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J Virol, August 1998, p. 6752-6757, Vol. 72, No. 8
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The High Mobility Group Protein 1 Is a Coactivator
of Herpes Simplex Virus ICP4 In Vitro
Michael J.
Carrozza and
Neal
DeLuca*
Department of Molecular Genetics and
Biochemistry, University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania 15261
Received 19 March 1998/Accepted 8 May 1998
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ABSTRACT |
ICP4 is an activator of herpes simplex virus early and late gene
transcription during infection and in vitro can efficiently activate
the transcription of a core promoter template containing only a TATA
box and an initiator element. In this study, we noted that the extent
of activation by ICP4 in vitro was highly dependent on the purity of
TFIID when recombinant TFIIB, TFIIE, and TFIIF were used as sources of
these factors. ICP4 efficiently activated transcription with a crude
TFIID fraction. However, when immunoaffinity-purified TFIID was used in
place of the less pure TFIID, ICP4 activated transcription to a
significantly lesser extent. This finding indicated that the crude
TFIID fraction may contain additional factors that serve as
coactivators of ICP4. To test this hypothesis, the crude TFIID
preparation was further fractionated by gel filtration chromatography. The TFIID that eluted from the column lacked the hypothesized coactivator activity. A fraction well separated from TFIID contained an
activity that when added with the TFIID fraction resulted in higher
levels of transcription in the presence ICP4. Further purification of
the coactivator-containing fraction resulted in the isolation of a
single 30-kDa polypeptide (p30). p30 was also shown to serve as a
coactivator of ICP4 with immunoaffinity-purified TFIID; however, p30
had no effect on basal transcription. Amino acid sequence analysis
revealed that p30 was the high mobility group protein 1, which has been
shown to facilitate the formation of higher-order DNA-protein
complexes.
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INTRODUCTION |
The herpes simplex virus (HSV)
infected cell protein 4 (ICP4) is the major transcriptional activator
of viral early and late gene expression (9, 16, 18, 19, 34,
42). This protein also serves as a transcriptional repressor of
some HSV promoters, including the ICP4 gene promoter (9,
35). The 175-kDa ICP4 polypeptide exists as a dimeric nuclear
phosphoprotein (8, 32, 39, 48) that binds to DNA
(17) and possesses discrete functional domains (11, 37,
38, 46). Although the DNA-binding activity of ICP4 appears to be
required for activation (38, 46), several studies have shown
that specific binding sites are not required (4, 14, 15, 23,
50). How the functional domains of ICP4 collectively function to
regulate the transcription of different HSV promoters is not completely
understood.
All HSV genes are transcribed from promoters recognized by the RNA
polymerase II (Pol II) transcription machinery (1, 5), which
consists of at least seven general transcription factors (GTFs): TFIIA,
-B, -D, -E, -F, and -H and Pol II itself (reviewed in reference
36). The GTFs form a preinitiation complex on class II promoters that enable Pol II to accurately initiate transcription (reviewed in reference 36). It has been shown that a
core Pol II promoter from the glycoprotein C (gC) gene, containing only a TATA box and initiator element (Inr), can be efficiently activated by
ICP4 in vitro (21). Although ICP4 can activate promoters containing only a TATA box, maximal levels of ICP4-activated
transcription are seen when both a TATA box and Inr are present
(7, 21), indicating that factors recognizing these elements
are involved in ICP4-activated transcription.
The observation that ICP4 interacts with TFIID (2), and
activates promoters with a TATA box possessing a low affinity for TATA
box-binding protein (TBP) to a greater extent than promoters with a
TATA box possessing a high affinity for TBP, suggests that ICP4 may
facilitate TFIID binding to the promoter (6, 25). It has
also been shown that activation of promoters by ICP4 is enhanced by the
addition of an Inr element, both in vivo (7) and in vitro
(21). Furthermore, ICP4 contacts TFIID through the
TBP-associated factor TAF250 in a manner dependent on the C-terminal
region of ICP4 (2, 10, 11), and this region of ICP4 is
required for efficient activation of transcription (2, 10,
11). TAF250 is an integral part of the TFIID complex (3, 45,
55) and has been shown to interact with Inr elements (33,
52). How the interactions between TBP, TAF250, and their respective cis-acting sites in combination with ICP4 result
in activation is not known. It is likely that additional cell factors that may function at start site regions and/or facilitate the formation
of ICP4 containing transcription complexes are involved in activation.
