From the Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9148
Received for publication, January 24, 2001
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ABSTRACT |
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In pancreatic acinar cells, the HOX-like factor
PDX1 acts as part of a trimeric complex with two TALE class homeodomain
factors, PBX1b and MEIS2b. The complex binds to overlapping half-sites for PDX1 and PBX. The trimeric complex activates transcription in cells
to a level about an order of magnitude greater than PDX1 alone. The
N-terminal PDX1 activation domain is required for detectable transcriptional activity of the complex, even though PDX1 truncations bearing only the PDX1 C-terminal homeodomain and pentapeptide motifs
can still participate in forming the trimeric complex. The conserved
N-terminal PBC-B domain of PBX, as well as its homeodomain, is required
for both complex formation and transcriptional activity. Only the
N-terminal region of MEIS2, including the conserved MEIS domains, is
required for formation of a trimer on DNA and transcriptional activity:
the MEIS homeodomain is dispensable. The activity of the
pancreas-specific ELA1 enhancer requires the cooperation of the trimer-binding element and a nearby element that binds the pancreatic transcription factor PTF1. We show that the
PDX1· PBX1b·MEIS2b complex cooperates with the PTF1 basic
helix-loop-helix complex to activate an ELA1 minienhancer
in HeLa cells and that this cooperation requires all three homeoprotein
subunits, including the PDX1 activation domain.
The HOX transcription factors are key mediators for establishing
the embryonic body plan and guiding organogenesis (1-3). The many
members of this large gene family have distinct roles in development,
despite having very similar DNA binding specificities and overlapping
patterns of expression. At least part of the increased binding
specificity required to distinguish the roles of the various HOX
proteins comes from their interaction with members of the TALE class of
homeodomain proteins (for review see Ref. 4). The TALE proteins are
characterized by the presence of a 3-amino acid loop extension between
Pdx1 is a member of the PARAHOX cluster of
homeobox genes implicated in the development of endoderm-derived organs
(11). Pdx1 is essential to the development of both the
endocrine and exocrine compartments of the pancreas (12-14). The
interaction of PDX1 with PBX is required for the expansion of the
various endocrine and exocrine cell types during development (15).
Although Pdx1 is expressed throughout the pancreas during
embryonic development, its expression is predominantly localized to the
Our previous studies identified the PDX1·PBX1b·MEIS2 trimer as one
of two pancreatic transcription factor complexes that cooperate in the
transcriptional activation of the ELA1 enhancer in acinar cells (23). The ELA1 enhancer comprises three functional
elements (A, B, and C) within 100 bp1 (24). The proper acinar
activity of the enhancer requires the synergistic cooperation between
the A and B elements (25, 26). The PDX1·PBX1b·MEIS2 complex
binds and mediates the acinar activity of the B element (10). The other
factor, PTF1, is a bHLH acinar cell-specific complex that binds and
mediates the activity of the A element (24,
27).2 PTF1 is essential for
the formation of the exocrine pancreas (28) as well as for the
transcription of the digestive enzyme genes (24, 29).
In this report, we show which domains of PDX1, PBX1b, and MEIS2b are
essential for the formation of the trimeric complex, for binding of the
complex to DNA, for transcriptional activity, and for cooperative
activation with PTF1. PDX1 contributes the only transcriptional
activation domain, even though the activity of the trimer is an order
of magnitude greater than that of PDX1 alone. PDX1 recruits PBX1b, and
they bind in tandem to adjacent PDX1 and PBX half-sites. PBX1b recruits
MEIS2b to the DNA-bound complex. MEIS2b is required for both the
transcriptional activity of the PDX1·PBX1b·MEIS2 trimer and its
cooperation with PTF1.
Oligonucleotides--
The ELAI B element has been
described previously (10); the top strand sequences of the B element
and its derivatives B2 and B7 are shown in Fig. 1A. The CRS1
element from the bovine CYP17 gene has the top strand sequence of
CCTCGAGACGTTGATGGACAGTGAGCAAGG, and the bottom strand
sequence of TCGACCTTGCTCACTGTCCATCAACGTCTCGAGGAGCT; the
adjoining PBX and MEIS binding sites are underlined
(30).
Antibodies--
Polyclonal rabbit antibodies against PDX1, PBX,
PBX1, and MEIS2 have been described previously (10). The polyclonal
rabbit antiserum specific for the a and b isoforms of MEIS2 proteins were raised against an 18-amino acid peptide, PMSGMGMNMGMDGQWHYM, corresponding to amino acids 377-394 of MEIS2b. Similarly, a 16-amino acid peptide, SVDPNVGGQVMDIHAQ, spanning amino acids 455-470 of MEIS2d, was used to produce the anti-MEIS2c/d. A 7-amino acid insert,
GFLLDPS, corresponding to amino acids 346-352 of MEIS2a and MEIS2c, is
specifically present in these two isoforms. The antibody DNA Constructs--
The B element reporter construct
(5B.EIp.hGH) has five head-to-tail copies of the B element (Fig.
1A) linked to the Ela1 minimal promoter from
The deletion series of PDX1, PBX1b, and MEIS2b were created by
polymerase chain reaction amplification of the corresponding coding
regions and cloning of the fragments into expression vectors. The
full-length and PDX1 deletion cDNAs were cloned into
pcDNA3.1/V5/His TOPO (Invitrogen Co., Carlsbad, CA). The encoded
PDX1 proteins contain V5 and His epitope tags at their C termini. To
synthesize the PBX1b deletion series by in vitro
transcription and translation (IVT), the cDNAs were cloned into
FLAG-pSP65, thereby adding a FLAG-tag at the N terminus of each
protein. Each PBX1b cDNA, together with the N-terminal FLAG-tag,
was transferred into pcDNA1.1/Amp (Invitrogen) for expression in
transfected cells. The MEIS2b deletion series was cloned into pET-28b
(Novagen, Madison, WI) under the control of the T7 promoter for IVT.
