regulatory regions in the promoters of the
bovine and human nuclear-encoded mitochondrial
F0F1-ATP synthase -subunit genes
(ATPA). One of these cis-acting regions
contains the sequence 5
-CACGTG-3
(an E-box), to which a number of
transcription factors containing a basic helix-loop-helix motif can
bind. This E-box element is required for maximum activity of the
ATPA promoter in HeLa cells. The present study identifies
the human transcription factor, upstream stimulatory factor 2 (USF2),
as a nuclear factor that binds to the ATPA E-box and
demonstrates that USF2 plays a critical role in the activation of the
ATPA gene in vivo. Evidence includes the
following. Antiserum directed against USF2 recognized factors present
in HeLa nuclear extracts that interact with the ATPA
promoter in mobility shift assays. Wild-type USF2 proteins synthesized from expression vectors trans-activated the
ATPA promoter through the E-box, whereas truncated USF2
proteins devoid of the amino-terminal activation domains did not.
Importantly, expression of a dominant-negative mutant of USF2 lacking
the basic DNA binding domain but able to dimerize with endogenous USF
proteins significantly reduced the level of activation of the
ATPA promoter caused by ectopically coexpressed USF2,
demonstrating the importance of endogenous USF2 in activation of the
ATPA gene.
Most cellular ATP is synthesized in the mitochondria through the process of oxidative phosphorylation. The mammalian mitochondrial oxidative phosphorylation system requires the functional interaction of gene products encoded by both the nuclear and the mitochondrial genomes (for reviews, see Refs. 1 and 2). The activities of the enzymes of the mammalian oxidative phosphorylation system vary greatly in response to a number of physiological conditions, including cell proliferation, hormonal stimulation, development, and differentiation (for reviews, see Refs. 3 and 4). The levels of these enzymes are controlled, at least in part, at a transcriptional level, although regulation at a post-transcriptional level also plays an important role in mitochondrial biogenesis (1-4).
During the past several years, analysis of mammalian nuclear encoded oxidative phosphorylation genes has resulted in the identification of a number of regulatory factors that contribute to the transcription of these genes. Characterization of these gene regulatory mechanisms should allow the delineation of signals involved in the control of cellular energy production and, possibly, in the coordinate expression of genes encoding proteins of the oxidative phosphorylation system.
Our laboratory has been analyzing the regulation of the nuclear gene
that encodes the -subunit of the mammalian mitochondrial F0F1-ATP synthase complex (ATPA) and
has identified several positive cis-acting regulatory
regions that are important for expression of this gene (5).
Furthermore, we have found that protein factor(s) present in HeLa
nuclei bind to these cis-acting regions. For example, we
have determined that the multifunctional regulatory factor, YY1, can
activate the ATPA promoter through an initiator-like element
(6). In addition, we have identified a protein(s), termed ATPF1, which
binds to the cis-acting region of the ATPA promoter which contains an E-box element (CACGTG) (5). This E-box
element is required for maximum activity of the ATPA
promoter in HeLa cells (5). A large family of regulatory proteins with a common structural feature, termed a basic helix-loop-helix motif (bHLH),1 bind to E-box elements (for
reviews, see Refs. 7 and 8). We were interested in isolating and
characterizing this regulatory factor(s), ATPF1.
In this paper, we describe the identification of the transcription factor, upstream stimulatory factor 2 (USF2), as a component of ATPF1. We also demonstrate that USF2 can trans-activate the ATPA gene through the E-box element. Furthermore, we show using a dominant-negative mutant (9) that USF2 plays an important role in the activation of the ATPA gene in vivo. USF was initially identified from HeLa nuclei and was shown to stimulate transcription from the adenovirus major late promoter through the core sequence CACGTG (10-12). Purification of USF activity from HeLa cells revealed two polypeptides of 43 and 44 kDa, termed USF1 and USF2, respectively (13, 14) Analyses of cDNA clones indicated that USF proteins are members of the Myc family of regulatory proteins characterized by a COOH-terminal bHLH-leucine zipper (zip) structure responsible for dimerization and DNA binding (13, 14). Other members of this family include the mammalian proteins, Myc (15), Max (16), Mad (17), Mxi1 (18), TFEB (19), and TFE3 (20). Transcriptional activation by USF1 and USF2 can be demonstrated both in vitro and in vivo, and USF proteins appear to play an important role in the transcriptional regulation of a number of different genes (see Ref. 14).
The following oligonucleotides were used in this
work. ATPA wild-type was
5-ACATCCGGGTGGCTGACT-3
containing the +23 to +46 bp
region of the bovine ATPA promoter (21). The wild-type E-box
element is underlined. ATPA mutE was
5
-ACATCCGGGCTTCGTGGGCTGACT-3
containing a mutated E-box element of
the ATPA promoter (5).
