Basic helix-loop-helix (bHLH) transcription
factors often function as heterodimeric complexes consisting of a
tissue-specific factor such as SCL/tal or MyoD bound to a broadly
expressed E protein. bHLH dimerization therefore appears to represent a
key regulatory step in cell lineage determination and oncogenesis. Previous functional and structural studies have indicated that the well
defined HLH domain is both necessary and sufficient for dimerization.
Most of these studies, however, have employed in vitro
systems for analysis of HLH dimerization, and their implications for
the requirements for in vivo dimerization remain unclear. Using multiple approaches, we have analyzed bHLH dimerization in
intact, living cells and have identified a novel domain in E proteins,
domain C, which is required for in vivo dimerization. Domain C, which lies just carboxyl-terminal to helix 2 of the HLH
domain, represents the most highly conserved region within E proteins
and appears to influence the in vivo conformation of the
adjacent HLH domain. These results suggest that HLH dimerization in vivo may represent a complex, regulated process that is
distinct from HLH dimerization in vitro.
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INTRODUCTION |
For several classes of transcription factors, such as members of
the basic helix-loop-helix, leucine zipper, and nuclear receptor families, dimerization represents a key, obligatory step prior to DNA
binding and transcriptional activation. This dimerization permits the
mixing and matching of factors with different DNA half-site binding
specificities and expands the repetoire of potential target sequences
that may be recognized. Basic helix-loop-helix (bHLH)1 factors in metazoan
organisms often regulate the expression of target genes as
heterodimeric complexes between tissue-specific factors, such as
SCL/tal or MyoD, and broadly expressed E proteins (1-3). The
composition of bHLH complexes is dictated in part by the dimerization
specificities of the constituents; in particular, tissue-specific bHLH
factors tend not to interact with one another but rather to bind
universally to E protein partners (4, 5). Likewise, the dominant
negative HLH proteins of the Id family exert their inhibitory effect
through preferential binding to E proteins (6-8). Thus a progenitor
cell during embryogenesis may contain an array of different cell
lineage-specific bHLH factors all competing with one another, as well
as with inhibitory HLH proteins, for dimerization with a common E
protein partner. Such competition would allow the progenitor cell to
make mutually exclusive, binary decisions with regard to lineage
commitment, proliferation, and terminal differentiation (2).
The structural basis for bHLH dimerization, according to x-ray
crystallography, resides in the formation of a parallel, four-helix bundle in which dimerization contacts derive from conserved hydrophobic residues clustered within a shielded core (9-11). However, the crystallographic structures have all been obtained with pre-formed, DNA-bound complexes and provide no information on the transition states
that occur in the process of dimerization in solution. Furthermore, the
data from the crystal structures provide no satisfactory explanation
for dimerization specificities: all HLH factors possess similar
hydrophobic dimerization contact residues, and yet tissue-specific bHLH
factors show highly restricted dimerization specificities while E
proteins demonstrate considerable promiscuity in dimerization. In
vitro biochemical studies suggest that nonconserved hydrophilic residues may somehow contribute to HLH dimerization specificity (12).
As an additional complication, in vitro dimerization of bHLH
factors does not appear accurately to reflect the dimerization process
in vivo. In most analyses, in vitro bHLH
dimerization, whether with crude extracts or with purified proteins, is
a highly inefficient process which requires subphysiologic temperatures and displays affinities in the micromolar range (13-16). Studies of
in vivo dimerization portray a process that appears to be
extremely efficient, with rapid heterodimerization occurring just prior to nuclear localization (17, 18).
To study factors influencing dimerization in vivo, we have
exploited a number of established systems applicable to bHLH
dimerization, including yeast two-hybrid, mammalian two hybrid, nuclear
redirection assays, and coimmunoprecipitation (4, 17-20). We found
that, contrary to findings in vitro, bHLH domains are not
sufficient for dimerization in vivo. In particular, E
proteins require an additional highly conserved domain, domain C,
located carboxyl-terminal to helix 2. Tissue-specific bHLH factors, by
contrast, require only the bHLH domain for heterodimerization with E
proteins. Therefore, intrinsic structural differences exist between E
proteins and tissue-specific bHLH factors, differences which may
explain their different dimerization specificities. In particular, the
role of domain C appears to be as an in vivo conformational
determinant, maintaining the bHLH domain of E proteins in a
"receptive" conformation for heterodimerization with
tissue-specific bHLH proteins.