Tandem His and T7 epitope tags were included at the N terminus of each
MEIS2b protein. For expression in transfected cells, the MEIS2b
cDNAs, together with the His and T7 tags, were transferred into
pcDNA1.1/Amp. An IVT expression plasmid for MEIS2d was generated by
polymerase chain reaction amplification of Meis2d cDNA using
Meis2d/pBS.SK Electophoretic Mobility Shift Assays--
Procedures for
preparing nuclear extracts, in vitro translation (IVT),
EMSA, and antibody supershift assays have been described previously
(10). In all EMSA reactions containing nuclear extract from 266-6
cells, a GATA binding site oligonucleotide Immunological Techniques--
Western blot analyses were
conducted as previously described (10). 30 µg of nuclear extracts, or
0.25 µl of the rabbit reticulocyte lysates containing the IVT
proteins were resolved by sodium dodecyl sulfate-10% polyacrylamide
gel electrophoresis and electrophoretically transferred onto Immobilon
polyvinylidene difluoride membranes (Millipore, Bedford MA). Immune
complexes were detected by enhanced chemiluminescence with Supersignal
West (Pierce, Inc., Rockford, IL). The exocrine cell lines were: 266-6 (ATCC CRL-2151) (32), C5-2E, (10), ARIP (ATCC CRL-1674) (33), and
AR4-2J (ATCC CRL-1492) (33). The
Co-immunoprecipitation of the PBX and MEIS proteins was performed as
follows. 10 µl of reticulocyte lysate containing equimolar amounts of
35S-labeled IVT protein and the unlabeled IVT partner was
incubated at 37 °C for 40 min. 5 µl of polyclonal antibody
specific for the non-labeled protein was added, and the reaction volume
was increased to 300 µl by adding buffer containing 10 mM
HEPES (pH 7.6), 250 mM NaCl, 0.1% Nonidet P-40, and 5 mM EDTA. After gentle mixing by rotation at 4 °C for
1 h, 20 µl of protein A beads (Santa Cruz Biotechnology Inc.,
Santa Cruz, CA) was added, and the mixing was continued overnight.
Following the incubation, the beads were collected and washed, then
resuspended in SDS-gel loading buffer. Samples were separated by
SDS-10% polyacrylamide gel electrophoresis, and the
35S-labeled IVT proteins were detected with a
PhosphorImager (Molecular Dynamics, Santa Clara, CA).
Cell Transfection--
Transfections of the RIN1046-38 The Trimeric Complex Binds Only to Sites Containing Both PDX1 and
PBX Half-sites--
We previously identified a trimeric homeoprotein
complex that binds to and mediates the activity of the B element of the
elastase I (ELA1) enhancer in pancreatic acinar cell lines
(10). The complex consists of a pancreatic HOX-like protein, PDX1, and
two TALE-class homeodomain proteins, PBX1b and an isoform of
MEIS2/MRG1. The trimeric complex binds an 11-bp region of the B element
containing overlapping half-sites for PDX1 and PBX but no recognizable
site for MEIS (Fig. 1A).
Mutations in either the PDX1 or PBX half-site eliminate trimer binding
(10). The central role of PDX1 in trimer binding was demonstrated by
mutation of a single nucleotide in the PDX1 half-site of the B element
(the A at position 7 of the PBX-HOX consensus): neither PDX1 nor the
PDX1-containing trimeric complex from 266-6 acinar cell nuclear
extracts bound detectably to the oligonucleotide containing this A to C
transversion (B7, Fig. 1B) and this B7
oligonucleotide was unable to compete for trimer binding in EMSA
competition assays (Fig. 1C). These results confirmed the
binding of PDX1 to its half-site and demonstrated that the formation of
the trimeric complex is dependent on an intact PDX1 half-site.
To be able to study the structure of the trimeric complex, it was
necessary to increase the PBX binding affinity of the B element without
altering the qualitative nature of the activity of the element in
cells. The binding affinity is low, because nucleotides at positions 1, 2, and 6 differ from the consensus binding sequence for PBX/HOX
heterodimers (37, 38). Changing the A at position 2 to T matches
the consensus PBX half-site (B2, Fig. 1A) and
increased the binding of the trimeric complex from acinar cell nuclear
extracts without affecting the binding of the PDX1 monomer (Fig.
1B). In competition assays the B2 element was ~5-fold more
effective than the unmodified B element (Fig. 1C). Antisera
specific for PDX1, PBX, and MEIS2 eliminated the B and B2 complexes
with equal effectiveness (Fig. 1D), confirming that the
homeodomain protein composition is the same for the two complexes. The
transcriptional activities of B and B2 in transfected cells were also
qualitatively the same (see Fig. 4, below). We subsequently used the
more effective B2 element to test the ability of various forms of the
three transcription factors to form complexes on DNA and activate transcription.
The Trimeric Complex Is Composed of PDX1, PBX1b, and
MEIS2b/MEIS2d--
To determine which of four MEIS2 isoforms are
present in the trimeric complex, we developed antisera against
distinguishing peptide regions of the isoforms. Four alternatively
spliced variants of Meis2 mRNA encode four distinct protein
products: 2a, 2b, 2c, and 2d (39). MEIS2a and 2b have a different
C-terminal tail (18 amino acids) than MEIS2c and 2d (94 amino acids);
MEIS2a and 2c share a common seven amino acid insert, GFLLDPS, which
MEIS2b and 2d lack. The antiserum specific for MEIS2a/b supershifted most of the trimeric complex from acinar cell extracts, leaving behind
a residual complex of slightly slower electrophoretic mobility (Fig.
