From the Department of Endocrinology and Metabolism, Hebrew University Hadassah Medical Center, 91120 Jerusalem, Israel
Received for publication, October 4, 2000, and in revised form, January 16, 2001
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ABSTRACT |
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The PDX-1 transcription factor plays a key role
in pancreas development. Although expressed in all cells at the early
stages, in the adult it is mainly restricted to the The mammalian pancreas develops by fusion of dorsal and ventral
buds which form as evaginations of the upper duodenal part of the gut.
Identification of the homeodomain-containing transcription factor
PDX-11 as the first molecular
marker temporally correlates with the pancreatic commitment of the
epithelial cells in this region (1-3). Targeted inactivation of this
gene in the mouse (4) as well as its mutation in man (5) results in
agenesis of the pancreas. The gene is expressed both in endocrine and
exocrine cells of the developing pancreas; however in the adult islet,
its expression is predominantly restricted to the Development, cell fate, and cell differentiation are complex events
that depend on switching on and off the expression of specific sets of
genes. Such regulation operates mainly at the transcriptional level by
the assembly of multiprotein complexes at the enhancer(s) and the
promoter regions of the gene. These complexes are formed and stabilized
through multiple protein-DNA and protein-protein interactions. Since
PDX-1 plays such a central role in To further identify key components of importance for the expression of
the human PDX-1 gene in Cell Cultures--
Hamster insulinoma HIT-T15, mouse insulinoma
Cell Transfections--
HIT-T15, HepG2, NIH3T3, COS, and CHO
cells were transfected using the calcium phosphate coprecipitation
method (15), and Plasmid Constructions--
Plasmids containing fragments of the
human PDX-1 promoter were kindly provided by Alan Permutt
(University of Washington, St. Louis, MO). The fragment spanning the
sequences from Preparation of Cell Extracts--
Nuclear extracts were prepared
as described (16). Whole cell extracts were prepared by resuspension of
the cells in high salt extraction buffer (400 mM KCl, 20 mM Tris, pH 7.5, 20% glycerol, 2 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, and 10 µg/ml leupeptin). Cell lysis was obtained
by freezing and thawing, and the cellular debris was removed by
centrifugation at 16,000 × g for 15 min at 4 °C.
Protein concentrations were determined by the Bradford method (17).
Gel Electrophoretic Mobility Shift Assay (EMSA)--
DNA
binding reactions were performed by incubating in ice for 20 min. 10 µg of whole cell extracts or nuclear extracts with 0.3 ng of
32P-labeled synthetic double-stranded oligodeoxynucleotides
spanning the E1 sequence in the presence of 10 mM Hepes, pH
7.9, 10% glycerol, 50 mM KCl, 5 mM
MgCl2, 5 mM dithiothreitol, 2 µg of
poly(dI·dC) and 0.1% Nonidet P-40. The E2 binding reaction
mixture contained 20 mM Hepes, pH 7.9, 10% glycerol, 20 mM KCl, 50 mM NaCl, 1 mM dithiothreitol, and 1 µg poly(dI·dC). Competitor
oligonucleotides were incubated in a 100-fold molar excess and
preincubated in the reaction mixtures for 10 min before the addition of
the radiolabeled probe. Oligonucleotides were end-labeled by a fill-in
reaction using the Klenow fragment of DNA polymerase I. For supershift experiments, 1 µl of antibodies were added during the preincubation period. The oligonucleotides used were: E1
(5'-TCTGCAAGCTCCGCCTCCTGGGTTCACG-3'), E1 mutant
(5'-TCTGCAAGCTCCGCCgaCTatGTTCACG-3'), E2
(5'-TTCTGGGTATTTATTTATATG-3'), E2 mutant
(5'-TTCTGGGTATgTAccTATATG-3'), SP1
(5'-CTAACTCCGCCCATCT-3'), and octamer (5'-CGTACTAATTTGCATTTCTA-3')
consensus binding sites.
DNase I Footprint Analysis--
For DNase I footprinting assays,
a fragment ( Human PDX-1 Sequences Involved in The Human PDX-1 Sequence DNase I Footprinting of the Human PDX-1 Members of the SP1 Family of Proteins Interact with the E1 Sequence
of the Human PDX-1 Enhancer--
Using the E1 sequence as a probe, two
binding complexes (a and b in Fig.