In this study, we determined whether additional factors were involved
in ICP4-activated transcription by observing the efficiency with which
ICP4 activated transcription in vitro, using purified GTFs and ICP4 as
well as HeLa transcription factor preparations differing in purity.
Using this system, we purified the cellular high mobility group protein
1 (HMG 1) from a crude HeLa transcription factor preparation based on
its ability to augment ICP4 activation.
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MATERIALS AND METHODS |
GTFs and ICP4.
Pol II and TFIID were obtained by
fractionating HeLa nuclear extract sequentially on phosphocellulose and
DEAE-Sephacel as previously described (12, 43). The DB
fraction was further fractionated by Superose 6 gel filtration
chromatography (Pharmacia) as described below, and TFIID-containing
fractions were identified by slot blot analysis with a polyclonal
antibody to TBP (Upstate Biotechnology). Hemagglutinin (HA)-tagged
TFIID (eTFIID) from the HeLa cell line LTR 
3 was
immunoaffinity purified to homogeneity by using the anti-HA monoclonal
antibody 12CA5 (Boehringer Mannheim) covalently coupled to protein
A-Sepharose (57). Recombinant human TFIIB (rTFIIB) was
purified from Escherichia coli according to the method of Ha
et al. (22). The recombinant 34- and 56-kDa subunits of
TFIIE were each purified from E. coli and the rTFIIE was
reconstituted in vitro (40). The recombinant TFIIF subunits, RAP30 and RAP74, were each purified from E. coli and
reconstituted to rTFIIF in vitro (53, 54).
ICP4 was purified from human embryonic lung fibroblast nuclei infected
with the wild-type HSV strain KOS as previously described (24,
28).
HMG 1 was purified by first applying the HeLa fraction DB (3 mg of
protein) to a 30-ml Superose 6 (Pharmacia) column in buffer
D (20 mM
HEPES [pH 7.9], 20% glycerol, 0.2 mM EDTA, 0.2 mM
phenylmethylsulfonyl
fluoride, 0.5 mM dithiothreitol) with 0.1 M KCl.
Fractions were
assayed for the ability to enhance ICP4-activated
transcription
in an in vitro transcription assay reconstituted with
purified
basal transcription factors as described below. Fractions were
also subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis
(SDS-PAGE) (10% gel) and silver stained. Fractions
exhibiting
the greatest enhancement of ICP4-activated transcription
were
pooled and applied to a 0.5-ml mini-Mono Q ion-exchange column
(Pharmacia) in buffer D with 0.1 M KCl. The column was eluted
with a
5-ml linear gradient from 0.1 to 0.5 M KCl in buffer D.
Fractions were
collected and dialyzed to 0.1 M KCl in buffer D.
Fractions were assayed
for enhancement of ICP4-activated transcription
by in vitro
transcription analysis as described below. A portion
of the peak ICP4
activation-enhancing fraction was also subjected
to SDS-PAGE (15% gel)
and Coomassie blue stained. The 30-kDa polypeptide
present in this
fraction was subjected to amino-terminal sequence
analysis by Edman
degradation (University of Michigan protein
and carbohydrate structure
facility).
In vitro transcription analysis.
Transcription reactions
were set up in a final volume of 30 µl containing 3 µl of fraction
CC which contains Pol II, 0.3 µg of rTFIIB, 0.16 µg of
reconstituted rTFIIF, 10.5 ng of rTFIIE p56, 6.43 ng of rTFIIE p34, 50 to 100 ng of ICP4, 10 fmol of template DNA, and either 1 µl of
fraction DB, 4 µl of immunoaffinity-purified TFIID, or 8 µl of
Superose 6-fractionated TFIID (called Superose TFIID). The amount of
TFIID in each preparation was normalized according to the relative
abundance of TBP present. This was determined by Western analysis using
a polyclonal antibody directed against TBP. Superose 6 and Mono Q
column fractions were assayed by adding 5 and 2 µl, respectively,
from the column fractions to transcription reaction mixtures by using
the Superose TFIID. The transcription template consisted of
superhelical plasmid DNA containing the gC core promoter with either a
wild-type (wt) or functionally mutant (mut) Inr. The mut Inr plasmid
contains a linker scanning mutation changing positions +1 to +6 from
ACTACC to GAGCTC.
