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Enhancer1
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Department of Molecular Genetics and Cell Biology and
Committee on Immunology, University of Chicago, Chicago, IL 60637
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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light chain gene enhancer has two unique essential
motifs, A and B. The transcription factors that bind the B
motif have been identified as Pu.1 and Pu.1-interacting partner (Pip).
We report here that the A site includes a binding site for the
myocyte-specific enhancer factor 2 (Mef2) family of transcription
factors. Mef2 proteins were first described in muscle cells and, in
vertebrates, include four known members designated A to D. Using a A
electrophoretic-mobility shift assay (EMSA), in conjunction with a high
affinity Mef2 binding site and anti-Mef2 Abs, we show that members
of the Mef2 family are present in nuclear extracts of -producing B
cells and bind the A site. Functional assays using the
chloramphenicol acetyltransferase (CAT) reporter construct containing
three copies of the A motif demonstrate that the A sequence can
function as an enhancer in conjunction with the thymidine kinase (TK)
promoter and is regulated by Mef2 proteins. Extrapolating from other
systems where transcriptional regulation by Mef2 has been studied,
other transcription factors may be involved along with Mef2 in
transcriptional regulation at the A site. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and kappa (
). The Ig H and
L chains are encoded by multiple segments that must be somatically
recombined to form a functional gene (1, 2). In B cells, expression of
Ig genes is strictly regulated for cell-type specificity and
development stage specificity. This is accomplished by multiple
cis-regulatory elements, promoters, and enhancers (2, 3, 4).
Additionally, these transcriptional elements have a significant role in
V(D)J recombination (1, 5, 6). Enhancers have been identified in the
J-C introns of both H (7) and genes (8) and also 3' of the C exons
of H (9, 10), and (11) genes. Unlike the H chain and
light chain gene locus, the light chain gene locus is
organizationally distinct (12). In the murine locus, two
transcriptional enhancers, E2-4 and E3-1, have been identified
and lie 3' of the J-C gene clusters (13). No intronic enhancers
have been found in the gene locus. E2-4 and E3-1, which are
>90% homologous, are thought to have evolved by gene duplication and
are believed to function similarly. Two distinct domains, A and
B, that are essential for function have been defined in the 2-4
enhancer (14, 15). Both A and B sites bind B cell-specific
factors in nuclear extracts (14, 15). The B site has two juxtaposed
but distinct binding sites that are bound by a pair of interacting
transcription factors (15). One of the composite elements is bound by
Pu.1, an Ets family transcription factor (15), while the other is bound
by Pip (Pu.1-interacting partner) recruited through specific
interaction with Pu.1 (16). Analysis of the human Ig enhancers has
also identified similar A and B domains (17).
Analysis of the A site using a transcription factor database
(Transcription Factor Search, 1995, Yutaka Akiyama, Kyoto University;
http://pdap1.trc.rwcp.or.jp/research/db/TFSEARCH.html) identified an
A+T rich consensus binding site for Mef2 (Fig. 1
A). The Mef2 transcription
factor was first identified as a protein that binds an AT-rich
sequence in the muscle-specific enhancer of the muscle
creatine kinase (MCK) gene (18). Mef2 factors bind as homo- and
heterodimers to the consensus sequence
CTA(A/T)4TA(G/A), which is found in the control regions
of numerous muscle-specific genes and growth factor-induced genes (19).
