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© 1991 Oxford University Press
Nucleic Acids Research, Vol. 19, No. 12 3221
Cooperation between upstream and downstream elements
of the adenovirus major late promoter for maximal late
phase-specific transcription
Guillaume Monctesert and Claude K6dinger*
Laboratoire de Gene"tique Moleculaire des Eucaryotes du CNRS, Unite 184 de Biologie Moleculaire
et de Genie Gen&ique de I'lNSERM, Faculty de M§decine, 11 rue Humann, 67085 Strasbourg
Cedex, France
Received April 12, 1991; Accepted May 13, 1991
ABSTRACT
Transcription from the adenovirus major late promoter
(MLP) is greatly stimulated during lytic infection, after
replication of the viral DNA has started. This replicationdependent activation has previously been shown to be
mediated by a positive regulatory cellular proteln(s).
Binding of this factors) to sequence elements (DE1 and
DE2), located between positions +76 and +124, with
respect to the MLP transcriptlonal startsite, is detected
only after the onset of DNA replication. Using a cellfree transcription system which mimics the late phase
induction of the MLP and DNA binding assays, we now
present evidence showing that maximal stimulation
also depends on the MLP upstream element (UE),
without Involving increased DNA binding activity of the
corresponding factor (UEF) during the lytic cycle. Our
results Indicate that the upstream and downstream
elements act cooperatively on transcription efficiency,
although no direct interactions between the cognate
factors could be demonstrated. These observations
strongly suggest that the elevated rate of transcription
originating at the MLP startsite, late in infection, results
from the simultaneous action of factors bound at the
upstream and downstream elements onto a common
target within the basal transcription machinery.
INTRODUCTION
Analysis of the molecular mechanisms underlying eukaryotic gene
control has largely relied on the development of in vitro
transcription systems using combinations of wild type and mutated
exogenous DNA templates. Such systems have led to the
identification of several protein factors which, together with RNA
polymerase, contribute to the setting up of active initiation
complexes and thereby contribute to the basal level of
transcription (1-3). In addition to these general transcription
factors, an increasing number of trans-acting factors have been
identified, which bind to specific DNA sequence elements located
at various positions with respect to the transcriptional startsite
• To whom correspondence should be addressed
(3—7). How these DNA-binding factors modulate basal promoter
activity is still unknown. Accumulating evidence indicates
however that, once bound to DNA, these factors achieve their
transcriptional effects by establishing protein-protein contacts
with the basal transcription apparatus, either directly or via
intermediary connections with adaptor proteins (see 8, 9 for
reviews). Temporal and tissue-specific transcriptional activation
or repression of a given promoter will occur only if a particular
combination of factors and cofactors has built up on it. The
understanding of the molecular mechanisms implicated in this
process clearly requires the identification and characterization
of the factors involved, as well as their relationships with the
other components of the transcription machinery.
The major late promoter (MLP) of human adenovirus 2 or 5
is one of the eukaryotic promoters that have been most extensively
studied (see 10, 11 for reviews). Efficient constitutive
transcription from this promoter has been found to essentially
depend on an intact TATA box centered at position - 2 8 , an
upstream sequence element (UE) between —67 and - 4 9 and an
initiator element encompassing the transcription startsite (12—18).
Additional elements, located further downstream, have also been
shown to contribute to basal promoter function (19, 20). The
specific trans-acting factors recognizing these elements have
subsequently been identified and their binding properties studied.
Thus, binding of the TATA box recognition factor, BTF1 (21)
or TFHD (22), appeared to be the prerequisite for die assembly
of the initiation complex, which is further stabilized by
interactions between ihllD and the factor bound to the nearby
upstream element, MLTF, UEF or USF (12, 16, 22). TFUD
also represents, in this promoter, the main target for direct
activation by the adenovirus E la or the pseudorabies IE proteins
(23, 24).
Although the MLP is active at early times in infection, a strong
stimulation occurs after the onset of viral DNA synthesis (10 for
review). This activation cannot just be ascribed to an increase
in the intracellular viral template copy number, since, as
previously suggested (25—27), replication is required to render
the template competent for transcription. Similar conclusions have
3222 Nucleic Acids Research, Vol. 19, No. 12
also been reached in the case of the activation by DN A replication
of SV40 (28), polyoma (29) and vaccinia virus late genes (30),
or of the Xenopus jS-globin gene (31), for example.