Transcription reactions were performed in a solution containing 40 mM
HEPES (pH 7.9), 60 mM KCl, 12% glycerol, 8.3 mM MgCl
2,
0.6 mM ribonucleoside triphosphates, 12 U of RNasin, and 0.3 mM
dithiothreitol. The reactions were incubated for 1 h at 30°C
mixtures,
and the reactions were stopped with 70 µl of transcription
stop
buffer containing 150 mM sodium acetate, 15 mM EDTA, and 150 µg
of tRNA per ml. Samples were then phenol extracted, chloroform
extracted, and ethanol precipitated.
For primer extension analysis, the pellets were resuspended in 10 µl
of a primer annealing mixture containing 10 mM Tris-HCl
(pH 7.5), 250 mM KCl, and 3 to 6 ng of 5'
32P-end-labeled primer
complementary to the nucleotides from +42
to +77 downstream of the
transcription start site. The primer
was annealed by heating at 65°C
for 30 min and then slowly cooled
to room temperature. The annealed
transcripts were subjected to
primer extension in a solution containing
50 mM Tris-HCl (pH 8.3),
75 mM KCl, 3 mM MgCl
2, 10 mM
dithiothreitol, 1 mM deoxynucleoside
triphosphates, 12 U of RNasin, 50 µg of actinomycin D per ml,
and 300 U of Moloney murine leukemia
virus reverse transcriptase
in a final volume of 40 µl. Reaction
mixtures were incubated at
42°C for 1 h, and the reactions were
stopped by adding 60 µl of
a solution containing 1 M ammonium acetate
and 20 mM EDTA. The
samples were phenol extracted and ethanol
precipitated. The pellets
were resuspended in 5 µl of gel loading
buffer containing 96%
formamide, 10 mM EDTA, 0.01% bromophenol blue
and xylene cyanol,
and 10 mM NaOH. The samples were separated on 6%
sequencing gels,
fixed, dried, and exposed to X-ray film.
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RESULTS |
Previous studies have shown that ICP4 efficiently activates
transcription in vitro with crude HeLa transcription factor fractions on a simple promoter containing only a TATA box and an Inr
(21). Mutation of the Inr impaired the ability of ICP4 to
efficiently activate transcription. Therefore, we hypothesized that
factors present in the HeLa transcription factor fractions and
dependent on an intact start site region were serving as ICP4
coactivators. In this study, we demonstrated the presence of an ICP4
coactivator in a crude HeLa TFIID preparation and then proceeded to
purify and identify the activity.
A crude TFIID preparation contains an ICP4 coactivator.
TFIID
is routinely prepared by fractionating HeLa nuclear extract on
phosphocellulose and DEAE-Sephacel (12, 43). This preparation is called the DB fraction. Although this fraction possesses
TFIID activity, it remains in a relatively crude state. In this study,
we first determined whether this fraction contained an ICP4 coactivator
activity that is separable from TFIID. As depicted in Fig.
1, we performed an in vitro transcription
experiment to compare the efficiencies of activation by ICP4 with
immunoaffinity-purified TFIID versus the DB fraction. TFIIB, -E, and -F
were added to all reaction mixtures as recombinant proteins purified
from E. coli, while Pol II was added within HeLa CC
fraction. The template used in this experiment was the gC core promoter
template, which contains only a TATA box and either the wt or mut Inr.
By using the wt and mut Inr templates to compare transcription
efficiencies, we were able to assess the role of this site in basal and
activated transcription as a function of the crude and purified TFIID
preparation.
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FIG. 1.
Abilities of different TFIID preparations to support
ICP4-activated transcription. In vitro transcription reactions were
performed with the Pol II fraction CC, rTFIIB, -E, and -F, and either
immunoaffinity-purified eTFIID or the crude TFIID fraction,
DB, in the absence and presence of purified ICP4. The quantity of each
TFIID preparation was normalized as described in Materials and Methods.
These conditions were tested on a gC core promoter template containing
a TATA box and either a wt or a mut Inr. Shown are the primer extension
products from reverse-transcribed RNA synthesized in the in vitro
reactions.