In the muscle cell, cooperative interaction between Mef2 factors and
myogenic basic-helix-loop-helix (bHLH) factors has been shown to
regulate muscle-specific transcription (20). Although Mef2 binding
sites are present in many muscle-specific promoters and are important
for skeletal and cardiac muscle development (18, 21), several findings
suggest that Mef2 proteins may play a role in nonmuscle gene
expression. Four different Mef2 genes have been identified by molecular
cloning experiments and are designated Mef2A,
Mef2B, Mef2C, and Mef2D (22, 23, 24). Mef2
proteins belong to the MADS family of transcription factors (25) (named
after the first four proteins in which the MADS domain was first
identified: minichromosome maintenance 1 (MCM1), which regulates
mating-specific genes in yeast; AGAMOUS and DEFICIENS, which have
homeotic function in flower development; and serum-response element
(SRE), which regulates serum-inducible and muscle gene expression) and
share a 56-aa N-terminal MADS box domain followed by a 27-aa Mef2
domain, which extends C-terminal of the MADS domain (26). These protein
domains together mediate DNA-binding, homo- and heterodimerization, and
interaction with bHLH proteins (27).
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In this paper we show that members of the Mef2 protein family bind the
A site of the enhancers of the Ig light chain gene. We report
here the fine analysis and functional properties of this
interaction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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J558L myeloma cells and NIH 3T3 fibroblast cells were routinely maintained in DMEM (HyClone, Logan, UT) fortified with 10% FCS (HyClone) and penicillin G (Life Technologies, Grand Island, NY) and streptomycin (Sigma, St. Louis, MO). A modified protocol based on the protocols of Schreiber et al. (33) and Dignam et al. (34), detailed in Eisenbeis et al., (16) was used for preparation of nuclear extracts. The nuclear extracts were aliquoted and quick-frozen in liquid N2 and stored at -70°C. Protein concentration was estimated by Bradford (35) assay using a kit from Bio-Rad (Hercules, CA).
EMSA
EMSAs were performed as described by Singh et al. (36). Probes
were made from complementary oligonucleotide pairs with XbaI
and BamHI overhangs, and their sequences are as follows
(complementary pairs are denoted as "top" and "bottom"). A
(top), 5'-GATCTTCCA CAAGCTAAAATTAGATCTGTGATAGG-3'; A (bottom),
5'-GATCCCTATCACAGA TCTAATTTTAGCTTGTGGAA-3'; B (top),
5'-GATCTGAAAAAGAGAAATAAAAGGA AGTGAAACCAAGG-3'; B
(bottom), 5'-GATCCCTTGGTTTCACTTCCTTTTATTTCTC TTTTTCA-3'; muscle
creatine kinase (MCK) (top), 5'-CTAGACTCGCTCTAAAAATAACC CTGTC-3'; MCK
(bottom), 5'-CTAGAGACAGGGTTATTTTTAGAGCGAG-3'; MEFmt1 (top),
5'-CGCTCTAAGGCTAACCCT-3'; MEFmt1 (bottom),
5'-AGGGTTAGCCTTAGAG CG-3' (mutated nucleotides
underlined). Annealed oligonucleotides were labeled by filling
them with labeled ([32P]dATP and [32P]dCTP)
and unlabeled (dGTP and dTTP) nucleotides and Klenow enzyme and
gel-purified on an 8% native polyacrylamide gel. Binding reactions
were conducted in 20-µl volumes containing 10 mM Tris (pH 7.5), 50 mM
NaCl, 1 mM DTT, 1 mM EDTA, 5% glycerol, 2 µg of poly(dI-dC)
(Pharmacia, Piscataway, NJ), 0.45 µg of sonicated salmon sperm DNA,
0.45 µg of denatured sonicated salmon sperm DNA, 30,000 to 50,000 cpm
of probe and 8 to 10 µg of nuclear extract or 2 µl of in vitro
translated protein. They were incubated at room temperature for 15 min
and electrophoresed on a 4% nondenaturing polyacrylamide gel at 200 V
for 2 h, using 0.5x TBE (1x TBE contains 100 mM Tris-borate and
2 mM EDTA). For competition assays, 100 ng of the respective annealed
cold competitor oligonucleotide was included and, for Ab supershift
experiments, nuclear extract or in vitro translated proteins were
incubated with 2 µl of preimmune or immune serum in 1x binding
buffer on ice for 30 min before the addition of the remaining
components of the binding reaction.
