© 1992 Oxford University Press
Nucleic Acids Research, Vol. 20, No. 11
2657-2665
The weak, fine-tuned binding of ubiquitous transcription
factors to the 11-2 enhancer contributes to its T cellrestricted activity
Bernd Hentsch, Athanasia Mouzaki1, Isolde Pfeuffer, Duri Rungger2 and Edgar Serfling*
Institute of Virology and Immunobiology, University of Wurzburg, Versbacher StraBe 7, D-87
Wurzburg, Germany, 1 Haematology, Geneva University Hospital, 25 rue Micheli-du-Crest, CH-1211
Geneva 4 and 2Station de Zoologie Experimentale, University of Geneva, 154 route de Malagnou,
CH-1224 Chene-Bougeries, Switzerland
Received April 8, 1992; Accepted April 30, 1992
ABSTRACT
INTRODUCTION
The T lymphocyte-specific enhancers of the murine and
human Interteukin 2 (11-2) genes harbour several binding
sites for ubiquitous transcription factors. All these sites
for the binding of AP-1, NF-kB or Oct-1 are noncanonical sites, i.e. they differ in one or a few base pairs
from consensus sequences for the optimal binding of
these factors. Although the factors bind weakly to these
sites, the latter are functionally important because their
mutation to non-binding sites results in a decrease of
inducible activity of the II-2 enhancer. Conversion of
three sites to canonical binding sites of Octamer
factors, AP-1 and NF-kB results in a drastic increase
in enhancer activity and the induction of the II-2
enhancer in non-T cells, such as B cell lines, murine
L cells and human HeLa cells. The Introduction of two
or three canonical sites into the enhancer leads to a
further increase of its activity, li-2 enhancer induction
Is also observed in B cells when the concentration of
AP-1 and Oct factors increases as a result of cotransfections with FosB and Octamer expression
plasmids. When II-2 enhancer constructs carrying
canonical factor binding sites were injected into
Xenopus oocytes the strong binding of ubiquitous
factors substantially overcomes the silencing effect of
negatively acting factors present in resting primary T
lymphocytes. These results suggest a fine-tuned
interplay between ubiquitous and lymphoid-specific
factors binding to and transact! vat ing the II-2 enhancer
and show that the binding affinity of ubiquitous factors
to the enhancer contributes to its cell-type specific
activity. Moreover, we believe that a dramatic increase
of transcriptional activity brought about by single point
mutations at strategic important factor binding sites
may also have relevance to the activation of nuclear
oncogenes.
A key event of tissue-specific expression of eukaryotic genes is
the control of their transcription which is mediated by numerous
transcription factors (see Ref. 1 for a review). Transcription
factors are nuclear proteins which bind to DNA and/or to other
factors and exert a positive or negative influence on the
transcriptional activity of RNA polymerase II (2). Over the last
years numerous eukaryotic transcription factors have been isolated
and analysed in detail (3). Cell-type restricted as well as
ubiquitous factors are involved in the transcriptional control of
tissue-specific genes. However, the exact way in which these
factors interact is not yet known.
The complexity of transcriptional control regions of eukaryotic
genes hampers the study of the interactions of transcription factors
involved in the control of eukaryotic genes. Very often the gene
control regions appear to be widely distributed, divided into
promoters, located just upstream of the transcription start and
enhancers which can be located several kilobases DNA away,
either upstream or downstream from the gene's transcriptional
start site or within the transcription unit of the gene (4). Even
in situations where the gene's control region spans only one
hundred or fewer base pairs of DNA, a large number of factors
can interact with these short DNA segments, and even a single
protein binding site consisting of a few base pairs DNA can be
recognized by multiple factors.
One example in this respect is the so-called Octamer motif
ATGCAAAT which was first detected as a conserved sequence
motif within the promoters of Immunoglobulin (Ig) genes and
later also within the promoters and enhancers of many other
genes, such as in those controlling the activity of such divergent
genes as histone genes, small nuclear RNA genes and viral genes
(5). In lymphoid cells two factors interact with the Oct motif,
the ubiquitous factor Oct-1 and the lymphoid-specific factor
Oct-2. The genes encoding both factors have been cloned and
their DNA binding and transacting properties have been analysed
(5, 6). Mainly due to its lymphoid-specific occurrence, Oct-2
* To whom correspondence should be addressed
2658 Nucleic Acids Research, Vol. 20, No. 11
has been implicated in the control of the activity of lymphoidspecific genes, e.g. the activity of Ig genes, whereas Oct-1 is
thought to control genes which are ubiquitously expressed, e.g.
the snRNA genes. Although direct functional tests have
demonstrated a preference of Oct-1 for the transactivation of
snRNA promoters and of Oct-2A for mRNA promoters (7),
several other parameters play an additional role in the gene control
mediated by Oct-1 and Oct-2.
