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Molecular and Cellular Biology, September 1998, p. 5478-5484, Vol. 18, No. 9
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Intramolecular Regulation of MyoD Activation Domain
Conformation and Function
Jing
Huang,1
Hal
Weintraub,2 and
Larry
Kedes1,*
Institute for Genetic Medicine and Department
of Biochemistry and Molecular Biology, University of Southern
California School of Medicine, Los Angeles, California
90033,1 and
Howard Hughes Medical
Institute, Fred Hutchinson Cancer Research Center, Seattle,
Washington 981042
Received 10 November 1997/Returned for modification 15 December
1997/Accepted 8 May 1998
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ABSTRACT |
The MyoD family of basic helix-loop-helix (bHLH) proteins is
required for myogenic determination and differentiation. The basic
region carries the myogenic code and DNA binding specificity, while the
N terminus contains a potent transcriptional activation domain.
Myogenic activation is abolished when the basic region, bound to a
myogenic E box, carries a mutation of Ala-114. It has been proposed
that DNA binding of the MyoD basic region leads to recruitment of a
recognition factor that unmasks the activation domain. Here we
demonstrate that an A114N mutant exhibits an altered conformation in
the basic region and that this local conformational difference can lead
to a more global change affecting the conformation of the activation
domain. This suggests that the deleterious effects of this class of
mutations may result directly from defective conformation. Thus, the
activation domain is unmasked only upon DNA binding by the correct
basic region. Such a coupled conformational relationship may have
evolved to restrict myogenic specificity to a small number of bHLH
proteins among many with diverse functions yet with DNA binding
specificities known to be similar.
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INTRODUCTION |
The skeletal muscle-specific basic
helix-loop-helix (bHLH) transcription factors (including MyoD, Myf-5,
myogenin, and MRF-4) are required for myogenic determination and
differentiation (34). Previous work has identified Ala-114
and Thr-115 of MyoD as comprising a myogenic code
crucial basic-region
residues of MyoD required for myogenic conversion and activation of
muscle-specific genes (6, 8, 35). Introducing just these two
residues from MyoD into corresponding positions in the ubiquitous bHLH
protein E12 allows the altered E12 to function as a myogenic activator
(9, 35) (see Table 1). Cooperative DNA binding was shown to
be affected in some basic-region mutants (3), but lowered
affinities or altered specificities of DNA binding by such
mutants complicated the interpretation. Thus, the molecular mechanisms
involved in deciphering the myogenic code remain unclear. Arg-111
of MyoD is highly conserved throughout the bHLH and bHLH/leucine
zipper families (see Fig. 1). However, in protein-DNA cocrystals
(12, 13, 21), it adopts a unique conformation due to the
small size of Ala-114, which is specific to the myogenic bHLH proteins: Arg-111 is buried in the major groove and contacts the N-7 of the final
G of CAGCTG. In other bHLH proteins, this arginine residue lies outside
of the major groove and position 114 equivalents are occupied by
bulkier amino acids, which themselves contact the G of CANNTG. These
observations raise the possibility that the exposure of Arg-111
prevents myogenic function. This paper demonstrates the long-sought and
fundamental mechanisms by which this important class of myogenic
regulatory proteins activates transcription.
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MATERIALS AND METHODS |
Plasmid construction.
Amino acids are numbered according to
full-length mouse MyoD throughout the text. PCR-based mutagenesis
(15) was used to generate the mutations in which alanine at
position 114 was replaced by asparagine (A114N), histidine (A114H), and
glycine (A114G) in the backbone of pEMSV-MyoD (10), a
eukaryotic expression vector for MyoD. Orientation was based on the
sense strand of MyoD. 
N-A114N was generated by replacing the
ScaI-PmlI fragment of pA114N with the
ScaI-AluI fragment from pEMSV-MyoD, which encodes two amino acids (aa) of MyoD. N-A114N-VP16 and A114N-VP16 were generated by replacing the ScaI-StuI fragment of
N-MyoD-VP16 with the ScaI-StuI fragments from
N-A114N and A114N, respectively. To create bacterial expression
vectors, an NdeI site encompassing the start codon for MyoD
was introduced by PCR mutagenesis. The NdeI-HindIII fragment containing the coding
region for MyoD was ligated into the backbone of pGM484 (gift from A. Klug, Medical Research Council) to generate pT7-MyoD. T7 vectors of
MyoD basic-region mutations were constructed by replacing the
PmlI-MluI fragment of MyoD in pT7-MyoD with those
from the corresponding mutants in the eukaryotic expression vectors.
