The PDX-1 Activation Domain Provides Specific Functions Necessary for Transcriptional Stimulation in Pancreatic ß-Cells
Mina Peshavaria1,
Michelle A. Cissell,
Eva Henderson,
Helle V. Petersen and
Roland Stein
Department of Molecular Physiology and Biophysics (M.P., M.A.C.,
E.H., R.S.) Vanderbilt University Medical School Nashville,
Tennessee 37232
Hagedorn Research Institute (H.V.P.)
DK-2820 Gentofte, Denmark
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ABSTRACT
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PDX-1 is a homeodomain transcription factor whose
targeted disruption results in a failure of the pancreas to develop.
Mutations in the human pdx-1 gene are linked to an early
onset form of non-insulin-dependent diabetes mellitus. PDX-1 binds to
and transactivates the promoters of several physiologically relevant
genes within the ß-cell, including insulin, glucose transporter 2,
glucokinase, and islet amyloid polypeptide. This study focuses on the
mechanisms by which PDX-1 activates insulin gene transcription. To
evaluate the role of PDX-1 in transcription of the insulin gene, a
chloramphenicol acetyltransferase reporter construct was designed with
a single yeast GAL4-DNA binding site in place of the A3/PDX-1 binding
element in the rat insulin II enhancer. In the presence of GAL4:PDX
chimeras containing N-terminal transactivation domain sequences, this
GAL4-substituted insulin construct was active in PDX-1-expressing
ß-cells and not non-ß-cells. PDX-1 activation was mediated through
three highly conserved segments of the transactivation domain. In
addition, when cotransfected together with the GAL4-substituted
insulin enhancer reporter gene in glucose-responsive MIN-6 ß-cells,
glucose-induced activation is observed with GAL4:PDX-1 but not with
fusions of the heterologous activation domains from herpes virus VP16
or adenovirus-5 E1A proteins. Using A3 element-substituted GAL4 insulin
enhancer reporter constructs containing mutations in two additional key
control elements, E1 and C1, we also show that full activation requires
cooperative interactions between other enhancer-bound factors,
particularly the E1 element activators. In contrast, the activity of
the VP16 activation factor was not dependent on the activators of
either the E1 or C1 sites. These results suggest that the PDX-1
transactivation domain is specifically required for appropriate
regulation of insulin enhancer function in ß-cells.
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INTRODUCTION
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Insulin gene expression is tightly restricted to the pancreatic
ß-cells within the islets of Langerhans. Tissue specificity is
conferred by a highly conserved enhancer region located within
approximately 350 bp of the insulin gene transcription start site;
multiple positively and negatively acting cis-elements
within the enhancer coordinately modulate insulin gene transcription
(1, 2, 3, 4). Detailed mutagenesis studies conducted on this region have
identified several key control elements, including the C2 (-317/-311
bp) (2), A3 (-201 to -196 bp) (5, 6), C1/RIPE3b (-118 and -107 bp)
(7, 8) and E1 (-100 to -91 bp) (9, 10, 11) elements. The core sequence
motifs for each of these elements are also found within the
transcription unit of all characterized mammalian insulin genes (12),
suggesting that similar transcriptional regulatory mechanisms are used
in control.
The A3 element contributes to both ß-cell-selective (5, 6, 10, 13)
and glucose-regulated transcription (13, 14, 15) of the insulin gene. The
ß-cell-enriched homeodomain factor, PDX-1 (pancreatic and duodenal
homeobox protein 1; also known as STF-1, IDX-1, IPF-1, IUF-1), controls
A3 element-directed transcription (6, 13, 16, 17, 18). Another PDX-1
binding site is present within the insulin transcription unit at -81
to -77 bp; however, this site appears to be much less important than
A3 in control (6). PDX-1 also activates expression of other islet
genes, including GLUT2 (19), islet amyloid polypeptide (IAPP) (20, 21),
somatostatin (22, 23), glucokinase (24), and HB-EGF (25).
The expression pattern of PDX-1 implicates this transcription factor in
the development of ß-cell identity. During embryogenesis, PDX-1
expression is observed in the ventral and dorsal walls of the primitive
foregut where the pancreatic buds will form (16). As development
proceeds, PDX-1 is detectable in islet cell precursors and,
transiently, in exocrine and ductal cells (26). In the adult pancreas,
PDX-1 expression becomes restricted to the islets (6, 16, 22, 23, 26),
where it is found in almost all (>90%) ß-cells as well as a subset
of
(3%) and
(15%) cells (6). PDX-1 expression is also
observed in the submucosal epithelium of the duodenum (22, 23, 26) and
antral stomach (27).
Germline deletion of the mouse pdx-1 gene also supports a
critical role for this protein in pancreatic development. Homozygous
knockout of the mouse pdx-1 locus results in pancreatic
agenesis (28, 29) and malformations of the rostral duodenum (29).
