From the
Previously we determined that the immunoglobulin kappa 3`
enhancer (
Immunoglobulin
An interesting feature of
Although various agents can modulate
Confirmatory results were obtained when these
mutant oligonucleotides were used as unlabeled competitors ( lanes
10-15). Mutants M.1 and M.6 completely abolished factor
binding to the wild type probe ( lanes 10 and 15).
Mutant M.2 competed complexes 1 and 2 but not 3 ( lane 11),
whereas mutant M.3 failed to compete any of the complexes ( lane
12). Finally, mutants M.4 and M.5 competed complex 3 but not
complexes 1 and 2 ( lanes 13 and 14). These results
suggest that the factor responsible for complex 3 binds between
nucleotides 404 and 411, and the factors responsible for complexes 1
and 2 bind between nucleotides 404 and 419.
Dimethyl sulfate
methylation interference assays were performed to define more narrowly
the protein-DNA contact sites (Fig. 5 A). Footprints were
generated with complexes 1 and 2, but unfortunately complex 3 did not
yield sufficient material for methylation interference analysis.
Studies with the upper DNA strand indicated that for complex 1,
methylation of guanine residues 407 and 413 interfered with binding. On
the bottom DNA strand methylation of guanines 409, 414, 417, and 418
inhibited complex 1 formation. Complex 2 exhibited partial contacts at
guanines 409 and 414 in the lower strand and showed contacts at
guanines 407 and 413 in the upper strand (Fig. 5 A). A
summary of the protein DNA interaction studies is presented in
Fig. 5B. Similar protein-DNA contact sites were observed
by in vivo footprinting of the human
In earlier studies, we identified two functional sequences
(PU.1/NF-EM5 and E2A) within the
We chose to focus our efforts on the linker scan B
mutation. This mutation reduced activity to near background levels when
assayed in the pre-B cell line 1-8 but reduced activity to only 30% in
S194 plasmacytoma cells. These results suggested a possible
developmental difference in the requirement for these sequences. This
functional region of the mouse
Our antibody and
mixing experiments (Figs. 6 and 7) indicate ATF-1 and CREM (or closely
related proteins) contribute to the formation of the protein-DNA
complexes observed with oligonucleotide 2. Rather than a supershift in
the EMSAs, new higher mobility complexes were observed with anti-ATF-1
and anti-CREM antibodies. The CREM antibody was prepared against
protein sequences that include the CREM DNA binding domain. Therefore,
this antibody most likely inhibits CREM DNA binding. The ATF-1
antibodies, however, were raised against the amino-terminal 30 amino
acids of ATF-1, a region distinct from the DNA binding domain.
Therefore, the formation of higher mobility complexes and the lack of a
supershift are somewhat surprising. One possible explanation is that
ATF-1 must interact with other proteins to bind efficiently to the
enhancer DNA sequences. Indeed, our mixing experiments (Fig. 7)
suggest that this is the case. The anti-ATF-1 antibodies may block the
ability of ATF-1 to interact with these proteins and therefore inhibit
ATF-1 from binding efficiently to enhancer DNA sequences.
It is
interesting that cotransfection of an ATF-1 expression plasmid
increased
This issue is complicated
further by the fact that the CREM gene can produce either activator or
repressor proteins depending upon its mode of RNA processing, selection
of transcription initiation site, or translation initiation site (see
below). The initially identified isoforms, CREM-
Our preliminary RNA analyses suggest that the CREM gene
is expressed differentially in the B cell lineage. We performed reverse
transcriptase PCR studies with RNA isolated from pre-B or plasmacytoma
cells and found that pre-B and plasmacytoma cells share some CREM
products but also express at least one distinct variant
each.
Our RNA studies
also indicated that ATF-1 RNA levels increase during B cell
development. ATF-1 was previously isolated by three independent groups
based on its ability to bind to the DNA sequence CGTCA (ATF/CRE site)
in a variety of viral and cellular genes
(27, 28, 37) . ATF-1 is also important for
Tax-mediated transactivation of the human T-cell lymphotropic virus
type I long terminal repeat
(38) . The activation or DNA binding
properties of ATF-1 can be modulated by heterodimerization with other
leucine zipper proteins (see for example, 39). Therefore, the ability
of ATF-1 to augment
The functional synergism between
the
-We thank our colleagues for critically reading
the manuscript and for helpful discussions. We wish to thank G. S.
McKnight for providing the expression plasmid for the catalytic domain
of protein kinase A, M. Yoshida for providing the full-length Treb36
(ATF-1) expression plasmid, P. Sassone-Corsi for providing CREM-
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
E3`) contains at least two functional DNA sequences
(PU.1/NF-EM5 and E2A) within its 132-base pair active core. We have
determined that the activities of these two sequences are insufficient
to account for the entire activity of the 132-base pair core. Using
site-directed linker scan mutagenesis across the core fragment we
identified several additional functional sequences. We used one of
these functional sequences to screen a
gt11 cDNA expression
library resulting in the isolation of cDNA clones encoding the
transcription factors ATF-1 (activating transcription factor) and CREM
(cyclic AMP response element modulator). Because ATF-1 and CREM are
known to bind to cAMP response elements (CRE), this functional sequence
was named the
E3`-CRE. We show that dibutyryl cAMP can increase
E3` enhancer activity, and in transient expression assays ATF-1
caused a 4-5-fold increase in the activity of the core enhancer
while CREM-
expression resulted in repression of enhancer
activity. RNA analyses showed increased levels of ATF-1 mRNA during B
cell development and some changes in CREM transcript processing. By
joining various fragments of the
E3` enhancer to the
E3`-CRE,
we observed that the
E3`-CRE can synergistically increase
transcription in association with the PU.1/NF-EM5 binding sites,
suggesting a functional interaction between the proteins that bind to
these DNA sequences. Consistent with this possibility, we found that
ATF-1 and CREM can physically interact with PU.1. The isolation of
activator and repressor proteins that bind to the
E3`-CRE may
relate to previous conflicting results concerning the role of the cAMP
signal transduction pathway in
gene transcription.