Gal-MyoD and Gal-A114N fusion plasmids were generated by fusing the
NdeI-HindIII fragments from T7 vectors in
frame at the region encoding the carboxyl terminus of Gal4 DNA binding
domain (aa 1 to 147) in the vector pGal0 (27). A PCR
fragment encoding MyoD residues 1 to 75 was inserted between the
NdeI and XbaI sites of pGal0 to generate
Gal-MyoD(1-75). pG5-LacZ and pG5-Luc were created by inserting a PCR
fragment, encoding five Gal4 DNA binding sites and the E1b TATA box
from pG5CAT (Clontech), into pNNN-
-Gal (16) devoid of
its promoter and pGL2-Basic (Promega), respectively.
Production of wild-type and mutant MyoD proteins.
Wild-type
and mutant MyoD proteins were expressed in Escherichia coli
BL21(DE3)/pLysS and purified to >90% homogeneity by standard
protocols (29), and E47 protein was purified as described previously (30).
DNA binding assays (gel mobility shift assays).
Oligodeoxyribonucleotides were prepared on an automated DNA synthesizer
(Applied Biosystems) using phosphoramidite chemistry. 1R contains the
right (R) site E box of the muscle creatine kinase (MCK) enhancer, and
2R contains two copies of the R site in tandem repeats. One strand of
the probe was end labeled with [
-32P]ATP by using T4
polynucleotide kinase annealed to a 10-fold molar excess of the
unlabeled complementary strand, and purified with a G-25 spin column
(Boehringer Mannheim).
For standard gel mobility shift assays, 5 × 104 cpm
of end-labeled DNA fragment (about 0.5 ng, or 10 to 35 fmol for a 20- to 30-bp probe) was incubated with 5 to 200 ng (as specified for separate experiments in the figures) of purified MyoD and/or E47 proteins for 15 min at room temperature in a solution containing 20 mM
HEPES (pH 7.6), 50 mM KCl, 3 mM MgCl2, 1 mM EDTA, 1 mM
dithiothreitol, 8% glycerol, and 1 µg of poly(dI-dC) in a final
volume of 20 µl. The reaction mixtures were resolved on nondenaturing
4% polyacrylamide gel with 1× Tris-borate-EDTA at 10 V/cm and room
temperature and were visualized by autoradiography.
Protease sensitivity assay.
Subtilisin (EC 3.4.21.14;
Boehringer Mannheim) was added to proteins both in the presence and in
the absence of specific DNA to final concentrations of 50 ng/µl and
incubated at 37°C for different lengths of time. The proteolysis
reactions were terminated by addition of 0.5 mM phenylmethylsulfonyl
fluoride (PMSF) (Sigma).
Cell culture and transfection assays.
Cell culture and
transfection procedures were performed as described previously
(16) except that transfections were done on 60-mm-diameter
plates with 4 µg of total plasmid DNA unless otherwise specified.
-Galactosidase (-Gal) and luciferase activities from cell lysates
were assayed with chemiluminescent substrates from Tropix.
Immunofluorescent staining of cells.
Cells were washed,
fixed in 4% paraformaldehyde in Tris-saline for 5 min, washed,
permeabilized with 0.1% Triton X-100 in Tris-saline for 5 min, and
washed again. Next, cells were stained with MF20 (mouse monoclonal
antibody to myosin heavy chain [MHC] at a 1:10 dilution) for
1 h. After being washed, cells were stained with goat anti-mouse
immunoglobulin G conjugated to fluorescein (1:500 dilution) for 1 h, washed, and examined by fluorescence microscopy. All washes were
done two or three times in Tris-saline.
In vitro protein association assay.
Purified MyoD and A114N
proteins were assayed for binding to glutathione
S-transferase-p300 beads as previously described (28). Binding to six-histidine-tagged dTAFII40
was carried out with Ni-nitrilotriacetic acid resin (Qiagen) according
to the manufacturer's protocol.