Western blotting
For Western blotting, 20 to 40 µg of nuclear extract or 5 µl of in vitro translated proteins were boiled for 5 min with an equal volume of SDS sample buffer and chilled on ice and resolved on an 8% SDS-PAGE followed by blotting onto nitrocellulose membranes (Hybond ECL, Amersham, Buckinghamshire, U.K.). Rabbit anti-Mef2A (cross-reacts with Mef2C), anti-Mef2B, and anti-Mef2D (kind gift of Dr. Ron Prywes, Columbia University, New York) were used as the first Ab followed by goat anti-rabbit-horseradish peroxidase (HRP) (Amersham) conjugate. Immunoblots were developed using a chemiluminescent ECL Western blotting kit (Amersham) and visualized by exposing to x-ray film.
In vitro transcription and translation
Mef2A and Mef2C cDNAs cloned into the CMV promoter-driven expression vector pcDNAI (Invitrogen, San Diego, CA) (a kind gift of Dr. E. N. Olson, Dallas, TX) were used. Mef2A and Mef2C proteins were made by T7-directed in vitro transcription-translation (TNT Kit, Promega, Madison, WI) in the presence of [35S]methionine and expression confirmed by SDS-PAGE.
Functional assays using a CAT reporter construct
The A3-TKCAT construct was made as follows. A trimer of
A cloned in pBluescript II KS (C. Eisenbeis et al., unpublished
observations) was amplified using the following PCR primers: (forward)
5'-CCCGCGGATCCCTAG TGGATCTTCCACAAGC-3' and (reverse)
5'-GCCCGGGGGATCCCTATCACAG-3'. PCR primers included BamHI
restriction sites to allow cloning into a BamHI site in
front of the thymidine kinase (TK) promoter that drives a bacterial
chloramphenicol acetyltransferase (CAT) gene in the TKCAT construct
(37). J558L cells were transfected by the DEAE-dextran method as
described by Eisenbeis et al. (15). Briefly, cells were grown to a
density of 10 x 105 cells per ml. A total of
107 cells were washed twice in TS buffer (137 mM NaCl, 5 mM
KCl, 0.4 mM Na2HPO4, 25 mM Tris, 1 mM
MgCl2, 0.7 mM CaCl2 (pH 7.4)) and resuspended
in TS buffer containing 20 µg TKCAT reporter construct, 5 µg of
ß-galactosidase reporter plasmid (pMC1924) (38), and 0.25 mg of
DEAE-dextran (Pharmacia) per ml. For coexpression studies, 20 µg of
expression plasmids for Mef2 proteins (respective cDNA cloned in pcDNAI
(Invitrogen), a kind gift of Dr. E. N. Olson, Dallas, TX) were
included, and in these experiments the total amount of DNA used for
transfection was normalized by using nonrelated plasmid DNA as a
"filler." After 20 min at room temperature, 15 ml of DMEM
containing 0.1 M chloroquine diphosphate was added, and the cells were
transferred to a 7.5% CO2 incubator at 37°C for 1
h. The transfected cells were then pelleted and resuspended in 40 ml of
tissue culture medium and incubated in 7.5% CO2. After
60 h, cell lysates were made from washed cells as described by
Gorman et al. (39). A sample of the lysate was removed to determine the
ß-galactosidase activity (40), and the rest was heated to 60°C for
10 min, chilled on ice for 5 min, and then centrifuged at 15,000
x g for 10 min. CAT assays were performed as described
previously (39). The volume of lysate used in each assay was normalized
for ß-galactosidase activity to control for variation in transfection
efficiencies. The TLC plates were analyzed and quantitated using a
PhosphoImager (Molecular Dynamics, Sunnyvale, CA). CAT activity was
calculated in terms of the percent of acetylated chloramphenicol over
the total chloramphenicol.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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enhancer A site sequence includes a Mef2 binding motif
The enhancers were identified, and essential binding sites and
regulatory domains were mapped in earlier studies (14). These results
are summarized in Figure 1
A, where the sequence of the
E2-4 and E3-1 enhancers of the Ig light chain gene are shown
with two E-boxes and the sequences of the unique A and B DNase I
footprinted sites. When the A site sequence was analyzed using a
transcription factor database (Transcription Factor Search, 1995,
Yutaka Akiyama, Kyoto University) an A+T rich consensus binding site
for Mef2 was identified. Figure 1B shows the sequence of the
A site with the Mef2 binding motif in bold face. It can be seen that
the A-footprinted region includes the entire Mef2 binding sequence.
