From the
JC virus causes the human demyelinating disease progressive
multifocal leukoencephalopathy by selective infection of glial cells.
This cell specificity results from glial-specific expression of viral
early genes (large and small T antigens). Analysis of transcriptional
regulation by the MH1 JC virus early promoter demonstrates that glial
specificity is directed by the basal promoter. Because T antigen
regulates the basal region of several viral and cellular promoters, we
investigated whether it controls the JC virus basal promoter in a
glial-specific manner. A JC virus T antigen expression plasmid
generated a 95-kDa protein which exhibited nuclear localization and
physical association with p53. T antigen repressed the JC virus and
SV40 early promoters 4- to 5-fold in glioma cells. Conversely, T
antigen induced 100- to 200-fold activation of the JC virus early
promoter in nonglial cells, whereas the SV40 promoter was repressed.
Activation required the JC virus TATA box sequence and a
pentanucleotide repeat immediately upstream of the TATA box, but was
independent of the upstream enhancer region. These data demonstrate
that the JC virus basal promoter is responsible for glial-specific gene
expression and suggest a mechanism for this regulation.
JC virus causes a fatal, AIDS
The JC virus early promoter
directs higher levels of expression of a reporter gene in glial cells
compared to nonglial cells
(4, 5) . The DNA sequence of
the JC virus promoter region varies considerably between isolates
starting immediately upstream of the viral basal promoter region, which
is highly conserved
(6) . The MH1 form of the JC virus promoter
contains a single basal promoter region, compared to the duplicated
TATA regions in the original Mad-1 JC virus isolate, and has
facilitated functional dissection of the elements involved in
transcriptional regulation (Fig. 1). In a transient transfection
analysis, the MH-1 JC virus early promoter activated 30- to 40-fold
more transcription in glial cells than in nonglial cells. Using a
sensitive luciferase assay, we found that upstream promoter elements
activated expression in both glial and nonglial cells
(5) . These
results implicated the JC virus basal promoter in the regulation of
glial specificity, and, therefore, we undertook a functional analysis
of the basal promoter region.
The early genomic region of Mad-1 JC virus
was inserted into a cytomegalovirus expression plasmid (pJC-T), and the
protein product generated by transient transfection of pJC-T in U87MG
glioma cells was examined. Western analysis of cell lysates from pJC-T
transfected cells demonstrated a 95-kDa protein
(Fig. 3A, lane 3) that migrated slightly faster
than the SV40 T antigen expressed from pSV-T (lane 2),
consistent with the predicted number of amino acids (JC virus =
688 amino acids; SV40 = 695 amino acids)
(3) , whereas
mock-transfected cells contained no reactive species (lane 1).
T antigen contained a functional nuclear localization signal, as shown
by strong nuclear staining with anti-large T antibodies in transfected
U87MG glioma cells (Fig. 3B), whereas mock-transfected
cells exhibited no staining (Fig. 3C). Finally, we
tested whether the pJC-T product could form a physical complex with
p53. U87MG cells were cotransfected with an expression plasmid
containing a human p53 cDNA and either pSV-T or pJC-T.
Fig. 3D shows that p53 coimmunoprecipitated with the T
antigens of both SV40 (lane 2) and JC virus (lane 3).
No p53 was detected in cells transfected with p53 expression plasmid
alone (lane 1). Thus, pJC-T directed expression of full-length
JC virus large T antigen that was functional by criteria of nuclear
localization and interaction with p53.
Comparison of the MH1 and Mad-1
basal promoter sequences revealed that the two promoters are identical
up through a repeated pentanucleotide sequence immediately upstream of
a polythymidine tract (Fig. 1B). A previous report
suggested that the pentanucleotide sequence mediates repression of the
early promoter in nonglial cells, based on relatively small changes in
CAT activity
(15) . To determine whether this sequence was
required for T antigen-induced transactivation, the DNA sequence
between the GA box and the polythymidine tract was replaced by an
irrelevant sequence (Fig. 7, promoter H). T antigen was
unable to stimulate a promoter containing the pentanucleotide mutation.
However, this mutation did not increase the level of transcription in
HeLa cells. Mutation of the GA box, which is immediately upstream of
the pentanucleotide sequences, did not abolish the ability of T antigen
to transactivate, demonstrating specificity for the pentanucleotide and
TATA box sequences (Fig. 7, promoter C).