One such additional parameter is the interaction of Oct-1 with
other transcription factors which help to enhance the relatively
poor transacting potency of Oct-1. The interaction of Oct-1 with
VP-16, a very strong viral transactivator, and an additional host
cell factor has been studied in detail (see Ref. 8 for a review)
and it has been shown that the interaction with VP-16 alters the
regulatory capacity of Oct-1. VP-16 acts as a transcriptional
modulator which allows Oct-1 to stimulate a new set of
promoters.
Further parameters which affect the transcriptional control of
Octamer factors are the binding affinity of their target sites and
the factor concentration in a given cell type. Functional tests of
Octamer DNA containing promoters in transient transfection
studies revealed a clear correlation between the binding affinity
of the site and its cell type-specific activity (9). Promoters
containing high affinity sites were found to direct transcription
in B cells as well as non-B cells, whereas the activity of promoters
with low affinity sites appeared to be restricted to B cells. The
activity of promoters with low affinity octamer sites could be
activated in non-B cells by co-transfection with Oct-1 or Oct-2
expression plasmids, i.e. by an enhancement of factor
concentration (9).
One of the most prominent factor binding sites within the
transcriptional control (enhancer) region of Interleukin 2 (11-2)
gene is a non-canonical Octamer binding site (10, 11). This site
plays an important role in the T cell-specific induction of D-2
A.
-200
I
-250
I
-60
I
-7
NF-kB: GGGACTTTCCC
TCEd: GGOA-TTTCAi
AP-l.TGAGTCA
UPS: TGTGTAATATGTAAAA
~
Cbt ATCCAAAT
AP-1: TGAGTCA
TREp:_AGAGTCA
B.
extract
nducbon
probe
C
EM
odract
+
+
inductor TREp 1SJT THEpl5JT
EM
-
-
+
+
UPS UPSQUPS UPSO
D.
«
EM
+
TCE* A/C T C E j
+
A/C
probe
Oct2B
Oct2A
2
3
4
Fig. 1. Scheme of the murine Interleukin 2 enhancer, the sequences and factor binding of non-canonical sites for ubiquitous factors. A. The murine 11-2 enhancer.
The most prominent protein binding sites are indicated by boxes. The two TATA boxes, TATA-1 and TATA-2, are shown as stippled boxes, the protein binding
sites for which no function has been detected so far (the proximal T cell element, TCEp, and the upstream sites, US-1 and US-2/3) by open boxes. The filled boxes
indicate protein binding sites which are involved in the establishing of inducible enhancer activity in E14 T lymphocytes. These are the upstream promoter §ite,
UPS, the two Purine boxes, Pu-bp and Pu-bd, the proximal TPA responsive element, TREp, and the distal J_cell element, TCEd (see Refs. 1 3 , 2 3 - 2 5 ) . The positions
and sequences of four non-canonical binding sites for ubiquitous factors are shown. Nucleotides which differ from canonical consensus sequences are underlined.
B. Binding of Oct factors to UPS DNA. EMSA. A radioactively labelled UPS probe (spanning the nucleotides from position - 6 4 to - 9 4 of 11-2 enhancerXlanes
1 and 3) or an U P S Q probe carrying a canonical Octamer binding site (see Materials and methods)(lanes 2 and 4) were incubated with 4 m protein of crude nuclear
extracts from uninduced ( - ) or induced EM cells ( + ) treated for 4 h with TPA/ConA (10 ng/2.5 ji% per ml) on ice followed by electrophoresis trough a non-denaturing
5% polyacrylamide gel. The positions of Oct complexes are indicated. C. Binding of AP-1 like factors to TREp DNA. A TREp-WT probe spanning the nucleotides
from - 1 4 3 to - 1 5 7 Oanes 1 and 3) or a 153T-TREp probe carrying a canonical TRE (lanes 2 and 4) were incubated with nuclear extracts of uninduced ( - ) or
induced EM cells ( + ) and treated as described above. The position of AP-1 complex is indicated. D. Binding of NF-kB like factors to the WT-TCEd spanning
the nucleotjdes from - 1 9 1 to - 2 1 5 Oanes 1 and 3) and the TCEd A/C probe containing a canonical kB-binding site (lanes 2 and 4). Note the strong binding of
NF-kB to the TCEd A/C DNA.
Nucleic Acids Research, Vol. 20, No. 11 2659
gene because mutations which impair the binding of Octamer
factors drastically decrease the D-2 enhancer activity (11;
I.Pfeuffer et al., submitted). Recently, we have identified a noncanonical AP-1 binding site which is closely linked to the Octamer
site within the enhancer's so-called upstream promoter she (UPS).
We will show elsewhere that the cooperative binding of Octamer
and AP-1 like factors to the UPS DNA contributes to the overall
activity of 11-2 enhancer (I.Pfeuffer et al., submitted). In this
communication we will show that the affinity of Octamer factors
binding to this site also contributes to the inducible D-2 enhancer
activity since the conversion of non-canonical Octamer site to
a canonical site leads to a drastic increase of the D-2 enhancer
activity in T cells and to its activity in non-T ceUs. Very similar
results were obtained when two other non-canonical binding sites
were converted to canonical sites which are bound by the
ubiquitous factors AP-1 and NF-kB. Moreover, in B cells the
activity of the otherwise non-active 11-2 enhancer could be
stimulated by co-transfections of factors recognizing the D-2
enhancer, such as Fos B and Oct-2. These and further
experimental results demonstrate that the binding affinity and
concentration of ubiquitous factors interacting with the D-2
enhancer contribute to its cell type-specific activity.