When analyzed by EMSA (Fig. 2), two major
complexes are formed by the labeled A oligonucleotide and J558L
nuclear extracts. They are denoted as JA1 and JA2, in decreasing order
of their m.w. (there are also minor complexes that we believe to be
breakdown products, as will be described later). To determine whether a
high affinity site for Mef2 could effectively compete with the A
site in an EMSA, we chose a sequence from the MCK gene, which had been
earlier identified as a functional element, binding muscle-specific
factors (18). This MCK oligonucleotide competes for both gel-shift
bands (JA1 and JA2), but a mutant MCK oligonucleotide, MEFmtl
(22), with mutations at the conserved site effecting Mef2 binding,
fails to compete (Fig. 2). As expected, unlabeled A oligonucleotide
also successfully competes whereas the B oligonucleotide, which does
not contain a Mef2 binding site, does not compete.
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A motif and MCK
oligonucleotide is the Mef2 consensus binding site, and mutations at
the conserved residues affecting Mef2 binding affect the ability of the
MCK oligonucleotide to compete for gel-shift bands obtained with the
labeled A site oligonucleotide. Together, these data suggest that
protein/s that bind the A site are capable of specifically binding
the MCK sequence and are therefore likely to contain one or more of the
Mef2 proteins or a highly related protein.
In vitro synthesized Mef2 proteins bind specifically to the A
site and give gel-shift bands that comigrate with the JA1 complex
To directly demonstrate that Mef2 proteins can bind the A
site, we performed EMSA with in vitro synthesized Mef2A and Mef2C, as
representative members of the Mef2 family. Mef2A binds the A site
and forms a major gel-shift complex that comigrates with the lower
mobility complex JA1, obtained with J558L nuclear extract (Fig. 3). Mef2C gives a gel-shift band that
runs faster than the JA1 complex, which may be an artifact of in vitro
synthesis. These Mef2A and Mef2C complexes are successfully competed
out by the MCK oligonucleotide (high affinity site for Mef2) but not
affected by the mutant site Mef2 mt1. The gel-shift complexes are
formed only with reticulocyte lysates programmed with Mef2A or C mRNA,
showing that it is a specific complex (data not shown). Furthermore,
the gel-shift complexes formed by Mef2A and Mef2C are clearly
supershifted by anti-Mef2 Ab (Fig. 4A, see below).
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, see below) (Fig. 2). Therefore, we
believe that the JA2 complex represents a breakdown product of Mef2
proteins and is unlikely to contain a different protein.
Mef2 proteins are present in gel-shift complexes obtained with the
A site
To determine whether Mef2 protein/s are indeed present in the JA1
and JA2 complexes, we used three rabbit antisera raised against
specific Mef2 family members: one raised against a peptide from Mef2A
that is specific for Mef2A but cross-reacts with Mef2C and two that
were reported to be relatively Mef2B and Mef2D specific (41).
Anti-Mef2A (Fig. 4A) and anti-Mef2D (Fig. 4C)
supershift the bands obtained with J558L nuclear extract and the
labeled A oligonucleotide whereas the anti-Mef2B Ab has no
effect (Fig. 4B). Anti-Mef2A Ab supershifts bands obtained
with both Mef2A and Mef2C proteins as expected, since it is known to be
cross-reactive. However, anti-Mef2D Ab also supershifts bands
obtained with Mef2A and Mef2C proteins and the labeled A
oligonucleotide, indicating that it also cross-reacts. These Abs do not
affect the gel-shift bands containing an unrelated protein (Pu.1 and
the labeled B oligonucleotide; data not shown).