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With this set of GTFs, ICP4 activated transcription, albeit poorly,
using immunoaffinity-purified TFIID (Fig.
1; compare lanes
1 and 2) in
an Inr-dependent manner (compare lanes 1 and 2 with
lanes 3 and 4).
This mutation in the Inr also reduced the number
of transcription start
sites from three to one (compare lanes
1 and 3). With the DB fraction,
ICP4 activated transcription four-
to fivefold better than with
purified TFIID (compare lanes 5 and
6 with lanes 1 and 2), also in an
Inr-dependent manner (compare
lanes 5 and 6 with lanes 7 and 8). These
results indicate that
a factor(s) present in DB allows ICP4 to activate
more efficiently.
In a previous report, we demonstrated efficient
ICP4-activated
transcription when immunoaffinity-purified TFIID was
substituted
for the DB TFIID fraction (
2). However, in this
study we used
a relatively crude HeLa TFIIE and -F preparation in
comparison
to the purified recombinant TFIIE and -F used for Fig.
1. It
is
likely that this crude TFIIE and -F fraction also contains
coactivators
that contribute to ICP4's ability to efficiently activate
transcription.
Also note that in lanes 5 and 6 it was not necessary to
add TFIIA
and -H to obtain efficient ICP4 activation. In the case of
TFIIA,
this was first observed in a previous study (
21).
ICP4 also activated transcription on the mut Inr template using the DB
fraction, although poorly in comparison to that seen
on the wt template
(Fig.
1; compare lanes 5 and 6 with lanes 7
and 8). This low level of
activation is reflective of ICP4-activated
transcription levels
observed with the purified TFIID (compare
lanes 1 and 2 with lanes 7 and 8), suggesting that a factor(s)
not associated with TFIID but
present in the DB fraction and dependent
on the start site region
serves as an ICP4 coactivator.
One study has demonstrated that this crude TFIID preparation contains
activities that function through the Inr (
28). It
is
believed that these activities allow TFIID to nucleate preinitiation
complexes through the Inr. However, these factors are not associated
with the TFIID multiprotein complex. The effect of this activity
is
evident in the experiment in Fig.
1. With the crude TFIID preparation,
the Inr had a threefold effect on basal transcription (compare
lane 5 with 7), indicating that a factor(s) in this fraction augmented
basal
transcription in an Inr-dependent manner. When purified
TFIID was
substituted for the DB TFIID preparation, this effect
was not observed
(compare lanes 1 and 3), indicating that the
Inr factor(s) is not
associated with TFIID.
Separation of TFIID from coactivator functions.
To clearly
demonstrate that this coactivator and basal Inr factor were activities
independent of TFIID, we further fractionated the DB fraction by gel
filtration chromatography on Superose 6. Since TFIID is approximately
700 kDa, we reasoned that gel filtration would resolve TFIID from the
many other proteins present in the DB preparation. The fractions
containing TFIID were identified by slot blot analysis with an antibody
directed against TBP. As expected, TFIID resolved early in the elution
of the column. As shown in Fig. 2A, the
Superose TFIID (lanes 5 and 6), like the immunoaffinity-purified TFIID
(lanes 1 and 2), lacked the basal Inr activity that was apparent when
the crude DB fraction was used (lanes 3 and 4). ICP4-activated
transcription was also reduced with the Superose TFIID preparation
(Fig. 2B, lanes 7 to 9), reflective of the levels of activation
observed with immunoaffinity-purified TFIID (compare lanes 7 to 9 with
lanes 1 to 3). The apparent lower level of activation seen with
Superose TFIID relative to eTFIID is simply a consequence of
the lower level of transcription with this preparation (lane 7). As
expected, the DB fraction supported efficient ICP4-activated
transcription (lanes 4 to 6). The experiments in Fig. 2 were performed
with equivalent amounts of TFIID, based on TBP content as determined by
Western analysis. These results indicate that the basal Inr and ICP4
coactivator activities are independent of TFIID, since TFIID
fractionated on the basis of size lacks both of these activities.
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FIG. 2.