2A, lane 3). The
antiserum specific for MEIS2c/d decreased the amount of the trimeric
complex slightly (lane 5 versus lane 4), although a supershifted band was not observed. Antiserum
against the MEIS2a/c-specific heptapeptide did not reduce the amount of the trimeric complex more than the preimmune serum (lanes 6 and 7). These results indicate that MEIS2b and possibly
MEIS2d are present in the complex, but MEIS2a and 2c are not. To
determine whether the residual slower mobility complex remaining after
treatment with
PDX1, PBX1b, and MEIS2 proteins synthesized by translation in
vitro can form trimeric complexes on DNA (Fig. 2B,
lanes 7 and 8). The complex formed with
recombinant PDX1·PBX1b·MEIS2b comigrates with the predominant
trimer from 266-6 acinar cells (Fig. 2B, lane 7),
whereas the PDX1·PBX1b·MEIS2d complex (lane 8)
comigrates with the slower mobility complex that remains after
The presence of MEIS2b and 2d in acinar cells was confirmed by Western
blot analysis (Fig. 3). Unlike PBX1b,
which is present in pancreatic acinar but not The Trimeric Complex Is More Transcriptionally Activate Than
PDX1--
Multimers of the B element are transcriptionally active in
the Domains of PDX1 Required for Trimer Binding and
Activity--
PDX1 contains three important functional domains
(Fig. 5A): an activation
domain (AD) at the N terminus (amino acid residues 1-79), a
pentapeptide motif (PP; residues 121-125) that directly contacts PBX,
and the homeodomain (HD) (residues 145-206) (9, 40, 41). A cryptic
C-terminal activation domain also functions in the transcriptional
activation of the somatostatin gene by PDX1 (40). We
constructed a series of PDX1 deletion mutants to test the function of
each domain in the context of the trimer (Fig. 5A). The
mutant proteins were synthesized by in vitro translation (IVT), and their ability to bind DNA was assayed by EMSA.
Full-length PDX1 binds to the B2 element as a monomer, a heterodimer
with PBX1b, and a heterotrimer with PBX1b and MEIS2b (Fig.
5B, lane 2). PDX1 mutants missing the regions
C-terminal to the HD or N-terminal to the PP motif had similar DNA
binding activities as the wild type PDX1 (lanes 3,
4, and 6). When the PP motif was also deleted,
however, the protein was unable to form heterodimeric and
heterotrimeric complexes on DNA (lane 5). Therefore, only
the DNA-binding domain and the PBX-interaction motif are required for
PDX1 to participate fully in forming the trimeric complex on DNA. These
are the same domains required for dimer formation with PBX1 (Fig.
5B and Ref. 9).
To investigate which PDX1 domains are essential for trimer function, we
tested the activity of the PDX1 deletion series by cotransfection with
PBX1b and MEIS2b into a non-pancreatic cell line (HeLa). HeLa has no
endogenous PDX1, although it contains both PBX1b (42) and MEIS2 (data
not shown). Multimers of the B2 element were inactive in HeLa in the
absence of cotransfected transcription factors (Fig. 4B).
Addition of PDX1 activated the B2 element at least 50-fold, whereas
addition of either PBX1b or MEIS2b alone or together had a barely
detectable activating effect. Addition of either PBX1b or MEIS2b in
cotransfections with PDX1 augmented the PDX1 activity 2- to 4-fold,
whereas cotransfection of all three transcription factors increased
activity 9-fold. The synergistic activity of the three proteins is
consistent with the cooperative binding of PBX1b and MEIS2b with PDX1.
As seen for the RIN-38
Reporter gene activation by the trimer, as well as by PDX1 alone,
required the N-terminal activation domain of PDX1 (Fig. 5C).
Both the PDX116-284 and PDX126-284 mutants missing the activation domain were inactive, either in the presence or absence of
cotransfected PBX1b and MEIS2, even though the PDX116-284 could form
DNA-binding trimers (Fig. 5B). These results imply that
neither PBX1b nor MEIS2b contribute an activation domain to the trimer.
Domains Important for the Function of PBX1b--
Three polypeptide
domains are uniquely conserved among the PBC class of homeodomain
proteins (mammalian PBX proteins, Drosophila Extradenticle,
and C. elegans Ceh-20 (6)). The PBC-A domain near the N
terminus of the proteins (Fig.
6A) mediates the interaction of PBX with MEIS and the closely related PREP1 protein (7, 43, 44). The
function of the PBC-B domain, located between the PBC-A and the HD, has
not been delineated. In addition, the C terminus of the HD is extended
by a fourth
In vitro synthesized, full-length PBX1b does not bind the B2
element, but does bind as a heterodimer with PDX1 and as a trimeric complex with PDX1 and MEIS2b (Fig. 6B, lane 2).
Illustrating the critical role that helix 4 plays in complex formation,
deletion of sequences C-terminal to it had no effect (PBX1b1-318,
lane 3), whereas further deletion to remove helix 4 abolished both dimer and trimer formation (PBX1b1-295, lane
4). Deletion of both PBC domains abolished the formation of the
trimer on DNA without affecting the interaction between PBX1b and PDX1
(PBX1b233-347, lane 5). However, the PBC-A domain alone is
not sufficient to bring MEIS into the trimeric complex, because
deletion of just the PBC-B domain also abolished the trimer formation
(PBX1b
PBX and MEIS can form stable dimers, even in the absence of DNA (42,
44), and the PBX·MEIS heterodimer binds a consensus sequence of
adjacent PBX and MEIS half-sites (7, 44). To confirm that the failure
of PBX1b233-347 and PBX1b
We analyzed the ability of each PBX1b mutant to participate in the
transcriptionally active trimer complex with PDX1 and MEIS2b in
transfected cells (Fig. 6D). The PBX1b1-318 mutant is only slightly less active than the full-length PBX1b, indicating that no
crucial domain is present C-terminal to the HD helix 4. Surprisingly, the PBX1b1-295 protein, although lacking helix 4 and unable to form a
trimer with PDX1 and MEIS2b in EMSA, is active in transfections. The
two PBC domain mutants, PBX1b233-347 and PBX1b Trimer Formation and Activity Requires the M1 and M2 Domains but
Not the HD of MEIS2b--
The HD and two N-terminal MEIS domains (M1
and M2) are conserved among mammalian MEIS proteins,
Drosophila Homothorax, C. elegans Ceh-25, and a
closely related protein pKNOX/PREP1 (5, 7). The functional significance
of these regions in DNA binding, protein interactions, and
transcriptional activation was evaluated with a series of deletion
mutants (Fig. 7).
MEIS2b does not bind the B2 element as a monomer or as a heterodimer
with either PDX1 or PBX1b (data not shown). Deletion of regions
C-terminal to the HD and N-terminal to the M1 domain had no impact on
formation of a trimer (MEIS2b1-338 and MEIS2b71-394, Fig.