light chain gene expression is under the
control of two enhancers: the intron and the
E3` enhancers. Both
the intron and the 3` enhancers are inactive in Abelson
virus-transformed pre-B cell lines in which the
locus is silent.
However, both enhancers are active at the B cell and plasma cell
stages. Previously we determined that a 132-bp
(
)
centrally located segment within the
E3` enhancer (the
enhancer core) contains nearly the entire enhancer activity when
assayed in plasmacytoma cells
(1) . This same core segment is
also active in pre-B cells, indicating the existence of flanking
negative acting DNA sequences that repress enhancer activity at the
pre-B cell stage
(2) . Within the enhancer core we previously
identified two functional DNA sequences. One sequence binds to the
transcription factors PU.1 and NF-EM5, and the other binds to the
transcription factor E2A
(1, 2, 4) . The
PU.1/NF-EM5 and E2A functional sequences were identified based on their
enhancer activity when assayed as multimers
(1) . Other
functional sequences may exist in the core segment which are not active
as multimers. These functional sequences may be revealed by a linker
scan mutagenesis approach.
gene
transcription is its inducibility at the pre-B cell stage. Although the
locus is transcriptionally silent in Abelson virus-transformed
pre-B cells, certain agents can induce
transcription. These
agents include bacterial lipopolysaccharide, interleukin-1, and
-interferon
(5, 6, 7, 8, 9, 10) .
Induction of
transcription in some cases appears to require
induction of the functional form of NF-
B
(8, 11, 12, 13, 14) . For
instance, lipopolysaccharide treatment is believed to cause
phosphorylation of I
B resulting in its dissociation from
NF-
B. NF-
B can then enter the nucleus where it can bind to
the intron enhancer and initiate expression of the
gene. Like the
intron enhancer the
E3` enhancer becomes active at the pre-B cell
stage when cells are treated with lipopolysaccharide. However, the
E3` enhancer does not require NF-
B for its activity
(1, 15) .
locus expression, conflicting results have been reported. For instance,
one report suggests that interleukin-1 and cAMP can increase
transcription, whereas others suggest an inhibitory effect by the same
compounds
(7, 9, 16) . This issue may be
clarified by identifying the nuclear factors that control
locus
transcription. In the studies presented here, we used linker scan
mutagenesis to identify additional functional sequences within the
E3` enhancer core. We show that one functional sequence binds to
the leucine zipper transcription factors ATF-1 and CREM. Because these
factors bind to cAMP response elements (CRE) we named this functional
segment of the
E3` enhancer the
E3`-CRE. We show that cAMP
levels can increase
E3` enhancer activity, and ATF-1 can activate
the
E3` enhancer while CREM-
can act as a repressor. We also
show that the
E3`-CRE can synergistically activate transcription
in association with the PU.1/NF-EM5 binding sites. In addition, ATF-1
and CREM can physically interact with PU.1. These factors may provide a
mechanism for the induction or repression of
gene transcription
in response to various agents.
Plasmid Constructs
To prepare plasmid
E3`coreTKCAT, the 132-bp AvaII- ApaLI core
fragment of 3` enhancer was blunted by Klenow polymerase and cloned
into the HincII site of pUC18. This fragment was excised by
HindIII- BamHI digestion and ligated into the
HindIII- BamHI sites of TKCAT containing sequences
109 to +51 of the herpesvirus thymidine kinase (TK)
promoter linked to the bacterial chloramphenicol acetyltransferase
(CAT) gene. PU.1+E2ATKCAT was prepared by cloning the E2A sequence
with BamHI- BglII restriction sites at the
BglII site adjacent to the PU.1/NF-EM5 sequence in a vector
containing the liver, bone, kidney (LBK) alkaline phosphatase promoter
(LBK
44 vector kindly supplied by T. Kadesch, University of
Pennsylvania). The PU.1/NF-EM5 and E2A sequence was excised by
BamHI- BglII digestion and ligated into the
BamHI site of TKCAT. Single copies of the PU.1/NF-EM5 and E2A
sequences with BamHI and BglII ends were cloned into
the BamHI site of TKCAT to yield constructs PU.1TKCAT and
E2ATKCAT, respectively. Oligonucleotides containing the
E3`-CRE+PU.1 or
E3`-CRE+E2A sites were synthesized
with BamHI and HindIII ends and cloned into the
BamHI- HindIII sites of TKCAT to yield constructs
E3`-CRE+PU.1TKCAT and
E3`-CRE+E2ATKCAT,
respectively. Linker scan mutants across the core fragment (A to M;
except B) were created by a polymerase chain reaction (PCR) method
(17) . For each mutation, divergent oligonucleotide primers with
10-bp overlaps (containing a new SalI restriction site) were
used. The sequences of the mutant oligonucleotides used for generating
the respective mutations are as follows. Linker scan mutant B was created by the method described by Kunkel
(18) using a 40-bp synthetic oligonucleotide containing the
mutant B sequences (5`-GTGTGACGGTAGCTAGCGTCGACCGTATCTTGGTCCATGG-3`).