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RESULTS AND DISCUSSION |
The MyoD A114N mutant fails to activate myogenic transcription upon
binding to DNA.
We sought to test the hypothesis that the exposure
of Arg-111 prevents myogenic function by decreasing the ability of
Arg-111 to easily fit into the major groove. We replaced Ala-114 in
MyoD with bulky amino acids, asparagine and histidine, that occur
naturally at the corresponding E-box-binding, basic-region positions of E47 and Max, respectively (Fig. 1). At
most a threefold decrease in DNA binding was detected for the
resulting A114N mutant homodimers (Fig.
2A). However, no differences in DNA
binding were detected when the mutant proteins (heterodimerized with
E47) were compared to the wild-type MyoD/E47 heterodimers for their
affinities for specific DNA sequences (Fig. 2B). In contrast, when
cotransfected with -Gal reporter plasmids driven by muscle-specific
promoters for either the MCK or human cardiac 
-actin (HCA) genes,
the A114N mutant activated the reporter gene to no more than 5%
of the level achieved by wild-type MyoD (Table
1). The A114H mutant showed a defect
similar to that of the A114N mutant. Another mutant, with Thr-115 of
MyoD replaced by Asn (present at the corresponding positions in
both E47 and Max [Fig. 1]) (T115N), exhibited a DNA-binding activity similar to that of the A114N mutant (Fig. 2) but retained at
least 60% of the transcriptional activity of the wild-type protein
(Table 1). Therefore, the detrimental effect of the A114N mutation on
transcriptional activation is highly specific and is not likely
engendered by subtle differences in DNA binding affinity but, rather,
must result from the effects of the bulky amino acid substitutions at
position 114. These mutant proteins were expressed in transfected cells
at the same levels as the wild-type MyoD protein as determined by
Western blot analysis (data not shown). Their nuclear localization
was not affected as determined by immunohistochemistry with an antibody
(Santa Cruz) specific to the carboxyl terminus of MyoD (data not
shown). This result is in accord with previous findings that MyoD
contains one nuclear localization signal in each of the basic region
and the H1 region and that disruption of both is required to abolish nuclear localization (32).
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FIG. 1.
Basic-region sequence alignment of the bHLH MyoD (mouse)
and E47 (human) proteins, MASH1 (mouse), and the bHLH-LZ Max (mouse)
protein. Amino acids are given in one-letter code; the numbering is
according to full-length mouse MyoD. The conserved yet conformationally
nonuniform arginines at the 111 positions are boxed; the residues at
position 114, which likely determine the conformation of Arg-111, are
shaded.
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FIG. 2.
DNA binding by wild-type and mutant MyoD proteins. (A)
Increasing amounts of MyoD proteins were incubated with labeled 1R
probe (0.5 ng) for 15 min at room temperature and analyzed by
nondenaturing 4% polyacrylamide gel electrophoresis. (B) Three hundred
nanograms each of MyoD protein and 20 ng of E47N (a truncated form of
E47 with a deletion in the N-terminal region) protein, together or
separately, were incubated at 37°C for 15 min prior to DNA binding
reactions carried out at room temperature for 15 min.
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With assays for myogenic activity, we found that the A114N mutant
dramatically loses the ability to promote myogenesis in
transiently
transfected NIH 3T3 cells: it produced 1.5% conversion,
in contrast to
30% conversion by MyoD. That the A114N protein
has retained any
residual myogenic activity may result from the
heterogeneous
conformation in this molecule (see below).
A114N mutation alters the conformation of the basic region.
To
examine the consequence of the A114N mutation for the conformation of
Arg-111, the critical residue, we made use of the fact that the basic
region of MyoD (which includes Arg-110 and Arg-111) happens to be a
processing-site motif for subtilisin (2). We tested the
subtilisin sensitivities of wild-type and mutant MyoD proteins (Fig.
3). Wild-type MyoD contains a core domain
(consisting of the bHLH domain [unpublished result]) that is
resistant to protease digestion in the presence of DNA, but in the
A114N mutant molecule the core domain is much more sensitive to
protease, even in the presence of DNA. These observations suggest that
the basic domains of the mutant and wild-type proteins have different
conformations. Although other possibilities remain, the simplest
explanation is that in the mutant, unlike in the wild-type MyoD,
revealed in the crystal structure (21), Arg-111 is no longer
protected by DNA and therefore may not have direct contact with DNA.
