Journal of General Virology (1995), 76, 2679-2692.
2679
Printed in Great Britain
PU box-binding transcription factors and a POU domain protein
cooperate in the Epstein-Barr virus (EBV) nuclear antigen 2-induced
transactivation of the EBV latent membrane protein 1 promoter
Anna Sj6blom, 1 Ann Jansson, 1 Weiwen Yang, ~ Sonia Lain, 2 Tina Nilsson ~ and Lars R y m o 1.
1Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital
and 2 Department of Medical Biochemistry, G6teborg University, S-413 45 Gothenburg, Sweden
Expression of the Epstein-Ban" virus (EBV) latent
membrane protein (LMP1) is regulated by virus- and
host cell-specific factors. The EBV nuclear antigen 2
(EBNA2) has been shown to transactivate a number of
viral and cellular gene promoters including the promoter
for the LMP1 gene. EBNA2 is targeted to at least some
of these promoters by interacting with a cellular DNA
binding protein, RBP-Jlc. In the present report we
confirm and extend our previous observation that the
LMP1 promoter can be activated by EBNA2 in the
absence of the RBP-Jtc-binding sequence in the LMP1
promoter regulatory region (LRS). We show that two
distinct LRS regions, - 106 to +40 and - 176 to - 136,
contribute to EBNA2 responsiveness. Site-directed
mutagenesis analysis of the upstream - 1 7 6 / - 1 3 6
EBNA2 responsive element revealed that two critical
cis-acting elements are required for full promoter
function. These same elements analysed by electro-
phoretic mobility shift assays define two binding sites
recognized by nuclear factors derived from B cells. An
octamer-like sequence ( - 1 4 7 to - 1 3 9 ) contained
overlapping binding sites for an unidentified transcriptional repressor on the one hand and a factor(s)
belonging to the POU domain family but distinct from
Oct-1 and Oct-2 on the other. An adjacent purine tract
( - 1 7 1 to - 1 5 5 ) held a PU.1 binding site, which was
also recognized by a related factor. The results suggest
that the POU domain protein and either of two PU boxbinding factors bind simultaneously to LRS, creating a
ternary complex that might be in part responsible for
mediating the transactivation of the LMP1 promoter by
EBNA2. There were no qualitative differences between
EBV-negative and EBV-positive cells with regard to
transcription factor binding to the octamer-like sequence
and the PU.1 recognition site, as revealed by electrophoretic mobility shift assays.
Introduction
multiple copies as autonomously replicating circular
episomes (for reviews see Liebowitz & Kieff, 1993;
Miller, 1990; and citations therein). Of the more than 80
genes encoded by the EBV genome, only a limited
number are consistently expressed in LCLs: a family of
six nuclear proteins, EBNA1 to -6; three membrane
proteins, LMP1, -2A and -2B; and two small R N A
molecules, EBER1 and EBER2. Six of these genes,
EBNA1, -2, -3, -5 and -6 and LMP1 have been shown to
be necessary for EBV-induced B lymphocyte growth
transformation, while the others seem to be dispensible
(Skare et al., 1985; Yates et al., 1985; Cohen et al., 1989;
Hammerschmidt & Sugden, 1989; Mannick et al., 1991 ;
Swaminathan et al., 1991 ; Longnecker et al., 1992, 1993;
Tomkinson & Kieff, 1992; Kaye et al., 1993; Tomkinson
et al., 1993).
EBNA2 is the first EBV-encoded protein expressed in
infected B lymphocytes and plays an important role in
the immortalization process. The results of Woisetschlaeger et al. (1991) suggest that the EBNA2 message
Epstein-Barr virus (EBV) is an ubiquitous human
pathogen that has a strong association with at least three
forms of cancer: African Burkitt's lymphoma (BL),
nasopharyngeal carcinoma and a subset of Hodgkin's
lymphomas (Henle & Henle, 1966; Herbst et al., 1991;
Young et al., 1989). In addition, states of immune
dysfunction, e.g. immunosuppression after organ transplantation, human immunodeficiency virus infection and
primary immunodeficiencies may result in EBV-provoked lymphoproliferative disorders. EBV infection of
human B lymphocytes in vitro leads to transformation
and the outgrowth of EBV-carrying lymphoblastoid cell
lines (LCLs) in which the virus genome is maintained in
* Author for correspondence. Fax
[email protected]
0001-3308 © 1995 SGM
+46
31 828458.
e-mail
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2680
A. Sjgblom and others
is transcribed from the Wp promoter at the initial phase
of infection but within a short period of time there is a
switch to a preferential use of the Cp promoter. The
molecular mechanism underlying the switch is still
unclear but the presence of EBNA2-responsive enhancer
elements in the Cp regulatory region suggests that
EBNA2 contributes either directly or indirectly (Woisetschlaeger et al., 1990; Sung et al., 1991). A number of
observations support the notion that EBNA2 exercises
its effect on the phenotype of the EBV-infected cell by
being a part of or by modulating the activity of regulatory
systems that control the expression of specific viral and
cellular genes. EBNA2 stimulates transcription from Cp
and of the LMP1, LMP2A and LMP2B EBV genes
(Abbot et al., 1990; FShraeus et al., 1990; Wang et al.,
1990a; Zimber-Strobl et al., 1993; Sung et al., 1991;
Woisetschlaeger et al., 1991). It activates the promoters
for the CD21, CD23, c-fgr and c-bcl-2 genes (Cordier et
al., 1990; Wang et al., 1987, 1990b, 1991; Knutson,
1990; Finke et al., 1992).
All EBNA2-inducible promoters characterized so far
contain a 5' G T G G G A A 3' motif which is part of the
recognition sequence for the cellular DNA-binding
protein RBP-JK (Tun et al., 1994). It has been shown that
RBP-JK binds to this motif in the context of EBNA2responsive elements (EB2REs) and that RBP-JK interacts
with EBNA2 both in the presence and absence of the
DNA binding site (Ling et al., 1993 ; Zimber-Strobl et al.,
1993; Henkel et al., 1994; Grossman et al., 1994;
Yalamanchili et al., 1994; Laux et aI., 1994a; Waltzer et
al., 1994; Johannsen et al., 1995). Thus, it remains an
attractive hypothesis that RBP-Jtc may target EBNA2 to
EB2REs. Recently, the macrophage- and B cell-specific
transcription factor PU. 1 (Klemsz et al., 1990; Goebl,
1990; Ray et al., 1992) was implicated in the EBNA2mediated transactivation of LMP1 (Johannsen et al.,
1995) and the bidirectional LMP 1/TP2 promoters (Laux
et at., 1994a). PU.1 was shown to bind to EB2RE in the
promoter region and mutation of the recognition
sequence completely abolished EBNA2 responses.
Another member of the ets gene family of transcription
factors, Spi-B, also bound to the same site (Laux et al.,
1994b).
We have continued our investigation of the molecular
mechanism by which EBNA2 activates the expression of
the LMP1 gene. LMP1 is encoded by the BNLF1 reading
frame transcribed in a leftward direction from a
promoter at position 169546 in the EBV B95-8 genome
(Baer et al., 1984; Fennewald et al., 1984). We have
previously shown that the LMP1 promoter is controlled
by positive and negative cis-elements in the Y-flanking
region of the promoter and that EBNA2 can activate the
promoter by overriding the effect of the putative
repressors (F~hraeus et al., 1990, 1993). In the present
report we confirm and extend our previous observation
that the LMP1 promoter can be activated by EBNA2 in
the absence of the RBP-JK-binding sequence in the
LMP1 promoter regulatory region (LRS). We show that
two distinct LRS regions, - 1 0 6 to +40 and - 1 7 6 to
-136, contribute to EBNA2 responsiveness. Within the
upstream - 1 7 6 / - 136 EBNA2 responsive sequence two
cis-acting DNA elements, recognized by PU.I and a
related factor and a POU domain protein, respectively,
are shown to be necessary in conferring optimum
promoter activity. Our results suggest that the POU
domain protein may assist in the targeting of EBNA2 to
the LMP1 promoter and that the POU domain protein
and the PU box-binding factors cooperate in the
transactivation of the promoter by EBNA2.
