(Received for publication, June 23, 1995; and in revised form, November 28, 1995)
From the
The GA-binding protein (GABP), a heterodimeric transcription
factor with widespread tissue distribution, has been found to be a
strong positive regulator of several ribosomal protein (rp)-encoding
genes. In such genes, e.g. the mouse rpL30 gene, the
GABP-binding sites are located 40-80 base pairs upstream of the
transcriptional start point. Potential GABP-binding sites are present
in the promoters of numerous other rp genes, not only in
upstream regions, but also in the immediate vicinity of the
transcriptional start point. The mouse rpS16 gene is an
example of the latter type. We demonstrate here that GABP binds to the rpS16 initiation region, and in so doing down-regulates rpS16 transcription both in vivo and in
vitro. Supplementation of cell-free extracts with GABP inhibits
transcription on rpS16 templates while concomitantly
stimulating transcription on rpL30 templates. The repressive
and stimulatory effects, which were proportional to the amount of GABP
added, required both the GABP subunit and either a
or
subunit. Mutations of the rpS16 GABP-binding sites that abolish binding increased rpS16 promoter activity in vivo and in vitro, whereas
mutations that strengthen GABP binding caused a reduction in promoter
activity. The binding of GABP to the rpS16 initiation region
does not significantly affect the positioning of the transcriptional
start points. Taken together with earlier studies, these new findings
indicate that GABP can have a dual role as repressor or activator of rp gene transcription.
GA-binding protein (GABP) ()(also called nuclear
respiratory factor 2 (NRF-2) or E4 transcription factor 1 (E4TF1)) is a
heteromeric transcription factor that has been shown to activate a
number of genes, including those encoding the mouse ribosomal proteins
L30 and
L32(1, 2, 3, 4, 5, 6, 7) .
One GABP subunit, termed
, is a member of the ets family
of DNA-binding proteins and binds weakly to DNA(1) . The other
subunit,
, contains ankyrin repeats and binds to DNA only
in the presence of the
subunit. There are two isoforms of the
subunit,
and
, which have
different carboxyl termini; there are also two variants of each of
these isoforms with differences in the internal portion of the
molecule(2, 8, 9) . The GABP
subunit,
together with either the GABP
or GABP
subunit, can form dimeric complexes on a single GABP consensus
motif (CGGAAR), as occurs in the mouse ribosomal protein (rp) L32 promoter. A tetrameric complex, composed of two
GABP
and two GABP
subunits, which is stabilized
by interactions through the
carboxyl termini, can
form when two GABP binding sites are present(1) . The binding
sites can be contiguous, as in the mouse rpL30 promoter (7) and herpesvirus ICP4 enhancer(1) , or separated, as
in the genes encoding the cytochrome c oxidase subunits IV and
Vb (CO4 and CO5b) (3, 5, 6) . The ability to
form tetrameric GABP complexes with noncontiguous binding sites is
afforded by the flexibility of the
subunits.
A previous study from our laboratory indicated that GABP is a strong positive regulator of the rpL30 and rpL32 genes (7) . Subsequently, a search for CGGAAR motifs in the proximal promoter regions of other mammalian rp genes revealed many potential GABP-binding sites(7) . While most of these sites are located upstream of the transcriptional-start point, as they are in rpL30 and rpL32, in five genes (mouse rpS16 and rpL7, human rpS6 and rpS14 and chicken rpS15) they are situated in the immediate vicinity of the transcriptional start point. It was previously observed that mutations in this region of the rpS16 promoter can influence the efficiency and precision of transcription both in vivo(10) and in vitro(11) . However, these mutations were not suitable for assessing the role of GABP in promoter function because they also disrupted the integrity of the polypyrimidine/purine initiator, which is a common feature of all vertebrate rp genes. We were therefore interested in determining whether GABP binds to the rpS16 initiation region and if so, how it influences the efficiency and precision of transcription.
