(Received for publication, June 14, 1996, and in revised form, November 18, 1996)
From the Laboratories of Biochemistry, Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6047
The mouse cytochrome oxidase (COX) Vb promoter
contains three sequence motifs with partial or full consensus for YY-1
and GTG factor binding and a CArG box, located between positions 480 and
390. Individually, all three motifs stimulated transcription of
the TKCAT promoter, and bound distinctly different proteins from the
liver and differentiated C2C12 nuclear extracts. Collectively, these
motifs, together with the downstream flanking sequence,
378 to
320,
suppressed the transcription activity of heterologous promoters,
thymidine kinase-chloramphenicol acetyltransferase (TKCAT) and
COXIV/CAT. The transcription activities of both TKCAT and COXIV/CAT
constructs were induced 3-4-fold during induced myogenesis of C2C12
cells. The downstream CArG-like motif binds transcription factor YY-1,
while the upstream YY-1-like motif binds to a yet unidentified factor.
Co-expression with intact YY-1, but not that lacking the DNA binding
domain suppressed the transcriptional activity. Mutations targeted to
the CArG-like motif abolished the suppressive effect of the negative
enhancer and the inducibility of the promoter during myogenic
differentiation. Our results suggest that the activity of the negative
enhancer may determine the level of expression of the COX Vb gene in
different tissues.
Cytochrome c oxidase
(COX),1 the terminal oxidase of the
mitochondrial electron transport chain, is a major site of regulation of oxidative metabolism (1, 2). The enzyme from mammalian tissues
contains 13 subunits, 10 of which are encoded by nuclear genes and the
3 largest catalytic subunits by the mitochondrial genes (3, 4). We have
recently characterized the mouse genes for nuclear encoded subunits IV,
Vb, and VIII (H) of the COX complex (5-9). The expression of the COX
Vb gene is particularly interesting, since it appears to involve a
large combinatorial array of DNA elements and protein transactivators
located at both the distal, as well as immediate upstream regions of
the transcription initiation sites (7, 9, 11, 12). Additionally, the
COX Vb gene encodes multiple mRNAs that exhibit 5 heterogeneity
due to transcription initiation at multiple initiator sites (5,
10).
The immediate upstream region of the mouse COX Vb gene consists of a
TATA-less GC-rich sequence characteristic of a number of
"housekeeping" genes. The basal promoter region has been mapped to
the 8 to +40 region of the gene, which contains several
protein-binding motifs. At least five of these protein-binding motifs
correspond to discrete transcription initiation sites (10). Initiation of RNA mapping to the +1 position of the promoter has been shown previously to depend upon a YY-1-binding initiator motif (12). Recently
we have found that the GA-binding protein factor-binding Ets
repeat sequence GTTCCCGGAAG at the +17 to +26 position also functions as an independent initiator for transcription of two clusters
of RNAs mapping to the +18/19 and +23/24 regions (10).
Although the COX Vb gene is constitutively expressed, its level of
expression varies in different tissues. C0X Vb mRNA is 10-20-fold
more abundant in the heart and kidney as compared to the liver.
Furthermore, the COX Vb mRNA level increases markedly during
induced differentiation of C2C12 myoblasts into myotubes. In the
present study we have investigated the contributions of various
protein-binding motifs from the 5 distal region of the promoter in the
overall transcriptional rates of the promoter. We have identified a
negative regulatory region, which encompasses a YY-1-like (YY-1
) and
CArG-like (CArG
) consensus motifs flanking a GTG element (13), a novel
enhancer found in the neu oncogene promoter. Results
presented in this paper show that factor YY-1 binding to the downstream
CArG
motif is important for the activity of the negative enhancer. Our
results therefore suggest that in addition to the multiple positive cis
acting elements (7-11), a negative enhancer region is involved in the
complex regulatory circuit of the COX Vb gene expression.
The 5 deletion promoter constructs were
derived from the
574 CAT DNA (5), which contains the mouse COX Vb
sequence
574 to +40, cloned in pCAT basic plasmid (Promega Biotech
Corp.) at the HindIII and blunt ended XbaI sites.
A series of 5
deletions of the
574 CAT DNA were generated by the
exonuclease III digestion of linearized plasmid, using the "Erase a
Base System" (Promega Biotech Corp.). The authenticity of all
mutations were determined by dideoxy sequencing (14).