4A) were obtained in HIT cell
extracts. Computer analysis for potential binding sequences (19)
revealed a GC-box element. To assess whether this motif could interfere
with the formation of the E1 complexes, excess unlabeled
oligonucleotide containing the SP1 consensus motif GGGCGG was added to
the binding reaction. The DNA complexes were competed away by the SP1
oligonucleotide (Fig. 4A, lane 3) but not by a
nonspecific one (Fig. 4A, lane 4). To confirm
that SP1 family members are involved in E1 complexes, specific SP1 and
SP3 antibodies were added separately (Fig. 4B, lanes
6 and 7, respectively) or simultaneously (Fig.
4B, lane 8) to the binding reactions. The results
demonstrate that the slower migrating complex a was
recognized by anti-SP1 (Fig. 4B, lane 6), whereas
complex b reacted with anti SP3 (Fig. 4B,
lane 7) antibodies.
The Transcription Factors HNF-3
To verify the presence of HNF-3 Combinatorial Effects of HNF-1
Mutant constructs were created to specifically alter the SP1 or
HNF-3 It is accepted that PDX-1 functions as a master regulator of the
exocrine and endocrine pancreatic programs. The pdx-1 gene is expressed early during development in cells of both origins, whereas
later it becomes restricted mainly to To identify the potential transcription factors that bind the distal
enhancer element, DNase I footprint analysis was performed, and two
protected regions (E1 and E2) were identified. The E1 area was found to
bind the transcription factors SP1 and SP3. These factors bind DNA with
similar specificity and affinity. SP1 is expressed in most tissues, and
targeted inactivation of the gene in the mouse results in retarded
growth of the embryos, which die early during gestation (20). Many
genes are controlled by SP1, which in some cases acts cooperatively
with other transcription factors (21). SP3 has been shown to function
either as an inhibitor by competing with SP1 for binding to DNA (22,
23) or as an activator, depending on promoter context and cell type
(23, 24). It has also been suggested that the relative amounts of SP1
and SP3 can vary during cellular differentiation, thus modulating the
response of target genes (see review, see Ref. 25). High levels of SP1
were found in hemapoietic stem cells, fetal cells, and spermatids (20).
We also observed about twice as much SP1 protein in pancreatic cells
than in fibroblasts but similar levels of SP3 in all tested cells (data
not shown). Furthermore, in this report we show that in transfected
fibroblasts, whereas excess SP1 has a stimulatory effect on the
enhancer transcriptional activity, SP3 shows a rather inhibitory effect
on transcription, mainly by inhibiting HNF-3 The E2 protected area is contained within an AT-rich sequence,
and factors binding this region were identified as HNF-3 Our findings that the enhancer element binds HNF-3 Since HNF-3 We demonstrate that an AT-rich and a GC-box sequences are the major
sites of regulation for the herein-described human PDX-1 enhancer element. We suggest that the transcriptional stimulation of
pdx-1 gene in Recently, mutations in genes coding for three members of the HNF family
of proteins have been identified in a subset of type 2 diabetes, MODY
(maturity-onset diabetes of the young), HNF-1-cell. To
characterize the regulatory elements and potential transcription
factors necessary for human PDX-1 gene expression in
-cells, we constructed a series of 5' and 3' deletion fragments of
the 5'-flanking region of the gene, fused to the luciferase reporter
gene. In this report, we identify by transient transfections in
-
and non-
-cells a novel
-cell-specific distal enhancer element
located between
3.7 and
3.45 kilobases. DNase I footprinting
analysis revealed two protected regions, one binding the transcription
factors SP1 and SP3 and the other hepatocyte nuclear factor 3
(HNF-3
) and HNF-1
. Cotransfection experiments suggest that
HNF-3
, HNF-1
, and SP1 are positive regulators of the
herein-described human PDX-1 enhancer element. Furthermore,
mutations within each motif abolished the binding of the corresponding
factor(s) and dramatically impaired the enhancer activity, therefore
suggesting cooperativity between these factors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cell (1-3),
where it acts as the mediator of glucose action on insulin gene
expression (6-9). In mice,
-cell-selective disruption of
pdx-1 leads to diabetes associated with reduced insulin and
glucose transporter 2 expression (3).
-cell differentiation and
function, the molecular basis of its regulation and, hence, the DNA
elements and the interacting proteins involved in this process must be
clarified. To this end, a 6.5-kb fragment upstream of the transcription
start site of rat pdx-1/stf-1 (10) and a fragment
extending from the
4.5 to +8.2-kb region of mouse pdx-1
(11) were shown to direct the expression of the
-galactosidase
reporter gene to pancreatic islet cells in transgenic mice. In
transiently transfected
-cells, appropriate expression of the rat
pdx-1 gene depended in part on a proximal E box that
predominantly binds the ubiquitous transcription factor USF1 (10).