A.
extract
induction
probe
extract
probe
ISJT-TREp
TREcd
competitor: l&JT-TREp
TREcol
UPSQ
OCT
competitor UPS,,
OCT
OCT
I
OCT 2B
OCT 2A
B14
+
+
t
f
+
+
+ +
+
+
I
c.
art-act
nducbon
_
probe.
TCEd A/C _
competitor TCEd AfC _
1
2
3
4
5
6
NF «B
Fig. 2. The factor binding to the 'converted' sites of ubiquitous factors within the 11-2 enhancer is very similar, if not identical to those of well-characterized binding
sites of ubiquitous factors. EMSA. A. The binding of Oct factors to the U P S Q motif (lanes 1, 3 and 4) and the Oct motif of Ig heavy chain enhancer (lanes 2,
5 and 6). Radioactively labelled U P S Q DNA (carrying an Oct consensus sequence within the 11-2 sequence from position - 6 4 to - 9 4 : see Materials and methods)
or the 51 bp Hinfl-Ddel fragment of Ig heavy chain enhancer (47) were incubated with 4 jig protein of crude nuclear extracts from uninduced (lanes 1 and 2) and
induced E14 cells (lanes 3 - 6 ) treated for 4 h by TPA/Con A (10 ng/2.5 Mg per ml). In lanes 4 and 6, an 100 fold molar excess of U P S Q DNA or Oct DNA
(corresponding to a 15 bp oligonucleotide containing an Oct consensus sequence) was added to the incubation for competition. B. Binding of AP-1 to the 153T-TREp
and the TREcoll motifs. A l53T-TREp probe (see Fig. 1 and Materials and methods)(lanes 1, 3 and 4) or a TREcoll DNA probe containing the AP-1 site of human
collagenase promoter (48) (lanes 2, 5 and 6) were incubated with nuclear proteins from uninduced (lanes 1 and 2) or induced EM cells (lanes 3—6). In lanes 4
and 6, an 100 fold molar excess of I53T-TREp or TREcoll DNA were added for competition. C. Binding of NF-kB like factors to the TCEd A/C DNA and the
canonical kB-site of Ig kappa enhancer (49). A TCEd A/C DNA probe (spanning the nucleotides from - 1 9 1 to - 2 1 5 with a point mutation at position - 1 9 9 )
(lanes I, 3 and 4) and the kB-site of Ig kappa enhancer (lanes 2, 5 and 6) were incubated with nuclear proteins from uninduced (lanes 1 and 2) and induced EI4
cells (lanes 3 - 6 ) . In lanes 4 and 6, an 100 fold excess of TCEd A/C DNA or of kB-DNA of Ig kappa enhancer were added for competition.
2660
Nucleic Acids Research, Vol. 20, No. 11
MATERIALS AND METHODS
Cell culture, DNA transfections and CAT assays
Murine E14 lymphoma cells, 70Z pre-B lymphocytes and A20J
B lymphoma cells were grown in RPMI medium supplemented
with 5% fetal calf serum to a density of about 2 X10* cells per
ml. About 2xlO 7 cells were transfected with 10 ng DNA
(purified by two CsCl/ethidium bromide density gradient
centrifugations) in a final volume of 1.2 ml using the DEAE
dextran protocol (12). After transfection, the cells were shocked
with 15% dimethylsulfoxide for 2 min and, 20 h later, they were
divided. One half of E14 cells was induced with the phorbolester
TPA (12-O-tetradecanoylphorbol-13-acetate; 10 ng per ml) and
the plant lectin Con A (Concanavalin A; 2.5 /tg per ml), the other
half was used as uninduced control. (In some EW cell experiments
the Ca++-ionophore Ionomycin (10 /tM) was used instead of
ConA). The B cell lines were induced by 50 ng per ml TPA.
The cells were incubated for another 20 h, harvested, sonicated
and their CAT activities were measured as described earlier (13).