The following synthetic double-stranded DNA oligomers were used for
cloning in different plasmid vectors, for PCR amplification of promoter
regions, or for competition in gel mobility shift experiments. These
double-stranded DNAs were synthesized either with 5 HindIII
and 3
SalI linkers (YY-1
, GTG, and CArG
) or 5
HindIII and 3
XbaI linkers (Mut1 and Mut2):
upstream YY-1-like motif (sequence
480 to
461), referred to as
YY-1
, 5
-agcttCAGAGTGGAGGGAATTg-3
; GTG element (sequence
456 to
431), 5
-agcttACTGTGGGGGGGGGGGTGTACg-3
; downstream CArG-like
motif (sequence
412 to
390), referred to as CArG
,
5
-agcttGGGCCATTGCAACAATTTGGTAACAACTAAGCg-3
; mutated YY-1 (+1
position) at the initiator site (sequence
8 to +31), referred to as
MUT1,
5
-tcgacGTCCCGCC
ATCTTGCTCAGCCTGTTCCCGGAAGTGCAT-3
; mutated YY-1 (+2 and +3 positions) at the initiator site (sequence), referred to as MUT2,
5
-tcgacGTCCCGCCC
CTTGCTCAGCCTGTTCCCGGAAGTGCAT-3
.
The 5
distal sequences
481 to
390 and
481 to
320 were amplified by
PCR using a sense primer containing HindIII and appropriate antisense primers containing SalI linker sequences. The PCR
products and also the YY-1
, GTG, and CArG
DNA sequences listed above were cloned in the HindIII and SalI sites of
pTKCAT plasmid, where the reporter gene expression is driven by the
thymidine kinase promoter. The blunt-ended
481 to
320 sequence was
also cloned in another heterologous promoter,
142 COXIV/CAT (7) at
the blunt-ended HindIII site, which is located at the 5
upstream most position of the promoter. A clone carrying the insert DNA in the right orientation was selected by DNA sequencing. Mutations at
the initiator region YY-1 (Mut1 and Mut2) were introduced in the
520/+40CAT
481/+40CAT promoter constructs by PCR amplification using a sense primer (sequence
480 to
460) containing a
HindIII linker and antisense Mut1 or Mut2 primers and the
resulting DNA was cloned in the HindIII/XbaI
sites of pCAT basic plasmid. Mutations at the internal sites (CArG
M1
and CArG
M2) were introduced by overlap PCR (15).
Mouse 3T3 fibroblast cells and COS cells were grown as
described before (12). Human hepatoma Hep 3B cell line was maintained in minimum essential medium with balanced Earle's salt, 10% fetal bovine serum, 2 µM glutamine, and 50 µg/ml gentamycin.
Mouse myoblast C2C12 cells were grown and induced to differentiate into
multinucleated myotubes as described recently (9). Cells were
transfected in replicate plates with CsCl gradient-purified CAT test
plasmids (5-10 µg/10-cm dish) and 2 µg/plate -galactosidase
expression plasmid, pCH110 (16), by the
Ca3(PO4)2 co-precipitation method (17).
In cotransfection experiments, 5 µg of wild type or mutant forms of
CAT reporter plasmids were used along with varying concentrations of
YY-1 cDNA expression plasmids (18). The total DNA concentration in
all cases was adjusted to 20 µg with CMV DNA as the filler. Cells
were harvested about 48 h post-transfection, and cell extracts were assayed for CAT activity using [14C]chloramphenicol
(19). The CAT activities were normalized to the -galactosidase
activities of the extracts. In cases where the expression plasmid DNAs
interfered with the
-galactosidase expression, CAT activities were
normalized to the protein contents of the extracts.
Nuclear extracts from mouse liver was prepared essentially as described by Gorski et al. (20). Nuclear extracts from cultured cells (3T3, COS, 3-1 pre-B, and C2C12 myotube) were prepared according to Dignam et al. (21). Binding assays were carried out essentially as described by Singh et al. (22), using 5-10 µg of nuclear extract or 200 ng of purified YY-1 factor (23) and 0.1-0.5 ng (5000-10,000 cpm) of 32P-end-labeled DNA probe. For competition experiments, stated amounts of unlabeled double-stranded DNA were preincubated with nuclear extracts or purified proteins for 10 min before the addition of labeled probes. DNA-protein complexes were resolved by electrophoresis on 4% polyacrylamide gels in a Tris-glycine buffer (50 mM Tris, 400 mM glycine, and 2 mM EDTA) or 0.25 × TBE (1 × TBE: 89 mM Tris base, 89 mM boric acid, and 2 mM EDTA, pH 8.0) buffer systems.