Here we show that GABP binds to the initiation region of rpS16 by virtue of interactions with a weak (nonconsensus) binding motif that overlaps the start points and a stronger motif that is immediately downstream of the start points (Fig. 1). By constructing various mutated versions of these motifs and studying their effects on both GABP binding and rpS16 promoter function in vitro and in vivo, we further demonstrate that occupation of the binding site by either tetrameric or dimeric GABP causes a significant reduction in rpS16 promoter activity, apparently by interfering with the formation of the transcriptional initiation complex. Consistent with these observations, we found that supplementation of in vitro transcription reactions with GABP represses rpS16 transcription while concomitantly stimulating transcription of rpL30. Thus, it would appear that GABP can have a dual role as repressor or activator of ribosomal protein gene transcription. To our knowledge, this is the first report of a repressive role for GABP in transcriptional regulation.
Figure 1: Schematic representation of the promoters of the rpS16 and rpL30 genes showing the locations of the DNA elements known to be important for promoter activity. The numbers below the line represent the approximate center of the elements as determined by methylation interference or DNase I footprints. The sequences of the GABP binding sites are shown with thick and thin arrows representing relatively strong and weak sites, respectively.
Figure 2:
Demonstration that GABP binds to the rpS16 promoter. A, recombinant and
GABP subunits (rGABP) were incubated with
P-labeled oligonucleotide probes derived from the rpS16 promoter (-11 to +18) or rpL30 promoter (-70 to -30) under conditions described for
EMSA. Reactants were subjected to electrophoresis on a 4%
polyacrylamide gel in 0.5
TBE. The complexes corresponding to
tetramers and dimers and
monomers are
indicated. B, various amounts (0, 2, 5, or 10 µl) of
native GABP (nGABP) from the second round of DNA affinity
chromatography were incubated with a
P-labeled rpS16 oligonucleotide probe (-11 to +18) under conditions
described for EMSA. The reactants were subjected to electrophoresis on
a 4% polyacrylamide gel in 0.5
TBE. The slower and faster
moving complexes are designated U and L,
respectively. C, aliquots (50 µl) from the first (A1) and second (A2) rounds of DNA affinity
chromatography were electrophoresed on a 10% SDS-polyacrylamide gel and
visualized by staining with silver. The relative molecular masses of
the candidate GABP subunits are indicated on the right side of the
panel. An aliquot (50 µl) of the nGABP A2 fraction was
electrophoresed on a 10% SDS-polyacrylamide gel in parallel with
bacterially synthesized
(rGABP
) or
(rGABP
) subunits, transferred
to Immobilon, and probed with antibodies against the GABP
subunit (D) or the GABP
subunit (E). In Panel
E, two exposures of the native GABP lane are presented in order to
illustrate clearly the four
subunit variants (marked by ticks).
Figure 3: Electrophoretic mobility of rpS16-protein complexes is supershifted by GABP-specific antibodies. EMSA of rpS16 oligonucleotide probe incubated with affinity purified native GABP (10 µl) in the presence or absence of preimmune serum or antibodies raised against recombinant GABP subunits. The positions of the tetrameric, dimeric, and monomeric complexes are indicated.
Figure 4:
DNase I footprint analysis of GABP binding
to wild-type and mutant rpS16 sites. rGABP subunits were
incubated with a DNA fragment labeled on the sense or antisense strand.
Free DNA and protein-bound DNA were partially digested with DNase I and
subjected to electrophoresis on a 4% polyacrylamide gel in 0.5
TBE. DNA was recovered from the tetrameric complexes by electroelution
and subjected to electrophoresis on a 5% gel containing 7 M urea. The positions of sequence markers are indicated at the left
and right of the panels. A, fragment corresponding to the
-51 to +29 region of wild-type rpS16. B, fragment
corresponding to the -51 to +29 region of rpS16 mutant cm9. At the bottom of the figures, the sequences of the
protected regions are indicated by a box. Large and small
arrows indicate the strong and weak GABP sites, respectively.
Hypersensitive sites are indicated by asterisks.
Figure 8:
Effect of mutations on GABP binding. Upper panel, rGABP was incubated with P-labeled
oligonucleotide probes containing wild-type sites or sites with point
mutations. The reactants were subjected to electrophoresis on a 4%
polyacrylamide gel containing 0.5
TBE. Complexes corresponding
to tetramers ((
)
), dimers (
),
and monomers (
) are indicated. Lower panel, summary of
GABP site mutants used in the study. For the ease of comparison, the
sequence of the rpS16 antisense strand is shown. The large and small arrows represent the major and minor rpS16 transcriptional initiation sites. Lower case letters indicate mutations; D and U refer to the
downstream and upstream sites, respectively; - and +
represent mutations designed to abolish or increase GABP binding,
respectively. The consensus binding motif for GABP (8) is shown
at the bottom of the figure (R =
purine).