7B, lanes 3 and 4). Deletion of the M1
and M2 domains in MEIS2b192-394 eliminated the formation of a trimeric
complex (lane 5). The MEIS2b HD has an inhibitory effect on
trimer formation: removing the HD in MEIS2b
Consistent with its inability to form a trimer, MEIS2b192-394 failed
to dimerize with PBX1b both in solution and on CRS1 (Fig. 7C, lane 5). The MEIS2b HD was not required for
the PBX·MEIS dimers to form in solution, but was essential for these
dimers to bind DNA (MEIS2b
The region containing M1 and M2 was also sufficient for a fully active
trimer in cotransfection experiments (Fig. 7D). All of the
deletion mutants that contained the MEIS domains, including the minimal
construct MEIS2b1-275, had approximately the same activity as the
full-length MEIS2b. The MEIS2b192-294, which lacks both MEIS domains,
had no activity.
The PDX1·PBX1b·MEIS2b Trimer Cooperates with a bHLH Complex to
Activate Transcription--
The synergistic interaction between the A
and B elements of the ELA1 enhancer (26) suggests that the
DNA-binding factor complexes that mediate their activities cooperate
within the context of the ELA1 enhancer. PTF1, which binds
the A element, comprises a bHLH heteromultimer that minimally contains
the acinar cell-specific p48 protein (29) and a ubiquitous E-box
binding protein (REB in rats, HEB in humans, ALF1 in
mice).3,4
A mini-enhancer construct bearing three head-to-tail copies of tandem A
and B elements (Fig. 8A) was
used to test possible cooperativity between the PDX1·PBX1B·MEIS2b
trimer and the p48·HEB dimer.
The PDX1·PBX1b·MEIS2b trimer activated the mini-enhancer nearly
4-fold in HeLa cells (Fig. 8B). Even though p48·HEB alone had no detectable effect on the mini-enhancer, it increased the activation by the trimer 2-fold. Addition of either PBX1b or MEIS2b to
PDX1 in the presence of p48·HEB had little if any stimulatory effect,
whereas adding PBX1b and MEIS2b together increased the activity of PDX1
with p48·HEB 4-fold. The increase in activity with the addition of
p48·HEB was dependent on the presence of the A element, because
p48·HEB had no effect on the activation of the B multimer by PDX1 or
the PDX1·PBX1b·MEIS2b trimer in either HeLa or RIN-38 cells (data
not shown). The A-element-dependent stimulation by
p48·HEB represents cooperative activation with the trimer.
Electrophoretic mobility shift experiments designed to detect a
cooperative binding of IVT PDX1·PBX1b·MEIS2b trimer with IVT
p48·HEB on adjacent B and A elements have been
unsuccessful.5
To determine which domains of PDX1, PBX1b, and MEIS2b are responsible
for the cooperative interaction of the heterotrimer with PTF1, key
deletion mutants of the homeoproteins were tested for their ability to
participate in activating the BA mini-enhancer (Fig. 8C).
The activation domain of PDX1 was required, whereas the region of PDX1
C-terminal to the homeodomain is entirely dispensable for activity. All
of the PBX1b and MEIS2b deletion constructs that formed heterotrimers
on the B element could participate in the activation of the BA
mini-enhancer. DNA binding by MEIS was not required for activity,
because the deletion of the MEIS HD and C terminus (MEIS1-275) did not
result in any loss of activity. These requirements mimic those of the
trimer for transcriptional activation through the B-element multimer,
which is independent of PTF1.
The trimeric complex of PDX1·PBX1b·MEIS2b binds a bipartite
PBX-PDX1 site and is an order of magnitude more transcriptionally active in HeLa cells than PDX1 alone. The transcriptional activity of
the complex is dependent on the activation domain of PDX1, as well as
domains that mediate the interaction between PDX1 and PBX1b and between
PBX1b and MEIS2b. Cooperative transcriptional activation with the
pancreas-specific bHLH complex PTF1 requires the same structural
domains and mimics the activity of the trimer within the context of the
ELA1 enhancer in pancreatic acinar cells.
Organization of the DNA-bound Trimer--
Based on the behavior of
the deletion mutants of the three proteins, we propose a model for the
organization of the PDX1·PBX1b·MEIS2b complex bound to DNA (Fig.
9). The trimer binds DNA through the HDs
of PDX1 and PBX1b. The HD of MEIS2b forms no sequence-specific DNA
interactions and is dispensable for DNA binding; indeed, DNA binding of
the trimer is enhanced by the absence of the MEIS HD. PBX1b is the
centerpiece of the complex; PBX1b interacts with PDX1 and MEIS2b
through separate polypeptide domains. PBX1b and PDX1 bind adjacent DNA
half-sites and interact directly through helix 4 of the PBX1b
homeodomain and the pentapeptide motif just N-terminal to the PDX1
homeodomain. PBX1b recruits MEIS2b into the complex through
interactions involving the N-terminal halves of the two proteins. Our
analyses did not reveal any direct interaction between PDX1 and
MEIS2b.
Our results are consistent with previous biochemical and structural
analyses of HOX·PBX and PBX·MEIS heterodimers. These analyses showed that when PBX and HOX proteins are bound at adjacent sites, the
HOX partner reaches across the DNA duplex and inserts the tryptophan of
the pentapeptide motif into a hydrophobic pocket formed by the PBX
homeodomain and helix 4 (45, 46). The PBC-A domain (7, 44) and the M2
domain of MEIS (7) are of primary importance in PBX·MEIS complex
formation; however, some interaction is maintained even in the absence
of the PBC-A domain (7). Indeed, we have found that deletion of the
PBC-B domain also disrupts PBX·MEIS interactions and eliminates
formation of the DNA bound trimer (Fig. 6).
Recently, trimeric complexes containing HOX-like, PBX-like, and
MEIS-like subunits have been reported with different modes of
binding to DNA. Similar to the PDX1·PBX1b·MEIS2b complex, a HOXB1·PBX1·PREP1 complex also binds DNA through the HOX and PBX HDs, and DNA binding does not require the homeodomain of PREP1 (43).
Other trimeric HOX·PBX-like·MEIS-like complexes require sequence-specific DNA binding of all three HDs (48-50), whereas at
least one trimeric complex requires only an HOX binding site (51).