Furthermore, selective removal of the pdx-1 gene in islet
ß-cells of mice has also clearly demonstrated that PDX-1 plays a key
role in vivo in generating functional ß-cells, at least in
part, through its actions on insulin, IAPP, and GLUT2 expression (30).
In this experiment, a critical region of the endogenous
pdx-1 gene, flanked by loxP sites, was conditionally removed
in ß-cells by expression of insulin enhancer/promoter-driven Cre
recombinase. Mice carrying this ß-cell-specific pdx-1
deletion developed diabetes (30). In humans, an inactivating mutation
in the ipf1/pdx-1 locus is linked to a form of early-onset
diabetes when inherited in a heterozygous state (31). Moreover, a
patient that is homozygous for this pdx-1 mutation failed to
develop a pancreas (32).
PDX-1 is analogous to most other transcription factors in possessing a
modular structure composed of separable functional domains. The highly
conserved homeodomain within the middle of the protein confers both
DNA-binding and nuclear translocation functions (33, 34), whereas the
N-terminal region provides most of the transactivation potential (17, 33, 35), although a cryptic activation domain in the C terminus may
also be involved in stimulating somatostatin gene expression in
-cells (33). Within the N terminus, at least three evolutionarily
conserved subdomains are critical for transactivation (35). PDX-1
mutants lacking these subdomains act as dominant negative inhibitors
due to their ability to translocate to the nucleus and bind DNA but not
stimulate transcription (35, 36).
Our current understanding of the amino acid sequences within the
N-terminal region of PDX-1 that are required for transcriptional
activation has been derived primarily from analysis of PDX-1 in
non-ß-cells or from GAL4:PDX-1 fusion proteins tested with simple
yeast GAL4 element-driven constructs in ß-cells (17, 33, 35, 37, 38).
These artificial constructs consisted of multimerized GAL4 binding
sites driven by the E1b promoter and allowed the mapping of the
activation domain in both ß- and non-ß-cell types. Although these
approaches have provided useful information about the general sequences
involved in activation, it is important to note that these experiments
do not measure transcriptional regulation within the context of the
native insulin enhancer in ß-cells. To define the factors regulating
PDX-1-activated transcription in this environment, the A3 element of
the rat insulin II enhancer was replaced with a single binding site for
the yeast protein, GAL4. This assay system allows us to study the
mechanisms involved in PDX-1 (i.e. GAL4:PDX-1) activation of
the rat insulin II enhancer in the ß-cell without interference from
the endogenous factor. The results from this study demonstrate that the
PDX-1 activation domain is specifically required for ß-cell-selective
and glucose-stimulated activation of the insulin gene by functionally
interacting with other insulin enhancer-bound factors. We discuss the
possible significance of these findings to other PDX-1-activated
genes.
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RESULTS
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GAL4:PDX-1 Only Stimulates -217 GAL Activity in ß-Cells
An insulin reporter gene in which the PDX-1 control region between
-205 to -189 bp was replaced with a single yeast GAL4 DNA binding
site was used to study how PDX-1 activates insulin gene transcription
(Fig. 1A
; -217 GAL). Replacement of the
A3 element with the GAL4 binding site reduced insulin
enhancer-mediated activity to the same extent as a site-directed A3
mutant that prevented PDX-1 binding (Ref. 6 and data not shown),
demonstrating that this mutation does not influence the activity of
other immediate 5'- or 3'-control elements. To test whether GAL4:PDX-1
fusion proteins could stimulate insulin enhancer activity, a fusion
construct representing the full-length [GAL4:PDX(FL)] and N-terminal
activation domain region [GAL4:PDX(1149)] of rat PDX-1 was
cotransfected with -217 GAL into either a ß- (HIT T-15, ßTC3,
INS-1) or non-ß- (HeLa, BHK) cell line. GAL4:PDX(FL) and
GAL4:PDX(1149) potentiated -217 GAL activity, but only in ß-cell
lines (Fig. 1
). The GAL4:PDX constructs were equally active,
stimulating approximately 3- to 8-fold, depending upon the ß-cell
line (Fig. 1
). We have previously shown that GAL4:PDX(FL) and
GAL4:PDX(1149) are effectively expressed, and at comparable levels,
in both HIT-T15 and HeLa cells (35), demonstrating that their
inactivity in non-ß-cells is not due simply to the lack of PDX-1.

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Figure 1. GAL4:PDX-1 Only Activates -217 GAL Activity
in ß-Cells
A, Schematic of the -217 to +2 bp region of the rat insulin II gene.
