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J Biol Chem, Vol. 274, Issue 50, 35662-35667, December 10, 1999
From the Department of Biochemistry, Purdue University,
West Lafayette, Indiana 47907-1153
Vaccinia virus has a DNA genome, yet replicates
in the cytoplasmic compartment of the cell. We previously described the
identification of a cellular protein having high affinity for vaccinia
virus late promoter DNA. Sequence substitutions in the vaccinia I1L promoter were used to define a 5-nucleotide block at the transcription initiation site as essential for interaction with the protein. Within
this sequence is the recognition motif for the nuclear transcription
factor YY1. This factor regulates a multitude of cellular promoters, as
an activator of transcription, as a repressor, or as an initiator
element-binding protein. Antibodies directed against YY1 were used to
show that YY1 copurified with the vaccinia late promoter-binding
protein and was present in late promoter-protein complexes in gel
supershift assays. Bacterially expressed YY1 also bound specifically to
late promoter DNA. A dinucleotide replacement within the YY1
recognition motif directly adjacent to the transcription start site
severely reduced the affinity of YY1 for the I1L promoter in
vitro and impaired I1L promoter-dependent
transcription in vivo. The intracellular localization of
YY1 was shown by immunofluorescence microscopy to shift from primarily
nuclear to the cytoplasm after vaccinia infection. These results
indicate that YY1 has a positive role in the regulation of vaccinia
virus late gene transcription and suggest that poxviruses have adapted
cellular initiator elements as a means of regulating viral gene
expression. This is the first identifiable cellular protein implicated
in poxvirus transcription.
Poxviruses are large DNA genome viruses that replicate in the
cytoplasm of host cells. Many species of insects, birds, and mammals
including humans are natural hosts for specific members of this large
family of viruses. As the laboratory prototype for poxviruses, vaccinia
virus is by far the best characterized of the group. Vaccinia has a
191-kilobase pair genome encoding some 200 different proteins (reviewed
in Ref. 1). Part of the complexity of the virus owes to its battery of
genes encoding anti-inflammatory and immunity-suppressing proteins
important for infectious spread in an animal host. Many other proteins
are made essential by the fact that the virus replicates outside the
nucleus in the cytoplasm of the cell. Vaccinia is able to synthesize
its mRNA and DNA outside the nucleus, suggesting that the virus
must either encode its own proteins participating in nucleic acid
metabolism or alternatively recruit nuclear proteins to replicate
virosomes in the cytoplasm. Because of the paucity of information on
host proteins participating in viral mRNA and DNA synthesis, it is
generally believed that vaccinia encodes the majority of proteins
functioning in these processes.
The life cycle of vaccinia virus is controlled at the level of the
timing of transcription of individual genes (2). Viral genes fall into
three classes: early, intermediate, and late. About half of the virus'
genes are early and half are late. Only five genes are known to be
intermediate. Each class has its own requisite set of transcription
factors that regulate the viral RNA polymerase that structurally and
functionally resembles its cellular counterparts. The products of the
intermediate genes A1L, A2L, and G8R are required for the subsequent
activation of the late class of genes (3), and the H5R gene product
stimulates late transcription in vitro (4). A cellular
protein has also been described as being essential for transcription
in vitro (5, 6). It is not yet known what proteins target
late promoters for initiation of transcription.
In a previous report, we described a cellular protein with vaccinia
late promoter binding activity (7). Prior indications were that the
protein interacts with DNA around the start site for transcription in a
viral late promoter and was capable of stimulating transcription
in vitro. Here we report that the late promoter-binding
protein is the transcription factor YY1. Evidence is provided for a
positive role for YY1 in transcription from a vaccinia late promoter
in vivo, and it is shown that vaccinia infection redirects
the subcellular localization of YY1 from the nucleus to the cytoplasm.