All mutations (A to M) were confirmed by DNA sequencing
(19) .
The PU.1-GST construct was prepared by inserting a
PvuII- BamHI fragment containing the entire PU.1
coding sequence, 4 bp of 5`-untranslated sequence, and 337 bp of
3`-untranslated sequence by blunt end ligation in the blunted
EcoRI site of pGEX-2TK (kindly provided by W. Kaelin, Dana
Farber Cancer Institute).
Cell Culture and Transfection
S194 and
1-8 cells were grown and transfected as described previously
(1) . Transfections generally contained 4 µg of test plasmid
and 1 µg of the -galactosidase expression plasmid pCH110
(20) to normalize the transfection efficiency. For
cotransfection experiments, 3-4 µg of reporter plasmid was
cotransfected with various amounts of expression plasmid. The total
concentration of DNA in each transfection was normalized with pUC18
plasmid DNA. Some transfected cells were incubated with
Bt
cAMP (Sigma) for 16 h before harvest. Cell extracts were
prepared by freeze-thawing and CAT assays and thin-layer chromatography
were performed according to Gorman et al. (21) .
Electrophoretic Mobility Shift Assay (EMSA) and
Methylation Interference Assay
Nuclear extracts were
prepared by the method of Dignam et al. (22) , and EMSA
was performed as described previously
(1) . Antibody treatments
were at room temperature for 20 min before addition of the probe. CREM
antibodies were a gift from P. Sassone-Corsi. ATF-1 antibodies were
obtained from Upstate Biotechnology Inc. CREB and c-fos antibodies were
obtained from Santa Cruz Biotechnology Inc. For methylation
interference assays, the E3`-CRE sequence with BamHI and
BglII ends was cloned into the BamHI- BglII
sites of the LBK
44 vector. This plasmid was cut with either
BamHI or BglII, dephosphorylated with calf intestine
phosphatase, labeled with [
-
P]ATP by
polynucleotide kinase, then digested with the appropriate second enzyme
to release the oligonucleotide insert. Labeled DNA fragments were
purified by polyacrylamide gel electrophoresis. DNA fragments were
processed for dimethyl sulfate methylation interference footprinting
with S194 nuclear extract as described previously
(23) .
Proteins made by in vitro translation were prepared from RNAs
transcribed in vitro from full-length cDNAs with T7 RNA
polymerase according to the manufacturer's specifications
(Stratagene), and proteins were translated in vitro using
nuclease-treated RNA-dependent rabbit reticulocyte lysates (Promega) at
30 °C for 60 min.
Screening the
A human
HeLa cell-derived gt11 Library
gt11 cDNA library (provided by P. Henthorn,
University of Pennsylvania) was screened with a multimerized
E3`-CRE probe according to the method of Vinson et al. (24) . The nucleotide sequence of the
E3`-CRE probe is
5`-AGCAACTGTCAATAGCTACCGTCACA-3` (upper strand);
3`-TGTGACGGTAGCTATTGACAGTTGCT-5` (lower strand).
RNA Analyses
Poly(A)RNAs isolated from 3-1, 1-8, MPC11, or S194 cells were
fractionated on 2.0% agarose-formaldehyde gels and transferred to
Nytran (Schleicher & Schuell) according to the manufacturer's
specifications. For reverse transcriptase PCR assays 1 µg of
poly(A)
RNA isolated from MPC11 plasmacytoma cells was
copied into cDNA using primer B (5`-CATGCTGTAATCAGTTCATAG-3`) and
reverse transcriptase in a 20-µl reaction containing 50 mM
Tris-HCl (pH 8.3), 150 mM KCl, 10 mM
MgCl
, 15 mM dithiothreitol, and 0.5 mM
dNTP at 42 °C for 1 h. Forty cycles of PCR were then performed with
primer B and primer A (5`-GATTGAAGAAGAAAAATCAGA-3`) using the following
cycle: 55 °C, 1 min; 72 °C, 2 min; 94 °C, 30 s. PCR
products were cloned into plasmid pGEM-T (Promega) for DNA sequence
analysis.
Preparation of GST-PU.1 Fusion
Protein
The GST-PU.1 fusion protein was expressed in
Escherichia coli BL21 (DE3) by induction with 1 mM
isopropyl 1-thio--D-galactopyranoside (Sigma) for 2 h.
Cells were harvested by centrifugation, resuspended in buffer
containing 20 mM Tris (pH 8.0), 100 mM NaCl, 1
mM EDTA, and 0.5% Nonidet P-40 (NETN buffer), and lysed by
ultrasonication with 30-s pulses four times. Debris was pelleted by
centrifugation (10,000 rpm, 30 min). The supernatant was mixed with
glutathione-agarose (Sigma) and rocked at 4 °C for 1 h. Beads were
then washed with NETN buffer to remove unbound proteins. An aliquot of
GST-PU.1 linked to glutathione-agarose was assayed by
SDS-polyacrylamide gel electrophoresis.