Murine LMTK" and human HeLa cells were grown in
Dulbecco's modified Eagle's medium containing 5% fetal calf
serum. They were transfected using the calcium phosphate coprecipitation method (14) and shocked with 25%
dimethylsulfoxide for 3 min. 40 /ig DNA was usually transfected
into two plates of 10 mm diameter each. The cells of one plate
were induced by TPA (50 ng per ml), the cells of the other were
ffff
Fig. 3. The conversion of non-canonica] binding sites of UPS-Oct, TREp and TCEd DNAs to canonical factor binding sites leads to a strong increase of enhancer's
activity in EI4 T cells and to its inducible activity in non-T cells. 10 /ig DNA of the construct pUCAT 2/1 + containing the wild type enhancer in front of the tk
promoter of CAT construct pBLCAT2 (13), designated as WT, or of constructs carrying a NF-kB consensus sequence (NF-kB ( - 199Q), an AP-1 consensus sequence
(AP-1 ( - 153T)) and an Oct-consensus sequence (Oct (-72T/-76T)), or two (AP-1 +Oct) and three (NF-kB+AP-1 +Oct) consensus sequences were transfected
into 2 x 107 EM cells, 70 Z pre-B lymphocytes or A20J B lymphoma cells using the DEAE dextran tranfcction protocol (12). 20 h after transfection, the cells were
divided. One half was used as uninduced control ( - ) , the other half of cells was induced by TPA/ConA (10 ng/2.5 /ig per ml) fora further 20 h period. The cells
were harvested, and their CAT activities (corresponding to the %-acety lation) were determined as descnbed (see Ref. 13 and Materials and methods). Note that for
the determination of enhancer activity in E14 cells only one fifth of the protein amount was used in contrast to that of other cells. 40 /ig of each construct was transfected
into two 100 mm plates of LMTK" cells or HeLa cells (left column diagram) using the calcium phosphate co-precipitation technique (14). In the HeLa cells of
the right column diagram, 5 /ig DNA were transfected per plate. The cells of one plate were used as uninduced control ( - ) , the cells of the other plate were induced
by TPA (50 ng per ml) for 20 h (+). The diagrams show the mean results of at least three independent transfection experiments.
Nucleic Acids Research, Vol. 20, No. 11 2661
used as uninduced control. In co-transfection experiments (see
Fig. 5) 10 /tg DNA of the indicator plasmid pUCAT 2/1 +
carrying the wild type D-2 enhancer (13) were transfected into
2 X 107 70Z pre-B cells or A20J B cells along with 10 /xg each
of effector plasmids encoding JunB, FosB, Oct-1 or Oct-2
proteins. The expression plasmids were the following: pRSVJunB (constructed by P.Angel) contains a complete copy of
murine junB cDNA (15) instead of the c-jun cDNA of expression
vector pRSV-cJ (16). pMSfosB-L carries a complete copy of fosB
cDNA (17) in the expression vector pMSE (18). The Oct-1 vector
(constructed by P.Matthias) contains the amino acid residues
1 -522 of Oct-1 (19) in the expression vector pEV3Sl (20). The
Oct-2 A vector corresponds to the expression vector pOEVl( + )
(21).
CAT activities were determined using the Phospholmager
(Image Quant 3.15) system of Molecular Dynamics and
normalized with regard to the protein content of cell lysates.
DNA cloning
All the recombinant DNA work was carried out according to
standard recombinant DNA techniques (22). The structure of
numerous 11-2 enhancer constructs mentioned in this study has
been described previously (13, 23-25). Point mutations were
introduced into the enhancer DNA using an oligonucleotidedirected mutagenesis system (Amersham), followed by dideoxysequencing. The following oligonucleotide primers were used to
convert the indicated sites to canonical binding sites of ubiquitous
factors:
A2OJ
A
Induction
probe
- .
+
I
+
70Z
.
.
I
+
+
LMTK
.
I
UPS,-:
153T-TREp:
TCEd A/C:
(-59) 5'-GGGTGTCACGATGATTTGCATATTACACATATTT-3' (-92)
(-64) 5'-CACGATGTTTTACATATGACICAT
ATTT-3' (-92)
(-143) 5'-CTGATGACTCACTGGAATTT-3'(-162)
(-188) 5'-ATGGATTTAGGGGAAATCCCTCT-3'(-21O)
The underlined nucleotides indicate the changes to canonical
factor binding sites. Two or three canonical sites were introduced
into the enhancer in the following way: (i) the 153TTREp/UPSo 11-2 enhancer (carrying two canonical sites, i.e. a
canonical TRE as well as Oct site) was constructed by fusing
the BamHI/AccI and Accl/Hind HI fragments of UPSQ and
153T-TREp enhancer constructs, respectively, (ii) The TCEd
AIC / 153T-TREp/UPSo D-2 enhancer construct (carrying three
canonical sites) was obtained by fusing the BamHI-Dral fragment
of 153T-TREp/UPSo enhancer construct with the Dral-Hindin
fragment of the construct TCEd A/C.
For the introduction of canonical binding sites into longer D-2
promoter/enhancer segments the BamHI-Dral fragment of 153TTREpAJPSo D-2 enhancer construct was fused with the DralHindm fragment of the D-2 CAT construct pDCAT 1 carrying
the upstream promoter DNA up to position —483 (13). A second
vector carrying all three canonical sites of D-2 enhancer and the
upstream DNA from -655 to about -2000 was constructed by
fusing the 1.4 kB long Dral-Hindlll fragment of plasmid
pILCATO (spanning about 2kb of upstream D-2 DNA)(13) to
the D-2 enhancer harboring three canonical binding sites.
HeLa
. + + . .