Protein Purification--
The protein was purified from HeLa
cells grown in suspension culture. Cell fractionation experiments
indicated that greater than 90% of the late promoter-binding protein
could be recovered in nuclear extracts relative to cytoplasmic
fractions. Nuclei were isolated and extracted with 0.6 M
KCl, 50 mM Tris, pH 8, 0.01% Nonidet P-40 (8). The extract
was applied directly onto nickel-agarose (Qiagen) and mixed for 2 h at 4 °C. The beads were washed three times in batch with 25 volumes of 10 mM imidazole in buffer A (150 mM
NaCl, 50 mM Tris, pH 8, 0.01% Nonidet P-40) and collected
by centrifugation. Late promoter-binding protein was eluted by three
extractions with 100 mM imidazole in buffer A. The eluates
were combined, diluted 5-fold in buffer B (50 mM Tris, pH
8, 0.1 mM EDTA, 1 mM dithiothreitol, 0.01%
Nonidet P40, and 10% glycerol), and applied to a phosphocellulose
column. The column was developed with a gradient of 0.05-0.5
M NaCl in buffer B. Late promoter binding activity eluted
at approximately 0.2 M NaCl. Phosphocellulose-purified
protein was subjected to velocity sedimentation on a 10-35% glycerol
gradient containing 0.2 M NaCl and buffer B in a SW41 rotor
at 40,000 rpm for 68 h at 4 °C. Catalase, hemoglobin, and
cytochrome c were used as sedimentation standards in a
parallel gradient.
Recombinant YY1 was expressed in Escherichia coli strain RR
harboring plasmid pHIS-YY1 (provided by Thomas Shenk, Princeton University) as described previously (9). Cells were lysed by freeze-thawing, and YY1 was purified from the soluble fraction on
nickel-agarose as described above.
Protein Assays--
DNA binding was assayed by electrophoretic
gel shift using the vaccinia virus I1L or H1L promoter DNA excised from
a plasmid or annealed synthetic oligonucleotides and end-labeled with
32P (10). Protein (5-10 ng for experiments utilizing
purified protein) and 1-2 ng of radiolabeled DNA probes were incubated together in the presence of 0.1 µg of poly(dI-dC), electrophoresed on
a low ionic strength 4% polyacrylamide gel, and visualized by
autoradiography (11). For gel supershift assays, 2 µg of anti-YY1
antibody (Santa Cruz Biotechnology) was included in the DNA binding
reaction prior to loading onto the gel. The antibody used in these
studies is a rabbit IgG directed against the C-terminal 20 amino acids
of YY1.
Western blotting of proteins was as described (12) after
electrophoresis on a 10% polyacrylamide gel containing 0.1% SDS. Blots were probed with 2 µg/ml anti-YY1 antibody.
Reporter Gene Experiments--
The I1L promoter (nucleotides
Immunofluorescence Microscopy--
BSC-40 cells were grown on
coverslips and infected with vaccinia virus at a multiplicity of 10 pfu/cell. Cells were fixed with 4% paraformaldehyde in
phosphate-buffered saline (PBS; 150 mM NaCl, 15 mM sodium phosphate, pH 7.0) for 10 min at 22 °C and were permeabilized by washing three times with 0.2% Triton X-100 in
PBS for 5 min (12). Nonspecific binding sites were blocked by a 20-min
incubation in 3% bovine serum albumin in PBS. Coverslips were
incubated with 2 µg/ml anti-YY1 antibody for 1 h and washed three times in PBS, followed by a 40-min incubation with 1 µg/ml fluorescein isothiocyanate-conjugated anti-rabbit antibody (Chemicon). Coverslips were exposed to 1 µg/ml DAPI (Sigma) in PBS for 1 min, mounted on slides with Vectashield mounting solution (Vector
Laboratories, Burlingame, CA), and photographed on an Olympus BX60
fluorescence microscope.