GST-PU.1 Affinity Chromatography
ATF-1
and CREM- proteins were prepared by in vitro transcription and translation in the presence of
[
S]methionine. The labeled ATF-1 and CREM-
proteins were incubated with 20 µl of glutathione-agarose beads
containing PU.1-GST or glutathione S-transferase alone for 1 h
at 4 °C. After incubation, the beads were washed three times with
NETN buffer, resuspended in SDS-polyacrylamide gel electrophoresis
buffer, boiled, and analyzed by 10% SDS-polyacrylamide gel
electrophoresis followed by autoradiography.
The PU.1/NF-EM5 and E2A Binding Sites Do Not
Account for All of the Core Enhancer Activity
Previously,
we determined that the sequences spanning nucleotide positions
391-523 of the 3` enhancer (numbering according to Ref. 25)
contain most of the activity of the intact 1.1-kilobase enhancer
(1) . Using nine multimerized 25-bp overlapping oligonucleotides
spanning this core fragment we localized two functional regions. The
first region binds to the transcription factors PU.1 and NF-EM5, and
the second region binds to transcription factor E2A
(1, 3, 4) . To test whether these two DNA
segments account for the entire activity of the core fragment, we
linked single copies of the PU.1/NF-EM5 plus E2A binding sites into a
vector containing the thymidine kinase promoter driving expression of
the CAT gene. Similarly, TKCAT constructs containing a single copy of
either the PU.1/NF-EM5 or E2A binding sites were prepared. These
constructs were transfected into S194 plasmacytoma cells, and CAT
activities were measured. No significant CAT activity was observed with
constructs containing single copies of either the PU.1/NF-EM5 or E2A
DNA binding sites (Fig. 1). However, the construct containing
both the PU.1/NF-EM5 and E2A binding sites exhibited 21% activity when
compared with the activity of the entire core fragment. These results
indicate that the PU.1/NF-EM5 and E2A binding sites can functionally
synergize to yield enhancer activity. However, other DNA elements must
be present in the core fragment which are necessary to yield 100%
enhancer activity.
Figure 1:
The PU.1/NF-EM5 and E2A
binding sites do not account for all of the E3` enhancer activity.
S194 cells were transfected with 4 µg of each DNA construct, and
CAT activities were determined. The results of representative
transfections are shown. Comparable results were obtained in at least
two to three independent experiments. Above each lane is
listed the transfected DNA construct, and the percent enhancer activity
is indicated below each lane.
Linker Scan Mutations across the Core
Fragment
To identify additional regulatory elements that
are important for activity of the E3` enhancer, we prepared a
series of 10-bp linker scan mutants (A to M; see Fig. 2) across
the 132-bp core fragment. These constructs were individually
transfected into 1-8 cells (a pre-B cell line) or S194 plasmacytoma
cells, and CAT activities were determined (Fig. 3, A and
B, respectively). It should be noted that although the entire
E3` enhancer is inactive in pre-B cells, the 132-bp core fragment
is active.
Figure 2:
Linker scan mutations within the E3`
enhancer core segment. The DNA sequences of the 10-bp linker scan
mutants (A to M) across the core fragment of the
E3` enhancer are
shown. The wild type sequence is shown on the top line.
Dashes represent nucleotide identities. Positions of the
E3`-CRE, PU.1, NF-EM5, and E2A sequences are
indicated.
Figure 3:
Identification of new functional sequences
in the E3` enhancer. 4 µg of each mutant construct was
individually transfected into either panel A, 1-8 pre-B cells
or panel B, S194 plasmacytoma cells, and CAT activities were
determined. Below each lane is indicated the linker scan
mutant used for transfection. CORE is the 132-bp
E3`
enhancer core cloned into TKCAT. Panel C, the relative
activities of the linker scan mutants in 1-8 pre-B cells ( solid
bars) and S194 plasmacytoma cells ( open bars) are
plotted. The activity of the wild type core fragment was considered as
100%, and the activity of each mutant construct is calculated relative
to this value. Data are from an average of two to three transfections.
The relative activity for each construct varied by less than
10%.
Consistent with our previous results, mutation of the DNA
sequences spanning the PU.1/NF-EM5 (mutations F, G, and H) and E2A
(mutations I and J) binding sites greatly reduced enhancer activity in
1-8 and S194 cells (Fig. 3, A and B). Linker
scan K also reduced enhancer activity in both cell types, but as yet no
protein binding to this DNA sequence has been identified
(1) .
In addition, linker scans A, B, and D showed reduced enhancer activity.
Linker scan B is particularly interesting because it reduced enhancer
activity to near background levels in 1-8 pre-B cells but only reduced
activity to 30% of wild type in S194 plasmacytoma cells
(Fig. 3 C). We demonstrated previously that multimerized
oligonucleotides containing wild type sequences of mutants A and B
(oligonucleotide 1), mutants B and C (oligonucleotide 2), and mutants D
and E (oligonucleotide 3) are inactive as enhancers when transfected
into S194 plasmacytoma cells
(1) . Therefore, these DNA segments
cannot exhibit enhancer activity on their own and must functionally
interact with other enhancer segments to be active. Our results here
with the mouse E3` enhancer are consistent with a mutational
analysis of the human 3` enhancer
(26) . These studies showed
that substitution of nucleotides in the human enhancer at sites similar
to linker scans B and D reduced enhancer activity to 21 and 19%,
respectively, in human Burkitt lymphoma cells
(26) .