+ +
WTCWTCWTCWTCWTCWTCWTC
WTC
Oct-1
—
0O-2BOct-2A
i
a extract
" • induction
probe
A20J
I
70Z
I
LMTK
I
HeLa
W T C W T C W T C W T C W T C W T C W T C
WTC
•tiv
extract
induction
probe
A20J
I
70Z
I
LMTK
i
HeLa
+
.
.
+
+
.
.
+
+
++
+
W T C W T C W r C W T C W T C W T C W T C
WTC
Oct-1
6
7
8
9
10
II
12
13
14
15
16
Ftg. 4. The factor binding to the non-canonical sites of UPS-Oct, TREp and TCEd DNAs (WT) and to their converted, canonical versions ( Q in nudear extracts
of non-T cells. EMSA. Crude nuclear extracts of uninduced ( - ) or induced ( + )
70Z pre-B lymphocytes, A20J B lymphoma cells, LMTK" cells and HeLa cells
were incubated with an UPSwr or U P S Q probe (the latter carries a canonical
Oct site) (A), with a TREp-WT or 153T-TREp probe (the latter carries a AP-1
consensus site) (B) or with a TCEd-WT or TCEd A/C probe (C).
0O-2A
JunB
FosB
Fig. 5. An increase of factor concentration leads to the inducible activity of 11-2
enhancer in B lymphocytes. Co-transfection experiment. 10 ng DNA of WTenhancer construct pDCAT 2 / 1 + (13) was transfected into 2 x l 0 7 70Z-pre B
lymphocytes or A2QJ B lymphocytes along with 10 )i% of the expression plasmids
pEV-Oct-1 (Oct-1), p O E V l ( + ) (Oct-2A), pRSV-JunB (JunB) and pMSE-FosB
(FosB) (see Materials and methods for the structure of vectors) using the DEAE
dextran transfection protocol (12). 20 h later the cells were divided. One half
of cells were used as uninduced control (—), the other half of cells were induced
by TPA (50 ng per ml) for a further 20 h period. Shown are the mean results
of three co-transfection experiments.
2662 Nucleic Acids Research, Vol. 20, No. 11
To avoid read-through from the vector's plasmid DNA we also
cloned the D-2 enhancers with canonical sites into the construct
pTKCAT containing two SV40 termination sequences in front
of the pBLCAT2 polylinker (26, 27). In these plasmids we also
replaced the long thymidine kinase (tk) promoter (reaching up
to -105) by a shorter tk promoter (reaching to -32).
Preparation of nuclear protein extracts and electrophoretic
mobility shift assays (EMSAs)
The crude nuclear protein extracts used in this study were
prepared according to a published protocol (28). In EMSA
experiments 4 /tg protein was incubated with 5000 cpm
(equivalent to about 0.2 ng) of a 32P-labelled oligonucleotide
probe and 2 ng poly dl • dC as unspecific competitor as described
(29). After incubation for 2 0 - 3 0 min on ice, the samples were
fractionated on nondenaturing 5% polyacrylamide gels at
200V/15 cm at room temperature followed by autoradiography
of the dried gel.
Xenopus Oocyte injections
The oocyte injections were done as described previously (30).
5 ng of 300 mM KCl-extracted SI00 proteins from resting splenic
T cells (see 30 for the extraction procedure) were injected into
the cytoplasm of oocytes (15 per group). 3 h later, 1 ng DNA
was injected into the nuclei of oocytes; i.e. a different DNA
construct per oocyte group. In parallel, batches of sibling oocytes
received the construct alone without T cell proteins, in order to
measure the constitutive transcriptional activity of 11-2
promoter/enhancer region in the oocytes. After an incubation for
16 h at 20°C, the oocytes were lysed and CAT assays were
performed as described (30).
RESULTS
The T cell-specific D-2 enhancer harbours numerous noncanonical, but functionally important binding motifs of
ubiquitous factors
The murine and human D-2 enhancers span about 275 bp which
harbour numerous binding sites for transacting factors (13,
23—25, 31—35). The sequences of these sites are highly
conserved between both enhancers. The most prominent factor
binding sites of the murine 11-2 enhancer are shown in Fig. 1.
These are the upstream promoter site, UPS, the two Purine boxes,
Pu-bp and Pu-bd, the proximal TPA responsive element, TREp,
and the distal T cell element, TCEd.
The UPS carries two closely linked non-canonical binding sites
for Octamer and AP-1 like factors (Fig. 1A and B). When one
or both sites are mutated and factors are unable to bind, the 11-2
enhancer activity decreases to about one third or less of its wild
type activity (I.Pfeuffer et al., submitted; see also 11). Although
in extracts of T and B lymphocytes the same proteins seem to
interact with UPS DNA, CAT constructs carrying five copies
of UPS DNA (5XUPS) were inactive in B cells but strongly
inducible in T cell lines, such as in murine E14 lymphoma cells
and human Jurkat lymphoma cells (24 and results not shown).
The TREp is a weak binding site of AP-1 or AP-1 like factors.