Methods
Plasmid construction. All constructs were verified by dideoxynucleotide chain termination sequencing utilizing the Sequenase system
(USB). The pEAA6, pgSVECAT, pgCAT, p g L R S ( - 5 4 ) C A T ,
p g L R S ( - 1 0 6 ) C A T , p g L R S ( - 1 4 6 ) C A T , p g L R S ( - 2 1 4 ) C A T and
p L R S ( - 106)CAT constructs have been described earlier (Ricksten et
al., 1987; Ffihraeus et al., 1990, 1993). The LMP1 regulatory sequence
is defined as nucleotides 169477 to 170151 of B95-8 EBV D N A (LRS),
which corresponds to positions - 6 3 4 to + 4 0 relative to the
transcription initiation site. We would like to point out that the EBV
sequence in the p g L R S ( - 146)CAT plasmid is identical to that of the
p g L R S ( - 1 4 4 ) C A T plasmid in our previous report (Ffihraeus et al.,
1993), in which 2 bp contributed to the EBV sequence by the linker
went unnoticed. To obtain a true - 1 4 4 construct the p g L R S
( - 214)CAT plasmid was cleaved with HindlII and N l a l l l , overhanging
3' ends were removed with T4 D N A polymerase and the appropriate
fragment was cloned in the p G e m - 3 Z f ( + ) vector (Promega), resulting
in the plasmid p g L R S ( - 1 4 4 ) C A T . The p g L R S ( - 1 4 6 ) ( - 2 1 4 /
- 145)CAT plasmid was made by cloning three sets of double-stranded
oligonucleotides with SalI ends in the SalI site of p g L R S ( - 146)CAT.
The pgLRS( - 106)(-- 1 7 0 / - 131)CAT plasmid was constructed by
directional cloning of ligated sets of synthetic oligonucleotides,
corresponding to the - 170 to - 131 region of LRS, into the PstI SalI
site upstream of the - 1 0 6 / + 4 0 sequence in the p g L R S ( - 1 0 6 ) C A T
construct. A series of mutated derivatives of the plasmid was
constructed using synthetic oligonucleotides in which consecutive 5 bp
segments of the normal - 1 7 0 to - 1 3 1 B95-8 EBV sequence were
replaced by segments of pnrine-pyrimidine transversions: - 1 3 5 to
- 131 (ml),
- 140 to - 1 3 6 ( m 2 ) ,
- 145 to - 1 4 1 ( m 3 ) ,
- 150 to - 146
(m4), - 155 to - 151 (m5), - 160 to - 156 (m6), - 165 to - 161 (m7)
and
- 170 to
- 166 (m8),
respectively.
Similarly,
the
pLRS( - 106)( - 181 / - 145)CAT plasmid was constructed by cloning
ligated sets of synthetic oligonucleotides corresponding to the - 181 to
- 1 4 5 region of LRS into the SalI site in the p L R S ( - 1 0 6 ) C A T
construct. Mutations were introduced as above, as 5 bp segments of
purine-pyrimidine transversions in the following positions: - 1 4 9 to
- 145 (ml),
- 155 to - 1 5 1 ( m 2 ) ,
- 160 to - 1 5 6 ( m 3 ) ,
- - 165 to - - 161
(m4), --170 to --166 (m5), - 1 7 5 to - 1 7 1 (m6) and - 1 8 1 to - 1 7 6
(m7), respectively.
The herpes simplex virus thymidine kinase (TK) promoter region in
our T K constructs correspond to nucleotides - 108 to + 51 relative to
the cap site (Edlund et al., 1985). The T K promoter-containing B a m H I
fragment was excised from the p T K C A T plasmid (Ricksten et al.,
1988) and inserted in a pGEM-3Zf( + ) vector in which the HindIII site
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EBNA2
had been changed to a BamHI site, resulting in the pgTKCAT plasmid.
LRS-containing derivatives were constructed by cloning sets of
synthetic oligonucleotides corresponding to selected regions of LRS in
the SalI site [pgLRS(-181/-145)TKCAT and pgLRS(-214/
-145)TKCAT] or between the PstI and Sall sites [pgLRS(-160/
-136)TKCAT, pgLRS(-194/-136)TKCAT and pgLRS(-217/
-136)TKCAT] upstream of the TK promoter in the pgTKCAT
plasmid.
To make a series of mutated reporter plasmids with deletions
covering the - 1 7 6 to - 1 4 7 region of LRS, PCR amplifications were
performed using the pgLRS(-217)CAT plasmid as a template and
primers that resulted in fragments with one end corresponding to
position +40 in LRS and the other end corresponding to positions
- 147, - 148, - 152, - 153, - 160 or - 176. The PCR fragments were
cloned into the TA cloning vector (Invitrogen). Taking advantage of a
synthetic HindlII site in one primer and a PstI site in the TA cloning
vector, the PCR fragments were then cloned between the HindIII and
PstI sites in the pgCAT plasmid. To generate the pgLRS(-217)CAT
plasmid a set of four double-stranded synthetic oligonucleotides
homologous to the - 2 1 7 to - 7 4 region of LRS with overlapping
single-stranded ends was annealed, creating an XmaI-PstI fragment.
This DNA fragment and an isolated HindIII XmaI fragment of LRS
corresponding to the - 7 3 to +40 region were cloned between the
HindIII and PstI sites of the pgCAT plasmid to create a continuous
-217 to +40 LRS sequence in the plasmid.
Cell culture, DNA transfections and CAT assays. DG75 is an EBV
genome-negative BL cell line (Ben-Bassat et al., 1977). The IB4 cell line
was derived by transforming human placental lymphocytes with the
B95-8 EBV strain (King et al., 1980). Cherry is an EBV-infected cell
line that was established by culture of lymphocytes from patients with
mononucleosis (Heller et al., 1981). Rael (Klein et al., 1972) and Raji
(Epstein et al., 1966) are EBV-positive BL lines. The CBC-Rael line was
obtained by infection of cord blood cells with the Rael virus strain
(Ernberg et aL, 1989). The lymphoid cells were maintained as
suspension cultures in RPMI 1640 medium (Life Technologies)
supplemented with 10 % fetal calf serum (Life Technologies), penicillin
and streptomycin. DG75 cells (5 x 106) were transfected with 10 lag
DNA of the reporter construct to be tested and 800 fmol DNA of the
EBNA2 expression vector pEAA6 or 800 fmol DNA of the pSV2gpt
vector using the DEAE-Dextran technique (Ricksten et al., 1988). Cells
were harvested after 48 h and aliquots of the cell lysates were assayed
for CAT activity (Ricksten et al., 1988).
Electrophoretic mobility shift assays (EMSAs). Nuclear extracts were
prepared as described by Dignam et al. (1983). The protease inhibitors
antipain (5 gg/ml), leupeptin (5 lag/ml) and aprotinin (2 gg/ml) were
added to the buffer in the final homogenization and dialysis steps.