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MATERIALS AND METHODS |
Plasmid Constructions--
Expression of LexA fusion proteins in
yeast employed the vector pEG202, kindly provided by Dr. Roger Brent
(Massachusetts General Hospital, Boston, MA) (21). Expression of B42
activation domain fusion proteins in yeast employed the vector pJG45,
also kindly provided by Dr. Roger Brent. PCR upon plasmid templates was
used to generate DNA fragments encoding the following: the bHLH domain
of SCL/tal (amino acids 186-242), the 20-kDa naturally occurring
isoform of SCL/tal (amino acids 176-331) (22), the bHLH domain of MyoD
(amino acids 108-163), the full-length coding region of MyoD, and the
various truncation mutants of E2-2 (encoding amino acids 467-588,
484-541, 467-541, 484-588, 484-563, and 484-548). PCR fragments
were cloned in-frame into pEG202 and pJG45 as
EcoRI-XhoI fragments. pJG-Id1 and pJG-Id2, yeast
expression plasmids encoding Id1 and Id2 as B42 fusion proteins, were
previously isolated from a HeLa cDNA library in the vector pJG45
(library provided by Brent laboratory). For mammalian expression of the
VP16 activation domain alone or fused to SCL/tal (amino acids
176-331), we used the plasmids pVP-HA1 and pVP16-TAL1, respectively,
both generously provided by Dr. Richard Baer (University of Texas
Southwestern Medical Center, Dallas, TX) (4). For mammalian expression
of the GAL4 DNA-binding domain fusion proteins, our laboratory
generated a parent vector pCMV-DB which contains the GAL4 DNA-binding
domain downstream of the CMV immediate early promoter. The starting
vector consisted of pCMV5 (23) from which the EcoRI site had
been eliminated, yielding pCMV5 R-. A
HindIII-PstI fragment encoding the GAL4
DNA-binding domain was released from the yeast expression vector pGBT9
(CLONTECH, Palo Alto, CA) and ligated into the
corresponding sites in pCMV5 R-. E2-2 fragments (encoding amino acids
484-541 and 484-563) with EcoRI-XhoI ends were
cloned into EcoRI-SalI sites of pCMV-DB, yielding
plasmids with in-frame GAL4-E2-2 fusions, pCMV-DB-E2-2 bHLH, and
pCMV-DB-E2-2 C. The G5E1bLUC reporter plasmid, with 5 GAL4-binding
sites upstream of the E1b TATA sequence followed by the luciferase
reporter gene, was generously provided by Dr. Richard Baer and has been
described elsewhere (4). pEMSV-MyoD, kindly provided by the laboratory
of Dr. Harold Weintraub (Fred Hutchinson Cancer Research Institute,
Seattle WA), was used for mammalian expression of the full-length MyoD
protein with its own activation domain. The mammalian expression
plasmid for the nuclear localization-deficient mutant of SCL/tal, pCMB,
has been previously described (24). For bacterial expression of MBP and GST fusion proteins, SCL/tal (encoding amino acids 176-331) and E2-2
(encoding amino acids 484-541 or 484-563), were cloned as EcoRI-XhoI fragments into pMAL-c2 (New England
Biolabs, Beverly, MA) and pGEX4T-1 (Pharmacia Biotech, Piscataway,
NJ).
Yeast Two-hybrid Techniques--
The yeast two-hybrid system
developed in the laboratory of Roger Brent was employed as we have
previously described (21, 25). The yeast strains (EGY48 and YPH499),
mating protocols, library screening protocols, and
-galactosidase
assays have all been described in an earlier publication (25). For
Western blot analysis of yeast expression of LexA fusion proteins,
equivalent quantities of yeast grown to mid-log phase in CM-URA, -HIS,
and -TRP media with 2% galactose, 1% raffinose were resuspended in SDS-PAGE loading buffer and boiled. Resultant Western blot membranes were probed with a rabbit polyclonal antibody to LexA, provided by Dr.
Erica Golemis (Fox Chase Cancer Center, Philadelphia, PA).
Library of Randomly Mutated SCL/tal--
Error-prone PCR
amplification of the bHLH encoding region of SCL/tal included 0.25 mM MnCl2 in a standard PCR reaction (with 2.5 mM MgCl2 and 200 µM dNTPs). After
30 cycles of mutagenic amplification, 0.1 µl out of a 100-µl
reaction was subjected to a second 30 cycles of mutagenic
amplification. Similarly, 0.1 µl of the latter reaction was subjected
to a third round of 30 cycles of mutagenic amplification. Equal
quantities of PCR products resulting from 30, 60, and 90 cycles of
mutagenic amplification were pooled and cloned into the
EcoRI-XhoI sites of pJG45. A library of 2 × 106 primary bacterial colonies was thereby generated. A
plasmid preparation of this library was then transformed into the yeast
strain YPH499, yielding 1 × 106 primary yeast
colonies.