The enhancer domain (-217 to -91 bp) contains a binding site for the
PDX-1 (A3, -201 to -196 bp), RIPE3b1 (C1, -115 to -107 bp), and
BETA2/NeuroD1:E47 (E1, -100 to -91 bp) activators. The A3 element was
replaced by a single GAL4 binding site (-217 GAL); the insulin
sequences are linked to the CAT reporter gene. The sequence of the A3
element (bold) is shown in comparison to the
GAL4-substituted element (bold and
underlined). A total of 16 nucleotides were changed by
the GAL4 substitution. B, The GAL4-substituted insulin
enhancer construct, -217 GAL, was cotransfected with vector alone (-)
or with vectors encoding fusions of the GAL4 DNA-binding domain with
full-length [GAL4:PDX(FL)] or amino acids 1 through 149
[GAL4:PDX(1149)] of PDX-1. Transfections were performed in ß (HIT
T-15, ßTC3, and INS-1) and non-ß lines (HeLa and BHK). The CAT
activity in each sample was normalized to the LUC activity from the
cotransfected pGL2RSV plasmid. Results are presented as relative to
-217 GAL cotransfected with the GAL4 DBD vector alone ±
SD; the -217 GAL alone point was arbitrarily set
to a value of 1.0. Each transfection represents an average of at least
four separate experiments.
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To further explore the ß-cell specificity of PDX-1, we directly
compared in ß (HIT T-15) and non-ß (HeLa) cells the ability of
GAL4:PDX-1 fusion proteins to activate GAL4 target genes in three
distinct promoter contexts: 1) a single GAL4 binding site within the
insulin enhancer (-217 GAL LUC); 2) a single GAL4-binding site linked
to the minimal E1b promoter [(GAL4)1E1b LUC];
and, 3) five GAL4 binding sites linked to the E1b promoter
[(GAL4)5E1b LUC]. GAL4:PDX-1 is a very poor
transactivator of (GAL4)1E1b LUC in either
HIT-T15 or HeLa cells, whereas (GAL4)5E1b LUC was
transcriptionally active in both ß- and non-ß-cells (though to
higher level in ß-cells) (Fig. 2
). In
contrast, GAL4:PDX-1 activated -217 GAL LUC effectively only in
ß-cells (Fig. 2
). These results clearly demonstrate that a single
PDX-1 binding unit cannot support cell type-specific transcription in
isolation, whereas a single PDX-1 site is sufficient in an insulin
enhancer context.

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Figure 2. Functional Interactions between PDX-1 and a
ß-Cell- Enriched Activator(s) of -217 GAL Are Crucial for
Transcriptional Stimulation
The -217 GAL LUC, (GAL4)1E1b LUC, or
(GAL4)5E1b LUC vectors were cotransfected into HIT-T15 and
HeLa cells either alone or with GAL4DBD, GAL4:PDX(FL), or
GAL4:PDX(1149). The LUC activity of each sample was normalized to the
CAT activity of cotransfected pRSVCAT. The relative activity ±
SD is calculated as the activity of reporter vector
plus the GAL4 expression construct divided by the reporter vector
alone. Note that the overall stimulatory pattern of GAL4:PDX with -217
GAL LUC and (GAL4)5E1b LUC is identical to that observed
with the analogous CAT reporter constructs [-217 GAL, Fig. 1 ;
(GAL4)5E1b CAT (17 35 )], although the magnitude of the
response differs.
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Collectively, the data suggest that the activity of PDX-1
transactivation domain per se is not ß-cell specific, but
requires other ß-cell-restricted enhancer-bound factors to promote
selective expression of the insulin gene. The following experiments
were designed to identify the sequences within PDX-1 that mediate
insulin enhancer activation, and to determine the role of this region
of PDX-1 in glucose-inducible insulin gene transcription. In addition,
the ß-cell-enriched activators that cooperate with PDX-1 to
stimulate insulin enhancer activity were identified.
The PDX-1 Transactivation Domain Mediates GAL4:PDX-1 Activity
To define the sequences of PDX-1 required for insulin enhancer
activation in ß-cells, N- and C-terminal GAL4 fusion mutants of rat
PDX-1 and the Xenopus homolog, XlHbox8, were assayed for
their ability to stimulate -217 GAL activity in HIT T-15 ß-cells
(Fig. 3
). The rat and Xenopus
proteins share 100% and approximately 50% identity in their
homeodomains [amino acids (aa) 146206] and N-terminal regions (aa
1145), respectively, whereas there is little conservation within the
C-terminal region (35). The activation domain of these proteins was
localized to aa 179 using a multimerized GAL4 binding
site-reporter-driven construct [(GAL4)5E1b; (17, 35)]. Transactivation is mediated by three highly conserved sequences
that span aa 1322 (subdomain A), 3238 (subdomain B), and 6073
(subdomain C) (35). Deletion of all three subdomains
[GAL4:PDX(1149
ABC)] results in 98% reduction of activity of the
(GAL4)5E1b reporter vector in HeLa cells (35).
Each of the GAL4:PDX fusion proteins analyzed here is expressed at
equivalent levels in transfected HIT T-15 (Ref. 35 and data not
shown).