Localization of the Protein Binding Site in the Vaccinia I1L
Promoter--
We previously described the identification of a late
promoter binding activity in extracts from uninfected HeLa cells (7). Prior nuclease footprinting experiments indicated that the protein bound the I1L promoter in the region surrounding the transcriptional start site (7). To delineate the sequences in the I1L promoter required
for interaction with the cellular protein, a series of promoter mutants
were constructed in which blocks of five nucleotides on the nontemplate
strand were replaced with C residues spanning nucleotides
The sequence immediately surrounding the nucleotides +2 to +6 of the
I1L promoter was searched for recognition motifs for known
transcription factors using the TRANSFAC program (17) accessed from the
WWW site at GBF-Brauschweig, Germany. The search revealed a core
recognition sequence for transcription factor YY1 (CCATT) on the
template strand. Two properties of YY1 supported its identity as the
late promoter-binding protein. First, SDS-polyacrylamide gel analysis
of column fractions generated in the purification of the late
promoter-binding protein often detected a co-purifying 65-kDa
polypeptide on SDS-polyacrylamide gels (data not shown). YY1 was
described previously as having an anomalous mobility on SDS-polyacrylamide gels, consistent with a 65-kDa polypeptide although
it is 414 amino acids in length (9). Also, it was previously reported
that human YY1 binds Ni2+, presumably through the 11 consecutive histidine residues in its amino acid sequence (18).
YY1 Antibodies Recognize the Late Promoter-binding Protein--
To
investigate whether the late promoter-binding activity was YY1, late
promoter-binding protein was tested for immunoreactivity with anti-YY1
antibodies. Nickel-purified promoter-binding protein was
chromatographed on a phosphocellulose column that was developed with a
NaCl gradient. Eluting fractions were assayed for I1L promoter binding
and YY1 by Western blotting with anti-YY1 antibody. Both the promoter
binding activity and a polypeptide with an apparent mass of 65 kDa
recognized by the anti-YY1 antibody eluted with a similar profile
peaking in fraction 32 (Fig. 2). Protein
from the peak fraction from the phosphocellulose chromatography
subsequently was subjected to velocity sedimentation on a glycerol
gradient. Again, both the promoter binding activity and the anti-YY1
antibody-reactive 65-kDa polypeptide were observed to co-fractionate,
peaking in fraction 20. The sedimentation rate of the promoter-binding
activity was essentially identical to that of the 4.46-S hemoglobin
sedimentation marker.
The identity of the late promoter-binding protein was confirmed by
antibody supershift assays. In electrophoretic mobility shift
experiments, the protein produced a characteristic complex with the I1L
promoter (Fig. 3). Inclusion of anti-YY1
antibody in the binding reaction resulted in a nearly quantitative
shift of the protein-DNA complex to a more slowly migrating complex. Antibody alone produced no complex with DNA. Essentially identical results were obtained with the vaccinia H1L promoter in which the
protein-DNA complex was observed to shift further in the gel when
anti-YY1 was included in the binding reaction. These results demonstrate that YY1 is a component of the protein-DNA complexes observed in gel shift experiments with late promoter probes.
A histidine-tagged version of YY1 was expressed in bacteria and
purified under native conditions by nickel-agarose chromatography. The
resulting protein preparations produced a protein-DNA complex in native
gel electrophoresis that had a slightly slower mobility than native
promoter binding activity purified from HeLa cell nuclei
(Fig. 4). The construct used to express
recombinant protein produces an 11-amino acid extension on the native
YY1 protein, possibly having a small effect on the mobility of the
protein-DNA complex. Binding reactions were conducted in the presence
of a 50-fold mass excess of the nonspecific competitor poly(dI-dC) to
ensure specificity of interaction. These results are consistent with
YY1 being the vaccinia late promoter-binding protein.
Importance of the YY1 Binding Site for Late Promoter
Function--
Replacement of nucleotides +2 to +6 in the I1L promoter
was shown above to abrogate binding to YY1. The last three nucleotides of the consensus YY1 core recognition sequence CCATT in the I1L promoter overlaps the conserved TAAAT motif previously shown to be
essential for the function of other vaccinia late promoters (19). We
sought a mutation that would affect the interaction of YY1 with a late
promoter without perturbing the TAAAT motif. An alteration of the YY1
binding site in the Rous sarcoma virus long terminal repeat promoter
initiator element corresponding to the 3'-T in the TAAAT motif and the
two following G residues resulted in inability to associate with YY1
(20). We therefore elected to replace the two G residues directly
downstream of the TAAAT motif with C residues to test for the
significance of the YY1 binding site. The double C mutant promoter was
found to have almost undetectable binding to YY1 in a gel shift assay
(Fig. 5). In addition, competition
binding experiments demonstrated that excess wild-type sequence I1L
promoter effectively competed with itself for binding to YY1, whereas
the mutant promoter had little capacity to compete with the wild-type
promoter in binding to YY1. Identical results were obtained with
bacterially expressed YY1 (data not shown).