Identification of Protein-DNA Contact
Sites
Previously we showed that oligonucleotide 2, which
contains the entire wild type linker scan B and C sequences and 4 bp of
linker scan D sequence (nucleotides 400-424), binds to factors
present in nuclear extracts of pre-B, plasmacytoma, and 3T3 fibroblast
cells
(1) . These factors result in the appearance of three
shifted complexes in EMSAs: a slowly migrating doublet (complexes 1 and
2), and a third faster migrating complex (complex 3; Fig. 4). All
bound complexes were efficiently competed by adding excess unlabeled
self-oligonucleotide (Fig. 4; lane 2) but not by a
nonspecific oligonucleotide ( lane 3).
Figure 4:
Protein
binding ability of wild type and mutant DNA probes. Various 4-bp mutant
oligonucleotides (M.1 through M.6) were labeled with P,
and EMSAs (with low ionic strength buffer) were performed using 3-1
pre-B cell nuclear extract. Parallel reactions were carried out with
the wild type oligonucleotide 2 sequence (see Fig. 5 B) as
probe in the presence of unlabeled mutant oligonucleotide competitors
(M.1-M.6), self-oligonucleotide ( SELF), or a nonspecific
oligonucleotide ( NS). DNA-protein complexes were resolved by
4% nondenaturing gel electrophoresis. Above each lane is
indicated the labeled probe and unlabeled competitor oligonucleotides.
Arrows point to complexes 1, 2, and 3. The wild type and
mutated oligonucleotide sequences are shown in the lower
panel.
To identify the
nucleotides responsible for binding to nuclear factors we prepared
consecutive 4-bp mutations across the 25-bp oligonucleotide 2 sequence
to yield six mutant oligonucleotides (M.1-M.6). Each mutant
oligonucleotide was used as a labeled probe in EMSA (Fig. 4,
lanes 4-9). Mutants M.1 and M.6 exhibited wild type
binding patterns ( lanes 4 and 9). Mutant M.2 showed
weak binding of complexes 1 and 2 but undetectable binding of complex 3
( lane 5; a faint, faster migrating complex was observed with
this probe as well as mutant M.3). Mutant M.3 was unable to yield any
of the specific complexes ( lane 6). Mutants M.4 and M.5
completely abolished binding of complexes 1 and 2 but exhibited
increased binding to the factor responsible for complex 3, perhaps
because of the increased amount of probe available ( lanes 7 and 8).
light chain 3`
enhancer (Fig. 5 C; 27).
Figure 5:
Identification of protein-DNA contact
sites. Panel A, the 25-bp oligonucleotide 2 sequence (see
panel B) was used in dimethyl sulfate methylation interference
assays. Lanes containing free ( F), complex 1
( 1), or complex 2 ( 2) are indicated. The positions of
the G residues that inhibited protein binding when methylated are
indicated by arrows. Panel B, summary of the
footprint and EMSAs. The oligonucleotide 2 sequence (nucleotides
400-424) is shown at the top, and the positions of
linker scan mutants B, C, and D are indicated. Asterisks show
protein contact sites identified by dimethyl sulfate methylation
interference assays. Below are shown the CRE-like motifs in the
sequence and the sequences of mutants M.1-M.6. The binding
activity of each mutant as measured by EMSA is summarized in the
lower right. Panel C, the protein contact sites of
the mouse enhancer are compared with the contact sites of the human 3`
enhancer determined by in vivo footprinting
(27).
The above analyses indicate
that protein contact sites within the oligonucleotide 2 sequence extend
across the linker scan B and C sequences. EMSA complexes 1 and 2 result
from factors that bind to sequences spanning linker scans B and C. On
the contrary, EMSA complex 3 results from factors binding to the linker
scan B region.
Isolation of cDNA Clones Encoding Proteins That Bind
to Oligonucleotide 2
A gt11 cDNA expression library
was screened with oligonucleotide 2 sequences (nucleotides
400-424) by the method of Vinson et al. (24) .
After several rounds of screening, two strong positive cDNA clones were
isolated. The DNA sequences of these clones indicated that they are
identical to ATF-1 (27; identical to Tax response element binding
protein, TREB36; 28) and CREM
(29) . These proteins are known to
bind to CREs. Inspection of the oligonucleotide 2 DNA sequence revealed
two regions with homology to CRE-like sites (30; see
Fig. 5B). We therefore named this segment of the
3` enhancer the
E3`-CRE.
Binding of CREM and ATF-1 to the
EMSA with oligonucleotide 2 DNA sequences and CREM
or ATF-1 protein prepared by in vitro transcription and
translation yielded inconsistent results. Faint high mobility complexes
were observed occasionally, suggesting that CREM and ATF-1 bind poorly
on their own but may bind more efficiently in the presence of other
nuclear proteins. To determine whether CREM or ATF-1 proteins were part
of the EMSA complexes observed with oligonucleotide 2 sequences and
nuclear extract proteins, we performed EMSAs with CREM and ATF-1
antibodies. We also included experiments with antibodies to two other
proteins, CREB and c-fos, which bind to CRE-like sequences. Whereas
preimmune, CREB, and c-fos antibodies had no effect on complexes 1, 2,
and 3 (Fig. 6, lanes 2, 5, and 6),
ATF-1 and CREM antibodies caused a loss of complexes 1 and 2 and the
appearance of higher mobility complexes ( lanes 3 and
4). The above antibodies had no effect on the DNA binding of a
distinct transcription factor, YY1 (data not shown). Therefore, ATF-1
and CREM, or closely related proteins, are associated with the
formation of complexes 1 and 2.