Besides these cis-acting alterations, the nature of which is still
unknown, sequences downstream of the MLP transcription
startsite have been shown by in vitro experiments to be essential
for promoter activation (32, 33). DNAse I footprinting
experiments combined with an in vitro transcriptional analysis
of MLP deletion mutants, have established that MLP activation
depends on sequence elements (DEI and DE2) located between
+76 and +120 and correlates with the increased binding of a
virus-induced 40 kD cellular factor to DEI (34, 35). Using a
series of nondefective adenovirus recombinants expressing
MLP—globin fusions, Leong et al. (36) have clearly established
the role of the MLP downstream elements in the late phasespecific induction of this promoter during the lytic cycle of
infection.
In this study, we further examined the function of the
neighbouring DEI (+86/+96) and DE2 (+113/ + 124) elements.
We show that these elements are functionally redundant and most
likely bind common proteins. In addition, our experiments reveal
that the late phase-specific stimulation of the MLP results from
a cooperative action of the upstream and downstream promoter
elements, although no synergistic DNA-binding activity of the
cognate proteins could be detected.
MATERIALS AND METHODS
Preparation of whole cell extracts
HeLa cells, grown in Eagle medium supplemented with 5% calf
serum, were infected with 10 PFU of adenovirus type 5 (wt) or
its Ela-defective dl312 derivative (dl) per cell. Cells were
harvested 20 h postinfection and extracts were prepared in parallel
from wt and dl-infected cells, to minimize extract to extract
variations. Experimental conditions were as previously described
(35), except that the final dialysis was against buffer B containing
20 mM HEPES-NaOH (pH 7.9), 5 mM MgCl2, 100 mM KC1,
1 mM EDTA, 1 mM dithiothreitol and 17% glycerol (37).
Recombinant plasmids
The BamHI fragment of pML553 (34) comprising the MLP
sequences between positions —259 and +553 was inserted into
the BamHI site of M13mp9. The resulting single-stranded
recombinant was used for oligonucleotide-directed mutagenesis.
The mutated MLP fragments were recloned into the BamHI site
of pBR322. The pG recombinant (34) contains the rabbit /3-globin
gene, between positions -425 and +1700. These MLP and
globin plasmids were used as templates for polymerase chain
reactions (see below).
In vitro runoff transcription
Transcription reactions were carried out as previously described
(35), with the following exceptions: i) final reaction volume was
32 y.\; ii) cell extracts (16 /tl) were preincubated for 15 min at
25 °C with sonicated salmon sperm DNA (200 ng); iii) the DNA
templates were obtained by polymerase-chain-reaction (PCR)
amplification of the wild type or mutated MLP sequences,
between positions -137 and +314 (with respect to the MLP
startsite), or the rabbit /3-globin gene sequences, between positions
—290 and +225 (relative the corresponding startsite). PCR
amplification was performed by incubating each plasmid (125 ng)
with appropriate pairs of 25-nucleotide primers (1 fig)
complementary to the borders indicated above, in a medium
(100 fi\ final volume) containing 2 U Taq polymerase, 0.2 mM
each dNTP, 100 mM Tris-HCl (pH 7.8), 10 jig/ml gelatin, 1.5
mM MgCl2, and subjecting the mixture to 30 cycles of (1 min
at 92°C, 2 min at 55°C and 3 min at 72°C) in a Cetus PCR
apparatus. After amplification, total DNA was purified by
phenol—chloroform extraction and used as template in the in vitro
transcription reactions. Transcripts were analyzed by
electrophoresis on 5% polyacrylamide-urea gels which were
vacuum-dried before exposure for autoradiography in the
presence of an intensifying screen. All transcription assays were
repeated at least three times, with independent template DNA
and extract preparations.
Electrophoretk band-shift assays
Gel retardation assays were performed essentially as previously
described (35). Briefly, about 0.3 ng (15,000 cpm) of
32
P-5'-end-labelled, double-stranded oligonucleotide probe (DEI
or UE, see Figure 1) were incubated with 2 y\ (10 ng protein)
of wt or dl extract, in the presence of 10 /tg of poly (dl-dC) as
nonspecific competitor, in a medium (10 /il final volume)
containing 50 mM KC1, 2 mM MgCl2, 10 mM EDTA and
2.5% Ficoll. After 10 min at 25°C, the complexes were separated
by non-denaturing polyacrylamide (4.5%, acrylamideibisacrylamide 80:1) gel electrophoresis. After the run, the gel was
transferred onto Whatman 3MM paper and vacuum-dried before
autoradiography.