The effect of the double C mutation on promoter activity was assessed
in a reporter gene experiment. The native I1L promoter was linked to
the E. coli Intracellular Localization of YY1 after Infection by Vaccinia
Virus--
YY1 has been documented as being located in the nucleus of
cells (22). The transcription of vaccinia late genes occurs in structures termed virosomes that are found exclusively in the cytoplasm
of the cell. For a nuclear protein to function in viral late gene
transcription, the normal intracellular targeting of the protein must
be altered. To address this issue, we examined the fate of YY1 after
vaccinia infection by immunofluorescence microscopy. The reactivity of
anti-YY1 antibodies with uninfected cells showed YY1 to be mostly
nuclear (Fig. 6), as reported previously (22). By 6 h after vaccinia infection, YY1 was distributed
throughout the cells in which nuclei often could not be distinguished
from the remainder of the cell. The cells had obvious DAPI staining of
cytoplasmic structures as evidence that they were indeed infected with
virus. The YY1 staining pattern changed further at later times
post-infection, in which distinct spherical structures in the cytoplasm
became evident by 16 h of infection. These results indicate that
infection by vaccinia virus had a profound effect on the intracellular
localization of YY1, causing significant quantities of the protein to
accumulate in the cytoplasm. The targeting of YY1 to the cytoplasm by
vaccinia infection is apparently not because of a general displacement
of DNA binding proteins from the nucleus because the immunolocalization
of the transcription factor CREB (cyclic AMP response element binding
protein) showed that its intracellular distribution was unaffected by
vaccinia infection (data not shown).
Our characterization of the vaccinia I1L promoter highlighted an
important sequence at the start site for transcription that implicated
the cellular transcription factor YY1 as a possible regulator of
vaccinia virus late gene transcription. The cellular protein that
targets vaccinia virus late promoters is shown here to react with
antibody directed against the transcription factor YY1. Bacterially
expressed YY1 interacted specifically with the I1L promoter, and a
simple dinucleotide replacement in the YY1 binding site downstream of
the conserved TAAAT late promoter motif resulted in both elimination of
YY1 binding and significant reduction in promoter strength in
vivo. These results provide a compelling argument for a role for
YY1 in activation of vaccinia virus late gene transcription. YY1 is a
zinc finger transcription factor belonging to the kruppel family of
proteins (reviewed in Ref. 23). This factor has been described as a
regulator of many cellular and viral promoters. YY1 can function as an
upstream activator, a repressor, or an initiator element regulator.
YY1 regulates several cellular genes in the capacity of an initiator
element-binding protein, i.e. the protein binds to the promoter overlapping the start site for transcription in a manner that
positively activates transcription (23). Contacts with the bases in the
recognition motif are made asymmetrically and have been proposed to
account for the directionality of transcription activated by YY1 (24).
The YY1 binding site in the vaccinia I1L promoter is also over the
start site for transcription, suggesting that YY1 may be acting as an
initiator element-binding protein in the vaccinia system. An important
distinction is that known cellular initiator elements have the YY1
recognition motif on the nontemplate strand, and vaccinia late
promoters have the motif on their template strand. The significance of
this distinction is unclear; however, it suggests that vaccinia may
utilize the protein for transcription in a way that may differ from
that which occurs in nuclei.
To our knowledge, YY1 is the first identified cellular protein
documented to function in poxvirus transcription. Rosales et al. (25) described a cellular protein required for transcription from vaccinia intermediate promoters; however, its identity has not
been reported. A cellular protein that activates late promoters in vitro has been described by Gunasinghe et al.
(6), but its identity has not been reported. YY1 would seem to be a
particularly suitable choice for adoption by vaccinia virus. Vaccinia
has a rather remarkable host range and can replicate in a wide variety, albeit not all, of mammalian cell lines. YY1 has been described as
being ubiquitous in terms of tissue distribution and the numerous mammalian cell lines that have been examined (23). Thus the cellular
distribution of YY1 would not likely impose a limit on the host range
of the virus.