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Figure 3. The N-Terminal Regions of PDX-1 Are Crucial for
Transcriptional Activation
Diagrammatic representation of rat PDX-1 and Xenopus
XlHbox8 (not drawn to scale). XlHbox8 sequences and homeodomain
sequences (HD, 146206) are indicated by hatched and
gray boxes, respectively. The activation domain (AD) is
located between amino acids 179. The deletion end points of PDX-1 and
XlHbox8 in the GAL4 chimeras are indicated. The transcriptional
activities of the full-length (FL) and mutant PDX-1 and XlHbox8 GAL4
fusion proteins with -217 GAL were examined in HIT T-15 cells. The
normalized CAT activity from -217 GAL plus GAL4:PDX-1 is reported
relative to -217 GAL cotransfected with pSG424 alone. Each value
represents an average of at least three transfections ± the
SE of the data.
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GAL4:PDX-1 and GAL4:XlHbox8 proteins lacking N-terminal sequences
[GAL4:PDX(76284) and GAL4:XlHbox8(91271)] were unable to
potentiate -217 GAL (Fig. 3
). In contrast, the GAL4 constructs
containing conserved N-terminal sequences of PDX1 or XlHbox8
[GAL4:PDX/XlHbox8(179/175), GAL4:PDX/XlHbox8(1149/1150),
GAL4:PDX/XlHbox8(1210/1210), and GAL4:PDX/XlHbox8(FL/FL)]
stimulated reporter gene activity effectively. GAL4:PDX(FL) and
GAL4:XlHbox8(FL) stimulate (GAL4)5E1b
chloramphenicol acetyltransferase (CAT) activity poorly relative to
GAL4:PDX(1149) (17, 35, 38), yet the homeodomain and C-terminal
regions of PDX-1 or XlHbox8 did not have any significant effect on
-217 GAL activation (Fig. 3
). To test this proposal more thoroughly,
the nonconserved C-terminal sequences of Xenopus XlHbox8
were substituted for those of rat PDX-1 in GAL:PDX(FL). As expected,
activation by this GAL4 chimera was similar to deletion mutants lacking
the C terminus, such as GAL4:PDX(1210) (data not shown).
Mutants within the conserved N-terminal subdomains of PDX-1 were next
tested for their ability to activate -217 GAL. This analysis included
aa 1322, 3238, 6073, and 8185 (i.e. subdomain D)
deletion mutants, as well as site-directed mutants within aa 117126
(i.e. subdomain E) that eliminate the potential interaction
of PDX-1 with PBX-1 or phosphorylation by protein kinase C. Deletion of
the A, B, or C subdomains individually reduced GAL4:PDX(1149)
activity by 2535% (Fig. 4
). In
contrast, little or no effect on -217 GAL activation was found upon
deletion of subdomain D or by preventing PBX-1 interaction or
serine/threonine phosphorylation within the aa 117126 region. The
latter result is consistent with a previous observation demonstrating
that the interaction of PDX-1 with PBX-1 is not required for insulin
transcription in ß-cells, although it does affect somatostatin
expression in islet
-cells (39). An N-terminal mutant spanning aa
76149 was unable to transactivate -217 GAL (Fig. 4
), further
emphasizing the requirement for PDX-1 activation domain sequences in
this process. Thus, it was not surprising that stimulation by
GAL4:PDX(1149
ABC) was severely impaired (i.e. by 70%;
Fig. 4
). However, GAL4:PDX(1149
ABC) activation of -217 GAL was
not entirely lost, as it was with (GAL4)5E1b CAT
(35), suggesting that as-yet-unidentified interactions, within either
less conserved sequences and/or subdomains D and E, are uniquely
required for transactivation of the insulin enhancer.

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Figure 4. Contribution of the Transactivation Subdomains to
the Activity of GAL4:PDX(1149)
The conserved amino acid segments (A, 1322; B, 3238; C, 6073; D,
8791; Pbx, 119123; PKC, 124125) and homeodomain sequences (HD,
146149) are indicated by black and gray
boxes, respectively. The deleted subdomains are indicated with
an X. HIT-T15 cells were transfected with -217 GAL vector alone and
either pSG424 (GAL4DBD), GAL4:PDX(1149) or a mutant form of
GAL4:PDX(1149). The results are calculated as a percentage of the
normalized CAT activity of the GAL4:PDX(1149) expression vector
± SEM.