These data demonstrate that Mef2 proteins are present and responsible
for the gel-shift bands JA1 and JA2 that are obtained with the J558L
nuclear extract and the labeled A oligonucleotide and suggest that
Mef2B is not involved but that Mef2A, Mef2C, or Mef2D appear to be
involved. These antisera exhibit cross-reactivity. This and other
considerations (see next section) suggest that only a subset of Mef2
proteins are present in the complexes with J558L nuclear extract.
Mef2A, -C, and -D are present in J558L nuclear extract
EMSA data suggest that one or more members of the Mef2 family
interact with the A site. To formally confirm the presence of Mef2
family members in J558L cells, we used Western blot assays. Although
Mef2 activity has been primarily characterized and studied in the
muscle cell, there are a number of reports of the presence of Mef2
proteins in different tissue and cell types (42), but there were no
previous reports of Mef2 proteins in J558L cells. In mammals, Mef2A and
Mef2D transcripts are expressed in many tissues, while Mef2C
transcripts are restricted to muscle, brain, and spleen (26). We
analyzed nuclear extracts from J558L myeloma cells and a non-B cell,
NIH 3T3 fibroblasts, using Abs against Mef2 proteins in Western blots
(Fig. 5). The immunoblot analysis shows
that both J558L and NIH 3T3 cells have Mef2A and/or Mef2C (since
anti-Mef2A Ab cross-reacts with Mef2C) and Mef2B. Another
laboratory, however, has failed to detect the presence of Mef2A in
J558L cells (43); it is therefore likely that the signal obtained with
the anti-Mef2A Ab could be entirely due to Mef2C. Both cells also
appear to have Mef2D although the anti-Mef2D signal could also be
due to the cross-reactivity it exhibits with Mef2A and Mef2C. The
appearance of multiple bands in the immunoblotting analysis done on
nuclear extracts has been documented by others (42)). This
heterogeneity is believed to be due to posttranslational modifications,
such as phosphorylation, and the presence of multiple Mef2 isoforms.
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A site.
Of the Mef2 family members, Mef2A is unlikely to be involved for the
reason discussed above. Mef2B does not seem to be present in the
gel-shift complexes (Fig. 4B). Although the Mef2C gel-shift
band runs faster than JA1, it is possible that this is a characteristic
of the in vitro synthesized protein and may not be true of the protein
made in the cell. That leaves Mef2C and Mef2D as possible candidates
for the gel-shift complexes formed with the J558L nuclear extract and
the A site.
Functional analysis of the A motif in the regulation of the
L chain enhancer
Ig enhancer elements, such as the octamer element (44, 45, 46),
the B site (40, 47), the µE3 site (48), and the B site (16),
form strong trans-activating elements upon multimerization
of single protein binding domains. For this reason we tested the
enhancer function of the A site, using it as a trimer. We have not
tested the A site as a monomer. In studies done in the muscle system
also, the Mef2 site is used as a dimer (22). To correlate the in vitro
ability of Mef2 proteins to bind the A site and in vivo activity of
the A domain of the E2-4 enhancer, we designed a CAT reporter
construct, A3-TKCAT. It consists of a bacterial CAT gene driven by a TK
promoter, with a trimer of A site oligonucleotides cloned upstream
of the promoter. A3-TKCAT reporter constructs were transfected into the
J558L B cell line, and their CAT activity was assayed (Fig. 6A). Compared with the
promoter-only construct, A3-TKCAT shows a greatly increased CAT
activity, demonstrating the ability of the multimerized A site to
act as an enhancer in conjunction with the TK promoter.