Thus, in different contexts the trimeric complexes of HOX and HOX-like
proteins with PBX-like and MEIS-like cofactors can require specific
binding sites for only the HOX, for HOX and PBX, or for all three proteins.
Transcriptional Activity of the Trimer--
The polypeptide
domains required for the increased transcriptional activity of the
PDX1·PBX1b·MEIS2b trimer, in comparison to PDX1 alone, include
those required for trimer binding to DNA (the PDX1 and PBX1b HDs) and
for protein·protein interactions necessary for formation of a stable
heterotrimer (the PDX1 pentapeptide, the PBX helix 4 and PBC domains,
and MEIS M1 and M2 domains). The N-terminal activation domain of PDX1
is essential for transcriptional activity of the trimer, because a
trimeric complex missing only this activation domain (PDX116-284) has
no transcriptional activity (Fig. 5C).
The absence of transactivation potential in the trimer lacking the PDX1
activation domain indicates that PBX1b and MEIS2b do not have
activation domains or that their activation domains do not function
without the PDX1 activation domain. Co-expression of PBX1 and MEIS1
fails to activate a reporter gene driven by a multimer of PBX·MEIS
binding sites (7). Other experiments also have failed to detect an
activation domain in PBX1b (52, 53). The presence of PBX1b and MEIS2b
does not appreciably increase the amount of PDX1 binding in gel shift
assays, so it is unlikely that the effect of PBX1b and MEIS2b is to
increase binding site occupancy by PDX1. Therefore, PBX1b and MEIS2b do
not appear to contribute either activation domains to the trimeric
complex or stabilization of PDX1 binding to DNA. Consequently, the
basis of the increased transcriptional activity of the trimer, in
comparison to PDX1 alone, is unclear.
Cooperative Transcriptional Activation with the p48·HEB
Complex--
The ELA1 enhancer comprises the A and B
elements, which bind the bHLH complex PTF1 and the
PDX1·PBX1b·MEIS2b trimer, respectively, as well as a
non-cell-specific element that binds unknown factors (10, 24, 26, 27).
The pancreatic activity of the enhancer is dependent on synergy between
the A and B elements in transgenic animals and transfected pancreatic
acinar cell lines (25, 26). Although PDX1 bears the only activation
domain within the PDX1·PBX1b·MEIS2b trimer, enhancer mutations that
eliminate the PBX1 half-site and trimer binding without eliminating
PDX1 binding are transcriptionally inactive (10). Thus, the requirement
for PBX and MEIS, as well as PDX1, for the cooperative activation of an
ELA1 mini-enhancer in HeLa (Fig. 8) is consistent with
previous results with the complete ELA1 enhancer.
The ELA1 mini-enhancer, comprising three copies of adjacent
PBX-PDX1 and PTF1 binding sites, directs high level expression in the
pancreas and gut of transgenic mice (26). The pancreatic expression of
this mini-enhancer is more than an order of magnitude greater than that
seen with a multimer of the PBX-PDX1 binding site or a multimer of the
PTF1 binding site. The activity of the mini-enhancer in HeLa cells is
dependent on the activation domain of PDX1, within the context of the
DNA-bound PDX1·PBX1b·MEIS2b complex (Fig. 8). The cooperative
activation of the mini-enhancer in HeLa cells upon co-transfection with
PDX1·PBX1b·MEIS2b and p48·HEB likely reflects the natural
interaction of these two complexes in the acinar pancreas to activate
the transcription of the transgenic mini-enhancer and the endogenous
ELA1 gene as well.
Cooperation between homeoproteins and bHLH complexes have been shown to
occur in two other instances. In the insulin gene enhancer, PDX1
contributes to synergistic activation in a manner different than its
action in the ELA1 enhancer. PDX1 binds to the A3/4 region
of the rat insulin I enhancer in the absence of PBX and MEIS proteins
and activates transcription by cooperating with the bHLH protein E47,
which binds to the adjacent E2 site (54). In this case, PDX1
contributes protein·protein interaction domains to recruit multiple
proteins, including E47, BETA2/NeuroD, and high mobility group protein
I(Y), to an activation complex on the E2A3/4 mini-enhancer. The
transcriptional activity of this complex is believed to result from
the clustering of multiple activation domains capable of interacting
with coactivators and the basal transcriptional machinery (54). In
addition, a PBX·MEIS/PREP1 heterodimer and heterodimers of E2A and
MyoD, myogenin, MRF-4, or MYF-5 exhibit cooperative DNA binding and
transcriptional activation (55). This interaction requires a conserved
peptide motif just N-terminal of the bHLH domain, which is not present
in p48 (55); and p48 cannot substitute for the myogenic
factors.5 Although the mechanism for the cooperation
between the PDX1 and p48 complexes is not known, it is likely that the
two complexes interact, either to stabilize each other's binding to
DNA or to recruit coactivators to facilitate transcriptional activation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helices 1 and 2 of the homeodomain (5) and include both the PBC
class (mammalian PBX proteins, Drosophila Extradenticle, and
Caenorhabditis elegans Ceh-20 (6)) and the MEIS-like TALE
factors (mammalian MEIS and PREP1 proteins, Drosophila
Homothorax (HTH), C. elegans Ceh-25 (5, 7)). Other
homeodomain proteins important in development also form complexes with
the TALE proteins, including the pancreas/duodenal homeobox-1
protein (PDX1) (8-10).
-cells of the islets of Langerhans of adult mammals (16, 17).
PDX1 has been implicated in the transcriptional control of a number of
-cell-specific genes, including insulin (16, 18), glucokinase (19),
islet amyloid polypeptide (20, 21), and glucose transporter type 2 (22). PDX1 also participates in the activation of an acinar cell-specific gene, elastase 1 (ELA1); in this instance it
acts as part of a trimeric complex with PBX1b and MEIS2 (10).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MEIS2a/c
was raised against a 14-amino acid peptide containing a tandem repeat
of the insert. All of the MEIS isoform-specific antibodies were raised
against peptide sequences common to mouse and human orthologues.