DNAse I footprinting assays
About 1 ng (10,000 cpm) of the BamHI (-259) -HindHI (+200)
MLP fragment of pML553,32P-3'-end-labelled at the HindHI
site (non-transcribed strand) was incubated in buffer B (40 fd final
volume), in the presence of 2 /tl wt extract and 1 /ig of poly (dldC), for 15 min at 25°C. Where indicated, the extract was
preincubated for 15 min at 25 °C with specific competitor doublestranded oligonucleotides, before addition of the probe DNA.
After digestion by DNAse I (15 min at 25° C with 100 Kunitz
units per assay), the DNA fragments were phenol—chloroform
extracted and separated on a 6% polyacrylamide sequencing gel,
next to DNAse I-treated (10 units per reaction) naked probe
DNA. After the run, the gel was vacuum-dried and exposed for
autoradiography.
RESULTS
DEI and DE2 are involved in the late phase-specific activation
of the MLP
In vitro transcription analysis and DNA binding studies of the
adenovirus MLP have established the requirement of a sequence
element spanning positions +86 to +96 (DEI), for the
replication-dependent activation of this promoter. The
contribution to this regulation of the nearby downstream element
(DE2, +113/+124) and the upstream promoter element (UE,
—67/-49) was examined by introducing site-directed alterations
into the corresponding MLP sequences (see Figure 1). To analyze
die effect of these mutations on the late phase-specific activation
of the MLP, the template efficiencies of the resulting mutants
were tested in the presence of extracts prepared from cells infected
for 20 h widi wild type adenovirus-5 (wt extracts). The
transcriptional activity directed by these late-infected cell extracts
was compared to that of extracts prepared 20 h after infection
with dl312 (dl extracts), an adenovirus-5 derivative which is
Nucleic Acids Research, Vol. 19, No. 12 3223
I Ttmplam |
DEI
DE2
1
WT
V//1
+86 + 9 6 + 1 1 3 + 124 + 3 1 4
iyy i
v//x
A2
m! A2
ItfV I
mUm1
X//A
+85 +96
_/\_rzza_
+85 +96
mUA12
Footprint proba |
C€1
(BamHO
DE2
(Hlndlll)
•259
OBgonudeotfdM
W/A
DE12
+ 124
+75
062
+104
(GTAGGCCACG)
(TTQTCAGTTT)
DEI
+75
(GTAGACTACG)
OEImi
+132
+104
- I XX I
+75
+104
(TTTTCACTTT)
~
~
Figure 1. Diagrams of the adenovirus MLP DNA fragments used for in vitro transcription and protein binding assays. The PCR-amplified DNA fragments used
as templates for run-off transcription are depicted, with promoter elements shown as boxes, and coordinates given relative to the startsite ( + 1 , arrow). The WT
template corresponds to the natural adenovirus type 2 sequence between positions -137 and +314. Point mutations (xx) correspond to G-to-A and C-to-T transitions
at positions - 6 2 and - 6 0 (in mU) and to G-to-T and G-to-C transversions at positions +88 and +92 (in ml), respectively (see below). Deletions spanning DEI
(Al), DE2 (A2) or both elements (A12) arc shown with corresponding coordinates. The BamHI-Hindlll fragment, excised from pML553 (see Materials and Methods)
was used as probe in the DNAse I footprinting experiments. The chemically synthesized double-stranded oligonucleotides used as probes or competitors in the bandshift and footprinting experiments are schematized at the bottom. Relevant nucleotide sequences are given next to corresponding wild type and mutated elements
(alterations relative to the natural sequence are underlined).
defective for Ela expression and whose DNA replication is
delayed compared to that of wt-infected cells. We have in fact
previously shown that the transcriptional activity of these dl
extracts was identical to that of extracts prepared from wt-infected
cells at 6 h post-infection (early phase) or at 20 h post-infection
from cells which were grown in the presence of cytosinearabinoside to prevent DNA replication (34).
Typical transcription assays, run in the presence of various
DNA templates, are shown in Figure 2. Since some of the specific
signals generated by the wt extracts were nearly saturated after
a 1.5 h exposure time (middle row of lanes), the corresponding
lanes were also exposed for a shorter time (0.5 h, upper row).