Accordingly, it is possible that in the A114N mutant Arg-111 is outside
the major groove most or all of the time, due to the bulkiness of Asn
at the 114 position, and thus becomes accessible for protease
digestion.
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FIG. 3.
Gel retardation assay. Six hundred forty nanograms of
bacterially purified MyoD and A114N proteins were treated with
subtilisin (50 ng/ml) both in the presence and in the absence of 0.5 ng
of 1R DNA probe. pre, proteins were treated with subtilisin at 37°C
for 1 min, the reaction was stopped by addition of PMSF, and then the
proteins were incubated with 1R probe at 30°C for 30 min; post,
proteins were incubated with 1R probe at 30°C for 30 min and then
treated with subtilisin at 37°C for 1 or 10 min, and the reaction was
stopped by addition of PMSF. Samples were analyzed by nondenaturing 4%
polyacrylamide gel electrophoresis.
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This model is consistent with the results of molecular modeling
analysis with the determined structural coordinates of the
MyoD-DNA
cocrystal (
21), which reveals that an Asn placed in
the 114 position would impinge on Arg-111 (see Fig.
5, upper panel),
and
suggests that Asn at 114 might in reality force Arg-111 to
remain
out of the major groove, similar to the situation in the
E47-DNA
cocrystal (
12,
21). In contrast, molecular modeling
with the nondeleterious T115N mutant did not suggest such friction
within the crystal structure. The final proof of the validity
of this
model must await X-ray crystallographic data for the mutant(s).
One prediction of the above-described model is that smaller amino acids
at position 114 would accommodate Arg-111 in the major
groove and allow
for myogenic activity. Thus, we generated the
A114G mutant. As is shown
in Table
1, it failed to induce myogenic
conversion, nor could it
activate HCA, MCK, or 4R reporter constructs.
Since glycine is known to
be a frequent "
-helix breaker," we
speculate that it probably
prevents the basic region from assuming
the
-helical structure
induced by cognate DNA sequences as occurs
normally. Interestingly,
when we compared the residues flanking
Gly114 to the
-helix
termination motifs deduced from protein
folding studies (
1),
we found that the MyoD sequences perfectly
matched the Schellman
motif
with Met-116 at the C" position of
the motif (requiring
an apolar residue), Arg-110, Arg-111, and
Lys-112 at C-4, C-3, and
C-2 (requiring apolar Lys or Arg), and
Ala-113 at C-1
(requiring a polar amino acid or Ala).
As a direct test of this model we attempted to obtain second-site
mutations at position 111 that could suppress the myogenic
defect in
the A114N mutant. To ensure the presence of all 20 possible
amino
acid substitutions and to increase the throughput of the
screening, we
used PCR to make four libraries of site-directed
random mutations at
Arg-111 by using GNN, ANN, TNN, and CNN in
place of the Arg codon. Each
library has a limited complexity
of 16, encoding five to seven
different amino acids, and is represented
by more than 200 independent
transformants. Thus, the confidence
of each different codon being
present in the library is 99.999%
(
7). We collected
transformants from each of the four libraries
as a pool, transfected
each pool of plasmid extracts into NIH
3T3 cells along with the
HCA-
-Gal reporter plasmid, and assayed
for transcriptional
activation of the HCA promoter and myogenic
conversion. If a library
contained a mutation that can restore
myogenic activity to A114N, it
should have been easily detectable
in this assay, because NIH 3T3
cells transfected by A114N plasmid
spiked with 1% wild-type MyoD
plasmid showed a level of activation
of the HCA-
-Gal reporter
significantly above the background level
produced by the A114N plasmid
alone, and muscle-like morphological
changes of the transfected cells
were visible. However, none of
the four libraries showed elevated
transcriptional activity or
myogenic conversion.