The YY1 binding site in the I1L promoter overlaps the TAAAT motif on
its distal side. Our data argue that the TAAAT and YY1 motifs are
overlapping but functionally distinct elements of vaccinia late
promoters. The importance of the TAAAT motif for late gene transcription is well documented (19). Replacement of any nucleotide in
this motif results in an inactive promoter. We have replaced the 5'-A
in the TAAAT motif of the I1L promoter and confirmed its importance for
transcription in
vivo;2 however, this
change has no effect on YY1 binding (7). Thus the three most
5'-nucleotides of the TAAAT motif serves some function other than
interaction with YY1. We replaced the two G residues immediately distal
to the TAAAT motif and observed a correlation between transcription
in vivo and binding to YY1 in vitro. The importance of nucleotides following the TAAAT motif in a natural vaccinia late promoter had not been described previously.
If nucleotides downstream of the TAAAT motif are essential for promoter
function, then is predicted that these sequences should be conserved in
vaccinia late promoters. The optimal binding sequence for YY1 has been
defined as (C/g/a)(G/t)(C/t/a)CATN(T/a)(T/g/c), in which uppercase
letters are the preferred nucleotide and lowercase letters are
tolerated to a lesser extent (26). In the context of the TAAAT motif of
vaccinia late promoters, the optimal YY1 site on the nontemplate strand
would be TAAATGGCG. Only in the first T residue does this sequence
deviate from the optimal YY1 site. This exact sequence is found in the
I1L promoter. The first G in this sequence is naturally conserved
because the vast majority of vaccinia late genes have translation
initiation codons at this location. The following G residue is also
highly conserved. An examination of the sequences of 51 late genes
revealed through the comparison of vaccinia and molluscum contagiosum
virus sequences (27) shows that 30 have G at this position, and 15 others have A.
For a cytoplasmic virus such as vaccinia to utilize a cellular protein
that is normally nuclear, a redirection of the trafficking of the
protein must occur. In agreement with others (22), we observed YY1 to
be predominantly nuclear in uninfected cells. After vaccinia infection,
YY1 clearly was distributed throughout the entire cell, and at later
times, was concentrated in cytoplasmic globules resembling virosomes,
large nucleoprotein complexes that are actively replicating DNA and
synthesizing late mRNA. Because the translation of all host
mRNA is believed to be inhibited shortly after vaccinia infection
(28), the redistribution of YY1 likely reflects an alteration in
intracellular trafficking of pre-existing protein. Whether this is a
passive process of binding of YY1 to the thousands of accumulating DNA
binding sites amplified by viral DNA replication in the cytoplasm or is
an active process induced by the virus is unclear.
It is of interest to point out that poxviruses can be added to the list
of DNA viruses that have been shown to require YY1 as a regulator of
transcription. Herpesviruses (29, 30), papillomaviruses (31, 32),
polyomaviruses (33), adenoviruses (34), parvoviruses (9, 35), and
retroviruses (20, 36) use YY1 in one of its various capacities as a
transcriptional regulator. Inclusion of poxviruses in this group
indicates that virtually all of the major DNA virus groups require YY1
function. The widespread utilization of this protein underscores its
fundamental importance in the transcription process.
We are grateful to Tom Shenk for the pHIS-YY1
plasmid and to Jonathan LeBowitz and John Maga for advice on
immunofluorescence and image processing.
*
This work was supported by a grant from the NIAID, National
Institutes of Health. This is paper number 16028 from the Purdue Agricultural Experiment Station.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
2
(X. Liu and S. S. Broyles, unpublished results.
The abbreviations used are:
PBS, phosphate-buffered saline;
DAPI, 4,6-diamidino-2-phenylindole.