Under our incubation conditions, a DNA fragment spanning the
wild type MLP sequence between positions -137 and +314
('WT' template, see Figure 1) was transcribed about 30-fold more
efficiently in wt extracts than in dl extracts (Figure 2A, lane 1),
while a control rabbit /3-globin fragment (glob) was transcribed
at roughly equal efficiencies in either extract (Figure 2A, lane
3224 Nucleic Acids Research, Vol. 19, No. 12
probe
Template.
$<E%
rue
• • • • • • Bamsma
rttainod DE1 • - - * - - •
LDE2 •
• • . - - •
•
DE1
UE
extract wt dl -
:
- wt dl
+ - + - . +
-UEF
wt
(0.5h expo)
I*
L
(1^h expo)
1 2
12
3 4 9 6 7
12
3 4
3
Free
1 2 3 4 5 6 7
4 5 6
Figure 2. Comparative mutational analysis of MLP activity in wt and dl extracts.
In vitro transcription was performed as described in Materials and Methods with
wt extracts (wt, top and middle series of lanes) or dl extracts (dl, bottom lanes),
in the presence of the PCR-amplified MLP templates (60 ng) or rabbit /3-globin
template (glob, 400 ng), as indicated. Intact (+) or altered ( - ) promoter elements
present in each MLP template are noted. The specific run-off transcripts were
separated by gel electrophoresis and visualized by autoradiography: reactions
carried out in the presence of wt extracts were exposed for 0.5 h (upper series
of lanes) and 1.5 h (middle), those run in parallel, but in me presence of dl extracts,
were exposed for 1.5 h (bottom). The longer exposure time (wt and dl, 1.5 h
expo) was chosen to visualize the overall extent of late phase-dependent stimulation
of the MLP activity. The non-saturating exposure time (wt, 0.5 h expo) allows
comparison of relative MLP activities in wt extracts. The major band in each
lane corresponds to the specific transcript, with the expected length (globin-specific
transcripts are indicated by arrow-heads). Panels A, B and C correspond to
independent experiments and illustrate the functional redundancy of DEI and DE2
and the role of UE, the cooperativity between the DE and UE elements, and
the ability of DEI and DE2 to separately cooperate with UE, respectively.
Figure 3. Comparative analysis of DEF and UEF binding activities in wt and
dl extracts. Standard band-shift assays were performed with the DEI (lanes 1 - 3 )
or UE (lanes 4 - 6 ) oligonucleotide probes (see Figure 1). The specificity of the
DEF and UEF complexes (arrows) was determined by experiments using
appropriate competitor oligonucleotides (not shown). The slower migrating bands
in lanes 1 and 2 correspond to ubiquitous complexes previously described (35)
UE TATA
DE1 D£2
I dl extract
Z
rrr~i m
I
1
8). Introduction of a double-point mutation into DEI or deletion
of the whole element (as in ml and Al, respectively; see Figure
1) did not significantly affect stimulation of the corresponding
templates by the wt extracts, under these in vitro transcription
conditions (Figure 2A, lanes 2 and 3; Figure 2C, lane 2).
Similarly, deletion of the DE2 element (as in A2) had no effect
on MLP activity (Figure 2A, lane 4 and Figure 2C, lane 7). By
contrast, simultaneous alteration or deletion of both elements (as
in mlA2 and A12) reduced about 4-fold MLP activity in wt
extracts, without affecting basal MLP activity as measured in
dl extracts (Figure 2A, lanes 5 and 6, Figure 2B, lane 2 and
Figure 2C, lane 4). These results clearly suggest that both DEI
and DE2 are involved in the late phase-specific activation of the
MLP. Furthermore, since either one of these elements mediates
by itself most of this activation, we conclude that DEI and DE2
are functionally redundant elements.
The MLP upstream element cooperates with the downstream
elements
Preliminary experiments (not shown) revealed that the late phasespecific stimulation of the MLP was substantially reduced if
binding of the upstream element factor (UEF) was titrated with
competitor UE oligonucleotides, prior to the transcription
reaction. To confirm the involvement of the UE element in the
late phase-specific MLP activation suggested by this observation,
we first investigated the effect of UE-directed mutations on this
phenomenon.