Polyclonal antiserum to USF1 and USF2 were obtained from Santa Cruz Biotechnology Inc., Santa Cruz, CA. Antibodies against E12/E2-2 were from Pharmingen, La Jolla, CA.
Oligonucleotide Screening of a Phage LibrarycDNA
clones encoding proteins with ATPA E-box binding activity
were isolated by screening a human HeLa cDNA expression library in
phage gt11 (Clontech; Palo Alto, CA). Approximately 106
phage plaques from the cDNA library were screened using a
concatamerized 32P-labeled double-stranded oligonucleotide
probe containing the ATPA E-box element by an in
situ filter detection method as described previously (22, 23). To
ensure the binding specificity of positive clones, filters were
rescreened with a concatamerized double-stranded oligonucleotide
containing a mutated E-site to detect clones that bound
nonspecifically. Only those positive plaques that did not bind to the
mutated site were analyzed further. The sequences of the
oligonucleotides containing the wild-type and mutated ATPA
E-sites are shown above.
Nuclear extracts were
prepared from cultured HeLa cells, as described (5, 23).
Self-complementary oligonucleotides containing the E-box of the
ATPA gene were used as a probe in mobility shift assays
after end filling with Klenow and [-32P]dATP. The DNA
binding reaction mixtures were performed at room temperature in binding
buffer (10 mM Tris, pH 7.5, 60 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, and 8% glycerol)
together with approximately 1-2 µg of HeLa cell nuclear extract, 2 µg of poly(dI-dC), and 0.1-0.5 ng of end-labeled oligonucleotide
(23). For supershift assays, 1-2 µl of antiserum was included in the
binding reactions before the addition of probe. For competition assays,
10 ng of cold oligonucleotide with the same sequence was used. Samples were analyzed by electrophoresis on 4% polyacrylamide gels using 1 × Tris borate-EDTA as the running buffer.
The ATPA-CAT
(chloramphenicol acetyltransferase) reporter constructs used in this
study have been described previously (5). Briefly, ATPA
(+23/+136 bp)/CAT contains the wild-type E-box element, ATPA
mutE (+23/+136 bp)/CAT contains a mutated E-box, and ATPA E (+49/+136 bp)/CAT has a deletion in the E-box element of the bovine ATPA promoter. All expression vectors were driven by
either the cytomegalovirus (CMV) immediate early promoter/enhancer or the SV40 promoter/enhancer. The human USF1 expression vector was a kind
gift from Dr. R. Roeder (24). The human USF2a and USF2b expression
cDNAs (25) and the mouse wild-type USF2a, mutant USF2a
N, and
mutant TDUSF2a
B expression cDNAs (9) were generous gifts from
Dr. Raymondjean and Dr. Sawadogo, respectively. The E12 expression
vector was generously provided by Dr. Kadesch (26).
HeLa cells were cultured as
described previously (5). HeLa cells were typically transfected with a
5-10 µg of the CAT reporter plasmid DNA together with various
concentrations of effector DNAs and 2 µg of a -galactosidase
expression plasmid (pCMV-
gal) by the calcium phosphate
coprecipitation method (5, 23). Cells were harvested approximately
48 h after transfection. CAT and
-galactosidase assays were
carried out as described previously (5, 6), and the CAT activities of
the extracts were normalized relative to the
-galactosidase
activities. Promoter activity values represent the average of at least
three separate transfections of three plates each.
We have determined previously that an E-box element (CACGTG) is required for maximum expression of the ATPA gene when tested in transient expression assays in HeLa cells (5). We have also determined that a protein factor(s) present in HeLa nuclei, termed ATPF1, binds to this E-box (5). To identify cDNAs that encode ATPF1, we screened a human HeLa cDNA expression library using a multimerized oligonucleotide containing the ATPA E-box as a probe. From this screening, we isolated two phage clones that hybridized to this probe but did not hybridize to a multimerized oligonucleotide containing a mutation in the ATPA E-element. These clones were plaque purified, and the nucleotide sequence of a portion of each cDNA was determined (23). Analyses of these sequences revealed that one cDNA encoded the regulatory factor, E12 (26) and the other cDNA encoded the transcription factor, USF2 (14, 25). Both E12 and USF2 have been shown to bind to E-box elements and to regulate the expression of a number of genes.