Methylation Interference AnalysisThe antisense strand of
the 90-bp DNA (sequence 480 to
390) was end-labeled using
[
-32P]ATP and T4 polynucleotide kinase. The labeled
DNA (2 × 106 cpm) was partially methylated using
dimethyl sulfate (24) and used as a probe for binding bacterially
expressed, purified YY-1 as described before (12). The protein-bound
complex was resolved by electrophoresis on a 4% polyacrylamide gel,
and the piperidine cleavage patterns of protein-bound and free DNA
probe were compared as described previously (24).
RNA from different mouse tissues and monolayer cell cultures were isolated and subjected to Northern blot hybridization using moderate stringency conditions as described before (6). The RNA bands were quantitated by scanning through an Agfa scanner and NIH Image analysis software.
Northern blot hybridization of RNA from different
mouse tissues showed that the level of COX Vb mRNA in the heart and
kidney is 10-20-fold higher than those detected in the liver, brain, Ehrlich ascites cells, or 3T3 fibroblasts. The levels of ubiquitously expressed COXIV mRNA, on the other hand, is known to vary only marginally in these tissues (6). The hybridization pattern in Fig.
1A is consistent with the S1 nuclease
protection data showing differences in COX Vb mRNA levels in
different tissues (10). Moreover, the level of Vb mRNA increased
nearly 5-fold during induced differentiation of murine C2C12 myoblasts
into myotubes (Fig. 1B). Thus, although the COX Vb mRNA
is expressed ubiquitously, its level of expression varies markedly in
different tissues.
Identification of a Negative Enhancer Sequence in the 5
Fig. 2 shows the
nucleotide sequence of the 574 to +40 region of the mouse COX Vb
gene, and potential protein-binding motifs both at the immediate
upstream and distal regions of the promoter. Previous studies (5, 10,
11) showed that the
319 to +20 region of the promoter contains a
number of sequence motifs that act as transcription activators, and
initiators. In the present study, the positive or negative effects of
the 5
distal region of the COX Vb gene was studied by a similar 5
deletion analyses.
Fig. 3 shows that the 574/+40 CAT DNA exhibits only
20% CAT activity in 3T3 fibroblast cells as compared to the activity of the previously analyzed
319/+40 CAT DNA construct. Similar 4-5-fold lower activities were also obtained in COS cells, Hep 3B
cells, and uninduced C2C12 myocytes. Progressive 5
deletions to
520
and
481 positions of the promoter had little effect on the
transcriptional activity in all four cell types, while deletions to
sequence
406 resulted in about 5-fold higher activity in all cells
tested. As shown in the illustration at the bottom of Fig. 3, deletion to nucleotide
406 eliminates the upstream YY-1
and GTG
motifs, in addition to disrupting the CArG
motif. Additionally, internal deletion of sequence
378 to
320 from the
520/+40CAT DNA
showed activity near 100% in 3T3 and C2C12 myoblasts (results not
shown). These results suggest that the
481 to
320 region of the
promoter containing at least four different potential protein-binding motifs functions as a negative enhancer in different cell lines tested.
To evaluate the activity of the negative regulatory region during
muscle cell differentiation, undifferentiated mouse C2C12 myoblasts
were transfected with various 5 deletion CAT constructs and the
relative CAT activities in undifferentiated myoblasts and
differentiated myotubes were assayed. Results of transient transfection
in Fig. 4 show that the activities of the
520 and
481 deletion constructs are induced about 3.5-fold in differentiated C2C12 myocytes as compared to undifferentiated myoblasts. The results
also show that deletion clones
406CAT,
319CAT and
174CAT exhibit
nearly 100% activity in both differentiated and undifferentiated myocytes. The internal deletion clone
(
378/
320) showed a similar activity in the range of 99-100% in both undifferentiated and differentiated myocytes. These results show that the negative modulatory effect of the enhancer region is reduced severalfold during
myogenesis, and provide a rational basis for the observed 5-6-fold
higher mRNA levels in induced C2C12 myotubes (presented in Fig.