Mammalian achaete-scute homologous protein 1 (MASH1), a neurogenic bHLH
protein that is incapable of inducing myogenic conversion
but can
partially activate transcription from a single muscle
promoter, the MCK
E box (
17), contains a bulky asparagine in
its basic domain
immediately adjacent to the Arg-111 site. However,
steric
considerations preclude a direct comparison between the
basic-region
peptides of MASH1 and MyoD. MASH1 has three fewer
residues in its basic
region, as it is missing amino acids corresponding
to those at 112 to
114 of MyoD, which aligns the asparagine with
T-115 of MyoD. We show in
this paper that the MyoD T115N mutation
not only transactivates but
also allows myogenesis. It is possible
that the activation of the MCK E
box by MASH1 is fortuitous, and
since activation of only one or a few
muscle genes is not sufficient
to cause myogenesis, such compromised
specificity might not have
been selected against in evolution. In fact,
that MASH1 has no
myogenic activity in vivo is most revealing.
These results suggest that the mechanism of myogenic activation at work
in MyoD involves the ability of Arg-111 to lie within
the major
groove and make direct contact with the guanine of the
E box. Since in
all the revertants we created with various amino
acids at position 111 there is still a bulky asparagine at position
114, it is unlikely that
any amino acids at position 111 can make
such a contact. Thus, it is
not surprising that these mutants
remain transcriptionally inactive.
Furthermore, since Arg-111
is invariant throughout all known bHLH
proteins, mutating it likely
abolishes basic-region structure
and/or DNA binding. In fact,
some mutations of MyoD (
8)
and E47 (
33) affecting this Arg
have been shown to
abolish DNA binding.
In the A114N mutant the altered basic-region conformation is
propagated to the whole molecule to affect transcriptional
activation.
In the basic region of the A114N mutant, an altered
conformation which does not alter its DNA-binding specificities might lead to a transactivation defect in at least two ways. First, the
altered basic-region conformation might lead to a profound change in
the conformation of the whole molecule that affects the function of the
activation domain(s). Second, the altered basic-region conformation
might disrupt binding to the basic region of a recognition factor, such
as myocyte enhancer factor 2C (MEF2C) (23). These two
mechanisms do not need to be mutually exclusive.
To investigate whether the basic-region mutation affects the
conformation of the whole molecule, we attempted to determine
whether the transcription activation domain in the A114N mutant
behaves differently than it does in MyoD by using two different
assays. First, we fused full-length copies of either MyoD or the
A114N mutant to the DNA-binding domain of Gal4 to generate Gal-MyoD
and
Gal-A114N and cotransfected each with a LacZ reporter gene
driven
by five Gal4 DNA binding sites into NIH 3T3 cells. Interestingly,
Gal-A114N activated transcription 10 times better than Gal-MyoD
(Table
2), even though the fusion proteins were
expressed and
appeared to bind to Gal4 DNA sites equally well
(data not shown),
indicating that the different conformation in
the basic region
of the A114N mutant affects the conformation and/or
the accessibility
of the activation domain(s). Secondly, we tested
the ability of
the activation domain in the A114N mutant to
interact with molecules
known to coactivate the MyoD activation domain.
It has been shown
previously that the MyoD coactivator p300 can target
both the
N-terminal activation domain (
28) and the bHLH
domain (
11)
(our unpublished data) of MyoD. There was no
detectable difference
between wild-type MyoD and the A114N mutant in
the ability to
bind to glutathione
S-transferase-p300 (data
not shown). In addition,
dTAF
II40 can also serve as a
coactivator for MyoD, and it targets
the amino-terminal
activation domain of MyoD (unpublished data).
The binding to
6His-TAF
II40 of MyoD was comparable to that of
A114N protein (data not shown). We found that while both p300
and
dTAF
II40 can potentiate the activation of myogenic
promoters
by MyoD in vivo, they do not potentiate the residual
activation
by the A114N mutant (Fig.
4B).
Thus, the altered conformation
of the basic domain of the mutant MyoD
protein appears to be propagated
to the activation domain at the N
terminus, rendering the molecule
incapable of responding to the
coactivators. These effects are
independent of any role of cofactors
such as MEF2C (see below).
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FIG. 4.