Purified TFIID lacks basal Inr activity and less
efficiently supports ICP4 activation. The DB fraction was applied to a
Superose 6 gel filtration column. The fractions containing TFIID were
identified by slot blot analysis of column fractions with an antibody
directed against TBP. The TFIID eluting from this column is designated
Superose TFIID. In vitro transcriptions were performed with Pol II
(CC), rTFIIB, -E, and -F, and either immunoaffinity-purified TFIID, DB
fraction, or Superose TFIID. The quantity of each TFIID preparation was
normalized as described in Materials and Methods. (A) Basal Inr
activity of three different TFIID preparations. Each TFIID preparation
was tested on the gC core promoter containing a TATA box and either a
wt or a mut Inr. (B) ICP4-activated transcription using the three
different TFIID preparations. Each reaction mixture received either 0, 50, or 100 ng of purified ICP4. Transcription reactions in lanes 1, 4, and 7 represent the basal levels of transcription with each of the
three TFIID preparations.
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It is clear that the more highly purified TFIID preparations were not
as efficient in supporting activated transcription as
the less purified
DB preparation and that there may be proteins
in the DB preparation
that facilitate activation. To test this
hypothesis, portions of each
fraction from the Superose 6 column
described above containing eluate
subsequent to that containing
TFIID were assayed for their effects on
ICP4-activated transcription
in vitro with the recombinant GTFs, Pol
II, and TFIID obtained
from the earlier-eluting fractions. Figure
3A shows the in vitro
transcription
results with fractions 23 to 28 of the 30 fractions
tested. Peak
enhancement of ICP4-activated transcription was apparent
in fraction
25.
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FIG. 3.
Gel filtration chromatography of the DB fraction further
fractionates an ICP4 coactivator. (A) Transcription in the presence of
ICP4 and gel filtration chromatography fractions. Superose 6 gel
filtration fractions were assayed by adding a portion of each fraction
to in vitro transcription reaction mixtures containing Pol II, rTFIIB,
-E, and -F, Superose TFIID, and the wt gC core promoter template. ICP4
was included in each reaction except in lane B, which represents basal
transcription levels with this combination of GTFs. The second lane
indicates the level of ICP4-activated transcription without the
addition of any column fractions. Only a subset (fractions 23 to 28) of
the fractions tested are shown. A total of 30 fractions were collected
from the column, with TFIID eluting in fraction 4. (B) Silver-stained
gel after SDS-PAGE analysis of Superose 6 fractions. A portion of each
fraction was loaded onto an SDS-10% polyacrylamide gel and silver
stained. Shown are fractions 17 to 27.
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The Superose 6 fractions were also subjected to SDS-PAGE and silver
stained (Fig.
3B). Fraction 25 contained two polypeptides
with apparent
molecular masses of 66 and 30 kDa. Although reduced
in abundance, these
polypeptides and coactivator activity were
also apparent in fraction
24. Fractions 24 and 25 were pooled
and further fractionated by Mono Q
ion-exchange chromatography.
Forty fractions were collected, and
aliquots of the flowthrough
and gradient were analyzed by using the in
vitro transcription
assay described above. Shown in Fig.
4 are the results of the
assay with the
flowthrough and fractions 18 to 24. The enhancement
of ICP4-activated
transcription exhibited an elution profile from
fractions 19 to 23, with the maximum appearing in fraction 23.
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FIG. 4.
Ion-exchange chromatography of Superose 6 ICP4
coactivator fractions. Superose 6 fractions 24 and 25 were loaded onto
a Mono Q ion-exchange column and eluted with a linear 0.1 M to 0.5 M
KCl gradient. Fractions were assayed by adding a portion of each
fraction to in vitro transcription reaction mixtures containing Pol II,
rTFIIB, -E, and -F, Superose TFIID, and the wt gC core promoter
template. ICP4 was included in each reaction except in lane B, which
represents basal transcription levels with this combination of GTFs.
The second lane indicates the level of ICP4 activated transcription
without the addition of any column fractions. Flowthrough (FT)
represents the material that did not bind the column at 0.1 M KCl.
Shown are the results with fractions 18 to 24.
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HMG 1 potentiates ICP4 activation with purified TFIID.
We next
determined whether Mono Q fraction 23 exhibited coactivator activity
with ICP4 and whether this fraction's activity was dependent on a
functional Inr using the immunoaffinity-purified TFIID. As shown in
Fig. 5A, ICP4 poorly activated
transcription using purified TFIID (lanes 1 and 2). The addition of
Mono Q fraction 23 significantly enhanced the ability of ICP4 to
activate transcription (lanes 5 and 6). This level of activation was
comparable to that observed when DB was substituted for purified TFIID
and Mono Q fraction 23 (compare lanes 5 and 6 with lanes 9 and 10).