USF2 Is Present in ATPA-HeLa ComplexesTo determine if E12 or
USF2 is a component of the ATPA-protein complexes in HeLa
cells, we examined the effect of specific antibodies raised against
these proteins using electrophoretic mobility shift assays. As shown in
Fig. 1, we found that addition of antibodies against
USF2 resulted in a supershift in the mobility of the
ATPA-HeLa complexes. In contrast, antibodies against
E12/E2-2 had no effect on the mobility of the ATPA-HeLa
complexes. Similarly, nonimmune serum also did not affect the mobility
of the ATPA-HeLa complexes. However, antibodies specific for
USF1 could also supershift the mobility of the ATPA-HeLa
complexes. To verify that the effects of the antibodies to USF2 and
USF1 were specific, we added an unlabeled oligonucleotide containing
the ATPA E-box as a competitor. These experiments
demonstrated that the supershifted USF-HeLa-ATPA complexes
were effectively inhibited by an excess of competitor oligonucleotide
(Fig. 1). These results indicate that USF2 and USF1 are components of
the endogenous ATPA E-box binding activity in HeLa nuclear
extracts.
USF2 Trans-activates the ATPA Promoter
The ability of USF2
and E12 proteins to function as transcriptional modulators of the
ATPA promoter was next analyzed using transient transfection
assays. These experiments demonstrated that cotransfection of the
ATPA/CAT reporter construct together with an expression
vector encoding USF2 resulted in a increased expression of the
ATPA promoter in a dose-dependent fashion (Fig. 2). In contrast, there was little or no effect of
cotransfecting E12 on expression of the ATPA/CAT construct
(Fig. 2). These results suggest that USF2, but not E12, can act as a
transcriptional activator of the ATPA promoter in
vivo.
To verify that trans-activation of the ATPA
promoter by USF2 was occurring through the E-box element, we examined
the response of reporter plasmids that contain either a mutation in the
ATPA E-box (ATPA mutE/CAT) or a deletion of the
E-box (ATPA E/CAT). As shown in Fig. 3, we
found that mutation of the E-box in the ATPA promoter
dramatically reduced trans-activation by USF2. However, especially at high concentrations of USF2, there was still some increased expression of the ATPA promoter in the mutant
construct. Similar data were obtained using an ATPA/CAT
construct that lacked the E-box element (Fig. 3). These results
indicate that USF2 can activate the ATPA promoter through
the E-box element as a well as some other sequence(s).
USF2a Isoform Also Activates the ATPA Promoter
Alternatively
spliced forms of USF2 resulting from the presence or absence of the
fourth exon have been described (9, 25). The previous
trans-activation experiments were carried out using the USF2
isoform that lacks the fourth exon, termed USF2b. Experiments were also
performed to examine the trans-activation potential of the
USF2a isoform on the ATPA promoter. The results of these experiments indicated that USF2a can also activate the ATPA
promoter, although not quite as effectively as USF2b (Fig.
4). Furthermore, the concentration of USF2a required to
achieve maximum activation of the ATPA promoter was lower
than that of USF2b (and higher concentrations were inhibitory). We also
determined that trans-activation of the ATPA
promoter by USF2a required the amino-terminal
trans-activation domains since a construct containing only
the bHLH-zip domains (USF2aN; 9) did not activate (Fig. 4). These
results suggest that USF2a and USF2b are activators of the
ATPA gene in vivo.
A Dominant-negative Mutant Demonstrates the Role of USF2 in Activation of the ATPA Gene in Vivo
The next series of
experiments were carried out to assess the direct involvement of USF2
in activation of the ATPA promoter in vivo since
the possibility remains that other bHLH-zip proteins more directly
stimulate this promoter. The strategy used relied on the use of a
dominant-negative mutant of USF2, TDUSF2aB. This mutant lacks the
basic region required for DNA binding but can still dimerize with
endogenous USF1 or USF2 proteins or with itself (9, 25). Since the
binding of USF to cognate sites requires dimers possessing two
functional DNA binding domains (13, 14, 28), the sequestration of
endogenous USF by ectopically expressed TDUSF2a
B allows the
identification of transcriptional processes that are directly dependent
upon USF2. The results of these experiments demonstrated that
expression of TDUSF2a
B significantly reduced the level of activation
of the ATPA promoter caused by ectopically coexpressed USF2a
(Fig. 5). These cumulative data argue forcefully that
the binding of USF2 to the E-box element in the ATPA
promoter is involved directly in the activation of the ATPA
gene in vivo.
Our laboratory has been analyzing the regulation of the nuclear
gene that encodes the -subunit of the mammalian mitochondrial F0F1-ATP synthase complex (ATPA).