Once a rare condition, PML is no longer infrequent since
approximately 5% of patients with AIDS develop this fatal brain
disease
(16) . The unique feature of PML is the selective
destruction of oligodendrocytes by JC virus and selective expression of
viral genes in astrocytes. Although it is likely that this cell-type
specificity is regulated at more than one point in the viral life
cycle, the best understood of these points is the glial-specific
activation of early viral gene expression. Based upon features of
transcriptional enhancers, it seemed likely that glial specificity
would be mediated by the tandemly repeated elements. However, our data
have demonstrated that the MH1 JC virus basal promoter region also has
a role in this specificity.
We therefore examined the role of T
antigen, which regulates the basal promoter, in JC virus early promoter
regulation. We detected a divergent, cell-type-specific response to T
antigen, and we found that these responses were mediated by sequences
in the basal promoter. Whereas JC virus T antigen induced repression of
the JC virus early promoter in glial cells, a different phenomena was
observed in HeLa cells, where the JC virus early promoter was strongly
stimulated.
Activation was dependent upon the sequence of the TATA
box. The function of the TATA mutant JC virus promoter in glial cells
in the absence of T antigen was not altered, demonstrating that the
loss of activation in HeLa cells was not due to a nonspecific loss of
promoter activity. Alterations in TATA sequences have been shown to
exert specific effects on function of promoters. For instance, the
sequence 5`-TATAA in the hsp70 promoter was crucial for activation by T
antigen and the adenovirus protein
E1A
(11, 17, 18) . Replacement of this sequence
with the SV40 TATA sequence 5`-TATTTAT abolished E1A-induced activation
but maintained heat inducibility, whereas mutation to an irrelevant
sequence resulted in loss of both E1A and heat inducibility. Upstream
elements were not required for activation by T antigen. Tissue-specific
expression from the myoglobin gene promoter required a specific TATA
box sequence in concert with a muscle-specific enhancer
element
(19) . The recent identification of TATA-associated
factors and the observation that T antigen binds to TBP suggest that
specificity directed by the basal promoter may be achieved through the
proteins that interact directly with TBP
(20) .
Mutation of
the pentanucleotide sequence adjacent to the TATA box also abolished
activation by T antigen. The pentanucleotide sequence is of particular
interest because it is the most upstream sequence shared by MH1 and
Mad-1 promoters. Others have reported the presence of a possible
repressor site in the JC virus Mad-1 promoter. Early gene expression in
JC virus-transformed hamster glial cells was abolished following fusion
of the glial cells with mouse fibroblasts, consistent with the presence
of a protein in the nonglial cells that repressed the JC virus early
promoter
(21) . Further evidence for a specific repressor site in
the JC virus early promoter came from studies in which an
oligonucleotide, containing sequences which overlapped the viral TATA
box, was introduced upstream of the SV40 basal promoter and was found
to stimulate reporter gene expression in glial cells (U87MG glioma),
but repress expression in mouse fibroblasts
(15) . This analysis
was performed using a heterologous promoter construct, and the changes
in CAT activity were very small, making interpretation of the data
difficult. Our pentanucleotide mutant did not exhibit increased
transcriptional activity in HeLa cells, possibly because of the extent
of the mutation.
Specific nuclear proteins bind to the
pentanucleotide repeats in the basal promoter
(22, 23) .
These experiments have employed a combination of gel mobility shift
assays and UV cross-linking of the protein to DNA to demonstrate the
binding of an approximately 50-60-kDa protein present in glial
and HeLa cells. The nature of this protein remains unknown.
Although
there are several potential mechanisms for these results, they are most
easily explained by the presence of a TBP-associated repressor that
binds to the basal JC virus promoter and that is displaced by T
antigen. A model for such a mechanism can be found with the adenoviral
protein E1A, which appears to activate the hsp70 promoter in a TATA
sequence specific manner by dissociating a 19-kDa repressor, Dr1, from
TBP
(14) . The hypothesis that the glial specificity of the JC
virus early promoter is regulated by a repressor acting on the basal
promoter in nonglial cells is attractive because it explains a number
of experimental observations: it explains why the basal promoter is
invariant, but the upstream enhancer areas vary between isolates
(6) and why extensive searches have failed to reveal a clearly
glial-specific element in the upstream promoter region. Finally, it
would also explain our recent studies showing that the upstream
enhancer regions activate the basal promoter in glial and nonglial
cells
(5) . Together with experiments demonstrating that the
glial-specific transcription factor Oct-6 regulates the JC virus basal
promoter
(24) , these results suggest that the mechanism of glial
specificity resides in basal elements instead of the enhancer region.