Mammalian Two-hybrid Assays--
K562 cells in mid-log phase
were resuspended at a concentration of 1 × 106
cells/ml in RPMI 1640 media with 5% fetal bovine serum. For each transfection, 1.6 × 106 cells were combined with 6 µg of DNA and 30 µl of DOTAP (Boehringer Mannheim, Indianapolis,
IN) premixed in 400 µl of Hepes-buffered saline. The 6 µg of DNA
consisted of 2 µg of GAL4 expression plasmid (pCMV-DB, pCMV-DB-E2-2
bHLH or pCMV-DB-E2-2 C), 2 µg of activation domain expression plasmid
(pVPHA1, pVP16-TAL1, or pEMSV-MyoD), and 2 µg of the G5E1bLUC
luciferase reporter plasmid. After overnight incubation, the cells were
resuspended in fresh RPMI 1640 with 10% fetal bovine serum and
cultured for an additional 24 h. To assay cells for luciferase
activity, the Luciferase Assay System kit (Promega, Madison WI) was
used, following the manufacturer's recommendations. In all cases,
equivalent numbers of cells were harvested for luciferase assays. Light
emission was measured on a Berthold Lumat LB 9501 luminometer (Berthold
Systems Inc., Pittsburgh, PA).
Immunofluorescent Nuclear Redirection Assay--
COS 7 cells
were seeded on glass coverslips in RPMI 1640 with 5% fetal bovine
serum at a density of 4 × 105 cells per
22-mm2 coverslip. Transfections were carried out overnight
with 5 µg of plasmids and 25 µl of DOTAP (Boehringer Mannheim) per
coverslip. The plasmids consisted of 2.5 µg of pCMB plus 2.5 µg of
GAL4 expression plasmid (pCMV-DB, pCMV-DB-E2-2 bHLH, or pCMV-DB-E2-2
C). Cells were incubated in fresh RPMI 1640 with 10% fetal bovine
serum for 72 h prior to fixation. The protocols for cell fixation
and indirect immunofluorescent staining for SCL/tal have been
previously described (17). The cells were visualized on an MRC-600
confocal laser scanning imaging system (Bio-Rad Molecular Bioscience
Group, Hercules, CA).
Coimmunoprecipitation Assay--
COS 7 cells grown to ~60%
confluency in 75-cm2 flasks were transfected with 5 µg of
pCMV-SCL/tal, an expression vector for full-length SCL/tal protein
(amino acids 1-331) (24). In addition, the cells received 5 µg of
either pCMV-DB-E2-2 bHLH or pCMV-DB-E2-2 C. Transfections were
accomplished overnight in Dulbecco's modified Eagle's medium with 5%
neonatal calf serum using 50 µg of DOTAP (Boehringer Mannheim) plus
10 µg of plasmid per flask. After supplying the cells with fresh
media consisting of Dulbecco's modified Eagle's medium with 10%
neonatal calf serum, the cells were incubated an additional 4 days
prior to harvesting. For harvesting, cells were gently scraped in room
temperature phosphate-buffered saline with 5 mM EDTA. Cell
pellets were then resuspended in 300 µl of ice-cold NETN (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 2 µg/ml leupeptin, 2 µg/ml
pepstatin, 0.2% aprotinin, and 200 µM
phenylmethylsulfonyl fluoride). After a 10-min incubation on ice with
intermittent inversion, insoluble cellular debris was eliminated by
pelleting. 50-µl portions of the extracts were saved for direct
immunoblot analysis. To the remaining 250 µl of extracts, 0.5 µg of
rabbit anti-GAL4 DNA-binding domain antibody (number sc-577, Santa Cruz
Biotechnology, Santa Cruz, CA) was added followed by incubation on ice
45 min with intermittent inversion. A 10-µl packed volume of protein
A-agarose beads, prewashed in phosphate-buffered saline with 1% bovine
serum albumin, were then added to each tube, followed by rotation at 4 °C for 30 min. The protein A-agarose beads were then washed 4 times with ice-cold NETN and resuspended in 50 µl of SDS-PAGE loading
buffer.
Immunoprecipitates and crude cellular extracts were then subjected to
SDS-PAGE on 12% gels followed by electrotransfer to nitrocellulose
membranes. Immunoblots were probed with the BTL-73 mouse monoclonal
anti-SCL/tal antibody that was kindly provided by Karen Pulford
(Oxford, United Kingdom) (26). BTL-73 was used as a one-half dilution
of a tissue culture supernatant. In addition, crude extracts from COS
cell transfectants were probed in parallel with the rabbit anti-GAL4
antibody at a 1/500 dilution (0.2 ng/ml). Western blots were otherwise
carried out as described previously (24).