The affinity of those to TREp DNA is at least lOfold less than
to a canonical AP-1 site, such as the AP-1 site of human
collagenase gene (Fig. 1C). The weak binding of AP-1 to this
site is, however, of functional relevance because introducing a
point mutation (at position —152 of TREp) which abolishes AP-1
binding results in a distinct decrease of inducible enhancer activity
to about one fourth of the wild type level (S.Muller-Deubert et
al., submitted). Very similar results have been reported for the
TREp of the human 11-2 enhancer (36).
The kB-like element of the 11-2 enhancer which we originally
designated as TCEd (for distal T cell element, since its activity
behaves in a T cell-restricted manner: 13, 25) is a poor binding
site of authentic NF-kB. In nuclear protein extracts of E14 T
lymphoma cells we detected the binding of three factors to this
element, one of which, designated as TCF-1, appeared to contain
the p50 component of NF-kB (25). The introduction of point
mutations into the TCEd which abolished TCF-1 binding led to
the inactivity of the D-2 enhancer whereas the conversion of the
TCEd to a perfect NF-kB binding site resulted in a drastic increase
of enhancer activity in E14 lymphoma cells (Fig. ID) and to its
activity in HeLa cells (25).
The conversion of non-canonical binding sites to canonical
sites results in a drastic increase of 11-2 enhancer activity in
T cells and to its activity in non-T cells
In order to investigate whether the conversion of weak binding
sites for ubiquitous factors leads, as reported for the TCEd (25),
to an increase of enhancer activity in T cells and its activity in
non-T cells, we converted the TREp and Octamer-UPS sequences
to canonical sites in the context of the D-2 enhancer (Fig. 1 A).
Then we compared the protein binding of 'converted' sites with
that of well-characterized, canonical sites in electrophoretic
mobility shift assays (EMSAs) and tested the activity of
corresponding CAT constructs in transient transfection assays.
Furthermore, we constructed D-2 enhancers carrying two or three
of the 'perfect' mutations, the activity of which was tested in
the same way.
As shown in Fig. 2, in EMSAs the converted Octamer-UPS
(UPS0), TREp (153T-TREp) and TCEd (TCEd A/C) probes
generated the same complexes as canonical binding sites upon
incubation with nuclear protein extracts of E14 T lymphoma cells.
This implies that the converted sites are recognized by very
similar, if not the same factors as canonical binding site, i.e. the
UPSQ DNA by the Octamer factors Oct-1, Oct-2A and Oct-2B
(as well as AP-1: see below), the 153T-TREp by AP-1 and the
TCEd A/C site by NF-kB.
The conversion of binding sites had a dual effect on the activity
of D-2 enhancer in E14 cells. As shown in Fig. 3, the presence
of canonical binding sites within the enhancer resulted in an
increase of its constitutive and inducible activity. Approximately
the same effect was observed for enhancers carrying two or all
three mutations. The strong binding sites also induced activity
of the enhancer in B lymphocytes and in non-lymphoid cells, after
transfection of D-2 enhancer CAT constructs into 70Z pre-B cells,
A20J B cells, L cells and human HeLa cells. Although in the
two B cell lines the constructs with a converted enhancer showed
a high constitutive activity, the activity of several of the constructs
was also induced by TPA. This is particularly the case for
constructs carrying two or three canonical sites. Similarly, these
constructs were found to be induced in L cells and, using different
DNA amounts, in HeLa cells (Fig. 3).
Incubation of non-canonical sites in parallel with their
converted, canonical versions with nuclear protein extracts from
B cells, L cells and HeLa cells revealed in EMSAs mainly
quantitative differences in factor binding. This is true for all three
sites, although, as shown in Fig. 4, the affinity of Oct factors
to the UPS and UPSQ differed by a factor of two, whereas that
Nucleic Acids Research, Vol. 20, No. 11 2663
of AP-1 to the TREp and 153T-TREp sites by at least one order
of magnitude (see also Figs. IB-ID).
In order to test whether the observed increase of enhancer
activity is not only a particular feature of the enhancer but a
general property of D-2 regulatory region, we introduced the
canonical sites into larger promoter fragments, spanning about
0.5 and 2kB of upstream DNA. We and others observed a
negative effect of upstream DNA sequences on the constitutive
activity of enhancer (13, 37). Thus, one could expect that these
sequences also exert a negative influence on the enhanced,
'unbalanced' activity of enhancers carrying canonical binding
sites. However, in transient transfections, CAT constructs
carrying two or three canonical binding sites behave like their
corresponding enhancer CAT constructs (not shown). These
observations suggest that the upstream DNA sequences exert at
most a marginal effect on the stronger activity of enhancers
carrying canonical factor binding sites.
We also tested the activity of D-2 enhancer with canonical sites
after their cloning in CAT vectors carrying two SV40 poly A
addition signals (in front of the tk promoter) and a truncated
version of tk promoter. Using such vectors, we intent to avoid
read-through transcription starting within the plasmid DNA and
a possible cooperation of 11-2 enhancer factors with the factors
binding to the tk promoter. However, no distinct differences in
the CAT activities were observed between such D-2 CAT
constructs and those used in the former experiments (not shown).