Tissue-specific regulation also appeared to require a distal enhancer
sequence located between the
6.2- and
5.67-kb region of the rat
pdx-1 gene. Analysis of the factors bound to this element
indicated that the endodermal factors HNF-3
and Neurod/Beta2
act cooperatively to induce pdx-1 expression in islet cells.
Furthermore, it was shown that glucocorticoids reduce pdx-1
gene expression by interfering with HNF-3
activity (12). Studies on
the mouse pdx-1 promoter revealed that the region from
2560 to
1880 bp regulates
-cell-specific transcription and
directs the appropriate developmental and adult-specific expressions in
transgenic animals. It was found that an HNF-3-like element contained
within this region is important for the
-cell specificity (11).
Additional studies indicate that two highly conserved sequences in the
5'-flanking region of the PDX-1 gene (PH1/area1 and
PH2/area2) confer
-cell-specific transcriptional activity (13, 14)
on a heterologous promoter. DNase I footprinting and binding analyses
revealed that both sequences bind and are transactivated by HNF-3
;
this is in accordance with the fact that its absence in mouse embryonic
stem cells had a dramatic effect on pdx-1 gene expression
(13). Thus, data from studies with the rat (12), the mouse (11, 13),
and the human (13, 14) promoters suggest that HNF-3
is an important
regulator of pdx-1 gene transcription. Interestingly, we
found that PDX-1 itself binds to the PH1/area 1 element and cooperates
with HNF-3
to activate transcription (14).
-cells, we sequenced about 4.5 kb
of the 5'-flanking region of the gene, constructed a series of 5'
deletion fragments fused to the luciferase reporter gene, and tested
them in
- and non-
-cells. In this report we provide evidence for
a novel
-cell-specific distal enhancer element that appears to be
specific to the human PDX-1 gene. DNase I footprinting analysis revealed two protected regions: one binding the proteins identified as SP1 and SP3 and the second, the transcription
factors HNF-3
and HNF-1
. These are thus candidate transcription
factors that are involved in regulating selective expression of the
human PDX-1 gene in the adult
-cell.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
TC6, and mouse glucagonoma
TC1 cells were cultured in Dulbecco's
modified Eagle's medium with 15% horse serum and 2.5% fetal calf
serum (FCS), AR42J, HepG2, CHO, HeLa, and NIH 3T3 cells with 10% FCS.
100 units/ml penicillin and 100 mg/ml streptomycin were added to the media.
TC1,
TC6, and AR42J cells were transfected using
the Fugene transfection reagent (Roche Molecular Biochemicals)
according to the manufacturer's recommendations with 1.5 µg of human
PDX-1 luciferase derivatives and 0.5 µg of the internal
control cytomegalovirus-
-galactosidase DNA plasmid (CMV-
Gal). In
co-transfection experiments 1.5 µg of the reporter plasmid and 0.1-1
µg (as indicated) of the expression plasmids HNF-3
, HNF-3
,
HNF-1
, HNF-1
, SP1, and/or SP3 were used. The cells were harvested
48 h after transfection, and about 100 µg of protein extracts
were used to measure luciferase activity with the luciferase assay
system (Promega, Madison, WI) and about 10 µg for the
-galactosidase assay as described (15). Luciferase activity was
measured with a luminometer (EG&G Berthold, Bad Wildbad, Germany) and
normalized to
-galactosidase values.
7 to +0.117 kb was subcloned in the pGL2 basic
luciferase vector (Promega). A series of 5' and 3' deletions were
performed. The PDX-1 enhancer element (Pen) was linked to
the minimal thymidine kinase promoter (TK) of the herpes simplex virus
subcloned into pGL2 vector. Mutations were created by polymerase chain
reaction, and each construct, mut-E1 and mut-E2, was validated by sequencing.
3707 to
3426) was labeled at either end by a fill-in
reaction using the Klenow fragment of DNA polymerase I and
[32P]dCTP to a specific activity greater than
104 cpm/ng of DNA. Probes were incubated with 20-50 µg
of whole cell extracts in a 50-µl reaction mixture containing 10 mM Tris, pH 7.8, 14% glycerol, 57 mM KCl, 4 mM dithiothreitol, and 0.2 µg of poly(dI·dC).