Aliquots were frozen in liquid nitrogen and stored at - 7 0 °C. EMSAs
were performed using a double-stranded synthetic oligonucleotide
corresponding to the - 173 to - 136 segment and - 176 to - 136
segment of LRS with single-stranded ends. The oligonucleotide was
labelled by a repair reaction in the presence of [~-a~P]dNTP (6000
Ci/mmol; Du Pont NEN) using the Klenow fragment of DNA
polymerase I (Boehringer Mannheim). Other probes used from the
LRS region were - 1 5 3 / - 114, - 1 4 6 / - 114 and - 1 4 4 / - 114. The
blunt-ended double-stranded oligonucleotides were labelled with [7a2P]ATP (6000 Ci/mmol; Du Pont NEN) using polynucleotide kinase
(Boehringer Mannheim). The labelled oligonucleotides were separated
from free isotope by electrophoresis in a 4% polyacrylamide gel
(acrylamide:bisacrylamide 30: 1) in 25 mM-Tris-HC1, 190 mM-glycine
and 1 mM-EDTA pH 8.3. The wet gel was autoradiographed and the
DNA fragments were excised, electroeluted by isotachophoresis
(Ofverstedt et al., 1984) and precipitated with ethanol. Binding
reactions (25 lal) contained 10 mM-Tris-HC1 pH 7.5, 50 mM-NaC1,
1 mM-DTT, 1 mM-EDTA, 5 % glycerol, 4 gg poly(dI-dC), 6 fmol 32p_
transactivation o f the L M P 1 p r o m o t e r
2681
labelled DNA (approximately 70000 c.p.m.) and 20 lag of nuclear
proteins (always added last). A 400-fold excess of competing oligonucleotide added before the 3~P-labelled probe was used for the
competition experiments. After incubation on ice for 25 min, the
samples were separated by electrophoresis on 5 % polyacrylamide gels
(acrylamide: bisacrylamide 30: 1) in 25 mM-Tris-HCl, 190 mM-glycine
and 1 mM-EDTA pH 8.3 for 3 h at 300 V.
Rabbit polyclonal antibodies against the transcription factors Oct-l,
Oc~-2, PU.1/Spi-1 and Spl were purchased from Santa Cruz
Biotechnology. The rabbit polyclonal antibody specific for POU
domain proteins was raised against the POU domain of Oct-1 and was
kindly provided by Dr Peter O'Hare (Marie Curie Research Institute,
Oxted, UK). The anti-Spl antibody was used as a negative control in
the PU. 1 supershift experiment. The supershift analyses were performed
as described above for the binding experiments except that after the
incubation on ice, 2 gl of the respective antibody was added. The
mixture was incubated at 4 °C for 60 min and then analysed on 5 %
polyacrylamide gels.
For in vitro expression, a fragment of pEAA6 containing the
EBNA2-encoding open reading frame, BYRF1, was cloned into the
plBI 31 vector (International Biotechnologies). The supercoiled DNA
template was sequentially transcribed and translated in the same
reaction mixture containing TNT rabbit reticulocyte lysate, amino acids
and the TNT T7 RNA polymerase, using the protocol described by the
manufacturer (Promega). Translated proteins were analysed by SDSPAGE. The EMSA binding reactions were performed as described
above except that 5 gl of the reticulocyte lysate was added together with
the DG75 nuclear extract.
Results
S e q u e n c e s in the - 2 1 4 / + 40 p a r t o f L R S responsible
f o r E B N A 2 responsiveness
We have previously demonstrated that EBNA2 can
transactivate the LMP1 promoter through sequences
d o w n s t r e a m o f p o s i t i o n - 2 1 4 in L R S ( F A h r a e u s et al.,
1990, 1993; S j 6 b l o m et al., 1993). I n o r d e r to m a p t h e
E B N A 2 - r e s p o n s i v e s e q u e n c e s in t h e - 2 1 4 / + 40 p a r t o f
L R S f u r t h e r , a series o f C A T r e p o r t e r p l a s m i d s c o n t a i n i n g 5' d e l e t i o n m u t a t i o n s o f L R S w a s c o n s t r u c t e d
a n d s u b j e c t e d to t h e E B N A 2 c o t r a n s f e c t i o n a s s a y in
D G 7 5 cells. T h e p l a s m i d t h a t c o n t a i n e d o n l y t h e - 54 to
+ 40 p a r t o f L R S s h o w e d E B N A 2 i n d e p e n d e n t a c t i v i t y
(Fig. 1). A d d i t i o n o f the s e q u e n c e s b e t w e e n - 5 4 a n d
- 1 0 6 r e s u l t e d in a s e v e n f o l d a c t i v a t i o n o f t h e r e p o r t e r
p l a s m i d b y E B N A 2 . T h e E B N A 2 r e s p o n s i v e n e s s was n o t
i n c r e a s e d f u r t h e r by t h e a d d i t i o n o f t h e s e q u e n c e b e t w e e n
-106 and -144. However, when an additional 2 bp
( - 1 4 5 a n d - 1 4 6 ) w e r e i n c l u d e d in t h e c o n s t r u c t , t h e
EBNA2 responsiveness was completely abolished. A
p l a s m i d w i t h a n u n i n t e r r u p t e d L R S s e q u e n c e u p to
position -214 showed a 47-fold activation by EBNA2.
I n s e r t i o n o f a 1 4 b p l i n k e r at p o s i t i o n - 1 4 6 in a
construct otherwise identical to pgLRS(-214)CAT
c o m p l e t e l y a b r o g a t e d the E B N A 2 r e s p o n s i v e n e s s o f the
p l a s m i d . W e c o n c l u d e f r o m t h e s e e x p e r i m e n t s t h a t at
least t w o s e p a r a t e d o m a i n s o f the - 2 1 4 / + 4 0
part of
L R S p a r t i c i p a t e in t h e E B N A 2 - d e p e n d e n t a c t i v a t i o n o f
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2682
A. Sjdblom and others
CAT activity (%)
+40
I
pgLRS(-54)CAT
I
-106
I
I
pgLRS( t46)CAT
[
pgLRS(-214)CAT
I
-EBNA2
+EBNA2
3.1 (0.23)
7-1 (2.1)
2.3 (0-70)
0.55 (0.05)
3.8 (1.0)
7.1 (1.9)
0.42 (0.05)
216 (0144)
6.4 (1.4)
0-25 (0-04)
0-32 (0-08)
1-2 (0-I4)
0-43(0.04)
19
47
0.27 (0-04)
0.25 (0.02)
0.95 (0.04)
0-25 (0-04)
0-42 (0.01)
1-3 (0.22)
-54
pgLRS( 106)CAT
pgLRS( 144)CAT
Inducibility
144
I
146
I
-214
l
pgLRS( 146)(-214~145)CAT I
(2.1)
(7.8)
-214
145
I
II
-146
pgCAT
Fig. 1. Identification of two regions in LRS that contribute to EBNA2 responsiveness in DG75 cells. CAT reporter plasmids containing
Y-deleted fragments of LRS were cotransfected with the EBNA2 expression vector pEAA6 ( + EBNA2), or an equivalent amount of
the pSV2gpt plasmid ( - E B N A 2 ) into DG75 cells. The CAT activity is given as percentage chloramphenicol acetylation. Inducibility
is defined as the ratio between the values obtained with the respective plasmids in the presence and in the absence of pEAA6. The value
shown is the mean of four transfections and the SEM values are indicated within parentheses.