An increase of FosB and Octamer factor concentrations leads
to an increase of D-2 enhancer activity in B cells
We were also interested to assess whether a higher concentration
of D-2 enhancer factors leads, like the higher affinity of factor
binding sites, to the activity of D-2 enhancer in non-T ceDs. For
this purpose, we co-transfected expression plasmids coding for
the AP-1 proteins JunB and FosB, Oct-1 or Oct-2A along with
wild type enhancer CAT constructs into 70Z and A20J B cells
(we chose JunB and FosB vectors because their expression
resulted in a strong transactivation of D-2 enhancer in E14 cells:
S.Miiller-Deubert et al., submitted). As shown in Fig. 5, the
expression of aU but the JunB coding plasmids had a stimulatory
effect on the D-2 enhancer activity in both ceD lines. The cotransfection with the Oct-2A vector led to a dramatic increase
of D-2 enhancer activity, an observation which supports
previously published data on the strong transactivating potency
of Oct-2A (38).
The strong binding of NF-kB and Octamer factors to the D-2
enhancer also overrides the inhibitory effect of repressor
proteins from primary T lymphocytes
Co-injections of D-2 promoter constructs with proteins from
resting T lymphocytes into Xenopus oocytes have revealed the
existence of repressor-like factors in resting, ex-vivo T cells.
These factors were found to act through the distal purine boxes,
Pu-bd. They are absent in unstimulated T cell lines and non-T
cells and seem, therefore, to be involved in the repression of
the D-2 enhancer in primary T lymphocytes (30; A.Mouzaki et
al., in prep.). These findings raise the question as to whether
the increase in D-2 enhancer activity, caused by the stronger
binding of ubiquitous factors to the D-2 enhancer, reflects the
artificial situation of cultured cell lines which lack the repressors
of primary T lymphocytes. The results of injections using D-2
CAT constructs with canonical binding sites show, however, that
the tighter binding of NF-kB to the D-2 enhancer is able to
A.
Protein
DNA
4
Fig. 6. The tight binding of NF-kB and Octamer factors to the 11-2 enhancer overrides the silencing activity of repressors in protein extracts from resting splenic
T lymphocytes. Xenopus oocyte injections. Oocytes were injected with 5 ng SESIOO proteins from resting splenic cells into their cytoplasm and, 3 h later, with
I ng DNA of the various CAT constructs (labelled by +). As controls (labeled by - ) , similar injections were carried out with sibling oocytes which received the
11-2 constructs alone (i.e. without the proteins from splenic T cells), to assess the basal activity of the 11-2 enhancer constructs in the oocytes. After an incubation
for 16 h the oocytes were lysed and their CAT activities determined as described (30). A. Autoradiography of a CAT experiment which shows that the tight binding
of NF-kB to the 11-2 enhancer carrying a NF-kB consensus sequence (NF-kB ( - 199C)) overrides the silencing activity of factors present in resting splenic T cells.
- , constitutive activity of 11-2 enhancer CAT constructs in Xenopus oocytes; +, the effect of proteins from resting splenic T cells (which were injected into Xenopus
oocytes prior the injections of DNA constructs) on the activity of constructs. B. The effect of stronger binding of NF-kB, AP-1 and Octamer factors on the silencing
effect of factors from resting splenic T cells. Shown are the mean values of injections into two different batches of oocytes.
2664 Nucleic Acids Research, Vol. 20, No. 11
substantially override the silencing effect of repressors from
primary T lymphocytes. The tighter binding of Octamer factors
was less effective in overcoming the silencing effect of repressors,
while no effect was observed for the 153T-TREp construct
carrying a converted AP-1 binding site (Fig. 6).
DISCUSSION
The results of numerous transient transfection studies have shown
that the enhancers of murine and human D-2 genes span about
275 bp DNA which harbour the most important DNA sequence
motifs for the establishment of inducible and T cell-restricted
transcription of the 11-2 gene (13, 31-35). Moreover, in
transgenic mice the D-2 enhancer (plus about 250 bp upstream
DNA) appeared to control the expression of a linked indicator
gene in a T cell-specific manner, i.e. the activity of the indicator
gene has only been detected in activated T cells (39). Taken
together, these results show that the T cell-specific transcription
of the D-2 gene is controlled by the transacting factors binding
to this immediate upstream (enhancer) region of about 300 bp.
The D-2 enhancer is recognized by a multiplicity of both
positively and negatively acting transcription factors. Among the
positively acting factors are several ubiquitously distributed
factors, such as Oct-1, AP-1 and NF-kB (10, 11, 13,36,40-42),
but also lymphoid-specific factors, such as Oct-2 (10), NFAT-1
(23, 43) and additional factors binding to the kB-like (TCEd)
motif of the enhancer (25). Among the negatively factors are
factors interacting with the Purine boxes, i.e. the binding sites
of NFAT-1 (30), and a Z n + + finger containing factor which has
recently been described to interact with DNA sequences located
just upstream of the UPS motif (44). Thus, it appears likely that
the T lymphocyte-restricted, inducible and transient expression
of the D-2 gene is mediated at the level of transcription by a finetuned interplay of positively and negatively acting protein factors
binding to the enhancer DNA. When the equilibrium of factors
binding to and transactivating the D-2 enhancer is changed the
proper regulation of enhancer activity becomes impaired. Such
changes can be brought about in several ways, such as by changes
of concentration or affinity of factors binding to enhancer DNA.