92
to +8 and fused to an hGH reporter gene (10). In the B2 and B7 multimer
constructs, sense strand and antisense strand oligonucleotides
containing five copies of the B2 or B7 elements (Fig. 1A)
were annealed and cloned into the Ela1 minimal promoter/hGH
reporter plasmid. The mini-enhancer construct 3(BA).EIp.hGH (26) has
three head-to-tail copies of adjacent B and A elements (PDX1·PBX1b·MEIS2b and PTF1 binding sites, respectively) from the
Ela1 gene inserted in front of the Ela1 promoter.
The Pdx1.luciferase reporter plasmid, used as an internal control in
RIN1046-38 cell transfections, has 4.5 kb of 5'-flanking sequence from
the Mus musculus Pdx1 gene (GenBankTM accession
number AF192495; a gift of Dr. Chris Wright, Nashville, TN) inserted
upstream of the luciferase gene in pGL3basic (Promega, Madison, WI).
(a gift of Professor Pierre Chambon, CNRS-INSERM,
Strasbourg, France) as template, and the insert was cloned into
pET-28b. The cDNA for the mouse p48 subunit of PTF1 (a gift of Dr.
Scott Rose, University of Texas Southwestern, Dallas) and the cDNA
for HEB (a gift of Dr. Richard Baer, New York, NY) were transferred to
pcDNA1.1/Amp for transfection experiments.
G1 (31) was added to
compete with GATA4 binding to the B or mutant B elements. IVT proteins
were incubated at 37 °C for 15 min prior to the standard EMSA protocol.
-cell lines were:
TC3 (34),
Ins1 (35), and RIN1046-38 (36).
-cell
line (RIN-38) (36) and HeLa (ATCC CCL-2) were performed with FuGene
(Roche Molecular Biochemicals, Indianapolis, IN) according to the
manufacturer's instructions. All of the plasmids for the expression of
transcription factors, as well as the
-galactosidase internal
control plasmid (pCMV
, CLONTECH, Palo Alto, CA)
utilized the CMV enhancer/promoter. All transfections contained the
same total amount of CMV enhancer/promoter, balanced as necessary by
the addition of insertless CMV vector (e.g.
pcDNA1.1/Amp). The activity of the hGH reporter gene was assayed by
radioimmunoassay (Nichols Institute, San Juan Capistrano, CA).
Transfections of HeLa cells were corrected for relative efficiency based on the activity of the cotransfected
-galactosidase reporter assayed with a Galacto-Light Plus kit (Tropix Inc., Bedford, MA). Transfections of RIN1046-38 cells were corrected for relative efficiency based on the activity of a cotransfected Pdx1.luciferase reporter, in which the luciferase reporter gene was under the transcriptional control of the Pdx1 gene promoter sequence.
Luciferase activity was assayed with a kit from Promega (Madison, WI).
Transfection results reported are the mean and standard error of a
minimum of four transfections, except as noted. For those deletion
plasmids of PDX1, PBX1b, and MEIS2b that had significantly less than
wild type activity in transfections, we confirmed the production of protein and its nuclear localization by immunohistochemical staining using monoclonal antibodies recognizing their peptide tags (PDX1 with
anti-V5, Invitrogen, Carlsbad, CA; PBX1b with anti-FLAG, Sigma Chemical
Co., St Louis, MO; MEIS2b with anti-T7, Novagen, Madison, WI). All were
expressed and localized to the nucleus at approximately wild type
levels, except for PDX116-206, which was found only in the cytoplasm
(data not shown).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
An acinar homeoprotein complex contains PDX1,
PBX1b, and MEIS2 and requires both PDX1 and PBX half-sites for DNA
binding. A, sequences of the ELA1 B element
and derivatives B2 and B7. The PBX and HOX/PDX1 binding half-sites are
in boldface and numbered, with the consensus
binding sequence shown on top. Dots indicate
identical residues. B, equal amounts of 266-6 nuclear
extract were analyzed for B, B2, and B7 binding by EMSA. The PDX1 and
trimer complexes are indicated. C, binding of the trimer
complex can be effectively competed by B2 and B oligonucleotides, but
not by B7 oligonucleotides. 30- or 150-fold excess unlabeled
oligonucleotides were added as competitors in the EMSA reactions as
indicated. D, antisera against PDX1, PBX, and MEIS2
eliminated the trimer complex bound to the B or B2 elements. Anti-PDX1
also eliminated the PDX1 complexes with the B and B2 elements.
MEIS2a/b antiserum contained MEIS2d, nuclear
extract was treated with a mixture of
MEIS2a/b and
MEIS2c/d
antisera (Fig. 2B, lane 4). The combination
eliminated all of the trimeric complex. In contrast, combining
MEIS2a/b and
MEIS2a/c did not eliminate the slower migrating
complex (lane 6), further showing that MEIS2c is not in the
trimeric complex.
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Fig. 2.
The MEIS2 isoforms in the acinar trimeric
complex are MEIS2b and MEIS2d. A, supershift EMSA using
isoform-specific MEIS2 antisera or the corresponding preimmune sera
(PI) showed that MEIS2b and possibly MEIS2d are in the
trimeric complex from 266-6 nuclear extract. B, supershift
EMSA with mixtures of MEIS2 isoform-specific antisera confirmed that
both MEIS2b and MEIS2d were in the 266-6 complex (lanes
1-6). Reconstituted trimeric complexes containing IVT PDX1,
PBX1b, and MEIS2b (lane 7) or MEIS2d (lane 8) had
electrophoretic mobilities similar to the 266-6 acinar trimeric
complex. Mobilities of the trimeric complexes containing MEIS2b or
MEIS2d are indicated by the short lines. The band of
intermediate mobility in the reconstituted complexes (lanes
7 and 8) is the PBX1·PDX1 heterodimer.
MEIS2b treatment. Thus, we conclude that the major molecular form of
the acinar trimeric complex comprises PDX1, PBX1b, and MEIS2b and that
MEIS2d replaces MEIS2b in a small fraction of the complexes.
-cell lines (10), the
MEIS2 proteins were detected in nuclear extracts from both. The levels
of the MEIS2 proteins were lower in
-cells than in acinar cells.