After 20 min of incubation at room temperature, 0.5-1 unit of DNase I
(Promega) diluted in 50 mM MgCl2 and 10 mM CaCl2 was added for 1 min. The reaction was
stopped by adding 150 µl of stop solution containing 200 mM NaCl, 20 mM EDTA, 1% sodium dodecyl
sulfate, and 5 µg of yeast tRNA. DNA was extracted with
phenol-chloroform, ethanol-precipitated, and analyzed on a denaturing
6% polyacrylamide gel. Sequencing reactions of each probe were
performed using the Maxam and Gilbert procedure (18).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Cell-specific Transcriptional
Activity--
To delineate the putative DNA sequences controlling
PDX-1 gene expression, we linked a fragment extending from
about
7 to +0.117 kb of the 5'-flanking region of human
PDX-1 to luciferase reporter gene and constructed a series
of 5' deletions, as depicted in Fig. 1.
The chimeric genes were transiently transfected into HIT-T15
-cells
and CHO cells. Expression was strongly
-cell preferential, as shown
in Fig. 1. Deletion of sequences between
7 and
3.7 kb led to an
approximate 2-fold increase in luciferase activity in HIT-T15 but not
in CHO cells, implying the removal of a negative regulatory element(s).
When an additional deletion of the distal region located between
3.7
and
2.3 kb was performed, the activity dropped by about 75%,
suggesting the presence of a strong positive regulatory element.
Further removal of sequences up to
160 bp had no significant effect.
In contrast, deletion of the proximal region between
160 and
100 bp
abolished the transcriptional activity in both cell lines (Rref. 10 and
data not shown). In summary, this data indicate that the 3.7-kb
fragment contains a strong positive regulatory region that confers
-cell-specific expression on the reporter gene.
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Fig. 1.
5'-Deletion analysis of the upstream
human PDX-1 promoter region fused to luciferase
reporter gene. The various constructs were transiently transfected
into HIT-T15 or CHO cells. Luciferase activity was normalized to the
control -galactosidase values. The human PDX-1 promoter
activity in HIT-T15 cells is given relative to the promotorless basic
luciferase plasmid. Numbers are relative to the S1 start site as
determined for the rat gene (10). The results represent the mean of
5-11 experiments (± S.E.).
3.7 to
3.45 kb Acts as a
-Cell-specific Enhancer Element--
To localize the sequences
responsible for the activity delineated in Fig. 1, the fragment
extending from
3.7 to 2.3 kb was subcloned directly upstream of the
minimal PDX-1 promoter (
160/+117) fused to the luciferase
gene. In transiently transfected HIT-T15 cells, an approximate 6-fold
induction in transcriptional activity was observed (Fig.
2A). However, 5' deletion of
sequences between
3.7 and
3.3 kb reduced the transcriptional
activity to basal promoter levels. To further delineate the regulatory
sequences contained within this 400-bp fragment, 3' deletions were
generated; transient transfections revealed the presence of a positive
regulatory element spanning the region from
3.7 to
3.45 kb. This
element had the characteristics of an enhancer as it strongly
transactivated the PDX-1 promoter when cloned in either
orientation upstream of the minimal thymidine kinase promoter (Fig.
2A). Confirming its role as a tissue-specific enhancer, this
250-bp fragment strongly stimulated
-cell-specific expression of the
luciferase reporter gene in transfected
-cells, HIT-T15 (80-fold)
and
TC6 (34-fold) versus non
-cells, the exocrine
AR42J (no activation), the glucagonoma
TC1 (3-fold), CHO (5-fold),
and the hepatoma HepG2 (10-fold) cells (Fig. 2B). From this
analysis it emerges that there is a new distal
-cell-specific
enhancer located between
3.7 and
3.45 kb. This element appears to
be specific to the human PDX-1 gene as no sequence homology
was found in the vicinity of this region in the mouse gene (not
shown).
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Fig. 2.