A previous analysis of tetrameric GABP complexes formed on the rpL30 promoter (7) showed a broad footprint, similar to that seen with the cm9 mutant of rpS16. Thus, a comparison of footprint data indicates that the GABP tetrameric complexes formed on the rpS16 promoter are less stable than those formed on the rpL30 promoter. A difference in stability was also evident from the results of a kinetic competition experiment (Fig. 5). In this experiment, 84% of the rpS16 tetramers dissociated by 0.5 min, compared to 44% dissociation of the rpL30 tetramers. By 8 min, the fraction of residual rpS16 tetramer was about 0.1 that of the rpL30 tetramer. The decay of the tetramers in this experiment was not strictly first-order, as one might expect on theoretical grounds, possibly due to inadequate mixing at the later time points. For this reason, it is not possible to obtain a precise quantitative estimate of the difference in stability from these data. Nevertheless, it is clear that the GABP tetramers formed on the rpS16 initiator region are relatively unstable compared to tetramers formed on the upstream binding site of the rpL30 promoter. Indeed, the stability of the rpS16 tetramers is similar to that of dimeric complexes formed on either the rpS16 or rpL30 promoters (data not shown).
Figure 5:
Analysis of the stability of tetrameric
GABPDNA complexes. Equimolar amounts of rGABP subunits were
incubated with rpS16 (-11 to +18) or rpL30 (-70 to -37)
P-oligonucleotides probes
under conditions described for EMSA. After a 15-min incubation,
protein-DNA complexes were competed with a 500-fold molar excess of the
corresponding unlabeled probe and reactants loaded onto a nondenaturing
gel at various time points. The relative amounts of tetrameric complex
were measured with a PhosphorImager. Results are presented as the
fraction of probe bound relative to zero
time.
Figure 6:
The
effect of supplementing in vitro reactions with GABP on rpL30 and rpS16 promoter activity. Increasing amounts
(4, 10, and 20 pmol) of either rGABP (lanes 2-4) or
rGABP
(lanes 5-7) or an equimolar
mixture of rGABP
and rGABP
(lanes
8-10) were added to in vitro transcription
reactions containing 3 µg of rpL30 (A) or rpS16 (B) templates. The RNA products were copied
into cDNA by extension from a primer complementary to cat vector sequences, and the cDNA was subjected to electrophoresis on
a 4% polyacrylamide gel containing 7 M urea. C, rpL30 and wild-type or mutant (cm13) rpS16 templates were transcribed in the same reaction in the absence (lanes 1 and 4) or presence (lanes 2, 3, 5, and 6) of GABP supplementation (4 or
10 pmol). The RNA products were processed as described above. In this
experiment, the amount of DNA templates was reduced to 2 µg each of rpL30 and rpS16.
We also observed inverse effects on rpS16 and rpL30 transcription when GABP and
subunits were
added to the reaction. Since
lacks the
homodimerization domain of the
subunit,
and
do not form a stable tetrameric complex on the rpS16 initiator region. Nevertheless, they form a readily
detectable dimeric complex (Fig. 7A). This binding
occurs at the stronger downstream site and is completely eliminated
when this site is destroyed in the cm8 mutant (see below). As shown in Fig. 7B, the GABP
dimers
repressed rpS16 transcription (lanes 5-7),
although not quite as effectively as the
complexes (lanes 2-4). As expected, rpL30 transcription was stimulated by the GABP
dimers under the same conditions. These results indicate that
dimeric complexes at the downstream site are sufficient to elicit a
repressive effect. The effect is apparently potentiated when
tetramers can be formed.
Figure 7:
GABP can repress rpS16 transcription. A, recombinant GABP
and
GABP
or GABP
were incubated with
P-labeled oligonucleotides corresponding to wild-type rpS16 sequences or mutant (cm8) rpS16 sequences under conditions described for EMSA. Reactants were
subjected to electrophoresis on a 4% gel in 0.5
TBE. B, rpS16 or rpL30 transcription reactions
were supplemented with either rGABP
and rGABP
(lanes 2-4) or rGABP
and rGABP
(lanes 5-7). The amount of rGABP used to
supplement the reactions was identical to that used in Fig. 6A. Lane 1 was not supplemented with GABP.