Transcription Factor YY1 Is a Vaccinia Virus Late Promoter
Activator*
,
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
29 to +16, relative to the 5'-A in the TAAAT motif) was ligated in
the form of an annealed double-stranded oligonucleotide into the
KpnI and NcoI sites of plasmid pTM1 to place the
-galactosidase gene behind the I1L promoter. Mutation of the
promoter was achieved by a 3-stage polymerase chain reaction strategy
(13). For reporter gene expression, 1 × 107 HeLa
cells were grown to 60% confluent monolayers and infected with
vaccinia virus WR strain at a multiplicity of 10 plaque-forming units
per cell. After 1 h of infection, cells were transfected with 3 µg of reporter gene plasmid using the transfection reagent Superfect
(Qiagen) per the instruction of the manufacturer. After 16 h,
cells were harvested by scraping, and -galactosidase activity was
determined spectrophotometrically as described (14). Protein concentrations were determined with the Bio-Rad protein assay. Primer
extension assays were performed as described (15) on 60 µg of total
RNA isolated from vaccinia-infected HeLa cells transfected with
reporter gene constructs as described. RNA was isolated using an RNeasy
mini kit (Qiagen), and primer extension was performed with an
end-labeled 30-mer oligonucleotide complementary to -galactosidase
mRNA and avian myoblastosis virus reverse transcriptase (Roche).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
30 to +11,
relative to the 5'-A in the conserved TAAAT motif at the start site for
transcription (Fig. 1). All of the
promoter variants were capable of binding the protein with the
exception of mutant 7 which has substitutions in nucleotides +2 to +6
overlapping the TAAAT motif at the start site for transcription. The
TAAAT motif is highly conserved in vaccinia late promoters and is
usually located at the initiation codon of late gene open reading
frames (16).
View larger version (60K):
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Fig. 1.
Scanning substitutional analysis of sequences
in the I1L promoter interacting with late promoter-binding
protein. Synthetic oligonucleotide probes having the wild-type I1L
sequence (lanes 1 and 2; panel B) or
the pentanucleotide substitutions (M1-M8) shown in
panel A were assayed for binding to the late
promoter-binding protein by electrophoretic mobility shift. DNA probe
bound to protein is indicated by the arrow. , probe alone; +, probe
plus 5 ng of purified promoter-binding protein.
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Fig. 2.
Co-fractionation of late promoter binding
activity and YY1. Nickel-agarose-purified protein was
chromatographed on a phosphocellulose column developed with a NaCl
gradient and assayed for I1L promoter binding activity by
electrophoretic mobility shift (panel A) and reactivity with
anti-YY1 antibody by Western blotting (panel B). The peak
fraction from the phosphocellulose chromatography was subjected to
glycerol gradient sedimentation and assayed for promoter binding
(panel C) and reactivity with anti-YY1 antibody (panel
D). The glycerol gradient was fractionated from bottom
to top, generating 32 total fractions. In panels
A and C, the mobility of the protein-DNA complex is
indicated by an arrow on the right. In
panels B and D, the mobility of the
antibody-reactive polypeptide is indicated on the left. The
mobilities of protein size standards are given on the
right.
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Fig. 3.
Detection of YY1 in association with vaccinia
late promoter DNA. Purified late promoter-binding protein was
assayed for DNA binding to the I1L promoter (panel A) and
the H1L promoter (panel B) by electrophoretic mobility
shift. Promoter DNA probes were incubated alone (lane 1),
with purified HeLa protein (lane 2), with purified HeLa
protein and anti-YY1 antibody (lane 3), or with antibody
alone (lane 4). The mobility of the protein-DNA complex is
indicated by a thin arrow, and the mobility of the
super-shifted antibody complex is indicated by a thick
arrow.
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Fig. 4.
Late promoter binding activity of bacterially
expressed YY1. Bacterially expressed YY1 was purified by
nickel-agarose chromatography, and DNA binding activity was determined
by electrophoretic mobility shift of the I1L promoter. Lane
1, DNA probe alone; lane 2, probe plus 10 ng of
purified HeLa YY1; lane 3, probe plus 20 ng of bacterially
expressed YY1. The bacterial preparation contains several contaminating
proteins, much of which appears to be YY1 degradation products (data
not shown). The mobility of the YY1-DNA complex is indicated by the
arrow.
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Fig. 5.
Effect of a dinucleotide replacement in the
YY1 binding sequence in the I1L promoter on interaction with YY1
in vitro and promoter activity in
vivo. The GG sequence immediately following the TAAAT
motif at the start site for transcription in the I1L promoter was
replaced with the sequence CC as shown in panel A. DNA
binding was determined by electrophoretic mobility shift (panel
B). Binding reactions contained wild-type I1L promoter alone
(lanes 1 and 7), or in combination with 4 or 10 ng of purified YY1 (lanes 2 and 3, respectively).