In agreement with earlier transcription analyses which used
partially purified transcription factors from uninfected HeLa cells
(16), a double-point mutation of the UE element (mU, see Figure
1), that abolished UEF binding (see below), reduced about 3-fold
J wt extract
mUA12
1
WT
(mU+A12)-mU412
0
20
40
60
BO
100
Relative activity {%)
Figure 4. Cooperativity between the UE and DE elements for late phase-specific
stimulation of MLP activity. MLP mutants were schematically depicted on the
left (see Figure 1). with altered promoter elements crossed (X)- Non-saturated
exposures of autoradiographs of the experiment in Figure 2 (and others not shown)
were scanned with a densitometer and the areas corresponding to the specific
transcripts generated in the presence of wt or dl extracts were diagrammed next
to the corresponding template, relative to the transcriptional activity of the WT
template in wt extracts (100%). The bottom line represents the calculated sum
of the transcription activities separately elicited by the mU and A12 templates.
The final value was adjusted for the basal promoter activity by deducting one
times the activity of the minimal promoter retained within the mUA12 template.
basal MLP activity, as measured in dl extracts. In addition, this
mutation reduced about 6-fold the activity measured in wt
extracts, thus decreasing the overall activation by about 2-fold
(compare the ratios of the signals generated by wt and dl extracts
in lanes 1 and 7, Figure 2A and see Figure 4). These results
support the conclusion that the UE element contributes to the
late phase-specific stimulation of the MLP. This could be
achieved either directly through the activation of the cognate
transcription factor (UEF) itself or through the cooperative
interaction between UEF, the DE-recognizing factor (DEF) and
the transcription machinery.
It has previously been shown by Leong et al. (23) that the
intrinsic in vitro transcriptional and DNA binding activities of
UEF were not affected by infection with pm975, an adenovirus-2
derivative expressing mainly the large Ela protein. In agreement
with this conclusion, identical amounts of UEF-specific retarded
Nucleic Acids Research, Vol. 19, No. 12 3225
B
Competitor (molar excess)
UEm
UE
DE12
DE2 --
--•
8
--8888
DE1m1
DE1
88
8 3 8 8 ! | | |
8888
; • : ' • > • •
Extract
-67-
UEC
UEm
tmn.
DE1 I
+86-
OE2
+124-
DE2
12
3 4
5 6
7 8
It
9 10
H
11 12
•MM
-«
12
3 4 5 6 7 8
910111213
14151617181920212223
2425
Figure 5. DNAse I footprinting of the MLP region. A) A comparative DNAse I protection analysis was carried out (see Materials and Methods) with the WT MLP
and the indicated mutant probes, in the presence of wt extract. The corresponding naked probe digestion pattern ( - ) is shown next to each protection assay (+).
The UE, DEI, and DE2 elements discussed in the text are positioned. Landmark coordinates are given, relative to the MLP startsite. B) Competition analysis of
the footprints on the WT MLP probe was performed by preincubating the wt extract with increasing amounts (molar excesses as mentioned) of the indicated doublestranded competitor oligonucleotides (depicted in Figure 1), before addition of the labelled probe DNA and DNAse I treatment. The uncompeted digestion pattern
is shown in lane 25, and naked probe patterns in lanes 1 and 24.
complexes were also observed by comparative gel-shift analysis
of dl and wt extracts (Figure 3, compare lanes 5 and 6). By
contrast, under the same probe-excess conditions, the level of
DEF DNA-binding aictivity was dramatically increased in wt
compared to dl extracts (Figure 3, compare lanes 1 and 2). Thus,
while activation of DEF clearly correlates with the late-specific
stimulation of MLP (35), no such a direct activation of UEF could
be detected.
We next examined the combined effect of alterations of the
UE and both the DEI and DE2 elements on basal MLP activity
(as tested in dl extracts) and on the extent of MLP activation by
the wt extracts. As shown in Figure 2B and depicted in Figure
4, mUA12, a mutant promoter simultaneously lacking functional
UE, DEI and DE2 elements (see Figure 1), displayed a 2 to
3-fold reduced basal activity compared to the WT template. This
reduction was in fact exclusively caused by the mutation of the
UE element, consistent with the low levels of DEF binding
activity in dl extracts. In agreement with this conclusion, an
alteration of the UE element alone (as in mil) produced the same
effect as the mUA12 mutation (Figure 2B, compare lanes 3 and
4), while a deletion of the whole DE region had no effect on
basal activity (Figure 2B, lane 2). By contrast, when assayed
in wt extracts, the mUA12 mutation had more dramatic effects
on template efficiency than mutations which separately destroy
either the UE (as in mU) or the DE elements (as in A12) (Figure
2B, compare lanes 2 and 3 with lane 4). As previously suggested
(23, 34), the residual activation of mUA12 transcription by the
wt extract most likely reflects the stimulatory effect of the Ela
gene products mediated by the intact TATA box retained in this
mutant. A quantitative analysis (Figure 4) of the results presented
in Figures 2A and B (and others not shown), reveals in fact that
the UE and DEI +DE2 elements cooperate for maximal promoter
activation: the effect of the DE and UE elements is about 3 times
more pronounced when these elements are present together
(activity of the WT template), than when present separately
(cumulative activity of the MU and A12 templates).