1B). Furthermore, the results show that in addition to the
481 to
406 sequence, the downstream flanking sequence,
378 to
319, is also necessary for the maximal effectiveness of the negative
enhancer.
Effects of the Negative Enhancer Region on the Activities of Heterologous Promoters
An examination of nucleotide sequence from
this region (see Fig. 2) shows the presence of a YY-1-like motif at
position 480 to
464 (YY-1
), a GTG element at positions
456 to
431, and a CArG-like protein-binding motif (CArG
) at position
412
to
390. The effects of these individual protein-binding motifs on the
transcription activity of a heterologous promoter, pTKCAT was tested in
3T3 fibroblasts, Hep 3B cells, and C2C12 myocytes. It is seen (Fig.
5A) that all three elements individually
function as transcription activators, although the relative extent of
stimulation in different cells varies markedly. For example, all three
sequence motifs induced transcription by 10-20-fold in both 3T3 and
C2C12 cells. In Hep 3B cells, however, both the YY-1
and CArG
motifs were minimally active, while the GTG sequence motif yielded over 15-fold increased activity. Interestingly, the 90-bp fragment (sequence
481 to
390) containing all three sequence motifs in the same order
as they exist in the COX Vb promoter, exhibited significantly lower
transcription stimulation in all three cell lines as compared to
activities obtained with the individual sequence motifs (Fig.
5A). Moreover, the activity of the TKCAT plasmid was reduced
3-4-fold in C2C12 cells when the sequence
481 to
320 of the COX Vb
gene was cloned upstream of the TK promoter. The same construct,
however, showed a marginal reduction in activity in 3T3 and Hep 3B
cells. In support of the internal deletion data in Fig. 5A,
these results suggest that in addition to sequence
480 to 390, the
downstream sequence up to
320 is also required for the effectiveness
of the negative enhancer activity. Results also suggest that three
different activator motifs, when placed in a tandem array with
appropriate downstream flanking sequence up to
320, function as a
transcription suppressor in both the COX Vb promoter as well as in
heterologous promoters.
Since the negative enhancer effect of the COX Vb promoter is reduced
during myogenesis (Fig. 4), we investigated the generality of this
effect using heterologous COXIV and TKCAT promoters, both of which are
ubiquitously expressed with no significant muscle predominance. Results
in Fig. 5B show that both TKCAT and COXIV/CAT promoters show
nearly similar transcription activities in both uninduced myoblasts and
induced myotubes. Constructs containing the COX Vb 481 to
319
sequence at the 5
upstream regions, however, show 5-6-fold reduced
activities in undifferentiated C2C12 myoblasts. These results further
confirm that the
481 to
319 region is involved in conferring the
negative enhancer activity, and that the effectiveness of the negative
enhancer is reduced during induced myogenesis.
Previous studies have shown that the CArG
sequence motif from the muscle-specific creatine kinase promoter binds
to transcription factors YY-1 and SRF, exclusive of each other (25,
26). The 90-bp DNA from the negative enhancer region of COX Vb gene
contains two potential YY-1-binding sites: the YY-1 site at the
480
to
461 position and a CArG
motif at the
412 to
390 position. We
have therefore tested both the YY-1
and CArG
motifs for binding to
bacterially expressed purified YY-1 factor. As shown in Fig. 6A, both of the motifs failed to bind any
detectable amount of the factor when the incubations were carried out
at room temperature, in the presence of added dI-dC. Surprisingly,
however, the 90-bp DNA probe containing all the three elements bound to
purified YY-1 factor under these binding conditions (see Fig.
6A). The gel pattern showed one major and a very minor slow
migrating complex, which probably represent the single and double
binding sites, respectively. The results also show that under
conditions that detect low affinity binding (incubation at 4 °C,
with added dG-dC) the CArG
motif, but not the YY-1
DNA bound to
purified YY-1 (Fig. 6B). In support of the gel mobility
shift data, methylation interference analysis with the YY-1-bound 90-bp
DNA (Fig. 6C) showed partial protection at nucleotides
403,
408, and
409, demonstrating that a region of the CArG
sequence motif is responsible for binding. These results suggest an
interesting possibility that the formation of high affinity YY-1
complex with the 90-bp DNA requires some of the upstream sequences in
addition to the putative CArG
region.