Surface Plasmon Resonance Analysis--
Production and
purification of bacterially expressed GST and MBP fusion proteins
followed previously described protocols (5). Surface plasmon resonance
studies employed a BIAcore biosensor device (Pharmacia Biosensor,
Piscataway, NJ) and followed previously described guidelines (27). In
particular, GST-E2-2 fusion proteins (encoding E2-2 bHLH (amino acids
484-541) or E2-2 C (amino acids 484-563)) diluted in 10 mM sodium acetate (pH 4.3) were covalently linked to the
carboxymethyl-dextran hydrogel matrix on a CM5 flow cell by the
standard amine coupling procedure described by the manufacturer
(Pharmacia Biosensor, Piscataway, NJ). For GST-E2-2 bHLH, 1800 resonance units were immobilized, and for GST-E2-2 C, 1700 resonance
units were immobilized. The soluble analyte, MBP-SCL/tal176-331 was used at a concentration of 4 µM in protein interaction buffer (20 mM
Tris-HCl, pH 8, 100 mM NaCl, 2.5 mM
MgCl2, 0.5 mM dithiothreitol, 100 µg/ml
bovine serum albumin, and 0.005% p20 surfactant). Just prior to
analysis, analyte preparations were centrifuged through
Microsep® 50,000 Mr cut-off
microconcentrators (Filtron Technology Corp., Northborough, MA) to
remove aggregates. The analyte was then flowed over the immobilized
ligands at a rate of 5 µl/min.
 |
RESULTS |
E2-2, but Not SCL/tal or MyoD, Requires Sequence Outside the bHLH
Domain for in Vivo Dimerization--
The minimal bHLH domains of E2-2,
SCL/tal, and MyoD have been well defined by alignment analysis of a
broad range of bHLH proteins from a wide variety of organisms (28). To
assay interaction of minimal bHLH domains in the yeast two-hybrid
system the amino acid sequences indicated in Fig.
1 were expressed in yeast as fusions with
either the LexA DNA-binding domain or the B42 transcription activation
domain. Interaction within yeast between coexpressed LexA fusions and
B42 fusions was reflected by activation of a
-galactosidase reporter
gene containing upstream LexA-binding sites. As shown in Fig.
2, LexA fusions with the minimal bHLH domains of MyoD and SCL/tal (LexA-MyoD bHLH and LexA-SCL/tal bHLH) displayed specific interaction with a larger fragment of E2-2 encompassing the carboxyl-terminal 121 amino acids (E2-2 467-588) fused to the B42 activation domain. Surprisingly, LexA-MyoD bHLH and
LexA-SCL/tal bHLH manifested no interaction with the minimal bHLH
domain of E2-2 (amino acids 484-541) fused to B42. As will be shown
below, similar results were obtained with E2-2 as the LexA fusion
component and were not attributable to poor expression of E2-2 bHLH
fusion proteins in yeast. These data suggest that the structural
requirements for dimerization in the yeast two-hybrid system differ for
tissue-specific bHLH proteins as compared with E proteins, the former
requiring only the minimal bHLH domain and the latter requiring
additional sequence.

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Fig. 1.
Alignment of the bHLH domains of E2-2, MyoD,
and SCL/tal. The sequences shown constitute the minimal bHLH
domains utilized in the yeast two-hybrid analyses shown in Fig. 2.
Alignments were performed using the program SeqVu 1.1.
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Fig. 2.
Relative quantitation of HLH interactions
using the yeast two-hybrid system. LexA DNA-binding domain fusions
were coexpressed in yeast with B42 activation domain fusions. Protein
interaction was reflected by activation of the LexA operator
-galactosidase reporter plasmid pSH18-34. LexA fusions include: LexA
only as a negative control, LexA fused to the minimal bHLH domain of
MyoD (as depicted in Fig. 1), and LexA fused to the minimal bHLH domain of SCL/tal (as depicted in Fig. 1). B42 fusions include: B42 fused to
E2-2 467-588 which includes the bHLH domain as well as flanking amino
acids, and B42 fused to the minimal bHLH domain of E2-2 (as depicted in
Fig. 1). Liquid -galactosidase assays were performed on three
separate occasions. Results shown are mean ± S.E.
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Requirement for Domain C for in Vivo Heterodimerization of
E2-2--
To identify additional sequence requirements for E2-2
heterodimerization, a number of E2-2 truncations were expressed in
yeast as LexA fusion proteins (Fig.
3A). These LexA-E2-2
truncations were analyzed for interaction with B42 fusions containing
the following HLH proteins: Id1, Id2, SCL/tal, and MyoD. As shown in
Fig. 3B, domain C, a stretch of 22 amino acids
carboxyl-terminal to the HLH domain (amino acids 541-563) is both
sufficient and necessary for heterodimerization of the E2-2 bHLH domain
with an array of HLH partners. Interestingly, domain A, an acidic
region upstream of the bHLH domain, which has been implicated in
selective heterodimerization of E12 with MyoD (29), had no influence on the heterodimerization of E2-2 with the various HLH partners.

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Fig. 3.