That the template efficiencies of mutants lacking either the DEI
(ml or Al templates) or the DE2 element (A2 template) were
nearly identical to those of the WT template (Figure 2A, compare
lane 1 with lanes 2—4) strongly suggests that each of these
downstream elements may separately cooperate with the UE
element to achieve maximal promoter activation. In agreement
with this conclusion, mutants either retaining the DEI or the DE2
element, but lacking the UE element (mUA2 and mUAl
templates, respectively) were as poorly responsive to the latespecific stimulation as a mutant (mU template) lacking only the
UE element (Figure 2, compare panels B and C).
DNAse I protection studies suggest that the DEI and DE2
elements bind the same factor
To examine the effect of the forementioned individual and
combined mutations on the protein binding activity of the
3226 Nucleic Acids Research, Vol. 19, No. 12
respective MLP elements, we performed DNAse I footprinting
experiments. A typical protection pattern of the WT MLP probe
by the wt extract is presented in Figure 5 A Qane 2), next to the
DNAse I digestion profile of the naked DNA (lane 1). The strong
protections spanning the UE, DEI and DE2 elements are
indicated. Additional, weaker protections, spanning the TATA
box region (between UE and the startsite) or the region directly
upstream of DEI, are observed. These protections, also found
with dl extracts (34), have not been further analyzed. Deletion
of either one of the DE elements Qanes 3 - 6 ) did not affect the
protection over the UE element, consistent with the UEF footprint
being detected in dl extracts which contain only very low DEF
binding activity (35). Similarly, an alteration of the UE element
which abolishes UEF binding (lanes 7-12) had no effect on
protein binding to the downstream elements. These results suggest
that efficient binding of either the UE or DE-specific factors can
occur independently from each other, in agreement with earlier
gel-shift or protection assays with crude or purified protein
fractions (12, 16, 17, 35).
We also performed competition experiments in which specific
footprints on the WT MLP template were competed with
increasing concentrations of selected synthetic oligonucleotides.
As shown in Figure 5B, when the reaction was challenged with
oligonucleotides spanning only the DEI or only the DE2 element
(DEI or DE2, see Figure 1), protections over both DEI and DE2
elements were simultaneously abolished in each case Qanes 2 - 5
and 10-13). The resulting DNAse I digestion patterns, within
the MLP downstream area, were indistinguishable from that of
naked DNA (lanes 1 and 24) or after competition of wt extracts
with an oligonucleotide (DE12, see Figure 1) spanning the whole
DE region (Figure 5B, lanes 14-17). Under the same conditions,
a mutated oligonucleotide (DElml, see Figure 1), used as nonspecific competitor, did not alter the digestion pattern (lanes
6 - 9 ) . These results suggest that the DEI and DE2 elements are
binding sites for the same factor (see Discussion).
Strikingly, the DE2 oligonucleotide also competed for the
footprint which spans the UE element (lanes 10—13), whereas
no such a competition could be obtained, under similar conditions,
with the DEI or the DEI2 oligonucleotides (see lanes 2—5 and
14—17). Sequence comparisons of the oligonucleotides used in
these experiments revealed in fact significant homologies between
part of the UE element ( - 6 3 / - 5 4 , non-transcribed strand) and
a region partially overlapping the DE2 element (+120/+129,
transcribed strand), which may explain these results. As expected,
the DE12 oligonucleotide which lacks most of the conserved
sequences because it only extends to position +124 (instead of
+132, as the DE2 oligonucleotide, see Figure 1), did not compete
for the UE protection. Similarly, the UE oligonucleotide, while
readily competing for UEF-specific binding (lanes 18—23), had
no detectable effect on proteins binding to the DEI or DE2
elements, whether used at the concentrations shown in this
experiment (lanes 18-23) or at higher concentrations (not
shown).
DISCUSSION
Previous studies have delineated sequence elements located
downstream of the MLP transcription startsite which are critical
for the late phase-specific activation of this promoter (34-36).