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The functional activity of the A3-TKCAT reporter construct
implies that transcription factors present in the J558L B cells are
capable of transcriptional activation via the A site. We now wanted
to see the effect of overexpressing Mef2 proteins in J558L cells. To
our surprise, cotransfecting with Mef2 cDNAs suppressed the CAT
activity of the A3-TKCAT reporter construct by about 50 to 60%, when
compared with the CAT activity of the cells transfected with the
A3-TKCAT reporter construct alone (Fig. 6A). Mef2 cDNAs
cotransfected with the promoter-only control plasmid TKCAT did not give
any significant CAT activity (data not shown). To determine whether the
suppression is specific, we repeated the cotransfection experiments
using a CAT reporter construct with a multimerized B site and cDNAs
for Mef2A and Mef2C. This reporter construct, B4-TKCAT, has four B
sites cloned upstream of the TK promoter and serves as an enhancer in B
cells but is not expected to be bound by Mef2 proteins (15, 16). Indeed
the B4-TKCAT reporter construct is not affected by overexpression of
Mef2A and Mef2C (Fig. 6B). Thus, the suppression of the
A3-TKCAT construct by overexpressing Mef2 proteins is clearly specific.
Furthermore, the effect of overexpression of Mef2 proteins is
dose dependent (Fig. 7). Increasing
amounts of Mef2C cDNA transfected into J558L cells translates into
increase in the suppression of the A3-TKCAT reporter activity. A
similar negative regulatory effect following overexpression of Mef2C
cDNA has been described in another system using the
c-jun promoter (see Discussion). Mef2
proteins therefore clearly affect the enhancer activity of the A
site, albeit in a negative manner, since the suppression is clearly
specific and dependent on the amount of the Mef2 protein expressed in
these cells. These data together with the EMSA analysis implicate Mef2
proteins to be functionally involved in the transcriptional regulation
at the A site.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A motif of the Ig
light chain enhancer, E2-4, includes a binding site for the Mef2
family of transcription factors and is bound in vitro by members of the
Mef2 family. Further, we show that the A motif functions as an
enhancer in conjunction with a TK promoter, and we show functional
evidence of the role of Mef2 in transcriptional regulation via the A
site. To our knowledge, this is the first time that Mef2 family
proteins, transcription factors essential for muscle cell
differentiation, have been shown to be involved in Ig gene expression.
Interestingly, the A3-TKCAT reporter construct, however, exhibits CAT
activity when transfected into a non-B cell line, namely NIH 3T3 mouse
fibroblast cells (data not shown), suggesting that the A site, though
essential, may not need any B cell-specific factors for its activity in
the B cell. The two Ig light chain enhancers, E2-4 and E3-1,
are highly homologous and are believed to function in a similar manner
(14). The A site is identical between both these enhancers except
for a single nucleotide difference that occurs at a variable position
in the Mef2 consensus sequence. We therefore believe that the A
motif of the E3-1 enhancer would behave in a similar fashion with
regard to the binding of the Mef2 proteins and enhancer activity in our
reporter construct assays.