Yeast two-hybrid analysis of E2-2 truncation
mutants binding to an array of HLH partners. E2-2 truncation
mutants fused to the LexA DNA-binding domain were coexpressed in yeast
with an array of B42 activation domain fusion proteins. Full-length coding sequences were employed for expression of Id1, Id2, and MyoD
fusions. The SCL/tal fusion encoded amino acids 176-331, which
constitutes the naturally occurring 20-kDa isoform of SCL/tal. Interactions were quantitated by liquid -galactosidase assays. Panel A graphically depicts the E2-2 truncation mutants used
as LexA fusions, indicating the E2-2 amino acids contributing to the
fusion proteins. Also depicted are the relative positions of domain A,
the basic domain, the HLH domain, and domain C. Panel B
depicts the relative -galactosidase activity associated with each
LexA fusion/B42 fusion combination.
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Domain C Influences the Conformation of the bHLH Domain in
E2-2--
To study further the role of domain C in the
heterodimerization of E2-2, a number of control experiments were
performed. First, Western blot analysis of LexA-E2-2 fusion expression
in yeast demonstrated insufficient differences in expression levels of the various truncation mutants to account for the differences in
interaction patterns (Fig.
4A). For example, LexA-E2-2
467-541 (Fig. 4A, lane 3) showed the same levels of
expression as LexA-E2-2 484-563 (Fig. 4A, lane 5), but only
the latter LexA fusion demonstrated heterodimerization with the various
HLH partners (Fig. 3B). Second, we isolated two independent
altered-specificity mutants of SCL/tal, SE1 and SS1, that exclusively
recognized forms of E2-2 lacking domain C (Fig. 4B). These
mutants were isolated from a library of randomly mutated SCL/tal bHLH
domains using a yeast two-hybrid screen for interaction with the
minimal bHLH domain of E2-2 fused to LexA. The selectivity of SCL/tal
mutants SE1 and SS1 for E2-2 lacking domain C rules out any trivial
explanations for the role of domain C, i.e. nuclear
localization or permitting DNA binding by LexA. In fact, these data
indicate that domain C influences the conformation of the E2-2 bHLH
domain such that the presence of domain C permits exclusive
heterodimerization with wild type HLH partners and the absence of
domain C permits exclusive heterodimerization with the SCL/tal mutants
SE1 and SS1.

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Fig. 4.
Panel A, Western blot analysis of
expression of LexA-E2-2 truncation mutants (graphically depicted in
Fig. 3A). The amino acids contributed by E2-2 are indicated
above each lane. Strains used for two-hybrid assays were
lysed and subjected to standard Western blotting using a rabbit
anti-LexA antibody. Panel B, yeast two-hybrid analysis of
interactions between LexA-E2-2 truncation mutants and B42 fusions with
the SCL/tal bHLH mutants SE1 and SS1. Interactions were quantitated by
liquid -galactosidase assays. The amino acids contributed by E2-2
are indicated in bold. The SCL/tal bHLH mutants SE1 and SS1
are altered specificity mutants obtained from screening a library of
random mutants for yeast two-hybrid interaction with the E2-2 minimal
bHLH domain.
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Domain C Is Required for in Vivo Dimerization in Mammalian
Cells--
To extend the findings in the yeast two-hybrid system,
three independent assays were employed to analyze in vivo
HLH dimerization in mammalian cells. In the mammalian two-hybrid
system, K562 cells were transiently co-transfected with vectors
expressing fusions with the GAL4 DNA-binding domain and with the VP16
activation domain. Also included in the transfection was the GAL5E1bLUC
reporter plasmid which contains GAL4-binding sites upstream of the
luciferase gene. As has been previously described, interaction of GAL4
fusions with VP16 fusions activates expression of the luciferase
reporter gene (30). As shown in Fig.
5A, a GAL4 fusion with the
minimal E2-2 bHLH domain (E2-2 amino acids 484-541) showed no
interaction above background with a VP16-SCL/tal fusion. However,
inclusion of domain C in the GAL4-E2-2 fusion (E2-2 amino acids
484-563) permitted interaction with VP16-SCL/tal. In Fig.
5B, GAL4 fusions were coexpressed with full-length MyoD.
Because MyoD has its own potent activation domains, it was not
necessary to express MyoD as a VP16 fusion. As with VP16-SCL/tal, MyoD
failed to interact with a GAL4 fusion with the minimal E2-2 bHLH domain
(E2-2 amino acids 484-541). As predicted, inclusion of domain C in the
GAL4-E2-2 fusion (E2-2 amino acids 484-563) restored the in
vivo interaction with MyoD.

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Fig. 5.
Mammalian two-hybrid analysis of E2-2
heterodimerization. Panel A, the VP16 activation domain
alone or fused to SCL/tal (amino acids 176-331) was coexpressed in
K562 cells with: (i) the GAL4 DNA-binding domain alone, (ii) GAL4 fused
to the E2-2 minimal bHLH domain plus domain C (E2-2 amino
acids 484-563), or (iii) GAL4 fused to the E2-2 minimal bHLH domain
minus domain C (E2-2 amino acids 484-541). Included in the
transient cotransfections was the G5E1bLUC luciferase reporter plasmid.