The gel mobility shift pattern in Fig. 7 shows that the
90-bp DNA probe forms six different complexes with the nuclear extract from differentiated C2C12 myotube, two of which comigrated with complexes formed with bacterially expressed YY-1. Consistent with the
YY-1 binding affinities of individual motifs in Fig. 6, the YY-1 motif
failed to compete with either of the complexes that comigrated with the
YY-1-bound complexes, while the CArG
DNA competed with complex 1 but
not with complex 3. However, this motif competed effectively with
complexes 4, 5, and 6. Surprisingly, the GTG motif also competed
effectively with complex 1 in addition to complexes 4-6. Finally, the
YY-1-binding motif from the Igk 3
enhancer region (23) competed with
both of the complexes that comigrated with the YY-1-bound complexes, in
addition to complexes 4-6. Although not shown, the 90-bp DNA
effectively competed out all of the complexes. These results suggest
the possibility that some of the slow migrating complexes are higher
order complexes formed due to protein-protein interaction.
The YY-1 factor binding to the 90-bp DNA was further ascertained by
including a YY-1-specific antibody in the mobility shift experiment. As
shown in Fig. 8A, affinity-purified antibody
caused a supershift of the complex obtained with bacterially expressed purified YY-1, while preimmune serum had no effect on DNA-protein complex formation. Similarly, the formation of YY-1-bound complex with
the C2C12 nuclear extract was affected by the antibody, although the
supershift of the complex is not readily apparent because of the
masking effect due to the presence of a number of slow migrating
complexes in the region (Fig. 8B). The electrophoretic patterns in Figs. 7 and 8 show significant differences mainly due to
the use of different buffers in these two experiments. The resolution
of DNA-bound complexes was better with the Tris-glycine buffer system
in Fig. 7, while the 0.25 × TBE buffer used in Fig. 8 was better
suited for the antibody reactivity.
Protein Binding Patterns with the Individual Sequence Motifs from the Negative Enhancer Region
Since the YY-1, GTG, as well as the
CArG
sequence motifs from the negative enhancer region individually
function as transcription activators in a heterologous promoter, the
patterns of protein binding with these motifs was compared using
nuclear extracts from liver and C2C12 myotubes. Fig.
9A shows that the GTG motif efficiently bound
to the liver nuclear extract to form three different complexes, which
were effectively competed by excess unlabeled GTG motif, but not by the
YY-1
or CArG
motifs. The nuclear extract from differentiated myotube,
on the other hand, formed a complex that was competed by excess
unlabeled GTG DNA, but not by the CArG
or YY-1
DNAs. In some lanes,
an additional nonspecific complex (NS) was also observed,
which was not competed by excess competitor DNAs. Although the
complexes obtained with the myotube extract appear to co-migrate with
complex 1 formed with the liver extract, currently the precise protein
compositions of these complexes remain unclear.
Fig. 9B shows the protein binding patterns of the CArG
motif with the liver and myotube nuclear extracts. The CArG
DNA formed two to three closely migrating complexes with the liver extract, all of
which were competed by excess unlabeled CArG
DNA but very poorly by
the YY-1
and GTG DNAs. The nuclear extract from C2C12 myotube, on the
other hand, formed a single complex that co-migrated with the fastest
migrating complex obtained with the liver extract. Additionally, the
complex formed with the myotube extract was competed with excess CArG
DNA but not with the YY-1
and GTG DNAs. The major complex with the
myotube extract migrated differently from the complex formed with
bacterially expressed YY-1 (Fig. 9B); in addition, it is not
supershifted by the YY-1 antiserum (results not shown), suggesting that
it does not represent a YY-1-bound complex. Finally, as shown in Fig.
9C, the YY-1
DNA motif formed two weak complexes with both
liver nuclear extract and myotube extract that were self-competed, but
not with the CArG
or GTG DNAs. The slow migrating complexes with the
liver and myotube extracts showed different migration on the gel,
suggesting that different tissue-specific proteins may be involved in
the formation of these complexes. These results together suggest
tissue-specific differences in protein binding patterns with all of the
three DNA motifs from the negative regulatory region.