Mef2 activity, though first identified in the context of muscle
specific enhancers, (18) is not restricted to the muscle cell. A Mef2
consensus site is present in the brain creatine kinase gene promoter
(49). Mef2 sites are also present in the promoters of two immediately
early gene promoters, c-jun and N10, that are activated in
quiescent cells following mitogenic stimulation (50). Mef2 proteins
have been shown to be present and capable of binding DNA in a number of
cultured cell lines, both lymphoid and nonlymphoid, of human and mouse
origin (42). However, until a recent report by Swanson et al. (43),
there were no reports of the presence of Mef2 in the -expressing
myeloma cell line J558L. Our data clearly show that Mef2 proteins are
present in J558L nuclear extracts. It is therefore formally possible
for any of these Mef2 proteins by themselves or as heterodimers to
regulate transcription at the A site. Using Abs specific for Mef2A
(which also cross-react with Mef2C, Fig. 4C), Mef2B (Fig. 4B), and Mef2D (cross-reacts with MEFA and Mef2C, we were
unable to identify a specific member in these Ab supershift experiments
since all these Abs, except anti-Mef2B, independently supershifted
the gel-shift bands. Since Mef2B is clearly present in the B cell
nuclear extract (Fig. 5) and the anti-Mef2B Ab does not supershift
the gel-shift bands (Fig. 4B), it appears that Mef2B may not
be involved in the regulation via the A motif. Based on our data and
the data from another laboratory (43) that failed to detect Mef2A in
J558L cells, Mef2C and -D are the likely candidates to bind and
regulate transcription via the A motif. In our EMSA experiments,
cell-free-produced Mef2C proteins gave a gel-shift band that runs
faster than JA1 complex. Furthermore, the A motif functions as an
enhancer in NIH 3T3 cells that do not express Mef2C (Fig. 5) (32). This
might suggest that Mef2D may be involved or that there is a degeneracy
enabling any Mef2 family member to bind and regulate transcription. It
is also possible that more than one Mef2 protein binds the A site as
a heterodimer or homodimer since there is evidence from the muscle
system that Mef2 proteins appear to bind DNA as dimers (51). Loss of
function assays have been done with Mef2 family members. Mef2B knockout
mice that were generated do not show any birth defects (32) while Mef2C
null mice show cardiogenic defects and embryonic lethal phenotype (27).
However, the current thinking on Mef2 proteins is that they have
overlapping function in the tissues in which they are expressed (E.
Olson, unpublished observations). It is therefore possible that
there could be a certain amount of degeneracy with regard to the
requirement of a specific Mef2 family member for transcriptional
regulation in the context of a specific enhancer.
In the transient transfection assays the A motif functions as
enhancer in conjunction with the TK promoter in the J558L B cell line,
indicating that functional transcription factor/s bind and regulate
transcription via the A site. Mutations in the region of the A
site that encompass the consensus Mef2 binding site have been shown to
significantly suppress the enhancer activity using a CAT reporter
construct E2-4 TKCAT2, with the full-length E2-4 enhancer
upstream of the TK promoter (14). These experiments were done with both
the E2-4 and E3-1 enhancers, suggesting the importance of the
A motif in transcriptional regulation. Although these mutations were
extensive and it could be argued that the mutations affected the
binding of other factors to the A site, they underscore the
importance of the Mef2 binding site, which was the main region that was
altered in these mutants.
In the transient transfection experiments, overexpression of Mef2
proteins had a negative effect on the transcriptional activation of the
reporter construct A3-TKCAT. All four Mef2 cDNAs that we tested
demonstrated this negative effect. The effect was specific because it
was seen only when the reporter construct had a Mef2 binding sequence
(Fig. 6). Suppression of A3-TKCAT by Mef2C was shown to be dose
dependent (Fig. 7). Overexpression of Mef2C has been shown to exert a
similar dose-dependent negative regulatory effect in an LPS-induced
c-Jun reporter system (52). The authors argue that the overexpressed
Mef2C competes with heterodimers of Mef2 proteins that have the ability
to activate transcription. However, at this time, there is no evidence
showing that heterodimers consisting of more than one Mef2 protein are
better trans-activating agents than homodimers formed by a
single Mef2 protein (E. Olson, unpublished observations).
The suppression of transcriptional activity by the overexpressed Mef2
proteins could be explained by proposing a model where the
overexpressed Mef2 proteins compete with a "functional transcription
complex" that binds and regulates transcription in vivo. This
"functional transcription complex" could be a posttranslationally
modified form of Mef2 protein, such as a phosphorylated form. The
overexpressed proteins may not be properly phosphorylated and therefore
may be transcriptionally inefficient. On the other hand, the
overexpressed Mef2 proteins may be identical to the endogenous Mef2
proteins, and the excess of Mef2 protein may sequester other factors
needed for transcriptional activation, making them unavailable for the
formation of the "functional transcriptional complex," a phenomenon
called squelching (53). Finally, the Mef2 cDNAs that were used for the
cotransfection experiments were obtained from muscle cells. Different
splice forms of Mef2 mRNA have been shown to be expressed in different
tissues (22, 23, 24, 30, 31, 32). It is possible that this muscle cell form of
Mef2 protein is not capable of transcriptional activation via the A
site but competes with the "functional transcription complex" for
binding to the A site. However, when we compared the Mef2C mRNA
species produced in mouse skeletal muscle and J558L cells, we found
that they are the same (data not shown). In summary, it could be
postulated that the overexpressed Mef2 protein is capable of binding
the A site but that it is unable to bring about transcriptional
activation or that it is able to squelch the "functional complex."