(
)
-associated
demyelinating brain disease (progressive multifocal leukoencephalopathy
or PML) by infection of oligodendrocytes. Oligodendrocytes are the only
cells in the body known to be lytically infected by JC virus, but viral
early genes (i.e. large and small T antigen) are also
expressed in astrocytes, which take on a bizarre, transformed
appearance
(1) . Attempts to isolate JC virus from PML brain met
with failure until it was discovered that human fetal glial cells
supported lytic infection in cell culture, further suggesting the
glial-specific nature of the viral infection
(2) . Molecular
analysis revealed JC virus to be a 5.1-kb double-stranded, circular DNA
virus in the Papovaviridae family with a remarkable degree of homology
to SV40
(3) . As with SV40, JC virus genes are physically divided
into two transcriptional units: those genes expressed early and late
after infection. A single, bidirectional transcriptional regulatory
region lies between these 2 units.
Figure 1:
Comparison of MH1 and
Mad-1 JC virus early promoters. A, schematic of promoter
structure. Open boxes represent tandemly repeated sequences
that comprise the enhancer regions, the striped box in the MH1
promoter is the GA box (Sp1 binding site), the black box is
the TATA box sequence, and the arrow marks the transcription
initiation site. Shaded boxes represent sequences with
homology to the large T antigen binding sites in the SV40 promoter.
Note that the Mad-1 promoter has duplicated TATA box regions.
B, DNA sequence of basal promoter regions of MH1, Mad-1, and
SV40. MH1 and Mad-1 diverge immediately upstream of a pentanucleotide
repeat indicated by the bracket.
Plasmid Construction, Transfections, and Expression
Assays
JC virus promoter-reporter plasmids were constructed by
inserting the promoter constructs described below into the luciferase
expression plasmid pAPLUC
(5) . The DNA sequence of
all inserts were verified by dideoxy sequencing (U. S. Biochemical
Corp.). Full-length JC virus promoter (pMH1luc, Fig. 7,
promoter B) was constructed using the MH-1 JC virus promoter
(promoter p2,
(6) ) and primers (JC6 5`-AGAAGCTTCACGTGACAGCT,
JC26 5`-AGACTGCAGTTAGCTTTTTGCAGCAA) to amplify a region that included T
antigen binding site I and the two upstream tandem repeats.
pMH1(-LTaI)luc, in which T antigen binding site I was deleted by
virtue of the location of a HindIII site between T antigen
sites I and II, was constructed by inserting a
HindIII/MscI fragment of the MH-1 JC virus promoter
into pA
PLUC. pMH1(Sp1 mut)luc (Fig. 7, promoter
C) contained mutations that abolish binding of Sp1
(5`-AGCGAATACCT; sequence alteration is underlined). Deletion of the
tandemly repeated enhancer sequences (pMH1(-TR)luc) was performed
by amplification of the promoter fragment including the GA box and T
antigen binding site I (primers JC26 and JC21 5`-TCCAGTTTTAGCCAGCTC;
Fig. 7
, promoter D). pMH1(-Sp1)luc, which was
identical with pMH1(-TR)luc except that the Sp1 site was also
deleted, was similarly derived (primers JC26 and JC24
5`-ACCTTCCCTTTTTTTA; Fig. 7, promoter E).