A protein (DEF), with an apparent size of 40 kDa, has previously
been identified, whose binding to at least one of these downstream
elements (DE) correlated with this transcriptional activation (35),
pointing to DEF as a potential positive transcription factor. In
this report, we demonstrate the participation of the MLP upstream
element (UE) in this replication-dependent stimulation. We show
that this contribution was not due to elevated binding activities
of the cognate UEF (or MLTF or USF) factor late in infection,
in contrast to DEF. Even though these factors bind independently
to their respective sites, our results suggest that they cooperate
to elicit the observed transcriptional effect, since the activation
by both elements together is greater than the sum of the effects
of each alone. DNA-binding competition experiments indicate
that the two major downstream elements (DEI and DE2) most
likely bind the same factor(s), a conclusion also supported by
the observation that these elements are redundant in their ability
to separately cooperate with UE and achieve maximal (or nearly
maximal) stimulation of the MLP, in vitro.
Synergistic promoter activation has previously been observed
under conditions where no cooperative DNA binding of the
activators occurred (38—41). From their results the authors
suggested that activators may cooperate not by directly interacting
with each other, but by simultaneously contacting a particular
component(s) of the transcription machinery. In this respect, it
may be relevant that UEF and BTF1 (or TFIID) stimulate MLP
activity by cooperatively binding to their recognition sites.
Whether DEF-mediated activation also involves BTF1, remains
to be established.
While the UE recognition factor (UEF), a protein which
belongs to the helix-loop-helix family of regulatory proteins and
binds as a dimer to its recognition site, is now well characterized
(42), still little is known about the protein(s) which bind to the
DE elements. Comparative band-shift, UV cross-linking and
south-western analyses (35) have indicated that a host-cell protein
of about 40 kDa (DEF) interacts with DEI. The observation that
DEI and DE2 may bind the same protein (36 and present study),
suggests that DE2, which shares no obvious sequence homology
with DEI, must contact a distinct domain of DEF. Thus, both
DE elements may interact with the same DEF molecule or
alternatively, each element may bind its own copy of the same
factor.
Whereas in our present in vitro transcription system DEI and
DE2 appeared as interchangeable and redundant elements, they
behaved as distinct elements, each one contributing to part of
the transcriptional effect, when assayed in vivo, after recombinant
adenovirus infection (36). The reason for the discrepancy between
the results of these in vitro and in vivo experiments is not clear,
but could reflect the involvement of additional factors, whose
effects would not be detected in the in vitro transcription system.
Such a possibility is in fact supported by our earlier observation
that a protein fraction, purified by chromatography on a
DEI-affinity column, produced footprints over both the DEI and
DE2 elements, but with a pattern over the DE2 element, different
from that generated by the starting material (35).
Despite a strong sequence homology (9/10) between the UE
element ( - 6 3 to -54) and a segment overlapping the 3' portion
of DE2 (+120 to +129), the UEF protein did not appear to bind
to this downstream region, since competition by the upstream
element had no visible effect on the protection pattern of the DE2
region, under our footprinting conditions (see Figure 5B).
However, we cannot exclude the interesting possibility that such
an interaction of UEF with the DE2 neighbourhood might occur
in vivo, which could at least partially account for the in vivo
transcriptional phenotype observed by Leong et al. (36). In this
respect, it is worth mentioning that a functional equivalent of
Nucleic Acids Research, Vol. 19, No. 12 3227
UEF, partially purified from duck erythrocytes, has been shown
to transactivate expression from the histone H5 gene by
interacting with an intragenic element (43).
Leong et al. (36) have detected an additional, but much weaker
protein binding site (Rl), located closer to the startsite, between
+37 and +68. It appears however from their results that deletion
of this element affects MLP activity, both at early and late times
after infection. If true, this would suggest that the Rl region is
not primarily involved in late phase-dependent events. We have
notrepeatedlyobserved this protection in our binding experiments
and have not analyzed it further. Interestingly however, there
exists a striking sequence homology (7/8) between segments in
Rl (+41 to +48) and DE2 (+117 to +124). The significance
of this homology remains to be clarified.