E3`
Enhancer
Figure 6:
ATF-1
and CREM bind to the E3`-CRE. Radiolabeled
E3`-CRE
oligonucleotide (oligonucleotide 2) was incubated with S194 nuclear
extract proteins. Samples were either untreated ( lane 1),
treated with preimmune sera ( lane 2), or treated with
antibodies against CREM ( lane 3), ATF-1 ( lane 4),
c-fos ( lane 5), or CREB ( lane
6).
We also performed mixing experiments
with ATF-1 and CREM and nuclear extract proteins to determine the
influence of ATF-1 and CREM on protein-DNA complex formation. For these
experiments, various amounts of nuclear extract proteins were assayed
alone or in combination with ATF-1 or CREM using the labeled
E3`-CRE as probe. Mixing of 2 µl of ATF-1 prepared by in
vitro translation with nuclear extract proteins caused a
significant increase in the intensity of all bound complexes. Adding
control rabbit reticulocyte lysate to the nuclear extract did not
change the binding pattern (Fig. 7), nor did addition of CREM
prepared by in vitro translation (data not shown). The above
antibody and mixing experiments suggest that ATF-1 and CREM bind to DNA
as a complex with other nuclear factors.
Figure 7:
ATF-1 enhances the binding ability of
nuclear factors to the E3`-CRE. Various amounts of nuclear extract
proteins (from 3-1 pre-B cells) were assayed alone, in the presence of
2 µl of ATF-1 prepared by in vitro translation, or in the
presence of control translation lysate ( RRL). The amount of
nuclear extract protein and the presence of ATF-1 or control lysate
( RRL) are indicated above each lane. Arrows indicate the positions of complexes 1, 2, and
3.
cAMP Levels Can Influence
Because CREM and ATF-1 are associated with the
cAMP signal transduction pathway, we sought to determine whether
increased cAMP levels could induce E3` Enhancer
Activity
E3` enhancer activity in pre-B
cells. One or two copies of the entire
E3` enhancer were cloned
into the TKCAT reporter plasmid. 1-8 pre-B cells were transfected with
each construct and either left untreated or treated with
Bt
cAMP (0.5 or 1 mM). Activity of the single copy
enhancer reporter was induced 3-fold by Bt
cAMP treatment
(Fig. 8). Activity of the double enhancer reporter was induced
11-fold by the same treatment (Fig. 8). Therefore, increased cAMP
levels can increase the activity of the
E3` enhancer in pre-B
cells.
Figure 8:
cAMP can
induce activity of the E3` enhancer in pre-B cells. 1-8 pre-B
cells were transfected with TKCAT constructs containing either a single
or double copy of the
E3` enhancer. After transfection,
Bt
cAMP was added to a final concentration of either 0.5 or
1.0 mM.
Effect of ATF-1 and CREM on
To test the influence of ATF-1 on the activity of
the E3` Enhancer
Activity
E3` enhancer, an ATF-1 expression plasmid was cotransfected
with
E3`coreTKCAT. A plasmid expressing the catalytic subunit of
protein kinase A was also included
(31) because ATF-1 function
is activated by protein kinase A. Transfections were performed in 1-8
pre-B and S194 plasmacytoma cells. Expression of ATF-1 at various
concentrations in 1-8 pre-B cells stimulated enhancer activity up to
5-fold (Fig. 9 A). In S194 plasmacytoma cells ATF-1
expression caused a 2-fold increase in enhancer activity (data not
shown). However, overexpression of ATF-1 in pre-B cells did not
activate the intact, normally silent
E3` enhancer (data not
shown). Therefore, reduced ATF-1 levels alone cannot be responsible for
the lack of
E3` enhancer activity at the pre-B cell stage.
Interestingly, activation of the core enhancer fragment was observed at
very low doses (0.5-2 ng) of ATF-1 expression plasmid. At higher
concentrations, ATF-1 caused repression of the core enhancer in both
cell types. Repression of enhancer activity at high ATF-1 doses
suggests squelching of interacting proteins.
Figure 9:
ATF-1 activates and CREM- represses
activity of the
E3` enhancer. Panel A, a representative
CAT assay is shown of 1-8 pre-B cells cotransfected with a reporter
plasmid containing the core fragment of the
E3` enhancer ( 132
CORE) along with the expression plasmids encoding ATF-1 or protein
kinase A ( PKA). Cells were transfected with 3 µg of
reporter plasmid and 1 µg of plasmid encoding the catalytic domain
of protein kinase A, 1 µg of
-galactosidase expression plasmid
pCH110, and various amounts of the plasmid expressing ATF-1 as
indicated. The CAT values at each concentration represent averages of
at least two to three independent experiments and are shown relative to
the activity of the reporter plasmid. Panel B, S194 cells were
cotransfected with 3 µg of the enhancer core reporter plasmid
( 132 CORE), 1 µg of internal control plasmid (pCH110;
-galactosidase), and various amounts of the CREM-
expression
plasmid indicated below the lanes. The percent CAT activity of
each construct is shown below the
lanes.