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Glucose Stimulates PDX-1 Transactivation Domain Function
Glucose-stimulated transcription of the insulin gene is mediated
through multiple enhancer elements, including A3 (13, 14, 15, 40), C1 (8, 41), and E1 (8, 41, 42). The A3 element of the insulin enhancer
contributes to glucose responsiveness in part through increased PDX-1
binding activity (13, 15, 40). To examine the contribution of the PDX-1
transactivation domain to glucose induction, GAL4:PDX(1149)
stimulation of -217 GAL was compared with GAL4 fusions of potent, but
unrelated, viral activation domains [GAL4:VP16 and
GAL4:E1A(121223)] in MIN-6 ß-cells. The wild-type insulin enhancer
(-238 WT CAT) was induced 4-fold in cells grown in 20 mM
(high) vs. 3 mM (low) glucose (Fig. 5
). As expected, replacement of the A3
element with the GAL4 binding site reduced the level of glucose
induction (Fig. 5
). GAL4:PDX(1149) fully restored glucose induction
of -217 GAL to wild-type levels (Fig. 5
). In contrast, the
transactivation domains of the herpes virus VP16 and adenovirus E1A
proteins were incapable of inducing -217 GAL activity in
glucose-stimulated ß-cells. These results indicate that the PDX-1
transactivation domain specifically potentiates insulin enhancer
activity in response to ß-cell activator factors and the glucose
signaling pathway.

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Figure 5. Transactivation by PDX-1 Is Potentiated Selectively
in Glucose-Stimulated ß-Cells
MIN6 cells were transfected with -238 WT, the wild-type insulin-driven
CAT reporter, or -217 GAL alone or plus GAL4:PDX(1149), GAL4:VP16,
or GAL4:E1A(121223) (GAL4:E1A). The results are calculated as the
ratio of the normalized CAT activity at 20 mM glucose
divided by the activity at 3 mM ±
SEM.
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Transactivation by PDX-1 Is Mediated by the Insulin Gene E1
Activators
The ß-cell specificity of GAL4:PDX-1 activation suggested that
another insulin enhancer factor(s) was required for stimulation by
PDX-1. To explore this hypothesis further, site-directed mutations
within the C1 and E1 elements were introduced into -217 GAL. The
binding of the BETA2/NeuroD1:E2A (43) and RIPE3b1 (7, 8) activators was
prevented in the mutants of E1 and C1, respectively. GAL4:PDX(FL) and
GAL4:PDX(1149) activation was reduced by 10-fold in the E1 mutant,
-217GAL(E1mt), relative to -217GAL in HIT T-15 cells, whereas
GAL4:VP16 stimulation was only inhibited by 2-fold (Fig. 6
). In contrast, GAL4:PDX stimulation
was potentiated by 2-fold in the C1 element mutant, -217GAL(C1mt)
(Fig. 6
). GAL4:VP16 activation was unaffected by the C1 mutation.
Activation by the GAL4:PDX-1 fusion proteins was completely abolished
in the C1/E1 double mutant, -217GAL(C1/E1mt), although stimulation by
GAL4:VP16 was only reduced by 2-fold (Fig. 6
). Analogous results were
obtained when these constructs were assayed in ßTC3 cells (data not
shown). The data indicate that functional interactions between PDX-1
and the E1-bound activators, BETA2/NeuroD1:E2A, are particularly
critical to insulin enhancer-mediated activation.

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Figure 6. Effect of Mutating the C1 and E1 Elements in -217
GAL on GAL4:PDX-1 Activation
The wild-type, E1 mutant (-217 GAL(E1mt)), C1 mutant (-217
GAL(C1mt)), or C1/E1 double mutant (-217 GAL(C1/E1mt)) -217 GAL
reporter plasmid was co-transfected into HIT T-15 cells in the absence
(-) or presence of GAL4:PDX expression vectors. The normalized CAT
activity from wild-type or mutant -217 GAL plus GAL4:PDX-1 is reported
relative to the insulin:GAL plasmid cotransfected with pSG424 alone.
The normalized CAT activity of -217 GAL, -217 GAL(E1mt), -217
GAL(C1mt), and -217 GAL(C1/E1mt)) transfected with pSG424 was
159,031 ± 14,667, 5,347 ± 330, 11,379 ± 1,741, and
6,474 ± 1,033 cpm, respectively.
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DISCUSSION
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PDX-1 activates transcription of genes associated with ß-cell
identity, including insulin, GLUT2, glucokinase, and IAPP. The
transactivation potential of PDX-1 has primarily been analyzed by
fusing the yeast GAL4 DNA-binding domain to various regions of the
PDX-1 protein and assaying the ability of these chimeric proteins to
transactivate an artificial promoter composed of multiple GAL4 binding
sites (17, 35, 37). In this study, the key PDX-1 binding site in the
insulin enhancer was replaced with a single GAL4 binding site and the
functional properties of chimeric GAL4:PDX-1 proteins were assessed in
this GAL4-substituted insulin enhancer-dependent transcription system.
Our results indicate that specific features of the PDX-1 activation
domain are required for appropriate regulation of ß-cell target
genes.