-140
-150
-160
170
-180
I
t
I
I
I
GCGGTGTGTGTGTGCATGTAAGCGTAGAAAGGGGAAGTAGAAAGCGTGTQT
106
pgLRS(- 106)(- 170/- 131)CAT ---t
octamer
+ D~ - >
<
P U box
Dfl
>
p
5.0
'
CAT activity (%)
15 20 25 30
10
i
Mutation: ml " ' - t ~
m2 "'-"1
m3 ----t
m4
m5
-{
---I
Inducibility
35
t
40
I
I
I
t::::::::::::::~
~:::-::::::::::r
f
t::::::::::::::l
m7 -.-]
m8 ---~
l
~::::::::::::::~-~::::::::::::::1
-106
pLRS(-106)(- 181/-145)CAT ---1
m3 ---I
m4 -- --1
m5 .---I
m6 ----I
m7 "'--I
pLRS(-106)CAT ----I
(11)
(10)
29
(11)
19
(5-8)
14
(2-6)
27
(3-2)
15
(3.6)
14
10
(2.5)
(4.2)
8.2 (1.3)
pgLRS( 106)CAT---I
Mutation: m l " ' 4
m2 . - 4
i
50
40
6-9 (0.65)
"~:::'::::::::::J
r:::::::::::::J
6.5 (0.49)
6-6 (1-2)
I:.:-:.::;::::1
t:::::,:::::q
I-EBNA2
7.8 (1.4)
O+EBNA2
6.8 (1-6)
8.5 (2-2)
t:::::::::,::::,
i:.:.:+:+:~
10
t:.:.:.:.:-:-:+--
(1-9)
8.9 (1-5)
%,
5.6 (1-6)
Fig. 2. Mutational analysis of EBNA2-responsive elements in the - - 1 8 1 / - 1 3 1 region of LRS. Stretches of purine-pyrimidine
transversions were introduced into the p g L R S ( - 1 0 6 ) ( - 1 7 0 / - 1 3 1 ) C A T and p L R S ( - 1 0 6 ) ( - 1 8 1 / - 1 4 5 ) C A T plasmids at positions
indicated by dotted boxes using synthetic oligonucleotides (see Methods). The reporter plasmids were cotransfected with pEAA6
( + EBNA2) or with an equivalent amount of pSV2gpt ( - E B N A 2 ) into DG75 cells. The CAT activity is given as percentage
chloramphenicol acetylation. Inducibility is defined as the ratio between the values obtained with the respective plasmids in the presence
and in the absence of pEAA6. The values shown are the mean of four transfections. The SEM values are indicated with error bars for
CAT activity and given as numbers within parentheses for inducibility by EBNA2.
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EBNA2 transactivation of the LMP1 promoter
2683
Table 1. Analysis of EBNA2-dependent activation of the TK promoter
mediated by the - 2 1 7 / - 131 LRS region
CAT activity (%)*
Reporter plasmid
- EBNA2
+ EBNA2
Inducibilityt
pgLRS(- 217/- 136)TKCAT
pgLRS(- 214/- 145)TKCAT
pgLRS(- 194/- 136)TKCAT
pgLRS(- 181/- 145)TKCAT
pgLRS( - 160/- 136)TKCAT
pgTKCAT
2.4 (0.30)
2-0 (0.17)
1.6 (0.18)
2.0 (0.28)
1-2(0-18)
2.1 (0.17)
33 (3.0)
7.7 (0.58)
15 (1.1)
5.2 (0.43)
II (0-73)
4.7 (0.26)
15 (3.1)
3.9 (0.17)
10 (1.6)
2-7 (0.26)
%6 (1.8)
2-3 (0.20)
* Expressed as percentage acetylation of chloramphenicol. The values shown are the mean of
four transfections; the SEMis given in parentheses.
t EBNA2 inducibility is calculated as the ratio between the CAT value obtained with and
without EBNA2. The SEMis given in parentheses.
the LMP1 promoter. One of the domains is located
upstream of position - 1 4 6 and the other closer to the
promoter between position - 1 0 6 and - 5 4 . The
-146/-54
region seems to contain two or more
negative cis-elements with the ability to repress the
constitutive activity of the promoter-proximal elements.
Interestingly, the insertion mutation only interfered with
the activating but not the suppressing functions of LRS
and prevented both upstream and downstream positive
elements from activating the LMP1 promoter.
The results of preliminary mutational analysis (data
not shown) and DNase I footprinting experiments
(Sj6blom et al., 1993) suggested that the element(s)
responsible for the major part of the EBNA2-induced
transactivation found above and around position - 146
of LRS were contained within the - 176 to - 136 region.
In order to define these regulatory elements in greater
detail, 5 bp stretches of purine-pyrimidine transversions
were introduced in this region, resulting in two different
series of mutated reporter plasmids, derived from
p g L R S ( - 106)(- 1 7 0 / - 131)CAT and p L R S ( - 106)( - 1 8 1 / - 1 4 5 ) C A T . The plasmids were cotransfected
with the EBNA2 expression vector in DG75 cells and
analysed for CAT activity. The results obtained with the
p g L R S ( - 106)(- 1 7 0 / - 131)CAT series revealed the
presence of at least two LRS subdomains important for
EBNA2 responsiveness (Fig. 2). For simplicity, the one
located at position - 1 5 0 / - 1 4 1 was designated D e
and the one at position - 1 7 0 / - 156 was designated Dfl.
D e contains a sequence with partial identity to the
octamer motif and Dfl contains a purine-rich sequence
resembling a P U box. It should be noted that although
the level of activity was higher, EBNA2 inducibility
(defined as the ratio between the activity obtained in
the presence and in the absence of EBNA2) of the
p L R S ( - 1 0 6 ) ( - 1 8 1 / - 145)CAT plasmid containing
only the Dfi region was similar to that o f the
p L R S ( - 1 0 6 ) C A T plasmid. This was also true for the
mutated plasmids belonging to this series due to the
fact that the mutations induced a proportional decrease
of the activity, both in the presence and absence of
EBNA2. In contrast, the EBNA2 inducibility of the
p g L R S ( - 106)(- 1 7 0 / - 131)CAT plasmid, which contains both the D e and Dfl regions, was increased about
sixfold compared with p g L R S ( - 106)CAT. Mutations in
either D e or Dflin the p g L R S ( - 106)(- 1 7 0 / - 131)CAT
series not only reduced total activity but also the
inducibility of the plasmids. The Dfl mutations reduced
the activity to the background [the p g L R S ( - 1 0 6 ) C A T
level]. We conclude from these experiments that (i) Dfl by
itself functions as a positive transcription element that is
not inducible by EBNA2; (ii) D e is not active in the
absence of Dfl; and (iii) elements in D e and Dfl cooperate
to create a fully EBNA2-responsive site.
To investigate whether the putative EBNA2-responsive element in the - 2 1 4 to - 1 3 6 region could confer
EBNA2 inducibility to a heterologous promoter in the
absence of the downstream EBNA2-responsive LRS
elements, fragments of this region were cloned in front of
the T K promoter in a reporter plasmid (Table 1). The
results showed that the - 2 1 7 / - 136 LRS region indeed
conferred EBNA2 responsiveness to the T K promotercarrying construct [pgLRS( - 2 1 7 / - 136)TKCAT]. Deletion of the - 1 4 4 / - 1 3 6 part of LRS resulted in
constructs [e.g. p g L R S ( - 2 1 4 / - 145)TKCAT] with only
about 20 % of the original activity, suggesting that this
sequence constitutes an important part of the EBNA2responsive element.
Detailed analysis of the De domain of LRS
The reason for the complete loss of EBNA2 responsiveness of the p g L R S ( - 146)CAT construct is not clear.
To clarify precisely at what point the repression was
relieved and the EBNA2 inducibility restored, a fine
mapping of the - 1 7 6 / - 144 LRS region was carried out
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A. Sj6blom and others
2684
EBNA2:
TLC
+
+
+
~I~ ~
~
+
+
+
+
+
+
+
+
the level obtained with p g L R S ( - 1 0 6 ) C A T . It might be
of significance that a complete octamer-homologous
motif was present in the p g L R S ( - 1 4 7 ) C A T plasmid.
Further addition of LRS sequence up to - 160 had only
minor effects on activity. However, when the complete
sequence downstream of position - 176 was included in
the construct a strong increase in responsiveness was
obtained. Addition of the sequence between - 1 7 6 and
- 2 1 7 had only a minor effect.