Mono Q fraction 23 did not affect basal transcription (lanes 1 and 5).
ICP4-activated transcription with the Mono Q fraction 23 was also
dependent on a functional Inr (compare lanes 5 and 6 with lanes 7 and
8). This was also reflective of the Inr-dependent nature of ICP4
activation with DB (compare lanes 9 and 10 with lanes 11 and 12).
However, this fraction did not display any Inr activity under basal
transcription conditions (compare lanes 5 and 7), suggesting that the
Inr activity present in DB may have eluted elsewhere in either the
Superose 6 or Mono Q column. Thus, Mono Q fraction 23 contains an
activity that helps ICP4 activate transcription dependent on an intact
Inr sequence and does not affect basal transcription.
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FIG. 5.
A 30-kDa polypeptide possesses ICP4 coactivator
activity. (A) In vitro transcription analysis of Mono Q fraction 23 with immunoaffinity-purified TFIID. In vitro transcription reactions
were performed with Pol II, rTFIIB, -E, and -F, and either
immunoaffinity-purified eTFIID or the crude TFIID fraction,
DB, in the absence and presence of purified ICP4 and Mono Q fraction
23. The quantity of each TFIID preparation was normalized as described
in Materials and Methods. Each condition was tested on a gC core
promoter template containing a TATA box and either a wt or a mut Inr.
(B) SDS-PAGE analysis of Mono Q fraction 23. A portion of Mono Q
fraction 23 was loaded onto an SDS-15% polyacrylamide gel and stained
with Coomassie blue. No additional bands were observed in this
preparation when an overloaded gel was stained with silver (data not
shown).
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SDS-PAGE analysis of Mono Q fraction 23 revealed the presence of a
single polypeptide with an apparent molecular mass of 30
kDa,
designated p30 (Fig.
5B). p30 did not interact with ICP4
in an
immunoaffinity assay using an HA-tagged ICP4 and the anti-HA
monoclonal
antibody 12CA5 (data not shown). Amino-terminal sequence
analysis
through 39 residues revealed that the sequence of p30
identically
matched the amino-terminal sequence of HMG 1 (Fig.
6). Therefore, HMG 1 can serve as a
coactivator of ICP4 in the
presence of an intact Inr sequence.
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FIG. 6.
p30 is HMG 1. p30 (Mono Q fraction 23) was subjected to
amino-terminal sequence analysis through the first 39 amino acids.
Shown is an amino acid sequence alignment comparing HMG 1 and p30.
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DISCUSSION |
In this study, we showed that the HSV activator ICP4 can activate
transcription from a core promoter element by using a relatively simple
set of cellular transcription factors. The level of activation was
lower than that observed with a less pure set of factors and was
dependent on a functional Inr element. This implied that there were
proteins present in the less pure system that helped ICP4 activate
transcription. One such protein was identified when the crude
TFIID-containing fraction (DB) was substituted for
immunoaffinity-purified TFIID in the transcription system. This
resulted in substantially higher levels of activation, indicating that
coactivators in addition to TAFs were present in this fraction and were
enhancing ICP4-activated transcription. With further fractionation of
the crude TFIID-containing fraction, it was possible to separate TFIID
from an activity that enhanced ICP4 activation. This activity was
purified to homogeneity based on the presence of a single 30-kDa
protein on SDS-PAGE profiles. This protein was shown to enhance ICP4
activation by using the purest set of transcription factors, which had
previously supported only low levels of activation. Amino-terminal
sequence analysis of the 30-kDa polypeptide identified it as HMG 1. Thus, we concluded that HMG 1 can serve as a coactivator of ICP4.