Deletion analysis of human PDX-1
distal regulatory sequences. A, the fragment
extending from 3.7 to
2.3 kb (solid line) was subcloned
upstream of the human PDX-1 minimal promoter (
160 bp)
(hatched) linked to the reporter luciferase gene. The
truncated plasmids were transiently transfected into HIT-T15 cells (see
legend of Fig. 1). Results represent the mean of 6-10 experiments
(±S.E.). B, the PDX-1 sequence from
3.7 to
3.45 kb acts as a
-cell-specific enhancer element. The distal
250-bp element (PEn for PDX-1 enhancer) was cloned upstream
of the thymidine kinase promoter linked to the luciferase reporter gene
as well as the parental TK-Luc and transiently transfected into
-cells, HIT-T15 and
TC6, and non-
-cells, AR42J,
TC1, CHO,
and HepG2. Luciferase activity was normalized to the control
-galactosidase values and shown relative to the basic TK-Luc vector.
The results represent the mean of 3-6 experiments (±S.E.).
-Cell-specific Enhancer
Element--
The transcriptional activity driven by the distal
enhancer element of human PDX-1 suggested the presence of
cis-acting regulatory elements in this region. To assess whether such
putative elements interact with specific proteins, we performed DNase I
footprinting analysis using the fragment extending from
3.7 to
3.3
kb as a probe and extracts from HIT-T15 and CHO cells. As shown in Fig. 3A, two protected regions were
obtained. The pattern of the first protected sequence E1,
3.473/
3.494 kb, shows a hypersensitive site in the presence of
HIT-T15 cell extracts. The second footprinted sequence, between
3.565
and
3.590 kb, E2, occurs within a particularly AT-rich region and
shows a slightly different digestion pattern in HIT-T15 and CHO cell
extracts. The sequence of the human PDX-1 enhancer element
(
3.7/
3.45 kb) is presented in Fig. 3B with the
footprinted regions underlined. To further characterize the trans-acting factors binding to the footprinted regions,
double-stranded oligonucleotides spanning these sequences were
synthesized and used as probes to detect HIT-T15 and CHO proteins by
electrophoretic mobility shift assay.
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Fig. 3.
DNase I footprinting analysis of the human
enhancer element. A, the analysis was performed using
the end-labeled fragment spanning the sequences between approximately
3.7 and
3.4 kb and incubated with no extracts (lanes 2,
3, and 9), with extracts from HIT (lanes
4, 5, and 10), or CHO (lanes 6,
7, and 11) cells. G+A and C+T sequencing
reactions were run alongside as a marker (lanes 1 and
8, respectively). B, nucleotide sequence of the
-cell-specific enhancer in the human PDX-1 gene. The E1
and E2 footprinted regions are indicated.
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Fig. 4.
SP1 and SP3 from HIT cells bind the E1
sequence of the human PDX-1 enhancer element.
A, EMSA was performed using CHO (lane 1) or HIT
(lane 2) cell extracts and a 32P-labeled E1
sequence. Two complexes were formed, labeled a and
b, indicated by arrows. Competition for binding
of HIT cell extracts to the labeled E1 sequence was performed with a
100-fold excess of an unlabeled oligonucleotide containing an SP1
consensus motif (SP1, lane 3) or with a nonspecific
oligonucleotide (ns, lane 4). B,
identification of SP1 and SP3 complexes. EMSA was performed with
HIT-T15 cell extracts incubated with the labeled E1 sequence
(lane 5) in the presence of antiserum against SP1
(lane 6) or SP3 (lane 7) or both (lane
8) or preimmune serum (PIS, lane 9).
and HNF-1
Interact with the
E2 Sequence of the Human PDX-1 Enhancer--
In EMSA, using the E2
sequences with HIT-T15 or
TC6 cell extracts, a faint complex labeled
a and a strong faster migrating b complex were
detected (Fig. 5A). The
b complex was observed in all pancreatic cells tested,
i.e. the glucagonoma
TC1 and the exocrine AR42J line as
well as in the hepatic HepG2 cells. In contrast, the a
complex was mainly observed in
-cells; a closely migrating complex
in AR42J and HepG2 cells runs slightly faster. E2 is contained within
an AT-rich region (Fig. 3B), and computer analysis for
transcription factors unveiled potential binding sites for several
homeodomain-containing proteins (19) including overlapping motifs for
HNF-3 and HNF-1. To determine whether HNF-3 or HNF-1 is involved in the
observed complexes, electrophoretic mobility shift assays were
performed using either the wild type E2 sequence (wt-E2) or the mutant
form containing a modified HNF-3/HNF-1 motif (mut-E2). The DNA
complexes a and b observed in HIT-T15 cells were
competed away by excess of unlabeled wild type oligonucleotide (Fig.
5B, lane 3). In contrast, the unlabeled
oligonucleotide containing the mutated HNF-3/HNF-1 motifs (Fig.