These results are consistent with the hypothesis that a
trans-acting factor with the ability to block the effect of
all positive elements in the - 2 1 4 / + 40 part of LRS binds
close to position - 146 in p g L R S ( - 146)CAT. The effect
of this putative repressor is eliminated in the
p g L R S ( - 1 4 4 ) C A T and p g L R S ( - 1 4 7 ) C A T plasmids.
To correlate the activity data with the potential binding
of transcription factors we performed EMSAs with
nuclear extracts of EBV-negative DG75 and EBVpositive Cherry cells. Radioactively labelled, doublestranded synthetic oligonucleotides corresponding to the
-153/-114,
-146/-114
and - 1 4 4 / - 1 1 4
LRS
regions were used as probes (Fig. 4). Three specific
complexes were recognized both in EBV-negative and
EBV-positive cells and localized to specific regions of LRS
+
CAT activity 47 11 6.4 0.3 4-4 4,8 6.6 60 13 68 59 0.6 >100
(% acetylation)
Fig. 3. Deletion analysis of the octamer-homologousregion of LRS for
EBNA2 responsiveness. Reporter plasmids with deletions covering the
- 217 to - 54 region of LRS (see Methods) were cotransfectedwith the
EBNA2 expression vector pEAA6 (+ EBNA2) into the EBV-negative
DG75 cell line. The CAT activity is presented as percentage
chloramphenicol acetylation. The experimentshown is a representative
example out of five transfections.
by 5' deletion mutation analysis (Fig. 3). As expected, the
- 146 LRS construct did not respond to EBNA2 in the
EBNA2 cotransfection assay in DG75 cells. Deletion of
2 b p [ p g L R S ( - 1 4 4 ) C A T ] or the addition of 1 bp
[ p g L R S ( - 147)CAT] restored EBNA2 responsiveness to
(a)
,v\ .v~ .@ ,~> > >
),
(b)
(c)
YY),Yy
LRS(-146/ 114)
LRS(-153/-114)
SIE~
~
SIE~
D~2=~
D~I~
~
....
D~2~
SIE
Dc~l
I
II
I
F
.1~
DG75 cells Cherry cells
DG75 cells
Antibody
OCT-1
OCT-2
POU
-
+
I
+
+
+
F
Cherry cells
+ +
DG75 cells
Antibody
OCT-1
OCT-2
POU
+
Cherry cells
+
+
+
+
+
Fig. 4. Analysis of transcription factors binding to the octamer motif-containing Dc~ region of LRS. EMSAs using nuclear extracts of
the EBV-negative DG75 and EBV-positive Cherry cells were performed with the - 1 5 3 / - 1 1 4 ,
- 1 4 6 / - 1 1 4 and
144/-114
fragments of LRS as probes. (a) The E M S A pattern obtained with the three probes and B cell extracts. The specific complexes are
indicated by black arrows and designated Dc~l, D~2 and SIE. A non-competable unspecific band is indicated by the hatched arrow.
The fastest migrating band is the free probe. Autoradiograms (b) and (c) are the result of E M S A supershift experiments using the
- 146/-- 114 and - 1 5 3 / - 114 LRS probes and rabbit polyclonal antibodies against the Oct-1 and Oct-2 factors and the P O U domain.
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2685
E B N A 2 transactivation of the L M P 1 promoter
(a)
1
2
3
4
LRS(-173/-136)
5 6 7 8 9
(b)
10 11
12
-136 -140
(I)
I
I
-150
-160
I
I
-170 -173
I
I
5 "-TCGACGTGTGTGTGCATGTAAGCGTAGAAAGC-W-_4~AAGTAGAAA- 3"
3 "-GCACACACACGTACATTCGCATCTTTCCCCTTCATCTTT- 5"
<-- D ~ --->
<
D~
>
[Dcd, Dill/Dil2]
(II) 5"-TCGACCACACACACGTACATTCGCATCTTTCCCCTTCATCTTT-3"
3" GGTGTGTGTGCATGTAAGCGTAGAAAGGGGAAGTAGAAA-5"
DcO
D/~
Dill
(]11) 5"-TCGACCACACACACGTACATTCGCATCTTTGGGGAAGTAGAAA-3"
3"-GGTGTGTGTGCATGTAAGCGTAGAAACCCCTTCATCTTT-5"
I
I}
-
5"-GTGTGTCACGTTGTAAGCGTAGAAA~AAGTAGAAA-3"
3"-CAGTGCAACATTCGCATCTTTCCCCTTCATCTTT-5"
(v)
5"-GTGTGTGTGCATGTAAGCGTTCTTTCCCCTTCATCAAA-3"
3"-CACACGTACATTCGCAAGAAAGC-C4~AAGTAGTTT-5"
t
DG75 cells
Mutation in competitor
(I) U n m u t a t e d
(II) -173/-136
(III) 160~136
(IV) 146/-142
(V) -170/-156
(w)
IB4 cells
+
+
+
+
+
-
-
+
+
. .
+
.
.
.
+
Fig. 5. Binding of factors in B lymphoid cells to the Dc~/Dil region. (a) A 32P-labelled double-stranded synthetic oligonucleotide
corresponding to the - 1 7 3 / - 136 LRS region was incubated with nuclear extracts from DG75 (lanes 1 6) and IB4 (lanes 7-12) cells
and subjected to EMSA. Lanes 1 and 7 in the autoradiogram show the binding pattern obtained with the nuclear extracts. Competition
reactions performed by adding a 400-fold excess of unlabelled competitor to the binding mixtures were analysed in the rest of the lanes
using oligonucleotides I to V with mutations in specific regions as indicated below the autoradiogram. Four specific complexes are
indicated by black arrows and designated Dc~1, Dill, Dfl2 and [D~ 1, Dill/Dil2]. Three non-competable, unspecific bands are indicated
by hatched arrows. (b) Nucleotide sequence of the double-stranded oligonucleotides I to V used in the competition experiment. Mutated
nucleotides in the oligonucleotides II-V are underlined.
by competition experiments (data not shown). Two of the
EMSA bands were due to factors that interacted with the
Dc~ region and were designated Dc~l and Da2 (Fig. 4).
The third complex corresponded to a factor that
interacted with the - 1 2 7 / - 1 1 9 LRS region, which
contains a sequence (5' T T C C C G A A A Y) with partial
identity to the binding site for the sis-inducible factor
(SIF; Sadowski et al., 1993). This complex, designated
SIE (sis-inducible element) in Fig. 4, was competed for
with an oligonucleotide that contained a consensus SIE
(Santa Cruz Biotechnology; data not shown). The
prominent band between the De2 and SIE bands
indicated with a broken arrow in Fig. 4 did not compete
with an excess of unlabelled probe and was therefore
judged as unspecific (data not shown). The D~2 complex
was formed when the - 1 4 6 / - 114 probe was used (Fig.
4, lanes 2 and 5). Interestingly, this complex was more
easily competed for with an oligonucleotide that extended to - 153 than - 146, suggesting that nucleotides
upstream of - 1 4 6 are involved in the protein binding
(data not shown). The D~2 complex was, however, more
or less completely replaced by the D~I complex in
EMSAs with the - 1 5 3 / - 114 probe (Fig. 4, lanes 1 and
4). Neither the D e l nor the Dcd complex was formed
with the - 1 4 4 / - 114 probe (Fig. 4, lanes 3 and 6). We
conclude from these results that D~ contains two
overlapping binding sites for the D e l and D~2 factors
with essential sequences located between position - 146
and - 1 4 4 for the D0~2 site and somewhat further
upstream for the D~I site. Under our EMSA conditions,
binding of the D~I factor was always dominant when the
complete Dc~ region was present, both with EBV-negative
and EBV-positive cell extracts. It should be noted that
the binding of the D~2 factor to the - 1 4 6 / - 114 LRS
probe correlated with the loss of EBNA2 responsiveness
of the corresponding p g L R S ( - 146)CAT plasmid. This
suggests that the D0d factor functions as a negative
regulator of LRS activity. In contrast, binding of the
D~I factor to the - 1 5 3 / - 1 1 4 probe was paralleled by
a high EBNA2 responsiveness of the corresponding
reporter plasmids, indicating the D~I factor is a
transactivating protein.