Reaction products were characterized as described in Fig. 6.
The
effects of the various mutations on GABP binding were determined by an
EMSA analysis (Fig. 8, top). As expected, no GABP
binding was detected with cm13 (lane 7), and tetramer
formation was eliminated in cm11 and cm12 (lanes 5 and 6). Interestingly, the cm8 mutation abolished dimer, as well
as tetramer, formation (lane 2; also see Fig. 7A), indicating that binding at the weak upstream
site requires the presence of a functional downstream site. Conversion
of the upstream site to the preferred consensus sequence not only
increased the stability of the tetrameric complexes (cm9, lane
3), but also enabled dimeric complexes to be formed at the
upstream site, even when the downstream site was nonfunctional (cm11, lane 5). Stronger GABP binding was also observed when the
wild-type sites were made contiguous (cm10, lane 4), probably
by enhancing the interaction between the subunits. The stronger
binding was confirmed by a DNase I footprinting analysis (data not
shown).
The effects of these mutations on promoter strength and start point selection were then analyzed by both transient transfection experiments and in vitro transcription assays (Fig. 9, Table 1). In contrast to the results of our previous mutational analysis of the rpL30 and rpL32 promoters(7) , the activity of the rpS16 promoter was not diminished when GABP binding was totally abolished (mutants cm8 and cm13). In fact, these mutants were about twice as active as the wild-type construct both in vivo and in vitro. Conversely, mutants cm9 and cm10, which bind GABP more tightly than wild-type, were even less active than the wild-type promoter. The in vivo activities of mutants cm11 and cm12, which form only dimeric GABP complexes, were similar to that of the wild-type rpS16 promoter, consistent with the idea that tetramer formation is not essential for the repressive effect of GABP. As might be anticipated, when the in vitro reactions were supplemented with GABP, mutants cm9 and cm10 were strongly repressed, while mutants cm8 and cm13 were relatively unaffected (Fig. 9B). Although the activity of mutant cm12 was significantly reduced by GABP addition, the cm12 template was consistently more active than the wild-type template in unsupplemented reactions. The reason for the relatively high in vitro activity of cm12 is presently obscure. None of the mutations had a marked effect on the location of the transcriptional start points; at most, displacements of only one or two base pairs were observed.
Figure 9:
Analysis of GABP site mutations on rpS16 promoter activity. A, in vivo. Individual rpS16 constructs containing wild-type or
mutant GABP binding sites were cotransfected with a wild-type rpL30 construct into S194 plasmacytoma cells. Cells were harvested 40 h
after transfection and cytoplasmic RNA was prepared. Approximately 50
µg of cytoplasmic RNA was analyzed by primer extension. B, in vitro. Individual rpS16 templates containing
wild-type or mutant GABP binding sites were transcribed in
unsupplemented reactions(-) or with reactions supplemented with 5
pmol each of GABP and GABP
(+). The RNA
products were subjected to primer extension and extension products
resolved by electrophoresis on 4% polyacrylamide gels containing 7 M urea. Quantitative data from these and other experiments are
summarized in Table 1.
The studies described above indicate that GABP can bind to
the rpS16 initiation region, and in so doing modulate the
activity of the rpS16 promoter. Because the consensus
recognition site for GABP, CGGAAR, is shared by many members of the ets family, it was important to establish that the recognition
sites in rpS16 are indeed specific for GABP. The best evidence
for this is the fact that affinity chromatography on rpS16 initiator-specific DNA columns resulted in the isolation of native
cellular proteins that were indistinguishable from bacterially
synthesized GABP and
subunits in size, antibody reactivity,
and ability to form tetrameric and dimeric complexes on the rpS16 recognition sites. Moreover, these complexes were shown by
electrophoretic mobility supershift experiments to react with
GABP-specific antibodies. These are essentially the same criteria that
were used to establish GABP specificity for binding sites in other
genes(3, 4) .