Interaction was reflected by luciferase activity, measured in relative
light units, in cellular extracts. Results are shown as the mean of
three independent experiments ± S.E. Panel B, the
full-length MyoD protein, using its own activation domain, was
coexpressed in K562 cells with the GAL4 fusions indicated. As in
Panel A, activation of the cotransfected G5E1bLUC
reporter plasmid was measured in RLU. As above, results are
shown as the mean of three independent experiments ± S.E.
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In the nuclear redirection assay, which has been previously described
(17, 18), COS cells were transfected with an expression plasmid for a
mutant SCL/tal which lacks a nuclear localization signal. In addition,
the COS cells were cotransfected with expression plasmids for either
E2-2 bHLH only or E2-2 bHLH with domain C, each fused to the GAL4
nuclear localization signal. Cells were then analyzed for subcellular
localization of SCL/tal by indirect immunofluorescence with confocal
microscopy. When coexpressed with only the GAL4 nuclear localization
signal, the SCL/tal mutant showed predominantly cytoplasmic
localization with perinuclear accumulation (Fig.
6A). When coexpressed with
E2-2 bHLH with domain C (E2-2 amino acids 484-563), the SCL/tal mutant
showed efficient nuclear localization as a result of its
heterodimerization with E2-2, a process referred to as nuclear
redirection (Fig. 6B). By contrast, no nuclear redirection
of mutant SCL/tal was observed with coexpression of the minimal E2-2
bHLH domain (E2-2 amino acids 484-541), indicating an absence of
heterodimerization (Fig. 6C).

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Fig. 6.
Nuclear redirection assay for interaction of
SCL/tal with E2-2 bHLH, with or without domain C. A mutant of
SCL/tal defective in nuclear localization was overexpressed in COS
cells along with the following. Panel A, the nuclear
localization signal of GAL4; panels B, E2-2 bHLH
plus domain C (E2-2 amino acids 484-563) fused to the
nuclear localization signal of GAL4; panels C, E2-2 bHLH
minus domain C (E2-2 amino acids 484-541) fused to the
nuclear localization signal of GAL4. 72 h post-transfection, COS
cells were fixed and subjected to indirect immunofluorescent analysis with a rabbit anti-SCL/tal antibody. In panels A and
C, cytoplasmic localization of SCL/tal indicates absence of
heterodimerization. Panel B shows efficient nuclear
redistribution of SCL/tal resulting from an interaction with the
coexpressed E2-2 partner.
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In coimmunoprecipitation assays (Fig. 7),
COS cells were cotransfected with expression vectors for full-length
SCL/tal (pCMV-SCL/tal) and either GAL4-E2-2 bHLH (pCMV-DB-E2-2 bHLH) or
GAL4-E2-2 C (pCMV-DB-E2-2 C). Transfectants were subjected to low
stringency immunoprecipitation with rabbit anti-GAL4 antibodies. Immune
complexes were then analyzed by immunoblot with the BTL-73 monoclonal
antibody specific for SCL/tal (26). As shown in Fig. 7, while no
detectable SCL/tal protein could be detected in complex with GAL4-E2-2
bHLH (lane 1), the 42 kDa full-length isoform of SCL/tal
could be detected in complex with GAL4-E2-2 C (lane 2). As
shown in the immunoblots in the lower panels of Fig. 7, crude extracts
from both COS cell transfectants contained similar quantities of
SCL/tal and GAL4-E2-2 proteins. The doublet observed on immunoblotting
for SCL/tal has been previously described (24). Thus, three independent
assay systems, mammalian two-hybrid, nuclear redirection, and
coimmunoprecipitation, all confirm the requirement for domain C for
in vivo heterodimerization of E2-2 in mammalian cells.

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Fig. 7.
Coimmunoprecipitation assay for interaction
of SCL/tal with E2-2 bHLH, with or without domain C. The
full-length SCL/tal protein (amino acids 1-331 encoded by the
expression vector pCMV-SCL/tal) was coexpressed in COS cells along with
the following: lane 1, GAL4-E2-2 bHLH minus
domain C (E2-2 amino acids 484-541, encoded by the vector pCMV-DB-E2-2
bHLH); lane 2, GAL4-E2-2 bHLH plus domain C (E2-2
amino acids 484-563, encoded by the vector pCMV-DB-E2-2 C). In the
upper panel, cellular extracts were subjected to
immunoprecipitation with a rabbit anti-GAL4 antibody, followed by
immunoblot analysis with a mouse monoclonal anti-SCL/tal antibody
(BTL-73). In the lower panels, the cellular extracts were
subjected directly to immunoblot analysis with either anti-SCL/tal or
anti-GAL4 antibodies. The positions of p42 SCL/tal, as well as
immunoglobulin heavy (H) and light (L) chains are
indicated by arrows.