Using a deletion analysis, we have identified several positive
cis-acting regulatory regions in the promoter of this gene
(5). By site-directed mutagenesis, we have determined that an E-box
element (CACGTG) located within one of these cis-acting
regions is required for maximum expression of the ATPA gene
(5). E-box elements have been found to be critical for the expression
of a number of different genes. A large family of transcription factors
with a common structural feature, termed the bHLH motif, binds to E-box
elements (for reviews, see Refs. 7 and 8). A subfamily of bHLH proteins
also contains a leucine zipper for additional dimerization potential
(7). In this paper, we demonstrate that the transcription factor, USF2, a bHLH-zip-containing protein, binds to the E-box sequence in the
ATPA promoter. Furthermore, we show that the binding of USF2 to the ATPA E-box activates this promoter. In addition, we
demonstrate that endogenous USF2 plays an essential role in the
activation of the ATPA gene. Evidence includes the
following. Specific antibodies against USF2 (or USF1) revealed that USF
proteins are components of the protein(s) in HeLa nuclear extracts
bound to the ATPA cis-acting region. Expression of wild-type
USF2 proteins had a stimulatory effect on the activity of the
ATPA promoter through the E-box sequence. These results
demonstrate that USF2 proteins functionally interact in living cells
with the E-box of the ATPA promoter. However, it is still
possible that ectopically expressed USF2 proteins supplant other
E-box-binding proteins that normally participate in the activation
process. The use of a dominant-negative mutant, TDUSF2a
B,
demonstrated that functional USF2 oligomers are important for
activation of the ATPA gene in vivo. This mutant
protein is able to dimerize through its HLH-zip motif but cannot bind
DNA because of a deletion in the basic region (9, 25). Consequently, in
cells transfected with the TDUSF2a
B mutant, functional USF2 oligomers are expected to be progressively replaced by defective oligomers unable to interact with the ATPA E-box. This
titration of functional USF oligomers by TDUSF2a
B resulted in a
decrease in the level of activation of the ATPA promoter
caused by ectopically coexpressed USF2a. The use of such
dominant-negative mutants has revealed previously that USF2 is a
regulator in vivo of the liver-type pyruvate kinase gene in
response to glucose (25) and of transcriptional activation by the
varicella zoster virus immediate-early protein IE62 (9).
USF activity in HeLa cells was initially described as a complex consisting of two polypeptides of 43 and 44 kDa, termed USF1 and USF2, respectively (13, 14, 31). Later, an additional level of complexity of USF2 was described since differential splicing generates mRNAs encoding polypeptides of 44 kDa (USF2a) and 38 kDa (USF2b) (9, 25). USF2b lacks an internal 67-amino acid domain present in USF2a because of splicing out of the fourth exon in the primary transcript. It has been reported that the trans-activation potential of the 38-kDa USF2b alternative spliced form depends on the promoter context (29, 30). For example, USF2b was found to be approximately three to four times less active than USF2a or USF1 on a minimal promoter depending on oligomerized USF binding sites (29). However, in the context of the liver-specific pyruvate kinase promoter, USF2b exhibited a trans-activation potential similar to that of USF2a or USF1 (29). In this paper, we demonstrate that USF2b is as effective (or slightly more effective) an activator of the ATPA promoter as USF2a.
The expression of both nuclear and mitochondrial genes that encode
proteins of the mammalian mitochondrial oxidative phosphorylation system changes in response to a number of conditions including cellular
proliferation, oxygen tension, neoplastic transformation, hormonal
stimulation, development, and differentiation (for reviews, see Refs. 3
and 4). The mechanism(s) by which a cell regulates and coordinates the
expression of these genes to meet cellular energy demands is not
understood. A number of transcription factors have now been identified
which are important for the expression of one or more nuclear genes
that encode proteins of the mammalian mitochondrial oxidative
phosphorylation system. Examples include: USF2 (this work), NRF-1 (also
termed -Pal; 32-34), GABP (34-37), OXBOX and REBOX-binding
factor(s) (38), Sp1 (39), and YY1 (6, 39). The question still remains
as to whether any of these regulatory proteins can respond to the
energy needs of a cell and, if so, by what mechanism(s). One
possibility is that the activity of these factors is affected by the
redox potential of the cell. Interestingly, it has been reported that
the DNA binding and activation potential of USF1 is strongly affected by redox changes (40). Furthermore, the DNA binding and
trans-activation potential of GABP (41) and the binding
affinity of the REBOX-binding protein(s) (38) have also been shown to
be sensitive to reducing conditions. It is therefore possible that
proteins such as USF, GABP, and the REBOX-binding factor(s) may
function as important links between the redox state of the cell and the
regulation of nuclear and mitochondrial gene expression.
We thank Drs. R. Roeder for the generous gift
of pCXUSF1; M. Sawadogo for pSVUSF2a, pSVUSF2aN, and pSVTDUSF2a
B;
M. Raymondjean for pCMVUSF2b and pCMVUSF2a; and T. Kadesch for pSVE12.
We also thank S. Anderson and W. Joiner for help with the transfections and CAT assays.