At this time we have no direct experimental evidence of other factors
being involved along with Mef2 proteins in transcriptional regulation
at the A site. However, the consensus emerging from muscle cell gene
expression studies strongly suggests that Mef2 acts as a cofactor for
myogenic bHLH proteins in the skeletal muscle differentiation program
but does not activate the program by itself (54). As described earlier,
members of the Mef2 family are fairly ubiquitous in their distribution
in several cell types. It is therefore possible that some other
protein/s is/are involved in rendering cell-type specificity to this
interaction of Mef2 proteins and the A site. In the muscle cell a
transcription complex containing Mef2 and a heterodimer of MyoD and
E12, a ubiquitous bHLH protein (55), regulates transcription (20). The
model proposed for this interaction suggests that a protein-protein
interaction between Mef2 and a MyoD/E12 heterodimer, with any one of
them binding to the DNA, is sufficient to activate transcription (20).
It is interesting to note that the A site is flanked by two E-boxes,
which can bind bHLH proteins such as E12 and E47 (55). One of the
E-boxes is located a mere 15 bp upstream. Earlier work using CAT
reporter constructs has shown that both E-boxes are essential for full
activity of the E2-4 and E3-1 enhancers in the mouse (14) and
also in humans (17). Extrapolating from the model proposed for the
muscle cell, it is possible that a protein-protein interaction may be
involved between Mef2 protein binding at the A site and bHLH
proteins, or with some other factor/s. However it is likely that one
would miss such an interaction in EMSA experiments, which are too harsh
to preserve these subtle protein-protein associations; this has been
seen in the case of muscle-specific enhancers (54). Given these
observations, it is likely that other factors are involved along with
the Mef2 proteins in transcriptional regulation at the A site.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Medicine, Northwestern University, Chicago, IL 60611.
3 Address correspondence and reprint requests to Dr. Ursula Storb, Department of Molecular Genetics and Cell Biology, 920 East 58th Street, Chicago, IL 60615. E-mail address:
4 Abbreviations used in this paper: L, light; H, heavy; EMSA, electrophoretic mobility shift assay; Mef2, myocyte specific enhancer factor 2; MCK, muscle creatine kinase; TK, thymidine kinase; CAT, chloramphenicol acetyl transferase; bHLH, basic-helix-loop-helix; MADS, minichromosome maintenance 1 (MCM1) + agamous + deficiens + serum-response element (SRE).
Received for publication April 23, 1998. Accepted for publication June 29, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain intron enhancer abolishes chain gene rearrangement in cis but not gene rearrangement in trans. EMBO J. 12:2392.
light chain gene intronic enhancer/matrix attachment retions impairs but does not abolish VJ rearrangement. Immunity 4:377.[Medline]
constant region. Mol. Cell. Biol. 13:1547. locus contains a second, stronger B cell-specific enhancer which is located downstream of the constant region. EMBO J. 8:1959.[Medline]
locus is duplicated and functionally independent of NFB. Genes Dev. 4:978. enhancers bind B-cell-specific factors. Mol. Cell. Biol. 12:309.2-4 enhancer. Mol. Cell. Biol. 13:6452. immunoglobulin enhancer is controlled by both positive elements and developmentally regulated negative elements. Mol. Immunol. 33:427.[Medline]
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