Since the D~ region contained a sequence with a
partial identity to the octamer motif we then asked if any
of the D~-binding proteins belonged to the oct gene
family of transcription factors. Supershift experiments
were performed with the - 1 5 3 / - 114 and - 1 4 6 / - 114
LRS probes and DG75 and Cherry cell extracts,
employing polyclonal antibodies against the Oct-1 and
Oct-2 factors and the POU domain, respectively. The
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2686
A. Sj6blom and others
activity and specificity of the antibodies were verified in
supershift experiments using a probe carrying the
octamer consensus sequence and B cell extracts (DG75,
Cherry, IB4; data not shown). The antibodies also
reacted with Oct-1 and Oct-2 proteins translated in vitro
(data not shown). In EMSAs with the LRS probes the
anti-Oct-1 and -Oct-2 antibodies did not detectably shift
the D e l or the De2 EMSA band (Fig. 4). The anti-POU
domain antibodies, on the other hand, very distinctly
depleted the D e l band, indicating that the corresponding
protein belongs to the POU domain family of transcription factors but is different from Oct-1 and Oct-2.
The De2 complex was not affected by the addition of
antibodies. The De2 factor is therefore different from
Del.
LRS(-176/-136)
Dcd, Dfll/Dfl2
...~.
Dal
D/~2
D/~I
Factors belonging to the ets gene family of proteins
interact with Dfl
Our results showed that elements in Dfl were necessary to
achieve maximum activity of LRS-containing reporter
plasmids in the presence of EBNA2 (Sj6blom et al.,
1993). To identify factors that interact with the Dfl
region, we performed EMSAs with nuclear extracts of
DG75 and IB4 cells using a radioactively labelled,
double-stranded synthetic oligonucleotide corresponding
to the - 1 7 3 / - 1 3 6 region of LRS as a probe (Fig.
5a, b). There was no consistent difference between the
EBV-negative and the EBV-positive cells with regard to
the pattern of complexes. Competition experiments with
unlabelled oligonucleotides I V (Fig. 5b) were carried
out in 400-fold molar excess over the labelled fragment
to define shorter sequences within the 37 bp LRS probe
that were involved in protein-DNA interactions. The
EMSAs revealed four specific complexes: D e l , Dill,
Dfl2 and [Del, Dfll/Dfl2] (Fig. 5a; lanes 2 and 8 as
compared to lanes 3 and 9). The Dill and Dfl2 bands had
very similar mobility and were only resolved under ideal
conditions. The weak bands indicated by broken arrows
in Fig. 5 were not competed for and were assumed to
represent unspecific complexes. Competition with an
oligonucleotide containing a mutated sequence between
position - 160 and - 136, which includes the D e domain
(Fig. 5a; lanes 4 and 10), or an oligonucleotide with a
mutated octamer motif between nucleotides - 1 4 6 to
- 1 4 2 (Fig. 5a; lanes 5 and 11) competed for all
complexes except D e l . Competition with an oligonucleotide that contained mutations covering the Dfl
region between - 170 to - 156, competed for all but the
Dill and Dfl2 complexes (Fig. 5a; lanes 6 and 12).
Notably, mutations that interfered with the formation of
either the D e l or Dfll/Dfl2 complexes also inhibited the
formation of the [Del, Dfll/Dfl2] complex. The results
suggest that DG75 and IB4 cells both contain a similar
DG75 cells
Antibody
Oct-1
+
Oct-2
+
POU
+
Fig. 6. Immunologicalanalysis of factors in B cells that bind to the De
domain. Nuclear extracts from DG75 cells were incubated under
binding conditionswith a 32P-labelleddouble-strandedoligonucleotide
correspondingto the - 176/- 136 LRS region followedby incubation
with rabbit polyclonal antibodies against Oct-l, Oct-2 and the POU
domain as shown underneath. The reaction mixtureswere analysed by
EMSA. Four specific complexes are indicated by black arrows and
designated D~I, Dill, Dfl2 and [Dc~l, Dfll/Dfl2]. A non-competable,
unspecific band is indicated by the hatched arrow.
set of transcription factors with affinity for the D e and
Dfl domains. Furthermore, the properties of the [Del,
Dfll/Dfl2] band suggest that it is formed by the
simultaneous binding of factors to the D e and Dfl sites
and that the complex is labile so that it is barely
detectable by EMSA analysis (see also Fig. 6 and 7).
Supershift experiments were performed with nuclear
extracts of different B cell lines and the - 1 7 6 / - 1 3 6
LRS probe, using specific antibodies against the transcription factors Oct-l, Oct-2 and PU.1 and the POU
domain protein family (Fig. 6; Fig. 7). The results
confirmed our previous observation that a protein
interacting with D e (the D e l factor) was recognized by
the anti-POU domain antibody. Furthermore, the [Del,
Dfll/Dfl2] band was also depleted by this antibody,
indicating that the complex contained a POU domain
protein. None of the complexes reacted with the anti-
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E B N A 2 transactivation of the L M P 1 promoter
2687
LRS~176/-136)
LRS(-176/-136)
D~I
Dfl2
D/~I
D~I
Dfl2
Dill
, Rael ~~ CBC,
Rael
+
+ +
+
+
DG75
Antibody
Control
PU.1/Spi-1
+
-
~t Cherry~ ~ Raji
IB
+
+
+
+
+
+
Fig. 7. Immunological analysis of factors that bind to the Dfl domain
in different B cell lines. Nuclear extracts from DG75, Rael, CBC-Rael,
IB4, Cherry and Raji cells were incubated under binding conditions
with a 32P-labelled - 1 7 6 / - 136 LRS oligonucleotide probe and with
control (anti-Spl) or anti-PU.1/Spi-I antibodies as indicated. The
reaction mixtures were analysed by EMSA. Three specific complexes,
D~I, Dill and Dfl2, are indicated by black arrows. A non-competable,
unspecific band is indicated by the hatched arrow.
DG75 cells
Oct-1 and -Oct-2 antibodies (Fig. 6). One of the factors
that interacted with Dfl was recognized by the PU. 1specific antibody, as shown by the elimination of the Dill
complex in all cell lines tested (Fig. 7). Furthermore, an
oligonucleotide that contained a consensus PU. 1-binding
motif inhibited the formation of both the Dill and Dfl2
complex in an EMSA competition experiment, suggesting that the Dill and Dfl2 factors belong to the same
family of DNA-binding proteins, the ets gene family
(data not shown). The Dill and Dfl2 factors were present
in both the EBV-negative and EBV-positive B cell lines,
although the relative amounts varied between the lines
(Fig. 7).
E B N A 2 is targeted to the L M P 1 promoter by the P O U
domain protein
Having shown that D~ can confer EBNA2 responsiveness to an heterologous promoter much more
efficiently than Dfl, we asked whether it would be
possible to demonstrate a direct interaction between
In vitro-translated
EBNA 2
Control
-
-
+
-
+
Fig. 8. Effect of the addition of in vitro-translated EBNA2 on protein
binding to the LRS probe. DG75 nuclear extracts were incubated with
the 32P-labelled - 1 7 6 / - 1 3 6 LRS oligonucleotide probe and aliquots
of reticulocyte in vitro translation reactions with control D N A or
EBNA2 D N A as indicated. The binding mixtures were analysed by
EMSA. The positions of the three previously identified complexes,
D~I, Dill and Dfl2, are indicated by black arrows. A non-competable,
unspecific band is indicated by the hatched arrow. The black arrow at
the top indicates a complex which was induced or strongly enhanced by
the addition of in vitro-translated EBNA2.