5B, lane 4) or a nonspecific (octamer consensus
motif) oligonucleotide showed no competition (Fig. 5B,
lane 5). When the mutated HNF-3/HNF-1 sequence was used as
probe, the a and b complexes were abrogated (Fig.
5B, lane 7).
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Fig. 5.
HNF-1 and
HNF-3
in
-cells
interact with the E2 sequence of the human PDX-1 enhancer
element. A, EMSA was performed using HIT-T15
(lane 1),
TC6 (lane 2),
TC1 (lane
3), AR42J (lane 4), HepG2 (lane 5), HeLa
(lane 6), CHO (lane 7), or NIH 3T3 (lane
8) cell extracts and a 32P-labeled E2 sequence. Two
complexes in
-cells, labeled a and b, are
indicated by arrows. NE, nuclear extracts.
B, competition for binding of HIT cell extracts to the wild
type labeled E2 sequence (Wt-E2) with a 100-fold excess of
an unlabeled oligonucleotide (lane 3), excess HNF-3/HNF-1
mutated site (E2mut, lane 4), or with a
nonspecific oligonucleotide (ns, lane 5). The E2
sequence carrying a mutated HNF-3/HNF-1 was used as probe with CHO
(lane 6) and HIT (lane 7) cell extracts.
C, The a complex corresponds to HNF-1
, and the
b complex corresponds to HNF-3
. EMSA was performed with
HIT-T15 cell extracts incubated with the labeled E2 sequence
(lane 1) in the presence of preimmune serum (PIS,
lane 2), or antiserum against HNF-3
(lane 3),
HNF-3
(lane 4), HNF-1
(lane 5), or HNF-1
(lane 6).
and HNF-1
in the E2
complexes, their ability to interact with a series of antibodies was tested. Fig. 5C demonstrates that the b complex
is specifically recognized by antibodies against HNF-3
(Fig.
5C, lane 4) but not with anti-HNF-3
(Fig.
5C, lane 3) or antibodies against the homeodomain
proteins PDX-1, Oct-4, cdx2/3, Nkx6.1, or isl-1 (data not shown).
Furthermore, the a complex interacted with anti- HNF-1
(lane 5) but not with anti-HNF-1
(lane 6)
antibodies. Cell extracts from COS cells transfected with an expression
plasmid for HNF-3
or HNF-1
were analyzed for their interaction
with the E2 sequence to establish that the protein contained in the
b complex corresponds to HNF-3
. Indeed, the HNF-3
and
HNF-1
complexes in COS cells migrated similarly to the a
or b complex in HIT cells, respectively, and were also
recognized by the corresponding antibodies (data not shown). Taken
together, these results demonstrate that the endogenous HNF-1
and
HNF-3
in HIT-T15 cells specifically bind the E2 sequence.
, SP1, SP3, and HNF-3
in the
Activation of the Human PDX-1 Enhancer Element--
To investigate the
effect of the above transcription factors on gene expression driven by
the enhancer element, we performed transient transfection experiments
in NIH3T3 cells. To this end, the PEn-TK-luciferase construct was
cotransfected with increasing amounts of the HNF-1
and HNF-1
plasmids separately or in combination with HNF-3
expression plasmid
(Fig. 6A). In parallel, we
carried out similar experiments with SP1 and SP3 expression plasmids
(Fig. 6B). As presented in Fig. 6A, HNF-3
and
HNF-1
but not HNF-1
separately stimulated the PDX-1
enhancer activity in a dose-dependent manner. Furthermore,
cotransfection with HNF-3
and increasing amounts of HNF-1
cooperatively activated the expression of the gene. Similarly, HNF-3
and SP1 individually activated the chimeric gene, and cotransfection
with a constant amount of HNF-3
and increasing amounts of SP1
significantly stimulated the expression of the gene in a more than
additive manner. In contrast, although SP3 lacked any effect on the
enhancer activity when acting independently, it dramatically suppressed
the HNF-3
-mediated transactivation (Fig. 6B).
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Fig. 6.
Interactive transactivation of the human
PDX-1 enhancer element by HNF-3 ,
SP1/3 and HNF-1
/
in
non
-cells. A, NIH3T3 cells
were transiently cotransfected with HNF-1
(dotted bars),
HNF-1
(hatched bars), or HNF-3
(black bar)
separately or cotransfected with a constant amount of HNF-3
and
increasing amounts of HNF-1
(gray bars) or
HNF-1
(dark hatched bars) and the wild type PEn-TKLuc.