Another adenovirus gene whose activation depends, at least
in part, on viral DNA replication is the peptide IX gene. The
mechanisms proposed for this promoter activation imply the
synthesis of a sufficient amount of new template molecules which
would result in the dilution of inhibitory DNA-binding proteins
and allow the redistribution of RNA polymerase and activating
transcription factors over the clean, newly synthesized DNA
templates. While RNA polymerase transit, from the nearby
upstream Elb unit, may by itself cause promoter occlusion by
preventing the attachment of necessary factors at the pIX
promoter (44), negative regulatory factors may also directly
repress transcription from this promoter on non-replicated
molecules. A binding site for such a repressor has recently been
identified, between positions +33 and +122 of the pIX gene
(45). In addition, this author has shown that the upstream
promoter element of the pIX gene suppresses the transcriptional
repression mediated by the downstream element. Whereas
promoter occlusion may similarly be invoked to explain the
replication-dependence of MLP activation in vivo, it is unlikely
that such a phenomenon could account for the results observed
in our in vitro system, since (i) the template fragments used in
the present study do not contain other promoters besides the MLP,
(ii) essentially no end-to-end transcription takes place on these
templates, (iii) the downstream element binding proteins act as
positive transcription factors and (iv) no specific repressor binding
sites have so far been identified within the MLP.
influence the binding of specific transcription factors to the
corresponding MLP sequences.
Transcription from the MLP in vivo has previously been shown
to pause or terminate prematurely around position +190, at late
but not early times after infection (49). It has recently been
reported that this termination site was promoter-specific since
it did not function efficiently when inserted downstream of a
heterologous promoter (50), suggesting that pausing required
interactions of the elongation complex with specific upstream
sequences or proteins bound to them. The possibility that the DE
elements identified here and the cognate factors may be involved
in the control of polymerase stalling at this site seems however
unlikely. Extended pausing in vitro was indeed not observed
under the standard incubation conditions used in our study, but
only in the presence of Sarkosyl (51 and our unpublished
observation). In addition, the results of Wiest and Hawley (50)
show that the MLP sequences between +33 and +133 were not
required for the Sarkosyl-dependent termination.
Transcriptional control from the long terminal repeat (LTR)
of human immunodeficiency virus provides an alternative,
intriguing example of cooperation between upstream and
downstream promoter elements. A number of studies have shown
that the virally encoded trans-activator Tat enhances transcription
of its own gene by interacting with a Tat-responsive element
(TAR), an RNA target located within the 5' region of the
transcript. This Tat-TAR complex seems in turn to act on
particular upstream promoter elements lwithin the LTR and
thereby elicit efficient transactivation (52-54). While it is clear
that DEF binds to DNA, it is not known at present whether it
exhibits, in addition, RNA binding activity.
The elucidation of the mechanism of action of DEF also implies
the understanding of the process leading to its own activation:
it will for instance be essential to determine whether the dramatic
increase in DNA binding activity observed late in infection
corresponds to increased DEF copy numbers or to posttranscriptional modifications of preexisting molecules, and to
identify the events responsible for these alterations. Clearly
answers to these questions await further characterization of the
DEF protein(s).
Reach et al. (46) have recently reported the construction and
transcriptional analysis of adenovirus mutants harboring
alterations within the natural MLP upstream region. Their results
indicate that mutagenesis of the UE element only weakly affected
(not more than 2 fold) MLP activity, late in infection. On the
other hand, these authors observed that transcription from the
MLP was markedly impaired when an additional mutation was
introduced into the inverted CAAT box located directly upstream
of UE, between positions - 7 6 and - 8 0 . We detected no
protections over this region, neither with cell extracts (Figure
5A, lanes 7—12) nor by genomic footprinting (47). It may
nevertheless be of interest to examine whether altering this CAAT element, which is retained in our template molecules, will
further enhance the dependence on UE of the late phase-specific
transcriptional stimulation seen in vitro.
Chang and Shenk (48) recently demonstrated the contribution
of the DNA-binding protein (DBP), encoded by the adenovirus
E2a gene, to the transcriptional activation of the MLP. The
stimulation observed in these experiments is clearly distinct from
the late phase-specific activation which we describe here, since
the MLP sequences tested by these authors did not extend
downstream of position +30. As proposed (48), the DBP may
ACKNOWLEDGMENTS
We thank P.Jansen-Durr for his initial contribution to this work
and helpful discussions and R.Mukherjee and B.Chatton for
critical reading of the manuscript. We are very grateful to
C.Hauss for excellent technical assistance, to B.Reimund for
advice in the mutagenesis, to the cell culture group for providing
cells and to the whole secretarial staff for help in preparing the
manuscript. This work was supported by grants from the CNRS,
the INSERM, the Association pour la Recherche sur le Cancer
and the Ligue Nationale Francaise contre le Cancer.
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