When the insulin enhancer A3 element is replaced with a single GAL4
binding site, the transcriptional activity of the enhancer is reduced
to a level that is consistent with that observed in an enhancer
carrying an A3 mutation (6). The chimeric GAL4:PDX-1 protein restores
activity of the GAL4-substituted enhancer in ß-cell lines, but not
non-ß-cells (Fig. 1
). This observation contrasts with the results
from the multimerized GAL4 element reporter gene that is activated by
GAL4:PDX-1 in both HIT T-15 and HeLa cells (17, 35). Thus, a single
PDX-1 transactivation domain is not sufficient to activate
transcription from the insulin enhancer but requires other
ß-cell-enriched factors. This conclusion is further emphasized by the
inability of GAL4:PDX-1 to activate expression from reporters driven by
either a single GAL4 binding site linked to a heterologous promoter or
the GAL4-substituted insulin enhancer lacking E1 and C1 activator
function. These results also suggest that the PDX-1 transactivation
domain per se is not ß-cell specific, but requires other
ß-cell-enriched factors to drive cell type-specific insulin gene
transcription.
The observation that a single enhancer-bound transactivator is unable
to activate transcription or mediate a response to a particular
stimulus is not uncommon. Many genes require "enhanceosomes" or
"response units" composed of multiple, distinct DNA-bound factors
to generate a complete transcriptional response (44, 45); it is
proposed that the various enhancer factors arrayed on a particular
promoter cooperatively create a unique surface that, in turn, recruits
the transcriptional machinery. For example, the interferon-ß (IFNß)
enhanceosome, which mediates transcriptional induction in response to
viral infection, requires three transcription factorsNF
B,
ATF2/c-jun, IRF-1as well as the chromosomal protein
HMG-I/Y bound to adjacent elements for synergistic activation of the
enhancer (46, 47). In vitro, the enhanceosome stabilizes
formation of a preinitiation complex composed of general transcription
factors (TFIID, TFIIA, and TFIIB) and the cofactor USA (47) through the
specific recruitment of TFIIB (48). In addition, the RNA pol II
holoenzyme is also targeted to the IFNß promoter by an interaction
between the enhanceosome and the transcriptional coactivator
CREB-binding protein (CBP)/p300 (48, 49). Importantly, although
the enhanceosome transcription factors can each individually associate
with CBP/p300 in vitro, in the context of the IFNß
enhancer, transactivation domains from all of the bound factors are
necessary for CBP/p300 recruitment (49). The need to assemble a
complex, multiprotein enhanceosome for efficient activation of IFNß
transcription ensures that the gene will only be expressed when the
multiple signals mediating the response to viral infection converge on
the promoter.
Similarly, the assembly of a PDX-1-dependent enhanceosome might require
other ß-cell-enriched factors for activity. Under such circumstances,
the aberrant expression of insulin in a PDX-1 expressing nonislet cell
types (i.e. epithelial cells of the duodenum) would be
precluded, despite the availability of PDX-1, by the lack of other
ß-cell-enriched factors. Since the E1 mutation in -217 GAL
drastically impaired PDX-1 activation (Fig. 6
), the insulin
enhanceosome appears to minimally depend on the PDX-1 and E1
activators. The E1 activator is a dimeric complex composed of basic
helix-loop-helix factors that are islet enriched [BETA2/NeuroD1 (43)]
and generally distributed [HEB (50) or the E2A encoded proteins, E12,
E47, or E2/5 (7, 51, 52, 53)], which is also incapable of supporting
significant activation of insulin gene transcription when isolated from
other insulin enhancer binding factors (7, 43, 54). Notably, stably
transfected PDX-1 can induce ectopic insulin expression in cultured
-cells that normally express the E1 element activators but not in
fibroblast cells that have no detectable E1 binding activity (55). E1
activator function is known to be mediated through interactions of
CBP/p300 with both BETA2/NeuroD1 (56, 57, 58) and the E47/HEB proteins
(58). Similarly, PDX-1 physically associates through N-terminal
conserved sequences with CBP/p300 in GST pull-down in vitro
and in vivo immunoprecipitation experiments (Ref. 59 and
data not shown). In addition, recent in vitro experiments
suggest that the PDX-1 homeodomain region may also interact directly
with the E1 activators (60). Together, the data indicate that much like
the IFNß gene, the assembly of an insulin enhanceosome requires both
activator:activator and activator:CBP/p300 co-activator interactions.
The results presented here stress the importance of the N-terminal
PDX-1 activation domain in communicating signals through CBP/p300 to
BETA2/NeuroD1 and the RNA polymerase II transcriptional
machinery.
The PDX-1 transactivation domain was dissected for the purpose of
identifying the motif(s) necessary for PDX-1 function in the context of
the insulin enhancer. Several studies have shown that the PDX-1
transactivation domain resides in the N-terminal region of the PDX-1
protein (17, 33, 35). As previously reported for the multimerized GAL4
binding site-driven construct (35), N-terminal sequences spanning amino
acids 1322, 3238, and 6073 are required for full transactivation
of the insulin enhancer. Whereas the C terminus and homeodomain
interfere with the transactivation potential of the N terminus when
assayed on the multimerized GAL4 construct (17, 35, 38), these regions
are largely dispensable for transactivation of the insulin enhancer by
the chimeric GAL4:PDX-1 proteins (Fig. 3
). This result further confirms
that a C-terminal cryptic activation domain that functions in
activation of the somatostatin PDX-1 binding site (33) is not required
for insulin enhancer expression.