The CC mutant promoter probe was incubated with 0, 4, or 10 ng of
purified YY1 (lanes 4, 5, and 6,
respectively). Lanes 8-11 depict a competition experiment
in which reactions containing 10 ng of YY1 and 0- (lane 8),
20- (lane 9), 50- (lane 10), and 100-fold
(lane 11) excess of nonradiolabeled wild type promoter DNA
as competitor. Lanes 12-14 show the results of a similar
experiment in which 20-, 50-, and 100-fold, respectively, excess of
nonradiolabeled CC mutant I1L promoter DNA was included as competitor.
The arrow indicates the protein-DNA complex. Panel
C, reporter gene activity of wild-type I1L promoter and CC mutant
constructs. -Galactosidase activity was normalized to protein
concentration. Filled bars represent averaged activities
among replicate samples, and open bars indicate the standard
deviation. Panel D, primer extension analysis of
-galactosidase mRNA. Lane 2 is analysis of RNA from
vaccinia infected cells transfected with the wild-type promoter
construct; lane 3 is from infected cells transfected with
the CC promoter mutant construct; and lane 1 is from
infected cells not transfected with plasmid. Lane 4 is
primer alone. The bracket indicates the cDNAs derived from the
population of RNAs arising from the promoter.
-galactosidase gene, as was the double C
variant promoter. Replacement of the first G in this sequence would
remove the first methionine codon in this construct; however, the
natural initiation codon for -galactosidase is located 18 nucleotides downstream of the first one and should serve as an adequate
replacement. The two promoter-reporter gene plasmids were transfected
into HeLa cells that previously were infected with vaccinia virus.
Reporter enzyme levels were found to be very high from the wild-type
I1L promoter (Fig. 5). Replacement of the two G residues immediately
downstream of the TAAAT motif resulted in a promoter with 8 ± 0.8% of wild-type promoter activity. Because the nucleotide
replacements disturbed the site at which translation initiation should
occur, we examined the -galactosidase transcripts produced in
transfected cells. Primer extension experiments detected -galactosidase transcripts in RNA isolated from cells transfected with the construct driven by the wild-type promoter. Vaccinia late gene
transcripts are heterogeneous at their 5'-ends and are, on average,
about 30 nucleotides longer than predicted from the site of initiation
because of apparent slippage of the RNA polymerase and polymerization
of nontemplate-encoded A residues as it initiates transcription within
the TAAAT motif (15, 21). From the length of the primer extension
products, we surmise that transcripts from the I1L promoter had a
10-30-nucleotide oligo-A leader at their 5'-ends. The double C mutant
promoter produced RNA with similar 5'-ends, but significantly reduced
transcript levels were evident. This result indicates that the reduced
reporter gene activity with the mutant promoter is because of reduced
promoter activity. We conclude that the two G nucleotides immediately
downstream of the TAAAT motif are important for both association with
YY1 and promoter activity in vivo, providing a correlation
between transcription activation and YY1 binding to the vaccinia promoter.
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Fig. 6.
Immunofluorescent microscopy of YY1
distribution in normal and vaccinia-infected BSC-40 cells. Cells
growing on coverslips were infected with vaccinia virus for 6 and
16 h and were stained with anti-YY1 antibody and DAPI. 0 h indicate cells not infected with virus. Merged pictures show the
DAPI-stained image superimposed on the YY1-stained image.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 765-494-0745;
Fax: 765-494-7897; E-mail: broyles@biochem.purdue.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Moss, B.
(1996)
in
Fields Virology
(Fields, B. N.
, Knipe, D. M.
, and Howley, P. M., eds)
, pp. 2637-2671, Lippincott-Raven Publishers, Philadelphia
2.
Moss, B.
(1994)
in
Transcription Mechanisms and Regulation
(Conaway, R. C.
, and Conaway, J. W., eds)
, pp. 185-205, Raven Press, New York
3.
Keck, J. G.,
Baldick, C. J. J.,
and Moss, B.
(1990)
Cell
61,
801-809[CrossRef][Medline]
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4.
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