Both situations were artificially created in experiments in this
study. By co-transfections of Oct and FosB expression plasmids
into B cell lines the activity of the D-2 enhancer could be induced
(Fig. 5). A similar effect was observed when the binding affinity
of three poor binding sites for ubiquitous factors was enhanced
by the conversion of non-canonical binding sites to canonical
ones. This was shown for a kB-like sequence element (the TCEd), an AP-1 site (the TREp) and an Octamer binding site (the
UPS).
Not in every case did the conversion of a non-canonical binding
site for ubiquitous factors to a canonical site lead to an increase
of D-2 enhancer activity. One exception was the non-canonical
UPS-TRE sequence (Fig. 1A). When this site was converted
within the enhancer to a perfect TRE by the introduction of two
point mutations (as shown in Fig. 1A) and tested in transient
transfections, the activity of the D-2 enhancer remained the same
as the wild type enhancer or was even reduced in its inducible
activity in E14 cells (I.Pfeuffer et al., submitted). The reduction
of D-2 enhancer activity is probably due to a weaker binding of
Oct factors to the converted UPS DNA. We will show elsewhere
that the Oct factors not only contact the nucleotides within the
Oct-like motif ATGTAAAA of UPS DNA but also several
nucleotides of flanking DNA, including a T residue which is
located at the opposite DNA strand and complementary to the
underlined A of the TRE-like sequence TGTGTAA at position
- 8 2 of enhancer DNA. When the UPS-TRE was converted to
a canonical TRE the A/T base pair was substituted by a C/G
base pair, which is not longer contacted by the Oct factors but
by AP-1. Thus, in this case the conversion of a non-canonical
AP-1 binding site does not only result in a stronger binding of
AP-1 but, in addition, to a decrease of the binding of Octamer
factors and 11-2 enhancer activity (I.Pfeuffer et al., submitted).
A dual effect on the factor binding has also been observed when
the kB-like TCEd sequence was converted to a canonical NFkB binding site (25). In crude nuclear extracts of induced E14
cells (and of other cells) NF-kB binds much more strongly to
a 'converted' TCEd carrying a canonical NF-kB site than to the
wild type TCEd (Fig. ID). In contrast, in enriched preparations
of TCEd factors from E14 cells two DNA binding factors,
designated as TCF-2 and TCF-3, were unable to bind to a NFkB site but specifically bound to TCEd DNA (25). A third, kBlike factor, designated as TCF-1, was able to bind to both
sequences. Since the binding of TCF-2 and TCF-3 to a DNA
sequence element was found to correlate with the loss of its
activity, we concluded that both factors might exert a repressorlike function (25). Thus, in the case of the TCEd motif, its
conversion to a canonical NF-kB site seems to result not only
in a tighter binding of a positively acting factor, but also in the
loss of binding of repressor-like factors.
We believe that the phenomenon described here for the murine
D-2 enhancer, i.e. the dramatic increase of its activity by one
point mutation at a non-canonical binding site for transacting
factors, might play an important role in the activation of nuclear
oncogenes, which code for transcription factors (see 45 for a
recent review). The occurrence of non-canonical binding sites
for ubiquitous factors is no special peculiarity of the D-2 enhancer.
Instead, such sites exist within the promoters and enhancers of
many eukaryotic genes (see the Refs. 5 and 46 for compilations
on the occurence of non-canonical Octamer and NF-kB sites,
respectively) and their conversion to canonical sites might also
lead to a similar enhanced activity as we observed for the D-2
enhancer. If one anticipates that such 'up' mutations occur within
the promoters of (nuclear) proto-oncogenes during the
differentiation of cells, cells will appear which contain a much
higher level of a certain transcription factor than normal cells.
The overproduction of such important factor proteins as Fos, Myc
etc. can lead to the activation (or repression) of novel sets of
genes and, in combination with other events, to profound changes
in cell growth and differentiation and, finally, in carcinogenesis.
ACKNOWLEDGEMENTS
For excellent technical assistance we wish to thank Elke Schorr.
We are indebted to Dr. Catherine Phillips for critical reading
the manuscript. For gifts of DNA constructs we thank Drs.
P.Angel (Karlsruhe), Iris Kemler (Zurich), P.Matthias (Basel),
W.Schaffner (Zurich) and M.Schuermann (Marburg). This work
was supported by grants of the Deutsche Forschungsgemeinschaft
(DFG), SFB 165 (University Wurzburg), the Bundesgesundheitsamt, research programme AIDS, and the Swiss National
Foundation grant 3.12 86 54.90.
Nucleic Acids Research, Vol. 20, No. 11 2665
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