The A3 element of the insulin enhancer contributes to glucose
responsiveness in part through increased PDX-1 binding activity (14, 37, 61). Recently, it has also been shown that the PDX-1
transactivation domain fused to the GAL4 DNA-binding domain can confer
glucose induction to a heterologous promoter composed of five GAL4
binding sites (37, 38). This suggests that, in addition to effects on
DNA binding, glucose also stimulates the activation potential of PDX-1.
In the current study, substitution of the PDX-1 cis-element
with a GAL4 binding site reduces glucose induction of the insulin
enhancer by 50%; the residual glucose effect is conferred by the
glucose-responsive C1 and E1 activator factors (8, 42). The glucose
induction is fully restored to wild-type levels by the addition of a
GAL4:PDX-1 transactivation domain-expressing construct, but not by
GAL4:activation domain fusions with the potent, but distinct, E1A or
VP16 transactivating proteins. These results confirm and extend the
previous conclusion by demonstrating that the PDX-1 transactivation
domain provides an essential and unique function to the insulin
enhancer in response to glucose that is independent of its ability to
bind the A3 element.
The mechanism of PDX-1 activity in native promoters appears to depend
on the context of its cognate DNA-binding element as well as on
interactions with other cell-specific factors. For example, PDX-1
activation of the TSEII element in the somatostatin enhancer is
potentiated by cooperative binding of a second homeodomain cofactor,
PBX (39). The activity of another PDX-1 binding site in the
somatostatin promoter, TSEI, is strongly enhanced by binding of
PBX1a/Prep1 heterodimers to an adjacent cis-element (62).
PDX-1 activation of the elastase-1 gene is similarly dependent on
cell context. In acinar cells, PDX-1 function in the elastase enhancer
requires formation of a trimeric complex between PDX-1, PBX1b, and
MRG1; furthermore, a multimerized PDX-1/PBX1b/MRG-1 binding site from
the elastase gene is inactive in acinar cells, indicating a dependence
on additional enhancer elements for PDX-1 function (63). In contrast,
PDX-1 cannot activate the elastase enhancer in ß-cells due to the
lack of PBX1b and MRG-1, although transcription from the multimerized
PDX-1 element of the elastase gene can be stimulated by uncomplexed
PDX-1 in ß-cells (63). Importantly, PBX does not influence the
activity of PDX-1 on the A element of the insulin enhancer (39),
suggesting that PDX-1 activation of the insulin gene may require a
distinct set of functional interactions.
The data in this report support the hypothesis that PDX-1
activation in the context of the insulin gene functions mainly through
a conserved transactivation domain in the extreme N-terminal region and
requires interaction with factors bound to adjacent elements, in
particular the E1 activators. The activity of the PDX-1 transactivation
domain, which has thus far been shown to be active in expression from
the insulin A3 element (this report and Ref. 17), the somatostatin
TAAT1 element (33), and a multimerized heterologous DNA binding site
(GAL4) (17, 35, 38), is likely to also be generally applicable to the
mechanism of PDX-1 activation of additional target genes such as GLUT2,
glucokinase, IAPP, and others. Our results provide further evidence
indicating that the interactions that the PDX-1 transactivation domain
make with additional DNA bound factors are flexible and depend heavily
on the context of the PDX-1 binding site in a target promoter. For
example, PDX-1 activation of the IAPP promoter may not be dependent on
an interaction with the E1 activators, just as PDX-1-mediated
activation of the insulin promoter does not require PBX interactions.
We speculate that in other PDX-1 regulated promoters, transcriptional
synergy will depend upon recruitment of common bridging factors, like
CBP/p300, to the promoter by PDX-1 and other DNA-bound factors.