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Fig. 3.
MEIS2 proteins are present in pancreatic
exocrine and -cell lines. Western blots
of nuclear extracts from pancreatic cell lines were probed with
antiserum that detects all four isoforms of MEIS2. The two left
lanes contained IVT MEIS2b and MEIS2d, respectively.
-cell line RIN1046-38 (RIN-38), due to the presence of
endogenous PDX1 in these cells (10). PBX1 is absent in these
-cells
(10), and MEIS2 is present at extremely low levels (Fig. 3). Adding either PBX1b or MEIS2b to the
-cell line by transfection had little
or no effect on the activity of the B multimer or on the B element with
the augmented PBX1b binding site (B2) (Fig.
4A). Co-expression of PBX1b
and MEIS2b in RIN-38
-cells increased the activity of either the B
or B2 multimer severalfold over the activity directed by the endogenous
PDX1 (7-fold for the B element; 4-fold for the B2 element). These
results indicate that the trimer complex is more transcriptionally
active in RIN-38 cells than PDX1 alone. The B7 mutation which
eliminated PDX1 and trimer binding in EMSA had no activity in the
-cell line, whether in the presence or absence of cotransfected
PBX1b and MEIS2b (Fig. 4A), confirming the central
importance of PDX1 binding to the activity of this element.
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Fig. 4.
The trimeric complex acts as a
transcriptional activator. A, RIN-38 cells were
transfected with plasmids bearing five repeats of B, B2, or B7 elements
driving an hGH reporter gene. The relative activity of the hGH reporter
was assayed with or without cotransfected PBX1b and/or MEIS2b.
Transfections were normalized based on the activity of a
cotransfected Pdx1.luciferase reporter plasmid. B, the
relative activity of the B2 multimer in HeLa cells was assayed with or
without cotransfected PDX1, PBX1b, and MEIS2b individually or in
combination. Transfections were normalized based on the activity of a
cotransfected CMV- -gal reporter plasmid. All values are the average
of four or more transfections. Error bars represent standard
errors of the mean.
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Fig. 5.
Domains of PDX1 required for trimer binding
and activity. A, schematic representation of the PDX1
deletion series. The activation domain (AD), pentapeptide
motif (PP), and the homeodomain (HD) are
indicated. B, binding of the PDX1 mutants as a monomer
(upper panel), a heterodimer with PBX1b (middle
panel), and a heterotrimer with PBX1b and MEIS2b (lower
panel) to the B2 element. Monomers, dimers, and trimers are
labeled as 1, 2, and 3, respectively.
Lane 1, reticulocyte lysate without added template.
Lanes 2-5, PDX1 test constructs as noted at the top
of the panel. Note that full-length PDX1 has approximately the
same mobility as the major complex endogenous to the reticulocyte
lysate. C, activation of the B2 multimer in HeLa cells by
co-transfected PDX1 mutants, alone (black bars) or in
combination with PBX1b and MEIS2b (gray bars). All
activities are expressed relative to the activity of wild type PDX1
(PDX1-284) alone. Because the PDX116-206 does not
accumulate in the nucleus of transfected cells (data not shown), its
inherent transcriptional activity could not be assessed.
cell line, the activity of the B2 element in the presence of the various combinations of transcription factors paralleled the activity of the natural B element (data not shown). The
increased PDX1 activity by cotransfection of MEIS2b, which was not
observed with transfected RIN-38 cells, is likely due to the presence
of endogenous PBX1b, which is absent in RIN-38 cells.
-helix, which forms part of the hydrophobic pocket that
binds the pentapeptide motif of HOX-like proteins (45, 46). We tested a
series of PBX1b deletion mutants to determine which of the
PBX1b-conserved domains are critical for trimer formation, DNA binding,
and transcriptional activation (Fig. 6).
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Fig. 6.
Domains important for the function of PBX1b
in the trimer. A, diagram of the PBX1b deletion series.
The relative positions of PBC-A, PBC-B, the HD and helix 4 (H4) are shown. B, binding of the PBX1b mutants
alone (upper panel; labeled as 1), in combination
with PDX1 (middle panel; dimers labeled 2), or
with PDX1 and MEIS2b (lower panel; trimers labeled
3) to the B2 element by EMSA. Lane 1:
reticulocyte lysate without added template. Lanes 2-6:
PBX1b test constructs as noted at the top of the panel.
C, interaction of PBX1b mutants with MEIS2b in solution and
bound to DNA. Upper panel: 35S-labeled IVT PBX1b
proteins were incubated with unlabeled IVT MEIS2b, precipitated with
anti-MEIS2, and the precipitated protein resolved by
SDS-10%-polyacrylamide gel electrophoresis. Lower panel:
binding of PBX1b·MEIS2b dimers to the CRS1 element. The PBX1b
monomers and PBX1b·MEIS2b dimers are labeled as 1 and
2, respectively. Lane 1: reticulocyte lysate
without added template. Lanes 2-6: PBX1b test constructs as
noted at the top of the panel. D, activation of
the B2 multimer in HeLa cells by PBX1b mutants in co-transfections with
PDX1 and MEIS2b. All activities are expressed relative to the activity
of PDX1 plus MEIS2b in the absence of co-transfected PBX1b.
B, lane 6). Thus, formation of the trimer on DNA
requires helix 4 of the PBX1b HD to recruit PDX1 and the PBC domains to
recruit MEIS2b.
B to form trimeric complexes was due to
their inability to interact with MEIS2b, we examined the formation of
PBX1b·MEIS2b dimers by coimmunoprecipitation and their ability to
bind a composite PBX·MEIS site (Fig. 6C). In the absence
of DNA, deletion of both PBC domains (PBX1b233-347) eliminated
dimerization with MEIS2b, whereas deletion of PBC-B alone (PBX1b
B)
reduced, but did not eliminate, dimer formation. To analyze the binding
of PBX·MEIS dimers on DNA, we used the CRS1 element from the bovine
CYP17 gene, which contains adjacent PBX and MEIS
half-sites (30). PBX1b
B and MEIS2b formed a very small amount of
bound complex, but PBX1b233-347 did not, paralleling the results in
solution. The dimer of PBX1b1-295 and MEIS2b, although fairly stable
in solution, did not bind DNA efficiently (lane 4). These
results agree with previous findings that the region immediately
C-terminal to the HD (i.e. helix 4) is important for stabilizing PBX binding to DNA and for interacting with HOX-like proteins (44, 45). Deletion of the entire region N-terminal to the HD
(PBX1b233-347) or just the PBC-B domain (PBX1b
B) permits binding to
both the B2 and CRS1 elements as a monomer (Fig. 6B, lanes 5 and 6; Fig. 6C, lanes
5 and 6). Relief of inhibition of DNA binding by
deletion of the PBC N-terminal region has also been observed by
Neuteboom and Murre (47).