pMH1(TATA-SV)luc (Fig. 7, promoter F) and
pMH1(TATA-irr)luc (Fig. 7, promoter G) contained the
entire JC virus early promoter sequences of pMH1luc, but the TATA
sequence was mutated either to that of SV40 (5`-TTTATTTATACA) or to an
irrelevant sequence (5`-TTTAGCGTCACA) by PCR site-directed
mutagenesis
(7) . A pentanucleotide mutant pMH1(-P)luc
contained irrelevant DNA sequence between the GA box and the
polythymidine tract (5`-CCCTCCTCATGCAGGTTTTTTTT; Fig. 7,
promoter H). pSVluc was constructed by ligating a
HindIII/NaeI fragment of the SV40 control region,
which contained all three T antigen binding sites, into
pA
PLUC. JC virus T antigen expression plasmid pJC-T was
constructed by ligating a NotI-linked
AciI/AvaI fragment of the early genomic region of the
Mad-1 JC virus into pCR/CMV expression plasmid (Invitrogen). The
orientation of the insert was confirmed by dideoxy DNA sequencing. SV40
T antigen expression plasmid (pSV-T; gift from Ulla Hansen, Dana-Farber
Cancer Institute) contained the genomic early coding region of SV40
downstream of the RSV promoter. pCMV-p53 was a wild-type p53 expression
vector
(8) . Transient transfections were performed by a standard
calcium phosphate method. 0.5
10
cells in 10-cm
dishes were transfected with 20 µg of plasmid DNA (15 µg of
promoter-luciferase plasmid, indicated amounts of T antigen expression
plasmids, and pBSK (Stratagene) as a carrier). In the experiment in
Fig. 1
, an SV40-CAT reporter plasmid was cotransfected to allow
normalization between cell lines as described
(5) . CAT activity
was determined by a standard two-phase assay. Luciferase assays were
performed in duplicate as described
(5) . Within T antigen
expression experiments, luciferase values were normalized by protein
concentrations as determined by a bicinchoninic acid assay (Pierce),
since T antigen regulates the promoters commonly used for internal
controls (e.g. RSV promoter). All transfection assays were
performed at least three times.
Figure 7:
Regulation of JC virus basal promoter by T
antigen. Transfections were performed using 0 or 4 µg of
pJC-T/plate, the cells were harvested 48 h after transfection, and
luciferase expression was measured and normalized to protein
concentration. Luciferase expression in the presence of T antigen was
divided by expression in the absence of T antigen, thus negative values
represent -fold repression of the JC virus promoter, whereas positive
values represent -fold activation. Promoter A is the wild-type
Mad-1 promoter, B is wild-type MH1 promoter, C contains mutations in the Sp1 binding site, D lacks the
upstream repeats, E lacks the Sp1 binding site and upstream
repeats, F and G contain mutations in the TATA
sequence to that of SV40 (F) and an irrelevant sequence
(G), and H has mutations across the pentanucleotide
sequence shown in Fig. 1.
Immunocytochemistry, Immunoprecipitation, and Western
Analysis
U87MG glioma cells were plated in 6-well plates and
transfected with either pJC-T or pBSK. After 48 h, the cells were fixed
in ethanol for 10 min, treated with 0.3% HO
for
10 min, blocked in 10% horse serum in PBS for 10 min, and incubated
with 10 µg/ml anti-large T antigen antibodies (PAb 416, Oncogene
Science) in 2% BSA/PBS for 1 h at room temperature. Following PBS
washes, cells were incubated in a 1:200 dilution of biotinylated horse
anti-mouse secondary (Vector) in 2% BSA/PBS at room temperature for 1
h, washed with PBS, incubated in avidin-biotin complex (Vector) for 1 h
at room temperature, and the chromogen was developed with
diaminobenzidine (Sigma). For Western analysis of T antigen, equivalent
amounts of 1% Nonidet P-40 or 1% Triton X-100 lysate protein were
denatured in SDS buffer and separated by 8% SDS-PAGE. Following
transfer to nitrocellulose, T antigen was identified using 15 µg/ml
primary antibody in 2% BSA/TBST (PAb 416 anti-large T antigen antibody,
Oncogene Science) and peroxidase-labeled horse anti-mouse secondary at
a dilution of 1:7,500 in 2% BSA/TBST. The peroxidase label was detected
by enhanced chemiluminesence (ECL, Amersham). Immunoprecipitations were
performed with 1% Nonidet P-40 cell lysates using an agarose-conjugated
anti-large T antigen antibody (PAb 416, Oncogene Science). Following
denaturation in SDS loading buffer, the immunoprecipitated complexes
were separated by 10% SDS-PAGE and transferred to nitrocellulose
(Schleicher and Schuell). Coimmunoprecipitation of p53 was detected by
incubation of the filter with
I-labeled anti-p53
antibodies (PAb 421, Oncogene Science), followed by autoradiography
(Kodak).