HMG 1 is member of a ubiquitous family of nonhistone chromosomal
proteins that share a DNA-binding region known as the HMG domain
(reviewed in reference 20). HMG 1 belongs to the HMG 1/2 subclass that characteristically contains multiple HMG domains and
binds DNA nonspecifically (reviewed in reference
20). The other subclass of HMG domain proteins
contains only one HMG domain and binds DNA more specifically. HMG 1 has
been shown to facilitate the formation of higher-order nucleoprotein
complexes, which is believed to be the result of a DNA-bending activity
shared by all HMG proteins. The role of HMG 1 as a coactivator, as
reported here, is not unprecedented. HMG 1 has been shown to enhance
Gal4-VP16-mediated activation in vitro (49) and p53
activation in transient transfection experiments (26). Thus,
it has been hypothesized that HMG 1/2 proteins, through DNA bending,
remodel promoter DNA conformation such that the interactions between
activators and GTFs occur more efficiently (49).
A model for ICP4-activated transcription.
While many HSV
promoters are considerably more complex than the core gC promoter, and
many do not contain a strong match to the consensus Inr element, the
studies described in this and previous reports (7, 21) allow
us to propose a mechanism for how ICP4 may function to activate a
relatively simple promoter. In an earlier study, we demonstrated that
ICP4 interacts with TFIID through TAF250 and that this interaction is
dependent on the C-terminal region of ICP4 (2). ICP4 did not
interact with any other GTFs in that study (2).
Additionally, it has been shown that the ICP4 C-terminal region is
important for activated transcription both in vitro and in vivo
(2, 10, 11). Since TFIID is the only GTF present in the
simplified transcription system used in the present study shown to
interact with ICP4, we believe that the weak but reproducible level of
activation is due to the interaction between ICP4 and TAF250.
Interestingly, this weak activation was dependent on a functional Inr
sequence (21), although Inr activity was not observed for
basal transcription. Therefore, ICP4 may exploit the Inr for activation
by a mechanism different from that utilized by the cell for basal
transcription.
How the cell utilizes the Inr element is unclear, and utilization may
occur by several mechanisms, involving a variety of
cellular factors
(
13,
27,
30,
31,
41,
44,
51,
56,
57), one of which may be
TAF250 (
33,
52). From DNase I footprinting
and
photo-cross-linking experiments, TAF250 has been shown to
contact the
Inr region (
33,
52). Furthermore, in
Drosophila,
TAF250 along with
Drosophila TAF150 (dTAF150) has been shown
to
be required for TFIID Inr-directed transcription (
52).
Unlike
dTAF150, the human homolog of dTAF150, known as CIF150, is not
a
stable component of TFIID (
28,
29). In the absence of
TAF150/CIF150,
TFIID Inr-directed transcription does not occur. Thus,
one hypothesis
is that TAF150/CIF150, although not recognizing the Inr
directly,
somehow stabilizes TAF250 recognition of the Inr and thereby
promotes
TFIID Inr-directed transcription (
29). Similarly,
one hypothesis
for Inr-dependent ICP4 activation in the simplified
transcription
system is that ICP4 through its interaction with TAF250
allows
TFIID to more efficiently bind and/or operate at the promoter
through the Inr. By this scenario, ICP4 would be functioning like
TAF150/CIF150, which would explain the lack of Inr function in
this
system in the absence of ICP4. Whether the addition of Inr-facilitating
functions provided by molecules such as TAF150/CIF150 further
augments
the activation function of ICP4 is an open question that
is currently
under investigation.
As described above, HMG 1 was shown to enhance activation by ICP4 and
has also been shown to serve as a coactivator in other
studies
(
26,
49). These studies proposed that HMG 1, through
its
DNA-bending activity, facilitates multiprotein complex formation
on DNA
by remodeling the promoter conformation. A change in the
DNA
conformation induced by HMG 1 may allow ICP4 to more efficiently
interact with TFIID through TAF250 and possibly other transcription
factors. Facilitation of the ICP4-TFIID interaction by HMG 1 may
enhance TFIID binding or further stabilize TFIID on the TATA box
and
Inr. This, in turn, would lead to increased preinitiation
complex
formation and increased synthesis of RNA.
| |
ACKNOWLEDGMENTS |
We thank Patricia Bates, Benoit Grondin, and William Hobbs for
helpful discussions and comments on the manuscript.
This work was supported by NIH grant AI30612.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: E1257 Biomedical
Science Tower, Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261. Phone: (412) 648-9947. Fax: (412) 624-1401. E-mail:
neal{at}hoffman.mgen.pitt.edu.
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J Virol, August 1998, p. 6752-6757, Vol. 72, No. 8
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