Transcripts of the CREM
gene can be alternatively processed to yield either transcriptional
activator or repressor isoforms. We performed cotransfection
experiments using expression plasmids expressing two isoforms of CREM
(CREM- and CREM-
). Consistent with its role as a repressor, a
CREM-
expression plasmid repressed enhancer activity in a
dose-dependent manner (Fig. 9 B). Similar results were
obtained when a plasmid expressing protein kinase A was included in the
transfection. No repression of TKCAT alone was observed. Expression of
the activator isoform CREM-
in pre-B cells had little effect on
the intact, normally silent
E3` enhancer but slightly induced core
enhancer activity in plasmacytoma cells (data not shown). Therefore,
the repressor isoforms of CREM can repress enhancer activity, but
activation of the
E3` enhancer at late stages of B cell
development is not controlled solely by the CREM-
isoform. The
above results indicate that ATF-1 and CREM proteins can influence the
activity of the
E3` enhancer.
ATF-1 and CREM Are Differentially Expressed in B
Cells
Poly(A)RNAs isolated from cell
lines representing pre-B cells (3-1 and 1-8) or plasma cells (S194 and
MPC11) were subjected to Northern blot analyses with ATF-1 or CREM DNA
probes. ATF-1 was found to be expressed in all cell lines with
5-6-fold greater expression in plasma cells
(Fig. 10 A). Thus, ATF-1 expression appears to increase
during B cell development. The same blot was stripped and rehybridized
with CREM cDNA sequences. These studies showed that the CREM gene was
also expressed in all cell lines studied but yielded a diffuse pattern
of hybridization, presumably because of the complex processing of CREM
transcripts (Fig. 10 B).
Figure 10:
ATF-1 and CREM expression in B cells.
Poly(A)RNAs isolated from 3-1, 1-8, MPC11, or S194
cells were subjected to Northern blot analyses and probed with ATF-1
sequences ( panel A). The same blot was stripped and
rehybridized with CREM sequences ( panel B). Panel C,
a diagram of the various CREM isoforms and reverse transcriptase PCR
primers. In.1 and In.2 represent glutamine-rich
domains, P-BOX represents a phosphorylation domain, and DNA
binding domains are represented as DBDI and
DBDII.
CREM transcripts can be
alternatively processed to yield a variety of products (see
Fig. 10C). To study CREM expression further, reverse
transcriptase PCR assays were performed with RNA isolated from MPC11
plasmacytoma cells using primers A and B (see ``Materials and
Methods''). The reverse transcriptase PCR products were cloned,
and five independent clones were sequenced. Two of the five clones were
identical to CREM-, and three represented a previously unpublished
splicing variant of the CREM gene. These three clones were identical to
CREM-
except that they also contained a deletion of the sequences
indicative of CREM-
(see Fig. 10 C). The
organization of this clone (CREM-
/
) is shown in
Fig. 10C. Definitive identification of all CREM isoforms
in the B cell lineage will require additional studies.
The
Previously, we demonstrated that
an oligonucleotide containing the E3`-CRE Functions Synergistically with the
PU.1/NF-EM5 Binding Sites
E3`-CRE was inactive as an
enhancer when assayed as a multimer in transient expression assays
(1) . To determine whether DNA sequences adjacent to the
E3`-CRE could functionally synergize in transcription, we linked
several core DNA segments to the
E3`-CRE and measured enhancer
activities after transfection into S194 plasmacytoma cells. First, an
additional 15 bp of 3`-flanking sequence that encompasses the wild type
linker scan D sequences (construct
E3`-CRE15) was linked to the
E3`-CRE. No activity was observed when this construct was tested
in S194 plasmacytoma cells (Fig. 11). We next tested whether the
E3`-CRE sequence could function synergistically with either the
PU.1/NF-EM5 or the E2A binding sites.
Figure 11:
The E3`-CRE sequence functions
synergistically with the PU.1/NF-EM5 binding sites in the 3` enhancer.
S194 cells were transfected with 4 µg of the reporter plasmids
indicated above each lane. Transfection efficiencies were
normalized by the level of
-galactosidase produced by the
cotransfected plasmid pCH110. The activity of the reporter construct
containing the PU.1 binding site ( PU.1TKCAT) was considered as
1, and the relative activity of the
E3`-CRE+PU.1TKCAT
reporter construct was determined relative to this
value.
Single copies of
oligonucleotides containing the PU.1/NF-EM5 or the E2A binding sites
were linked adjacent to the E3`-CRE motif in the TKCAT vector.
Reporter constructs containing the PU.1/NF-EM5 or E2A binding sites
alone were used for comparison. Single copies of the PU.1/NF-EM5 or E2A
binding sites were incapable of activating the thymidine kinase
promoter (Fig. 11). Similarly, no activity was observed when the
E3`-CRE was linked to the E2A binding site. However, the construct
containing the
E3`-CRE sequence adjacent to the PU.1/NF-EM5
binding sites resulted in 3.7-fold activation (Fig. 11).
Therefore, the
E3`-CRE interacts with the PU.1/NF-EM5 binding
sites in the
E3` enhancer to activate transcription
synergistically.
ATF-1 and CREM Physically Interact with
PU.1
The transcriptional synergy between theE3`-CRE
and PU.1/NF-EM5 binding sites suggested that the proteins binding to
these sequences might interact physically to elicit transcriptional
activity. To test whether CREM and ATF-1 can physically interact with
PU.1, we performed GST-fusion protein experiments.