EBNA2 and factors bound to the D~ or Dfl region.
Accordingly, we assayed binding of EBNA2 expressed in
a reticulocyte lysate translation system to a - 1 7 6 / - 136
LRS probe by EMSA in the presence of transcription
factors furnished by DG75 nuclear extract (Fig. 8). The
presence of recombinant EBNA2 in the binding mixture
resulted in a specific elimination of the D~I band
corresponding to the POU domain protein, which was
not seen when a reticulocyte lysate containing the vector
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2688
A. SjdbIom and others
control only was added. The PU. 1-related complexes in
the Dfl region were not affected. The addition of the
EBNA2 protein also resulted in an increase in mass in the
EMSA pattern at a position corresponding to a slower
migrating band (Fig. 8). Competition experiments
showed that this complex was specific (data not shown),
but the nature of its constituents is not clear. Our
interpretation of these data is that EBNA2 can make a
direct contact with the POU domain protein but the
molecular consequences of the interaction remains
unknown.
Discussion
It is now well established that a major way by which
EBNA2 exerts its effect on the EBV-infected cell is by
regulating the activity of certain viral and cellular
promoters. We and others have previously reported that
LRS contains cis-elements that mediate the EBNA2induced up-regulation of LMP1 expression (F~hraeus et
al., 1990, 1993; Ghosh & Kieff, 1990). Our previous
characterization of LRS indicated that the region
between - 2 1 4 and +40 is sufficient to direct EBNA2induced transactivation of LMP1 promoter-containing
plasmids in B cells. We now provide evidence that two
different regions of this part of LRS, - 176 to - 136 and
-106 to +40, contribute to EBNA2 responsiveness of
reporter plasmids independently of each other. We have
defined transcriptional elements and factors in the
- 1 7 6 / - 1 3 6 region that participate in the EBNA2induced transactivation of the LMP1 promoter.
The upstream region of the EBNA2-responsive LMP 1,
LMP2A, Cp and CD23 promoters all contain the DNA
sequence 5' GTGGGAA 3'. A number of recent reports
demonstrate that this motif in the context of an EBV
DNA promoter sequence specifically interacts with a
DNA-binding cellular protein, RBP-J~c, that can target
EBNA2 to its responsive element, and that this interaction results in promoter activation (Ling et al.,
1993; Zimber-Strobl et al., 1993; Grossman et al., 1994;
Henkel et al., 1994; Meitinger et al., 1994; Waltzer et al.,
1994; Yalamanchili et al., 1994; Johannsen et al., 1995).
The core sequence of the RBP-J~c-binding site is present
at positions - 2 2 3 / - 2 1 7 and - 2 9 8 / - 2 9 2 in LRS,
which were not included in the sequence analysed in this
report. Our previous results using deletion analysis and
site-directed mutagenesis have, however, very clearly
demonstrated that EBNA2 can activate the LMP1
promoter in reporter constructs lacking the RBP-JKbinding site (F~hreaus et al., 1990, 1993; Sj6blom et al.,
1995). This has recently been confirmed by Johannsen et
al. (1995). It is also consistent with the observation that
RBP-JK has a much lower affinity for its binding site in
the LRS context than for the corresponding sites in the
Cp or CD23 promoters (Ling et al., 1994). On the other
hand, it has been reported by Laux et al. (1994a) that
LRS sequences between - 2 3 2 and - 1 9 9 are essential
for EBNA2 responsiveness of the LMP1 promoter in B
cells. The reason for this discrepancy remains unexplained.
Our results show that the - 1 7 6 / - 1 3 6 LRS region
contains two domains, here denoted De ( - 1 5 0 / - 1 4 1 )
and Dfl ( - 1 7 0 / - 156) (Fig. 2), of particular importance
for EBNA2 responsiveness. Constructs that contained
both De and Dp had a higher EBNA2 inducibility than
the corresponding deleted plasmid [pgLRS(- 106)CAT]
and mutations in either element reduced EBNA2
inducibility. In contrast, although the level of activity
was higher in plasmids that contained only Dfl as
compared with the deleted construct, EBNA2 inducibility was the same. This was true regardless of whether
Dfl was mutated or not. Our interpretation of the results
is that (i) elements in De and Dfl must cooperate to
achieve maximum EBNA2-responsiveness of the
- 1 7 6 / - 1 3 6 region; (ii) in the absence of De the Dfl
element functions mainly as a positive element, amplifying the activity of elements in the - 1 0 6 / + 4 0 LRS
region.
It should also be noted that the - 176 LRS construct
had about the same EBNA2 inducibility as that which
contained the complete region up to - 2 1 7 (about 47fold induction), showing that no essential elements were
deleted (Fig. 3). Essentially the same result was obtained
with a -217 LRS construct in which the - 2 1 7 / - 1 9 5
sequence had been mutated (Sj6blom et al., 1993). On
the other hand, Johannsen et al. (1995) have reported
that a -215 to +40 LMP1 promoter construct was
EBNA2-responsive but a - 2 0 5 / + 40 construct was not.
They also mapped binding sites for unidentified transcription factors to the - 2 1 5 / - 2 0 5 region (LBF3, 5, 6
and 7 in B cells) which were assumed to contribute to
EBNA2 responsiveness (Johannsen et al., 1995). In iine
with the latter observation, we have recently obtained
evidence from experiments with deletion mutants of
EBNA2 which suggest that part of the transactivating
effect of EBNA2 might be mediated through sequences
in the - 2 1 4 / - 195 LRS region (A. Sj6blom, A. Nerstedt,
A. Jansson & L. Rymo, unpublished results). The reason
for the inconsistency between these results is not clear.
We are presently investigating if differences in reporter
constructs, expression vectors, cell lines or transfection
conditions may play a role. It is conceivable that the
artificial rearrangement of promoter elements or the
introduction of mutations associated with in vitro studies
might have had consequences that do not properly reflect
the function of the promoter in the native state.
Protein-binding studies employing EMSAs suggested
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EBNA2
that both EBV-negative and EBV-positive cells contain
apparently similar sets of transcription factors that bind
to the D~/Dfl region. Furthermore, it was not possible to
identify EBNA2 as a component of the EMSA complexes
obtained with IB4 nuclear extracts by supershift experiments with monoclonal antibodies (data not shown).
The only detectable difference with regard to proteinbinding between B cells that contained EBNA2 and
those which did not was a somewhat diminished
protection of the Dc~ domain when studying the IB4 cell
extract in the DNase I footprint analysis (Sj6blom et al.,
1995). Consistent with this difference in the Dc~ domain,
a somewhat changed intensity of the D~I complex was
seen when compared in the EMSA studies using EBVnegative and EBV-positive EBNA2-containing cells. This
might be an indication of a decreased stability or a
conformational change of the complex between the
elements in the Dc~ domain and cellular factors in
EBNA2-containing cells.
The sequence at position - 1 4 7 / - 1 4 0 in Dc~ with
partial identity (6/8) to the octamer motif suggested that
a member of the Oct family of transcription factors
might be responsible for the effect of the D0~ element on
LMP1 promoter activity. We have, however, not been
able to demonstrate the binding of the Oct-1 or Oct-2
factors, which are expressed in B cells, to this site with
specific antibodies. The Oct factors belong to a larger
family of transcription factors designated POU domain
proteins (Wegner et al., 1993). These proteins share a
conserved DNA-binding domain (the POU domain)
consisting of the POU-specific 75 to 82 amino acid
domain, a short variable linker region and the homeodomain. Using an antibody against the POU-specific
domain we were able to demonstrate that both EBVpositive and EBV-negative B cells contain a distinct
factor that binds to the octamer motif in the context of
surrounding LRS sequences. If sequences upstream of
position - 146 in the binding site were deleted, the POU
domain protein was replaced by an unidentified factor,
suggesting that D~ contains two overlapping proteinbinding sites. Interestingly, the binding of the unidentified factor correlated with a complete loss of EBNA2
responsiveness. Deletion of a further 2 bp resulted in the
loss of binding of the unidentified factor, which correlated with the restitution of EBNA2 responsiveness to
almost the same level as when the POU domain protein
was bound (provided no elements upstream of D~ were
present in the promoter constructs). It is, however, not
immediately obvious what role this putative silencer
might play in the regulation of LMP 1 promoter activity,
since the binding of the POU domain protein to a DNA
fragment that contained an unmutated D~ always
dominated over that of the unidentified factor both in
EBV-positive and EBV-negative B cells. It is of course
transactivation o f the L M P 1 p r o m o t e r
2689
possible that our assay conditions do not properly reflect
the in vivo situation.