 |
MATERIALS AND METHODS
|
---|
Cell Culture and Transfections
Monolayer cultures of HIT T-15 (64), ßTC-3 (54), MIN-6 (37),
HeLa (64), and BHK (65) cells were grown under conditions described
previously. INS-1 cells were cultured and transfected in RPMI 1640
(-glucose) medium supplemented with 10% (vol/vol) FCS, 10
mM HEPES, 1 mM sodium pyruvate, 2
mM L-glutamine, 50 µM
ß-mercaptoethanol, and glucose to a final concentration of 4
mM. All media were supplemented with 50 µg/ml each of
streptomycin and penicillin. The insulin:CAT constructs were
transiently transfected by the calcium phosphate coprecipitation [HIT
T-15, BHK, and HeLa, (64), MIN-6 (37)] or lipofectamine (ßTC3 and
INS-1; 1 x 106 cells) procedures
(LipofectAMINE Reagent, Life Technologies, Inc.,
Gaithersburg, MD). The precipitates (12.5 µg total) used for the HIT
T-15, MIN-6, HeLa, and BHK cell transfections contained 1.25 µg CAT
reporter vector, 2.5 µg GAL4 fusion protein expression vector or CMV4
plasmid (66), and 1.25 µg pGL2RSV. The Rous sarcoma virus (RSV)
enhancer-driven luciferase (LUC) expression plasmid, pGL2RSV
(Promega Corp., Madison WI), was used as a recovery
marker. HIT T-15 and BHK cells were treated with 20% glycerol (2 min)
4 h after the addition of the calcium phosphate DNA precipitate.
After transfection, the MIN6 cells were grown in RPMI 1640 (10% FCS)
containing either 3 or 20 mM glucose for 16 h. ßTC3
and INS-1 cells were transfected with a ratio of 6 µl of
LipofectAMINE reagent to 1 µg DNA using the conditions described by
the manufacturer (i.e. 1 µg CAT reporter vector, 2 µg
GAL4 fusion protein expression vector or CMV4 plasmid, and 1 µg
pGL2RSV). Extracts were prepared either 16 (MIN6) or 48 h after
transfection and chloramphenicol acetyltransferase (CAT) (67) and
luciferase (68) enzymatic assays performed. The CAT activity from the
reporter construct was normalized to the LUC activity of the
cotransfected internal control plasmid. Each experiment was carried out
at least three times with two or more independently isolated plasmid
preparations.
Plasmid DNAs
The -238 WT CAT and -217 WT CAT vectors contain rat insulin II
gene sequences from -238 bp to +8 bp or -217 bp to +2 bp,
respectively (69). PCR was used in the construction of -217 GAL (70);
the insulin A3 element (-205 to -189 bp) in -217 WT was replaced
with a single palindromic GAL4 DNA binding site
(5'-CGGAGGACTGTCCTCC-3'). This mutant exchanges 16 nucleotides of
insulin sequence with a GAL4 binding element of the same length and
causes a mutation in the core A3 element binding site (i.e.
TAATT) as well as 4 nucleotides 5' and 7 nucleotides 3' to the core.
The -217 CAT (E1mt) (69) and -238 CAT (C1mt) (8) plasmids served as
the PCR templates in generating -217 GAL(C1mt), -217 GAL(E1mt), and
-217 GAL(C1/E1mt) plasmids. The -217 GAL LUC vector was made by
subcloning the insulin GAL4 sequences from -217 GAL just
upstream of the LUC gene in pSV0ARPL2L (71). The
(GAL4)1E1b LUC vector was a gift of M. Daniels
and D. K. Granner (Vanderbilt University, Nashville TN). All of
the GAL4:PDX-1 or GAL4:XlHbox8 fusion protein expression vectors and
their derivatives were previously reported (35) except the
GAL4:PDX(149
Pbx) and GAL4:PDX(1149
PKC) vectors that were
prepared in GAL4:PDX(1149) by PCR using an oligonucleotide spanning
the site of mutagenesis at either the Pbx interaction motif (aa
119123 FPWMK mutated to AAGGQ)
(5'-GCGCAAGCTTACGCGTGAGCTTTGGTGGACTGGCCGCCGGCGGCAGGGAGATGAACGCGGCT-3')
or the potential PKC phosphorylation site (aa 124125 ST mutated to
AA) (5'-GCTTTTCCACGCGTGAGCTTTGGCGGCTTTCATCCACGGGAAAGGGAG-3'). The
GAL4:VP16 (72), GAL4:E1A(121223) (73), and the GAL4 DNA binding
domain [termed GAL4 DBD; pSG424 (73)] expression vectors were
previously described. Cloning and isolation of plasmids were performed
by standard protocols. The sequence of all plasmid constructs was
confirmed by DNA sequence analysis.
 |
ACKNOWLEDGMENTS
|
---|
The authors thank Mr. K. Gerrish and Drs. M. Gannon, Y. Qiu, S.
Samaras, and C. Wright for assistance and scientific discussions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Roland Stein, Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Light Hall, Room 708, Nashville, Tennessee 37232. E-mail: roland.stein{at}mcmail.vanderbilt.edu
This work was supported by a grant from the NIH (NIH RO1 DK-50203 to
R.S.) as well as partial support from the Vanderbilt University
Diabetes Research and Training Center Molecular Biology Core Laboratory
(Public Health Service Grant P60 DK-20593 from the NIH).
1 Present address: Ontogeny Inc., 45 Moulton Street, Cambridge,
Massachusetts 02138-1118. 
Received for publication December 9, 1999.
Revision received August 16, 2000.
Accepted for publication August 22, 2000.
 |
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