S-Labeled CREM-
and ATF-1 were prepared by in
vitro transcription and translation and were incubated with
GST-PU.1 fusion protein bound to glutathione-agarose beads. Similar
incubations were carried out with GST protein alone as a control. These
experiments indicated that indeed CREM-
and ATF-1 interact
physically with PU.1. No interaction of CREM-
or ATF-1 was
observed with the GST protein alone (Fig. 12). These results are
interesting because the synergistic activity observed between the
E3`-CRE and PU.1/NF-EM5 binding sites might be a result of
protein-protein interactions between PU.1 and ATF-1 or CREM proteins.
Figure 12:
Interaction of ATF or CREM- with
GST-PU.1 fusion protein.
S-Labeled ATF-1 or CREM-
proteins were incubated with either GST ( lanes 1 and
2) or GST-PU.1 fusion protein ( lanes 3 and
4) attached to glutathione-agarose beads. After incubation,
beads were washed, suspended in SDS-sample buffer, and the bound
proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis
and detected by autoradiography.
E3` enhancer core which can
enhance transcription when present in multiple copies
(1) . In
the present study, we used a linker scan mutagenesis approach to
identify additional functional enhancer sequences. This approach
confirmed the importance of the PU.1/NF-EM5 and E2A binding sites for
enhancer function. In addition, this linker scan approach revealed the
existence of other functionally important enhancer sequences. In
particular, linker scans A, B, D, and K showed reduced enhancer
activities. None of these DNA sequences is capable of supporting
enhancer activity when assayed as multimers
(1) . Therefore, in
the context of the
E3` enhancer, these DNA motifs most likely bind
to proteins that require interaction with other proteins to enhance
transcription.
E3` enhancer appears to be
comparable to the DR sequence identified in the human 3` enhancer
(26) . We used the mouse functional sequence as a probe to
screen a cDNA expression library. This screen resulted in the isolation
of the transcription factors ATF-1 and CREM. Because these factors bind
to CRE elements, we named this functional sequence within the
E3`
enhancer the
E3`-CRE. Indeed, Bt
cAMP was shown to
increase
E3` enhancer activity in pre-B cells.
E3` enhancer activity while a CREM
expression
plasmid resulted in repression. The fact that repressor as well as
activator proteins bind to the
E3`-CRE may help to explain why
previous studies yielded conflicting results concerning the role of the
cAMP signal transduction pathway in
gene transcription. For
instance, some reports indicated that elevation of cAMP levels within
pre-B cells increased
gene expression while others reported
lowered
expression in response to increased cAMP levels
(7, 9, 16) . Our studies reported here suggest
that the variety of nuclear factors that can bind to the
E3`-CRE
may result in complex regulatory pathways.
/
/
,
antagonize cAMP-induced gene expression, whereas CREM-
is a
transcriptional activator
(29, 32, 33, 34) . In addition, a
repressor form of CREM-
was identified (S-CREM) which uses an
internal AUG translation initiation site
(35) . Recently, it was
shown that the CREM gene can generate an additional product, ICER, by
alternative usage of an intronic promoter in response to the adenyl
cyclase signal transduction pathway
(36) . ICER has been shown
to be a strong repressor of cAMP-induced transcription. Therefore,
differential production of the various CREM isoforms may determine
whether a particular binding site functions as an activator or a
repressor.
(
)
By DNA sequence analysis, we have
identified at least two CREM isoforms expressed in plasmacytoma cells
(CREM-
and CREM-
/
). One of these isoforms,
CREM-
/
, represents a previously unpublished CREM splicing
variant. A more thorough analysis will be necessary to identify all of
the CREM isoforms expressed in the B cell lineage.
E3` enhancer function could be influenced by
the presence of other leucine zipper proteins within the cell. For
instance, repressor isotypes of CREM could predominate at the pre-B
cell stage when
E3` enhancer activity is very weak. These
repressive forms of CREM could potentially be replaced by an activator
form of CREM at later stages of B cell development when enhancer
activity is maximal. Alternatively, increased levels of ATF-1 may help
to override inhibitory influences of the CREM gene products. The
complex mode of regulation and the multiplicity of factors that bind to
CRE sites
(40) may explain why conflicting results have been
obtained concerning the role of cAMP and
gene expression
(7, 9, 16) .
E3`-CRE and the PU.1/NF-EM5 binding sites is interesting.
Recently, it was shown that the interleukin-6 response element of the
junB promoter contains an ets binding site and a CRE-like
site, which are important for efficient activation of the junB promoter by interleukin-6
(41) . These results suggest a
functional interaction between leucine zipper and ets proteins.
Similarly, the
E3` enhancer contains a CRE-like sequence
(
E3`-CRE) and an ets binding site (PU.1; 42), which are located
adjacent to one another. Our transfection assays indicate a functional
interaction between these sequences. In addition, our glutathione
S-transferase chromatography experiments (Fig. 12)
indicate that PU.1 can physically interact with the proteins that bind
to the
E3`-CRE (ATF-1 and CREM). Therefore, the interaction of
leucine zipper and ets proteins may be a common mechanism for
transcriptional regulation. It will be interesting to determine the
precise mechanism of PU.1 interaction with ATF-1 and CREM and how these
interactions relate to the developmental control of
E3` enhancer
function.
and CREM-
expression plasmids and CREM antibodies, and P. Henthorn
for providing the cDNA expression library. We appreciate the help of
Sujatha Nagulapalli in preparing some of the plasmid constructs. We are
grateful to Tom Kadesch, Narayan Avadhani, and John Pehrson for helpful
comments.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.