It has recently been reported that the PU.1 transcription factor binds to the PU box present in Dfl (Laux
et al., 1994b; Johannsen et al., 1995). PU.1 is expressed
specifically in haematopoietic tissues, particularly in the
monocytic and B lymphoid lineages (Klemsz et al., 1990;
Goebl, 1990; Ray et al., 1992). Our protein-binding
studies showed that two discrete factors bound to an
element in the DE domain (the D/~I and Dp2 complexes
in Figs 5 and 7) and competition experiments with an
excess of a PU box consensus oligonucleotide resulted in
the removal of both complexes (data not shown). EMSA
supershift experiments using an anti-PU.1 antibody
depleted the complex corresponding to the Dill band in
all B cell lines tested, confirming the conclusions of Laux
et al. (1994b) and Johannsen et al. (1995). In addition,
Laux and colleagues showed that a second member of
the Ets family of transcription factors, Spi-B, also
recognized the same PU.1 recognition site (Laux et al.,
1994b). Thus, it seems reasonable to assume that the D~2
complex in our study is formed by the binding of Spi-B
to DE. Spi-B is expressed in all human haematopoetic cell
lineages except T cells and has been shown to transactivate reporter plasmids containing PU boxes (Ray et
al., 1992).
EMSAs revealed that a relatively abundant factor in B
cells bound to the - 1 2 7 / - 1 1 8 LRS region which
contains a sequence with partial identity to the binding
site for SIF. An oligonucleotide that contained a
consensus SIE inhibited the binding. SIF is a DNAbinding protein that is induced by polypeptide growth
factors and the presence of an SIE in a promoter region
is sufficient under certain circumstances to mediate
growth factor-activated transcription (Sadowski et al.,
1993). However, this factor did not seem to be essential
for the regulation of LMP1 promoter activity, at least
not in the context of the - 1 4 4 / + 4 0 LRS region, since
deletion of SIE had little effect on EBNA2 responsiveness
[compare pgLRS(-144)CAT and pgLRS(-106)CAT constructs in Fig. 1].
How does EBNA2 contact LRS? Evidence accumulating from several groups strongly indicates that the
EBNA2 protein does not bind directly to a specific DNA
sequence but acts via protein-protein interactions with
the LMP1 promoter regulatory region. EBNA2 might
also act at a distance. We have recently reported evidence
suggesting that one of the functions of EBNA2 might be
to inhibit dephosphorylation by protein phosphatase 1
leading to a modulation of the activity of transcription
factors involved in the regulation of the LMP 1 promoter
(F~hraeus et al., 1994). On the other hand, there are
several possible candidates for the targeting of EBNA2
to the promoter: RBP-Jtc at the Jtc site (Laux et al.,
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2690
A. Sj6blom and others
1994a), the PU box-binding proteins including PU.1
(Johannsen et al., 1995), the POU domain protein at the
Dc~ site (this investigation) and unidentified factor(s)
binding to promoter-proximal ( - 1 0 6 / + 4 0 ) sites (A.
Sj6blom, A. Jansson, W. Yang & L. Rymo, unpublished
results). In the present investigation, the PU box in Dfl
and PU.1 could not convey EBNA2 responsiveness
alone since a construct containing only Dfl [Table 1;
p g L R S ( - 1 8 1 / - 1 4 5 ) T K C A T ] was not induced by
EBNA2. This lack of response was also seen in the series
of plasmids derived from p L R S ( - 1 0 6 ) ( - 1 8 1 /
- 145)CAT, which contain Dfl but not D~ and showed
the same level of responsiveness as the - 1 0 6 LRS
construct (Fig. 2), irrespective of whether Dfl was
mutated. On the other hand, the POU domain protein
and D~ seem to be instrumental in mediating the
EBNA2 effect since a ( - 1 6 0 / - 136) LRS DNA fragment
conveyed responsiveness to a basal TK promoter [Table
1; p g L R S ( - 1 6 0 / - 1 3 6 ) T K C A T ] . However, we have
not so far been able to demonstrate a direct physical
association between EBNA2 and the POU domain
protein with EMSA supershift experiments (data not
shown). Similar results have been reported by Sauder et
al. (1994). They identified a protein-binding region in a
D N A fragment of the LMP1 promoter that corresponds
to the Dc~/Dfl region studied in the present investigation.
No interaction between EBNA2 and the LMP 1 promoter
fragment in EMSA supershift experiments could be
found under conditions that clearly showed binding of
EBNA2 to the LMP2A promoter. However, analysis by
sucrose gradient centrifugation provided evidence that
the LMP1 promoter-binding proteins form a complex
that sediments with a higher velocity in EBNA2-positive
cell extracts. They suggest that EBNA2-positive cells
might contain specific complexes bound to the LMP1
promoter that are too labile to be detected by EMSA
(Sauder et al., 1994).
The regulation of the immediate early (IE) genes by
VP16 in the herpes simplex virus (HSV) system (Thompson & McKnight, 1992) has been used as a model to
explain EBNA2 regulation of EBV promoters via RBPJK and its binding site (Laux et al., 1994a). A similar
model could be made for the interaction of EBNA2 with
the POU domain protein and the PU. 1 factor and the D~
and Dfl elements. The enhancers associated with the
HSV IE genes contain one or more copies of two
different cis-regulatory sequences: 5' TAATGARAT 3'
(' tatgarat') and the direct repeat 5' CGGAAR 3' (' cigar')
motifs. The tatgarat motif forms a complex with Oct-l,
VP16 and a host cell factor and the repeated cigar motif
binds a tetrameric complex consisting of the subunits of
an ets gene family member, the GA-binding protein.
These neighbouring protein complexes then engage in
protein-protein interactions with each other, creating a
functional multicomponent complex. The PU.1 transcription factor (and/or Spi-B) bound to Dfl and the
POU domain protein at the octamer site in D~ and
EBNA2 might interact in an analogous way.
In conclusion, our findings are consistent with the idea
that efficient EBNA2 transactivation of the LMP1
promoter requires the cooperation of multiple factors
that mediate their effect through different DNA sequences. We have identified a POU domain protein with the
ability to target EBNA2 to a promoter. Functional
cooperation between the POU domain protein and PU. 1
(and/or Spi-B) may contribute to the B cell-specific
activation of the LMP1 promoter. It is possible that
EBNA2 might stabilize the association between the POU
domain protein, PU. 1 and EB2RE by forming a complex
that cannot easily be detected by EMSA. It is also
possible that post-transcriptional modifications of either
PU. 1 or the POU domain protein, or both, may occur.
We thank Carina Str6m and Jane L6fvenmark for skilful technical
assistance. We are very grateful to Dr Peter O'Hare for the POU
domain-specific antibodies. This study was supported by grants from
the Swedish MRC (project 5667), the Swedish Cancer Society, the
Inga-Britt och Arne Lundberg Foundation and by National Cancer
Institute Grant 28380-08. S. L. was a recipient of an EMBO Long Term
Fellowship.
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