B, had no detectable activity in transfections, indicating that a principal role for PBX1b
in trimer activity is the recruitment of MEIS2b.
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Fig. 7.
Domains important for the function of MEIS2b
in the trimer. A, schematic of the MEIS2b deletion
series. The positions of the MEIS domains, M1 and M2, as well as the HD
are indicated. B, formation of the trimeric complexes with
the MEIS2b mutants on the B2 element. 3 denotes the trimers
and 2 in lane 2 indicates the PDX1·PBX1b dimer.
Lane 1: reticulocyte lysate without added template.
Lanes 2-7: MEIS2b test constructs as noted at the top
of the panel. C, analyses of the interaction of MEIS2b
mutants with PBX1b. Upper panel: 35S-labeled IVT
MEIS2b proteins were incubated with unlabeled IVT PBX1b, precipitated
with anti-PBX1N, and the precipitated protein resolved by
SDS-10%-polyacrylamide gel electrophoresis. Lower panel:
binding of the PBX1b·MEIS2b heterodimers to the CRS1 element.
Lane 1: reticulocyte lysate without added template.
Lanes 2-7: MEIS2b test constructs as noted at the top
of the panel. D, activation of the B2 multimer in HeLa
cells by MEIS2b mutants in co-transfections with PDX1 and PBX1b. All
activities are expressed relative to the activity of PDX1 plus PBX1b in
the absence of co-transfected MEIS2b.
HD, and MEIS2b1-275
enhanced the formation of the trimers (lanes 6 and
7). These results showed that the MEIS2b HD is dispensable
for the formation of a heterotrimer on DNA. This is in agreement with
the absence of a MEIS consensus site in the B element and with the
absence of any sequence requirements beyond the PBX-PDX1 binding site
for trimer binding and transcriptional activity (10).
HD and MEIS2b1-275, lanes 6 and 7). PREP1 can also dimerize with PBX in the absence of
its HD, but, as for MEIS, the HD is required for DNA binding (43).
Therefore, the N-terminal region containing the M1 and M2 domains is
sufficient for trimer formation. The M2 domain is known to interact
with PBX (7). Our deletion analysis did not unveil any direct
interaction between MEIS2b and PDX1.
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Fig. 8.
The PDX1·PBX1b·MEIS2b trimer and the
p48·HEB dimer of PTF1 cooperate in activating transcription from an
ELA1 mini-enhancer in HeLa cells. A,
schematic of the 3(BA).EIp.hGH mini-enhancer. B, the
relative activity of the 3(BA).EIp.hGH reporter gene in HeLa cells was
assayed with or without cotransfected PDX1, PBX1b, MEIS2b, and
p48·HEB individually or in various combinations as indicated. The
gray line indicates the activity of the 3(BA).EIp.hGH in the
absence of added transcription factors. This level of background
activity was the same as that of a promoter-only EIp.hGH construct
(data not shown), which was unaffected by added transcription factors
in any combination. All values are the mean of six or more
transfections, except for the pairwise combinations of PDX1 and PBX1b,
PDX1 and MEIS2b, PBX1b and MEIS2b, and p48 and HEB, which represent the
mean of two transfections. Error bars represent standard
errors of the mean. C, the relative activity of PDX1, PBX1b,
and MEIS2b deletion proteins in the cooperative activation of the BA
mini-enhancer by the homeodomain heterotrimer and PTF1. The presence of
full-length PDX1, PBX1b, and MEIS2b is indicated by +; the presence of
particular PDX1, PBX1b, and MEIS2b deletion mutants is indicated by
their identifying numbers from Figs. 5, 6, and 7. Values are the mean,
with standard error, of four or more transfections.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Model of the trimeric complex. PDX1 and
PBX1b bind to adjacent half-sites on the B2 variant of the
ELA1 enhancer B element. Protein·protein interactions
occur between the PDX1 and PBX1b HDs, and between the pentapeptide
(PP) of PDX1 and helix 4 (H4) of PBX1b. PBX1b and
MEIS2b interact through their N-terminal domains, with the M2 domain of
MEIS, and the PBC-A and PBC-B domains of PBX particularly important to
the interactions. Binding of the MEIS2b HD to DNA is not required for
trimer formation or transcriptional activity. PDX1 contains the only
transcriptional activation domain (AD) in the complex.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Scott Rose, Christopher Wright, Richard Baer, and Pierre Chambon for providing cloned transcription factors, and Dr. Peter Wellauer for communicating results prior to publication. We thank Dr. Mike Peyton for many helpful discussions and communication of unpublished results.
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FOOTNOTES |
---|
* This research was supported by Grant DK-55266 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
Biology, University of Texas Southwestern Medical Center, 5323 Harry
Hines Blvd., Dallas, TX 75390-9148. Tel.: 214-648-1942; Fax:
214-648-1915; E-mail: swift@hamon.swmed.edu.
Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M100678200
2 S. D. Rose, R. J. MacDonald, M. J. Peyton, R. E. Hammer, and G. H. Swift, manuscript in preparation.
3 P. Wellauer, personal communication.
4 S. Rose and R. J. MacDonald, unpublished.
5 M. J. Peyton and R. J. MacDonald, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: bp, base pair(s); bHLH, basic helix-loop-helix; kb, kilobase(s); IVT, in vitro transcription and translation; EMSA, electrophoretic mobility shift assay; CMV, cytomegalovirus; AD, activation domain; HD, homeodomain; PP, pentapeptide motif.
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