The A114N mutant fails to respond to coactivators of
MyoD. (A) Schematic maps of the MyoD wild-type and mutated peptides
used to acquire the data shown in panels B, D, and E.  , amino-terminal activation domain;
 , DNA binding domain;  , HLH
dimerization domain. The grey box and white box do not symbolize any
specific functional domains. (B) Residual transactivation activity of
the A114N peptide cannot be potentiated by p300 or TAFII40;
1.5 µg of each plasmid was transfected. Vec, vector; N,
N-MyoD~(1-75). (C) The A114N mutant and MyoD interact with MEF2C
equally well. Proteins synthesized by in vitro
transcription-translation were mixed and immunoprecipitated with an
antibody against the carboxyl terminus of MyoD. (D) Interaction of
A114N with MEF2C [GalM2C(1-174)] is not reconstituted in a mammalian
two-hybrid assay; 1 µg of each plasmid was used. (E) The A114N mutant
fails to transactivate even when supplemented with VP16 activation
domain. All transfections were done in NIH 3T3 cells and repeated at
least three times; 2 µg of each plasmid was used. N-MyoD and
N-A114N, MyoD and A114N proteins each lacking N-terminal aa 3 to 56;
N-MyoD~VP16, N~MyoD with VP16 activation domain (aa 412 to
490) inserted between MyoD residues 173 and 174; N-MyoD~(1-75)
and N-A114N~(1-75); N-MyoD and N-A114N with MyoD activation
domain (aa 1 to 75) inserted between MyoD residues 173 and 174.
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MEF2 proteins are known to be cofactors for myogenic bHLH proteins
(
18,
23). To test whether the basic-region conformation
of
the A114N mutant affects binding of MEF2C, we first examined
whether
MEF2C can be coimmunoprecipitated with the A114N protein.
Either wild-type or A114N mutant MyoD was mixed with E47 protein
and
MEF2C protein and immunoprecipitated with an antibody against
the
carboxyl terminus of MyoD (Santa Cruz). All of the proteins
were
synthesized with an in vitro transcription-translation reagent
(Promega). The wild-type protein and A114N mutant interact with
MEF2C
equally well in this assay (Fig.
4C). In a mammalian two-hybrid
assay,
MEF2C coding sequences were fused to the Gal4 DNA binding
domain, while
regions of MyoD or the A114N mutant were fused to
the VP16 activation
domain; these constructs were cotransfected
with a luciferase reporter
gene driven by five Gal4 DNA binding
sites. We found that MyoD
reconstituted the two-hybrid activation,
presumably as a heterodimer
with endogenous E proteins, as it
was reported that E12 was required to
support MyoD-MEF2C interaction
(
23), whereas the A114N
mutant failed to do so (Fig.
4D). We
obtained similar results with E12
overexpression (data not shown).
Together, these data indicate that
although MEF2C and the A114N
mutant interact, the mutant locks up the
VP16 activation domain
(see below). Since MEF2C does not differentiate
between wild-type
MyoD and the A114N mutant, it is unlikely that MEF2C
would be
the postulated recognition factor (if there is any) that
interprets
the myogenic code in the basic region of MyoD. This is
consistent
with the fact that the MyoD-E12basic mutant can also
interact
with MEF2C in the presence of E12 (
4).
Furthermore, the addition of the VP16 activation domain to the A114N
mutant cannot rescue its diminished transactivation of
muscle specific
promoters (Fig.
4C), suggesting that, in the presence
of the A114N
mutation, neither the wild-type activation domain
nor a substitution
VP16 activation domain is functional. Note
that this result is not to
be confused either with the original
finding that addition of the VP16
activation domain to the A114I
mutant and the MyoD-E12basic protein
restored some of their activities
on an artificial, multimerized single
E-box promoter, 4R-TK, (
9,
34) or with recent results that
confirmed such events on the
artificial multimerized E-box promoter
(reference
4 and our
unpublished data). The 4R-TK
promoter can be activated equally
well by several nonmyogenic bHLH
proteins, including E12, and,
as such, does not appear to represent an
appropriate reporter
for assaying conformational requirements of
myogenic activation.