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Domain C Does Not Enhance in Vitro Dimerization--
To examine
the role of domain C in a highly controlled system of in
vitro dimerization, we employed surface plasmon resonance analysis
of bacterially expressed, purified proteins. In the data shown in Fig.
8, GST-E2-2 bHLH (E2-2 amino acids
484-541) and GST-E2-2 C (E2-2 amino acids 484-563) immobilized at
similar densities were employed as the solid phase ligands. The soluble
analyte consisted of MBP-SCL/tal (SCL/tal amino acids 176-331) at a
concentration of 4 µM. As shown in Fig. 8, a weak
association (>1 µM) was detectable between MBP-SCL/tal
and GST-E2-2 bHLH. Surprisingly, virtually no association was
detectable between MBP-SCL/tal and GST-E2-2 C. Similar findings were
obtained with SCL/tal as the immobilized ligand and the E2-2 proteins
as the soluble analyte (data not shown). Therefore, for in
vitro heterodimerization, domain C, rather than a requirement,
appears to behave in an inhibitory fashion.

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Fig. 8.
Analysis of in vitro E2-2
heterodimerization with SCL/tal by surface plasmon resonance
(BIAcore). GST-E2-2 bHLH (E2-2 amino acids 484-541) and GST-E2-2
C (E2-2 amino acids 484-563) were immobilized on separate CM5 chips at
densities of 1800 and 1700 resonance units, respectively. The soluble
analyte in both assays consisted of MBP-SCL/tal (SCL/tal amino acids
176-331) at a concentration of 4 µM. The indicated
sensorgrams were obtained at room temperature with a flow rate of 5 µl/s.
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Domain C Is the Most Highly Conserved Module within E
Proteins--
An alignment of the human E proteins, E2-2, HEB, E12,
and E47, with E proteins from a range of organisms including
Drosophila and zebrafish shows the extremely high
phylogenetic conservation of domain C. Fig.
9 highlights blocks of phylogenetic
identity, i.e. regions of 100% conservation, within E
proteins. Domain C represents by far the largest block of phylogenetic
identity within E proteins, with 100% conservation over 20 amino acids
in organisms ranging from fly to human. To determine if this entire
conserved block was required for in vivo dimerization,
domain C was subjected to carboxyl-terminal truncation at the proline
residue highlighted in Fig. 9. As shown in Fig.
10, inclusion of only the first 7 residues of domain C (E2-2 amino acids 484-548) sufficed for
heterodimerization of E2-2 with SCL/tal in the yeast two-hybrid system.
Thus, despite its striking evolutionary conservation, the entirety of
domain C appears not to be required for in vivo
heterodimerization.

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Fig. 9.
Phylogenetic alignment of E proteins with
highlighting of amino acids conserved in all species from
Drosophila to human. The positions of the various
domains are indicated above the aligned sequences. The
conserved proline residue in the center of domain C is highlighted in
blue.
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Fig. 10.
Carboxyl-terminal truncation of domain C at
the conserved central proline residue does not eliminate function.
Yeast two-hybrid analysis was employed to analyze the in
vivo interaction of the indicated LexA-E2-2 fusions with
B42-SCL/tal (SCL/tal amino acids 176-331). E2-2 484-541 consists of
the minimal bHLH domain. E2-2 484-563 consists of the bHLH domain plus
domain C. E2-2 484-548 consists of the bHLH domain plus the first
portion of domain C up to the central proline residue. Liquid
-galactosidase assays were performed in three separate experiments.
Results shown are mean ± S.E.
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DISCUSSION |
The basic helix-loop-helix domain has been well defined both by
functional studies as well as multiple sequence alignment analysis
(28). The most compelling functional studies have shown sufficiency of
a minimal bHLH domain of MyoD, encompassing 68 amino acids, for
myogenic conversion of fibroblasts (31). Thus, for MyoD the HLH domain
appears to suffice for both in vitro and in vivo
heterodimerization. Our data similarly show that the minimal bHLH
domains of MyoD and SCL/tal can support in vivo
heterodimerization (Fig. 2). For E proteins a discrepancy exists
between requirements for in vitro and in vivo
dimerization. Our own in vitro studies on E protein
dimerization, as well as those of other laboratories, have clearly
demonstrated sufficiency of the minimal bHLH domain sequence shown in
Fig. 1 (see surface plasmon resonance data in Fig. 8) (10, 16). By
contrast, our findings for in vivo heterodimerization of
E2-2 indicate a requirement for additional sequence outside the bHLH
domain. Most likely this discrepancy is due to fundamental differences
between in vitro and in vivo HLH dimerization. In particular, in vitro HLH dimerization, as studied by surface
plasmon resonance, appears to be a highly inefficient process with slow association rates and rapid dissociation rates (see Fig.