Since YY-1 is known to be involved both in positive as
well as negative modulation of transcription activity with different promoters (27-34), the effects of YY-1 overexpression on the activity of the COX Vb promoter was studied. We have addressed the role of the
initiator site YY-1-binding site and also distal upstream CArG motif,
which also binds YY-1, on the activity of the negative enhancer. The
520/+40CAT construct (
520/CAT) and also constructs carrying
mutations at the initiator YY-1 site (
520/MUT1 and
520/MUT2) were
co-transfected with CMV/YY-1 expression construct in C2C12 myoblasts
and the transcriptional activities were assayed. As shown in Fig.
10A, co-expression with 1 and 2 µg of YY-1
cDNA construct caused about 60% and 84% inhibition, respectively.
Both MUT1 and MUT2 constructs yielded essentially similar patterns of
inhibition, although the extent of inhibition was about 10% lower as
compared to the wild type construct.
The role of negative enhancer region in the YY-1-mediated transcription
down-regulation was further ascertained using heterologous promoter
TKCAT and COXIV/CAT constructs, which lack YY-1-binding sites. Results
in Fig. 10A show that YY-1 had negligible to marginal (0.7-10%) inhibitory effect on the transcription of both TKCAT and
COXIV/CAT constructs. Activities of constructs containing the COX Vb
520 to
320 sequence in both cases were inhibited by YY-1 in a
concentration-dependent manner. A control experiment in
Fig. 10B shows the levels of immunodetectable YY-1 in
untransfected 3T3 cells and cells transfected with 1-10 µg of YY-1
construct. The Western blot data show that the level of YY-1 in
untransfected control cells is below the limit of immunodetection,
while immunodetectable protein bands are seen in cells transfected with
1, 5, and 10 µg of YY-1 cDNA. These results provide evidence that
the
520 to
319 region of the promoter contains a negative enhancer
activity, which is modulated by YY-1.
To ascertain the role of the CArG motif in the YY-1-mediated
down-regulation of transcription, we generated two mutant constructs,
481/CArG
M1 and
481/CArG
M2. Mutations were targeted to the CArG
region of the
481/MUT1CAT plasmid, which has mutated YY-1-binding site at the +1 position. As shown in Fig. 10C, both M1 and
M2 mutant forms of CArG
DNA motifs, whose sequences are shown below
the gel shift pattern, failed to compete significantly with CArG
DNA
binding to YY-1. In direct binding assays (Fig. 10C), the
CArG
M1 DNA bound to YY-1 at a vastly reduced level, while the CArG
M2 DNA did not bind to YY-1. Transcriptional activities of the parent and
mutant constructs were compared by transfection in C2C12 myblasts as
well as differentiated myotubes. As expected, the parent plasmid construct,
481/MUT1 exhibited about 8-fold higher transcriptional activity in induced myotubes as compared to myoblasts (see Fig. 10D).
481/CArG
M1 and
481/CArG
M2 constructs yielded
9-11-fold higher activities in uninduced myoblasts as compared to the
481/MUT1 construct, suggesting that mutations in the CArG
sequence
drastically reduced or abolished the negative enhancer function of the
319 to
481 sequence of the promoter. Additionally, in C2C12
myoblasts, co-transfection with 1 µg of CMV/YY-1 cDNA construct
inhibited the activity of the
481/MUT1 construct by over 70%, while
similar co-transfections had marginal inhibitory effects (10-18%) on
the activities of the
481/CArG
M1 and
481/CArG
M2 constructs.
Surprisingly, both of these mutant constructs showed 70-80% reduced
transcription activity in induced C2C12 myotubes, suggesting that in
addition to functioning as a component of the negative enhancer, this
region might also function as a transcription enhancer under some
cellular conditions. These results provide evidence that the
YY-1-binding CArG
region of the negative enhancer plays an important
role in modulating the transcriptional activity of the promoter.