The MH1 JC Virus Basal Promoter Directs Glial-specific
Gene Expression
We compared the ability of JC virus early
promoter mutants to activate gene expression in U87MG glioma cells and
HeLa cervical carcinoma cells in a transient transfection assay. An
SV40CAT reporter plasmid was cotransfected to allow for normalization
of transfection efficiency and to allow a semiquantitative comparison
of expression between the two cell lines
(5) . Fig. 2shows
the ratio of normalized luciferase expression between U87MG and HeLa
cells for each promoter construct. Wild-type JC virus early promoter
pMH1luc was 34-fold stronger in glial cells than in nonglial cells. A
promoter with mutations in the GA box (Sp1 binding site) was 19-fold
stronger in glial cells, and a deletion mutant which contained the Sp1
binding site but lacked the upstream repeats remained 19-fold stronger
in glial cells. Together with our earlier observation that the upstream
tandem repeats activated gene expression in both U87MG and HeLa cells
(5), this demonstrated that the basal region of the JC virus early
promoter directed glial-specific expression of a reporter gene.
Figure 2:
The MH1 JC virus basal promoter directs
glial-specific expression of a reporter gene. Ratios of normalized
luciferase expression in U87MG glioma and HeLa cells are shown.
Promoter A is the wild-type MH1 JC virus promoter, B contains mutations that abolish binding of Sp1, and C is
the proximal promoter region including the Sp1
site.
Functional Analysis of the JC Virus Basal
Promoter
We examined the function of the JC virus basal promoter
by expressing a transcription factor that might regulate this region.
The MH1 JC virus promoter has notable similarity to the SV40 promoter,
which is strongly active in a wide variety of cell types and is
repressed by its early gene product large T antigen (T antigen) through
cooperative binding to three sites in the basal promoter
(9) . T
antigen activates the human hsp70 promoter in a manner that is
dependent upon the exact sequence of the TATA
box
(10, 11) , and there is in vitro evidence
that T antigen physically interacts with TATA binding protein
(TBP)
(12) . Based on these observations, it appears that T
antigen regulates the basal promoters of viral and cellular genes
during initiation complex formation. Because T antigen regulates
various basal promoters in a divergent fashion, and because the TATA
box sequences of JC virus and SV40 are different (Fig. 1), we
used T antigen to investigate regulation of the glial-specific JC virus
basal promoter region.
Figure 3:
JC
virus T antigen expression in U87MG glioma cells. A, Western
analysis revealed a 95-kDa protein in U87MG cells transfected with
pSV-T (lane 2) or pJC-T (lane 3). Mock-transfected
cells (lane 1) showed no immunoreactivity. B,
immunocytochemistry with anti-large T antigen antibody revealed strong
nuclear staining in cells transfected with pJC-T. C, absence
of staining in mock-transfected U87MG glioma cells (methyl green
counterstain, 200). D, physical association between
p53 and JC virus T antigen was demonstrated by immunoprecipitation with
anti-large T antigen antibodies, followed by Western analysis with
I-labeled anti-p53 antibodies. Lane 1 contained
no transfected T antigen, lane 2 was a cotransfection of pSV-T
and p53, and lane 3 was a cotransfection of pJC-T antigen and
p53.
Regulation of the JC Virus Early Promoter by T
Antigen
To determine the transcriptional effect of JC virus T
antigen on the JC virus early promoter, pJC-T was cotransfected with JC
virus promoter-reporter plasmids into U87MG glioma cells. SV40 T
antigen is known to repress its own expression by binding to three T
antigen binding sites in the SV40 early promoter
(9) . The JC
virus early promoter pMH1luc was repressed 5-fold by JC virus T antigen
in a concentration-dependent manner (Fig. 4). Fig. 4also
shows that JC T antigen, which has 72% amino acid identity to SV40 T
antigen, repressed the SV40 promoter 5-fold. Repression was partially
dependent upon the presence of binding site I, since deletion of site I
in pMH1(-LTaI)luc resulted in weaker repression. Similar levels
of repression were seen with transfections performed in another glioma
cell line (T98G glioblastoma cells, data not shown).
Figure 4:
Transcriptional repression of the JC virus
(pMH1luc) and SV40 (pSVluc) early promoters by JC virus T antigen
measured at 48 h after transfection into U87MG glioma cells. Repression
was dependent upon binding site I, since a promoter in which this site
has been deleted, pMH1(-LTaI)luc, was repressed less strongly
than wild-type promoter.