8).2 Recent NMR studies of
E47 homodimers showed a high degree of dynamics within the HLH domain
and poor definition of helix 1, both findings suggestive of a high
degree of dimer instability (32). The low affinity and relative
instability observed in HLH dimers in vitro is incompatible
with previous observations of high efficiency HLH dimerization in
vivo (17, 18).
Domain C appears to function in vivo as a cis-acting
conformational determinant for the adjacent HLH domain of E2-2. The
concept that regions outside the HLH domain can influence dimerization has support from several earlier studies. Klein et al. (33) identified a dimerization-defective isoform of the rat E protein, REB,
generated by alternative splicing. The dimerization-defective REB
differs from the dimerization-competent isoform REB
only in that
REB
possesses an additional 24-amino acid ankyrin-like domain
located 180 amino acids amino-terminal of the bHLH domain. Presumably
the ankyrin-like domain of REB
makes contacts in cis with the bHLH
domain, thereby masking its dimerization potential. Shirakata and
Paterson (29) have demonstrated an influence of domain A in E12,
located just upstream of the bHLH domain, in preventing
homodimerization and promoting heterodimerization with myogenic bHLH
proteins. Wright et al. (34) have shown that antimyogenin monoclonal antibodies recognizing epitopes amino-terminal of the bHLH
domain inhibit the heterodimerization of myogenin and E12. Hara
et al. (8) have shown that Cdk2-dependent
phosphorylation of Id2 at a serine residue in the amino terminus,
approximately 30 amino acids upstream from the HLH domain, completely
blocks the ability of Id2 to heterodimerize with E12. Thus ample
precedent exists for the presence of external cis-acting domains which
influence the accessibility and/or the conformation of the HLH
domain.
The means by which domain C influences the conformation of the adjacent
HLH domain remains undefined. One possibility is that domain C simply
represents an extension of helix 2. This possibility is unlikely for
two reasons. First, multiple algorithms for the prediction of protein
secondary structure suggest that helix 2 of E2-2 ends amino-terminal to
domain C (35). Second, domain C does not promote, and in fact inhibits,
in vitro dimerization (see Fig. 8); as a simple helical
extension, domain C would be predicted to promote in vitro
dimerization. Another possibility is that domain C, at its central
proline residue, bends back on top of helix 2 and induces a
conformational change. This cis-acting model for domain C is unlikely
for two reasons. First, carboxyl-terminal truncation of domain C at the
central proline residue does not eliminate its function (Fig. 10).
Second, the cis-acting model also predicts that domain C should enhance
in vitro dimerization, the opposite of what has been
observed (Fig. 8). A third possibility is that domain C serves as a
docking site for a chaperonin which induces the conformational change
in the HLH domain. This chaperonin-docking model is appealing for three
reasons: 1) the unique requirement of domain C for in vivo
but not for in vitro dimerization; 2) the exquisite
evolutionary conservation of domain C; and 3) the significant precedent
for regulation of transcription factors by chaperonins. With regard to
the last point, heat shock protein 90 has been shown to regulate the
DNA binding functions of E12 and MyoD (36) as well as the
ligand-inducibility of the bHLH dioxin receptor (37). Using the potent
and specific inhibitory compound macbecin I (38), we have ruled out a
major role for heat shock protein 90 in HLH dimerization in yeast (data
not shown). However, numerous other chaperonin systems remain to be
tested.
Analyses of bHLH structures in solution indicate that prior to
dimerization, monomeric bHLH domains are either completely disordered
(14) or in an antiparallel hairpin-like conformation (39, 40). Thus the
transition from monomeric to dimeric bHLH molecules requires major
structural alterations from either a disordered or antiparallel
conformation into a highly structured, well organized parallel
conformation. This transition may occur slowly and inefficiently
in vitro permitting some observable dimerization. However,
for efficient in vivo dimerization, there is most likely active refolding of bHLH domains into conformations receptive for
dimerization. Our observations suggest that domain C is a cis-acting
determinant which permits the refolding of E proteins into
dimerization-competent monomers. In such a conformation, the E proteins
may then be capable of heterodimerizing with a wide array of
tissue-specific HLH factors. Thus domain C may contribute to the unique
ability of E proteins to bind a diverse array of HLH partners.
Special thanks to Dr. Richard Baer for
providing mammalian two-hybrid constructs, to Drs. Roger Brent and
Erica Golemis for providing yeast two-hybrid reagents, to Dr.
Karen Pulford for monoclonal anti-SCL/tal antibodies, and to the
Weintraub and Kadesch laboratories for MyoD and E2-2 cDNAs. We also
thank Dr. Scott Vande Pol, Dr. Fred Racke, Dr. Menachem Shoham, and Dr.
Peter Harte for helpful discussions.