The specificity of the YY-1-mediated inhibition of transcription was
further ascertained using various YY-1 deletions expressed as Gal4
fusion proteins. Recently, Bushmeyer et al. (18) have systematically mapped various functional domains of YY-1 using the Gal4
promoter system. The Gal4 fusion constructs (18) were co-expressed with
the COX Vb 520/+40CAT construct in 3T3 cells. Results presented in
Fig. 11A show that Gal4-YY-1 inhibits the transcription activity by about 80%, indicating that it is as effective as the parent YY-1 cDNA construct in down-regulating the
COX Vb promoter activity. The
(16-99)Gal4-YY-1 construct (deleted
amino acid residues 16-99), which contains almost all the functional
domains, excepting the transcription activation domain, inhibits the
activity by about 85%. The 1-256/Gal4-YY-1 construct (deleted
residues 257-414), which lacks the DNA binding as well as
transcription suppressor domains (18), has no inhibitory effect,
suggesting that DNA binding is important for its negative modulatory
activity. The construct 201-414/Gal4-YY-1 (deleted residues 1-200),
which contains both intact DNA binding domain and transcription
repressor domain inhibits the activity at a rate comparable to the
intact Gal4-YY-1 construct. The Western blot presented in Fig.
11B shows that the various Gal4-YY-1 fusion proteins are
indeed expressed in C2C12 myoblasts under the co-expression conditions
described in Fig. 11A. As reported previously (18), the YY-1
N-terminal domain fusion protein, 201-414/Gal4-YY-1, shows an
abnormally faster migration on SDS-polyacrylamide gels in comparison to
other Gal4 fusion proteins. These results demonstrate that both DNA
binding and transcription repressor domains of the YY-1 are essential
for the negative enhancer activity of the protein.
Subunit Vb of the mammalian COX is classified as a ubiquitously
expressed subunit of the enzyme complex (2). Results presented in this
study, along with previous results (5, 10), demonstrate that the
relative abundance of the COX Vb mRNA in different tissues varies
markedly; the mouse kidney and heart contain about 10-20-fold higher
level of the mRNA than the liver (Fig. 1A). Furthermore, a 4-6-fold higher level of the mRNA is detected during induced differentiation of C2C12 myocytes. Our results suggest that widely varying levels of Cox Vb mRNA transcription in different tissues are due to the presence of an unusual negative regulatory element at
the 5 distal region of the promoter, whose suppressor activity appears
to vary in different cell types and during induced myogenesis. The
activity of this negative enhancer is regulated by transcription factor
YY-1 in conjunction with other protein factors that bind to this region
in a sequence- and tissue-specific manner.
Results of deletion analyses (Figs. 3 and 4) show that the negative
regulatory region spans sequences 481 to
321, and consists of two
distinct domains. A region between
481 and
390 contains three
different sequence motifs showing partial consensus to the YY-1-binding
site (NF-E1
), a GTG enhancer element, and a CArG-like element (CArG
).
The second region between
390 and
321 contains an Ets sequence
motif, which binds to an unknown factor from 3T3, liver, and C2C12
nuclear extracts (results not shown). All three upstream region
sequence motifs, YY-1
, GTG, and CArG
, stimulated transcription of the
TKCAT promoter in 3T3, Hep 3B, and C2C12 cells at different levels
(Fig. 5). All three DNA motifs exhibited distinctly different patterns
of protein binding with nuclear extracts from liver and C2C12 myotubes
(Fig. 9), suggesting that the cell- or tissue-specific binding patterns
may reflect upon the differing levels of transcription activation with
these activator motifs in different cells. The three motifs placed
together in a tandem array, as they exist in the COX Vb promoter,
nearly completely reversed the transcriptional activity obtained with
the individual sequence motifs. The transcriptional down-regulation of
the heterologous promoter was more pronounced when further downstream
sequences up to
320 were linked to the TKCAT promoter or the mouse
COXIV promoter (Fig. 5). It is therefore likely that the 90-bp region, containing the three protein-binding motifs, together with the flanking
downstream sequence is involved in conferring the negative regulatory
activity.
An interesting observation of this study relates to the unusual nature
of YY-1 binding to the negative regulatory region. Gel mobility shift
patterns in Fig. 6 show that the YY-1 and CArG
sequences fail to bind
to bacterially expressed purified YY-1 factor under conditions that
detect only complexes with slow off rates. Under conditions that detect
rapidly dissociating complexes, however, the CArG
sequence motif, but
not the YY-1
motif binds to the YY-1 factor (Fig. 6). The 90-bp DNA
probe containing all three sequence motifs, on the other hand, binds to
the YY-1 factor under slow off rate conditions and the bound complex
yields a single-site methylation footprint, which maps to the CArG
sequence region. Thus, although the CArG
sequence region appears to
provide the site for binding, the overall binding efficiency appears to be modulated by a still unknown sequence or structure within the 90-bp
DNA sequence. YY-1 is a multifunctional factor, which functions both as
a transcriptional activator and a repressor (18, 27-35) of different
promoters under different cellular conditions and in different cell
systems. Additionally, YY-1 binding is known to induce DNA bending in
some cases (35). In the present case, YY-1 binding may induce similar
DNA bending, facilitating specific interactions between protein-bound
complexes at both upstream and downstream regions of the COX Vb
negative enhancer.