Three-fold
stimulation of the Rous sarcoma virus (RSV) promoter was seen in this
assay, demonstrating that repression of the JC virus early promoter was
not the result of a nonspecific reduction in gene expression. Analysis
of the time course of transcriptional repression of the JC virus early
promoter by T antigen (0.5 µg or 4 µg/plate at 0, 6, 12, 24,
and 48 h following a 12-h transfection) revealed progressively stronger
repression over time with maximal repression apparent between 24 and 48
h (data not shown). Although the promoter constructs contained the JC
virus origin of replication, detectable plasmid DNA replication did not
occur over the 48 h of the transient transfection assay as determined
by Southern analysis of low molecular weight DNA extracted from the
transfected cells (data not shown). Thus, transcriptional repression
was not dependent on DNA replication. In conclusion, T antigen produced
JC virus early promoter repression in U87MG glioma cells that was
dependent upon concentration of T antigen and the presence of T antigen
binding site I.
Comparison of JC Virus and SV40 T Antigens
The
experiments in Fig. 4demonstrated that JC virus T antigen
repressed both JC virus and SV40 early promoters. To compare the
function of JC virus and SV40 T antigens, we tested their regulation of
the SV40 promoter in U87MG glioma cells. Fig. 5A shows
that both viral T antigens produced transcriptional repression of the
SV40 early promoter, and that 50% repression occurred with lower
amounts of transfected pJC-T than with pSV-T. The pMH1luc JC virus
promoter was repressed by both T antigens in a similar manner (data not
shown). Quantitative Western analysis also suggested that JC virus T
antigen was a more potent repressor than SV40 T antigen
(Fig. 5B); however, since the relative affinity of the
antibody for the two T antigens is not known, this conclusion remains
tentative. Thus, in glial cells, JC virus and SV40 early promoters and
T antigens were functionally interchangeable.
Figure 5:
Comparison of transcriptional repression
of the SV40 early promoter (pSVluc) by JC virus and SV40 T antigens in
U87MG glioma cells. A, titration of JC virus and SV40 T
antigens revealed that both proteins repress the SV40 early promoter.
50% repression occurred with transfection of lower amounts of JC virus
T antigen expression plasmid (pJC-T) compared to pSV-T. B,
quantitative Western analysis of T antigen also suggested that JC virus
T antigen was a more potent repressor.
Cell-type Specificity of T Antigen Promoter
Regulation
We examined the possibility that repression was
specific to glial cells. The SV40 promoter was repressed by T antigen
in HeLa cells (Fig. 6), but T antigen produced 100- to 200-fold
activation of the JC virus early promoter. A second nonglial cell line
(JEG3 choriocarcinoma cells) gave similar results, although the level
of activation was lower. SV40 T antigen also produced stimulation of
the JC virus early promoter. There was no evidence of plasmid
replication as determined by Southern analysis (data not shown).
Activation was dependent upon a direct interaction between T antigen
and the promoter, since pMH1(-LTaI)luc showed only slight
stimulation (Fig. 6).
Figure 6:
In HeLa cells the JC virus early promoter
(pMH1luc) was stimulated strongly by T antigen, whereas the SV40
promoter (pSVluc) was repressed. Very weak activation was observed with
the JC virus promoter lacking T antigen binding site I,
pMH1(-LTaI)luc.
Promoter Elements Required for Transcriptional
Activation
Having found that T antigen regulated the JC virus
early promoter in a cell-specific manner, we sought to define promoter
elements that were required for activation in HeLa cells. Activation
was independent of the tandemly repeated enhancer region, since a
reporter construct containing only the proximal promoter was still
activated by T antigen (Fig. 7, promoters D and
E). Recent data have suggested that T antigen activates the
hsp70 promoter in a TATA sequence-dependent manner
(10, 11) and that it interacts with TATA binding protein (TBP) (12),
and TBP-associated repressors (e.g. Dr1) have been implicated
in TATA sequence-dependent transcriptional
regulation
(13, 14) . We thus tested whether T antigen
activation of the JC virus promoter was TATA-dependent. T
antigen-induced repression was still seen in U87MG glioma cells
following mutation of the JC virus TATA sequence to that of the SV40
promoter or to an irrelevant sequence (promoters F and
G). In HeLa cells, however, T antigen failed to activate the
pMH1(TATA-SV)luc promoter and the pMH1(TATA-irr)luc promoter,
demonstrating that transactivation by T antigen was indeed dependent
upon the sequence of a TATA box.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.