B, same as in A where transfections were carried
out with SP1 (dotted bars), SP3 (hatched bars),
or HNF-3
(black bar) separately or HNF-3
co-transfected with increasing amounts of SP1 (gray
bars) or SP3 (dark hatched bars). Luciferase activity
was normalized to the control
-galactosidase values and shown
relative to the basic PEn-TKLuc vector. The results are the mean of
four representative experiments. C, effects of mutations in
the SP1 and HNF-3
motifs on the human PDX-1 enhancer
activity. The PEn-TKLuc (empty bar) and the derived
constructs carrying mutations in the SP1 (mut-E1,
dotted bar) or the HNF-3
(mut-E2,
hatched bar) binding sites were transiently transfected in
HIT-T15 cells. The results represent the mean of seven experiments
(±S.E.). D, transactivation with HNF-3
in non-
-cells.
CHO cells were transiently cotransfected with 0.1 µg of HNF-3
expression plasmid (black bars) or empty vector
(hatched bars) and either the wild type PEn-TKLuc or the
derived mut-E2 constructs. Luciferase activity was normalized to the
control
-galactosidase values and shown relative to the basic TK-Luc
vector. The results represent are mean of n = 4 (±S.E.).
/HNF-1 motifs in the context of the human PDX-1
enhancer element (Pen) linked to the luciferase reporter gene and
driven by the minimal TK promoter. The mutation that eliminated SP1/SP3 binding (mut-E1) caused more than 90% reduction in enhancer activity in HIT-T15 cells (Fig. 6C). Mutation in the overlapping
HNF-3
/HNF-1 motifs (mut-E2) also caused an 80% decrease in enhancer
activity. The effect of HNF-3
on enhancer activity was further
examined by cotransfection experiments in CHO cells using the wild type reporter construct (PEn) or the E2 mutant form carrying a modified HNF-3
/HNF-1 site (mut-E2) together with an HNF-3
expression vector. About 10-fold activation was detected using the wild type enhancer, but only 2-fold increase was detected with the mutant reporter construct (Fig. 6D). Similar experiments showed
that HNF-1
transactivated the wild type enhancer by about 2-fold but had almost no effect on the mutated element (not shown). The data presented thus suggest that the transcription factors HNF-3
, HNF-1
, SP1, and SP3 are regulators of the herein-described human PDX-1 enhancer element.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells. Fragments containing
6.5- and
4.5 kb- sequences of the rat (10) and the mouse (11)
pdx-1 gene, respectively, were sufficient in targeting its
expression to rodent islet cells. Therefore, to further characterize
the potential regulatory elements of the human PDX-1 gene,
we analyzed about 4.5 kb in the 5'-flanking region of the gene. By
transient transfections of
-cell and non-
-cell lines with
different 5' and 3' deletions, a strong
-cell-specific enhancer
element located between
3.7 and
3.45 kb was demonstrated. The
4.5-kb 5'-flanking sequences of the human and mouse pdx-1 genes are markedly different, apart from the conserved proximal promoter region; only three short highly homologous areas located 3' of
the herein-described enhancer element are observed. No homology was
observed between this enhancer and the one previously described further
upstream in the rat gene (12). These observations suggest that several
regulatory elements in pdx-1 gene contribute to its
-cell-specific expression.
-mediated transactivation.
and HNF-1
. The important role of HNF-3
in transactivating the
conserved regulatory elements in the mouse and human pdx-1
5'-flanking region was recently shown (13, 14), and its absence in
mouse embryonic stem cells dramatically impaired pdx-1 gene
expression (13). Moreover, the distal enhancer element identified in
the rat pdx-1 (stf-1) gene binds HNF-3
and
Neurod/Beta2 factors, which cooperatively induce its expression
in islet cells. It was further shown that glucocorticoid-induced
reduction of pdx-1 expression was mediated by impaired
HNF-3
activity (12). HNF-3
belongs to the forkhead/winged helix
family of transcription factors and is essential for endodermal cell
lineages (26-28). Since HNF-3
is not restricted to
-cells, selective transcription of pdx-1 must an require additional
factor(s). It is believed that HNF-3
, by its structural similarity
to histone H5, may alter nucleosomal structure, thus facilitating gene
transcription by opening the chromatin structure and thereby providing
access to other transcription factors (29, 30). Whereas HNF-3
binds as a monomer, members of HNF1 family of transcription factors bind DNA
as homo- or heterodimers (31). HNF-1
and HNF-1
are hepatocyte-enriched transcription factors that are also expressed in
other tissues like in the pancreas. The relative abundance of these two
proteins differs markedly between the different pancreatic cell lines
(Fig. 5A and data not shown). It is conceivable that the
relative levels of HNF1 subtypes may also be one of the factors contributing to the tissue specific expression of the PDX-1
gene, as it has been recently shown for the regulation of glucose
transporter 2 (Glut2) gene in hepatocytes and
-cells (32). The
relative levels of these two proteins have also been reported to affect vitamin D binding protein gene transcription. Although HNF-1
had a
stimulatory effect, HNF-1
acted as an inhibitor of
HNF-1
-transactivating potency (33).