In fact, although addition of the VP16 activation
domain rescues
the basic-region mutants on a 4R-TK transcription
reporter, its
addition to the same mutant MyoD molecules fails to
rescue them
when myogenesis is used as an assay. In these instances it
is
possible that the A114I and MyoD-E12 basic proteins exhibit lowered
affinity or cooperativity in binding to the 4R-TK promoter. However,
even such low-level DNA interactions might well be compensated
by the
addition of the VP16 activation domain, whereas changes
of the
conformation of the protein in the A114N mutant are not
restorable by
addition of the VP16 activation domain. Thus, the
activation domain
cannot function properly unless there is an
intact myogenic code in the
basic region. Upon binding to the
proper E-box DNA sequences the
myogenic code determines the proper
conformation of at least two
separated domains of the molecule:
the centrally located basic domain
and the amino-terminal activation
domain (Fig.
5C and D). In this light, it is important
to note
that when it is fused to Gal4, the basic domain is an extremely
potent inhibitor of the N-terminal activation domain and that
with the
basic domain deleted, the activity of the activation
domain fused to
Gal4 increases remarkably (Table
2). This mechanism
ensures that
the activation domain is unmasked only upon DNA binding
by the
correct basic region. bHLH proteins exhibit specificity,
but
specificity based upon primary sequences is clearly not enough
(
5,
16,
24). Thus, cells likely use this
clever unmasking
strategy to control both the
activity and the specificity of transcription
factors. Such
a mechanism may have evolved to restrict myogenic
capability
and specificity to a small number of bHLH proteins
among many
with diverse functions yet with DNA binding specificities
known
to be similar.
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FIG. 5.
(A and B) Molecular modeling reveals different
basic-domain conformations in wild-type MyoD (A) and the A114N mutant
(B). -Carbon traces of residues 111 and 114 are presented. Note the
drastically different conformations of Arg-111. (C and D) Schematic
representation of changes in MyoD structure following binding to DNA.
Binding of MyoD basic domain (striped area) to a target DNA site (C) is
associated with a distinctive change of conformational structure that
is propagated to the activation domain (AD), allowing interaction with
transcriptional coactivators (labeled as "C" in panel D). In panel
C, the AD is sketched as interacting with the basic domain, an
inference drawn from the inhibitory effects of basic domain elements
shown in Table 2. DNA binding by the A114N mutant also keeps the AD in
the conformation shown in panel C.
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Intramolecular conformational regulationa general
theme?
Cellular factors in trans have previously
been reported to negatively target the activation domains of
transcriptional activators to affect cell physiology, e.g., Gal80
masking of the Gal4 activation domain when yeast cells grow in glucose
medium (20) and MDM2 oncoprotein masking of the activation
domain of the tumor suppressor protein p53 in certain tumors (22,
25). In cis, the unliganded steroid binding domain of
glucocorticoid receptor (GR) was reported to inhibit both DNA binding
and transcriptional activation by GR (14, 26). However,
masking in cis of an activation domain by the DNA binding
domain has not been documented, although it has been conjectured from
previous work done with GR (19) and MyoD (35).
Here we report an example of such intramolecular masking, mediated by
the DNA binding domain of the activator, as a means of regulating the
activation domain function and executing tissue specificity. This is
also the first demonstration of a conformational difference in the DNA
binding domain of a transcriptional regulator directly affecting the
conformation and activity of the transcriptional activation domain. It
is reminiscent of the properties of the yeast pheromone-receptor
transcription factor, which changes conformation upon binding to
a-specific but not to -specific genes (31). It
will be interesting to find out whether this strategy is also employed
by other transcription factors that play crucial roles in controlling
cell proliferation and differentiation.
| |
ACKNOWLEDGMENTS |
We thank D. Livingston, R. Tjian, L. Comai, G. Gill, and E. Olson
for providing reagents, L. Nekludova and C. Pabo for a theoretical analysis of A114N mutant structure, and M. Stallcup, K. Yamamoto, and
S. Tan for advice and discussion.
This work was supported in part by grants (to L.K.) from the National
Institutes of Health.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute for
Genetic Medicine and Department of Biochemistry and Molecular
Biology, University of Southern California School of Medicine, 2250 Alcazar St., Los Angeles, CA 90033. Phone: (323) 442-1145. Fax: (323) 442-2764. E-mail: kedes{at}hsc.usc.edu.
J.H. dedicates this paper to the memory of Man-Hua Wu.
| |
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Molecular and Cellular Biology, September 1998, p. 5478-5484, Vol. 18, No. 9
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