As illustrated in Fig. 8, one of the rpS16 recognition sites is suboptimal: the upstream site, which overlaps the transcriptional start points, has a nonconsensus G at position 1. DNase I footprinting analysis, kinetic competition experiments and mutational studies demonstrated that tetrameric GABP complexes formed on these sites are relatively unstable compared to tetrameric complexes formed on a tandem pair of consensus sites, e.g. those in rpL30. Since the upstream site is considerably weaker than the downstream site, the dimeric complexes that form on the rpS16 promoter are located almost exclusively on the downstream site.
Given that GABP serves as a transcriptional activator of several other genes, we were initially surprised to find that it has a repressive function for rpS16. Earlier mutational studies of the rpS16 initiation region (10, 11) were inconclusive on this point because the mutations that abolished GABP binding also obliterated the polypyrimidine tract that spans the start points. In the present study we have eliminated this ambiguity, and find that transcriptional activity is either increased or decreased relative to the wild-type gene depending on whether the mutations eliminate or strengthen GABP binding. Moreover, when GABP was added to a cell-free transcription system, transcription from the wild-type rpS16 promoter decreased in a dose-dependent manner while transcription from the rpL30 promoter concomitantly increased. The magnitude of the repressive effect of GABP, estimated by comparing the activities of mutants cm8 and cm13 with that of wild-type in vivo or in unsupplemented in vitro reactions, is about 2-fold. Although this is not a large effect in absolute terms, it is comparable to the contribution of any single transcriptional activator to the strength of an rp promoter(7, 11, 15, 16, 17) , and is therefore likely to be biologically significant.
Our studies
suggest that occupation of the downstream GABP site would be sufficient
to cause repression. This was indicated by the fact that repression
could be elicited by dimers, which form stable
complexes only at the stronger downstream site. Thus in the wild-type rpS16 promoter, the combination of relatively unstable
tetramers and residual dimers on the downstream site effectively
modulates the transcriptional activity. The binding of GABP does not
significantly affect the selection of the transcriptional start points.
Although the mechanism by which GABP regulates transcriptional
activity is not fully understood, there is general agreement that the
DNA binding specificity resides mainly in the subunit, while the
activation function resides exclusively in the
subunit(1, 4, 9) , apparently in a region
that is common to the various
isoforms(8, 9) .
This region contains evenly spaced clusters of hydrophobic residues,
which could mediate interactions with components of the basal
transcriptional apparatus(18, 19) . In agreement with
this general concept, we also observed that both
and
subunits are necessary to elicit the regulatory function of GABP, both
as an activator of rpL30 and as a repressor of rpS16 (Fig. 6). The particular components of the transcriptional
apparatus that interact with GABP have not yet been identified, and it
is presently unclear whether the activator and repressor functions
involve the same or different constituents of the
subunit. It
seems reasonable to suppose that similar interactions might be involved
in both functions and that the distinction between activation and
repression is determined by the context of the GABP sites within the
promoter architecture. Thus, depending on precise spatial
relationships, the GABP interactions could either facilitate or impede
the assembly of a transcriptional initiation complex.
A relationship between GABP function and binding site context might be inferred for the four rp genes that have been analyzed to date: an activation function for rpL30, rpL32, and Xenopus rpL14(7, 20) , where the sites are located 50-75 base pairs upstream of the transcriptional start points, and a repressive function for rpS16, where the sites overlap the transcriptional start points. Yet, in the CO4 and CO5b promoters, GABP bound to sites that overlap with transcriptional start points apparently has an activation function(3, 5) . Thus, other contextural features besides proximity to the start points may determine whether GABP will have a positive or negative effect on transcription.
The fact that a single transcription factor can serve as both an activator and repressor is not unprecedented(21, 22, 23) . The use of this principle for the regulation of rp gene transcription would make sense if there is not a large variation in GABP content from cell type to cell type. In this case, the opposing effects could be part of a fine-tuning mechanism, which helps ensure relatively uniform rates of transcription among the many unlinked rp genes(16) . However, if the level of GABP were to vary greatly among different cells, then the opposing effects could actually widen differences in rp gene transcription rates. Although GABP is known to have a widespread tissue distribution(8) , virtually nothing is known about its relative abundance in different tissues. Without this information, it is presently difficult to judge whether the activator/repressor function of GABP is advantageous for the coordinate regulation of rp genes or whether it is simply a tolerated consequence of evolutionary tinkering.