/HNF-1
and
SP1/SP3 and that mutations in each site dramatically impair its
transcriptional activity suggest cooperativity between these factors.
Cooperativity between SP1 and members of HNF3 family has also been
demonstrated for the surfactant protein B (34) and for the
uteroglobin/CC10 (35) genes in the lung epithelium. Hence, SP1 could
function as a bridging factor between the hepatic nuclear factors and
the basal transcriptional machinery, or conversely, HNF-3
may
facilitate HNF-1
and SP1 binding by bending the DNA.
/HNF-1
and SP1 seem equally important for the human
PDX-1 promoter activity, the synergism and possible
interactions between these factors need to be analyzed. Nevertheless,
HNF-3
, HNF-1
, and SP1 are also present in other pancreatic and
hepatic cells, and yet the human PDX-1 enhancer activity was
low in these cells, pointing to the possibility that other accessory
proteins and/or an additional level of transcriptional control may
contribute to the
-specific transcriptional activity of the
PDX-1 gene. Transcriptional regulation appears to be a
multistep process that may also involve chromatin remodeling,
e.g. the methylation status of CpG sequences in a
control element (36, 37). Findings showing that methylation of SP1
sites might be a relatively common and physiological mechanism of gene
repression have been reported for leukosielin (CD43) (38, 39), cyclin
D1 (40), and lung epithelial T1
genes in nonexpressing cells (41).
SP1 elements have also been shown to play a key role in protecting a
CpG island in the adenine phosphoribosyltransferase (APRT) gene from
de novo methylation (37, 42). The effect of methylation on
the identified enhancer element transcriptional activity needs to be tested.
-cells is mediated by a unique combination
of protein-protein interactions and that separate modules in its sequence could be active at a given stage by binding a specific set of
transcription factors. Indeed, the transcription factors HNF-3
,
HNF-1
, HNF-1
, SP1, and PDX-1 itself, which regulate the
expression of the PDX-1 gene, have been previously shown, mainly by knockout in mice, to be important developmental regulators.
(MODY3) (43), HNF-1
(MODY5) (44), and HNF-4
(MODY1) (45). Thus, genes coding for the
transcription factors controlling PDX-1 gene expression may
be candidates for susceptibility to diabetes.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Alan M. Permutt (Washington
University) for the human PDX-1 gene. We are infinitely
grateful to Dr. Robert Costa (University of Illinois at Chicago, IL)
for the gifts of HNF-3 and HNF-3
expression vectors and
antibodies, Dr. Guntram Suske (Marburg, Germany) for SP1 and SP3
expression vectors and antibodies, Dr. Moshe Yaniv (Paris, France) for
HNF-1
and HNF-1
expression vectors and antibodies, and Dr.
Ariella Openheim for SP1 consensus oligonucleotide. Our sincere thanks
to Rahel Oron for excellent technical help and Dr. Leora Havin for
helpful discussion.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Juvenile Diabetes Foundation International and the Israel Science Foundation and the European Commission (BIOMED2).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 225952.
To whom correspondence should be addressed: Dept. of Endocrinology
and Metabolism, Hadassah University Hospital, P. O. Box 12000, Jerusalem 91120, Israel. Tel.: 972-2-677 83 98; Fax: 972-2-643 79 40;
E-mail: Danielle@md2.huji.ac.il.
Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M009088200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PDX, pancreatic duodenal homeobox; kb, kilobase(s); bp, base pair(s); Luc, Luciferase; HNF, hepatocyte nuclear factor; CHO, Chinese hamster ovary; TK, thymidine kinase; EMSA, electrophoretic mobility shift assay.
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