Experiments in Fig. 10 (A and D), demonstrating a
60% suppression of transcription in 3T3 and C2C12 myoblasts by
co-expression with YY-1 cDNA, provide evidence for its involvement
in the negative regulation of the COX Vb gene under transient
transfection conditions. Several lines of evidence suggest that the
YY-1-mediated inhibition is specific. First, as shown in Fig. 10, the
YY-1-mediated inhibition is dose-dependent. Results of
cotransfection with various deletion constructs of YY-1 (expressed as
Gal-4 fusion proteins) in Fig. 11 demonstrate that the DNA binding and
repressor domains of YY-1 are essential for transcription suppression
of the COX Vb promoter, while the transcription activation domain is
not necessary. Third, mutations targeted to the CArG region of the
promoter resulted in vastly reduced YY-1 binding and reduced
effectiveness of the negative enhancer (Fig. 10, C and
D). It is therefore likely that the relative levels of YY-1
in a given cell type may determine the level of expression of the COX
Vb gene. It is also likely that other tissue-specific factors binding
to the CArG
or other flanking motifs might affect the binding or the
activity of the YY-1 factor. It was recently reported that although
nuclear extract from differentiated chick embryonic myocytes contained
YY-1 protein, its DNA binding efficiency was reduced, possibly due to
interference from another muscle-specific factor (36). It is known that
a number of different nuclear protein factors, including Sp1 (37), E1A
(38), FKBP12 (39), and c-Myc (40), interact with YY-1 and modulate its
functional properties. Our results showing a reduced transcription
activity of the
481/CArG
M1 and
481/CArG
M2 constructs in
differentiated myotube suggests that in the context of the COX Vb
promoter, the CArG
sequence functions as a transcription activator in
differentiated myocytes, and mutations in this region may affect the
binding of myotube-specific transcriptional activator protein(s).
A number of different negative enhancer mechanisms have been reported
(41, 42), including the repression due to competition for DNA-binding
sites, the quenching mechanism, where the repressor interferes with the
function of activator(s) without inhibiting its binding ability, and
the direct repression where the negative regulatory factor blocks the
activity of the basal transcription complex. It appears likely that the
COX Vb negative regulatory region represents a mechanism similar to the
quenching phenomenon. The activity of the negative enhancer may require
specific interactions between proteins bound to the upstream 90-bp DNA
sequence with those bound to sequences downstream of the 390 region,
although the details of this interaction currently remain unknown. We
also postulate that positive acting sequence motifs like YY-1
, GTG, and CArG
, when correctly juxtaposed to the downstream regulatory region, alter the activity of various transactivator proteins, with the
net effect being the transcriptional repression of the promoter. Recent
studies have shown that in a number of different promoters, regulatory
regions consist of alternatively positioned positive and negative
acting regulatory elements. In the case of the low density lipoprotein
receptor gene, the sterol-dependent binding of a protein to
a regulatory sequence inhibits or alters the activity of an adjacent
Sp1 factor-binding site (43). In several other cases, including the
mouse IgH (44), the T cell receptor gene locus (45), rat collagen II
gene (46), and cystic fibrosis transmembrane conductance regulatory
gene (47), transcriptional regulation involves interaction between both
positive and negative elements. Currently the nature of proteins
binding to the individual sequence motifs of the COX Vb negative
regulatory region, the nature of interaction between the various bound
complexes, and the precise role of the YY-1 in mediating these
interactions remain unknown. Further experiments are under way to
elucidate these mechanisms.
We are thankful to Drs. Michael Atchison and Sarah Bushmeyer for providing the CMV/YY-1 and Gal4-YY-1 cDNA clones and YY-1 antibody. We also thank Drs. Michael Atchison and Robert Carter for critically reading the manuscript.