From the
The gastrin gene is transiently expressed in fetal pancreatic
islets during islet neogenesis but then switched off after birth when
islet cells become fully differentiated. Previous studies identified a
cis-regulatory sequence between -109 and -75 in the human
gastrin promoter which binds islet cell-specific activators and a
nonspecific repressor and thus may act as a molecular switch. The
present study identified another cis-regulatory sequence
(
Pancreatic islet endocrine cells share common evolutionary and
developmental origins with gut endocrine cells
(1) . This
relationship is emphasized by the transient expression of hormones such
as gastrin, peptide-YY, and secretin in fetal islets even though these
peptides are expressed largely in the luminal gastrointestinal tract in
the adult
(2, 3, 4) . The transient expression
of gastrin may serve to promote islet proliferation and
differentiation, which occur concurrently with pancreatic gastrin
expression
(5, 6, 7, 8) . Although
little gastrin is expressed in the adult pancreas, it can be
re-expressed when islets undergo neoplastic transformation. Therefore,
the molecular mechanisms controlling gastrin gene transcription in
islet cells represent an interesting example of developmental control
of gene expression.
Transgenic mouse studies have shown that 1300
bp
These elements were
identified by deletional analysis, a method which may fail to detect
other upstream sequences that regulate gastrin transcription. For this
reason, we carried out systematic scanning mutagenesis of the 200-bp
sequence upstream of the transcriptional start site and identified a
new cis-regulatory domain from -163 to -140 which controls
gastrin transcription in islet cells. There was mutually exclusive
binding of two nuclear proteins to this domain, in which binding of one
factor blocked the transcriptional activation by the other. Thus, the
gastrin promoter comprises a tandem array of switch-like cis-regulatory
elements in which activation can be blocked by factors binding to
adjacent sequences. This tandem array of regulatory sequences may
provide the molecular mechanism to explain the transient activation and
subsequent repression of gastrin transcription in fetal islets.
Minimal
promoter constructs containing the wild-type (WT) or point mutant
elements spanning -163 to -140 bp (see legend in
Fig. 3
) were created by ligation of kinased double-stranded
synthetic oligonucleotides into the BamHI site of the
heterologous thymidine kinase promoter vector pT81 with a luciferase
reporter
(17) , as described previously
(12) . All
constructs were sequenced to confirm proper head-to-tail orientation of
multimeric inserts.
Gel mobility shift
assays were performed by incubating the various extracts with 4 fmol of
double-stranded oligonucleotide probe (40,000 cpm) end-labeled with
[
Probes
for footprinting assays were derived from the wild-type gAT-Sp1 pT81
plasmid construct. Probes were 3` Klenow end-labeled on the lower
strand with all 4 [
Linker Scanning Mutagenesis Identifies Important Gastrin
Regulatory Elements Between -160 and -141 bp-To
identify additional cis-regulatory elements controlling gastrin
transcription in islet cells not seen by 5`-deletional analysis, a
series of linker scanning mutations was constructed. Prior deletional
analysis demonstrated that 200 bp of 5`-flanking DNA of the human
gastrin gene contained the regulatory elements that conferred most of
the transcriptional activity in gastrin-expressing RIN 38A insulinoma
cells
(11, 12) . The linker scanning mutations
systematically substituted 10-bp blocks from -170 to -61
bp. The 11 linker scanning mutant constructs were confirmed by direct
sequencing and then transiently transfected into 38A islet cells.
Reporter gene activity was compared to that of the wild-type gas200
pXP2 vector.
Fig. 1 shows the functional domains identified within
the proximal 200 bp of the gastrin promoter which regulate
transcription in islet cells. The first includes block mutants 2 and 3,
which correspond to an AT-rich sequence CTAAATGAAA (-160 to
-151 bp) and an Sp1 consensus binding site GGGCGGGGCA (-150
to -141 bp), neither of which has been described previously as an
important regulatory element in the gastrin gene. Mutations in these
elements led to a 25-30% fall in transcriptional activity. The
second domain is defined by block mutant 6 which mutates the CACC
element, known to be a transcriptional activator in 38A cells
(12) . There was an augmentation of activity with block mutant
7, corresponding to a de-repression of the inhibitory GNE
(11) .
The final domain spans blocks 9 and 10, which mutate the previously
characterized gastrin E-box
(11) and another Sp1 binding site
which interacts with the gastrin-epidermal growth factor response
element
(25) . This linker scanning data corroborates earlier
analyses which characterized these aforementioned promoter elements.
Prior studies, however, had not identified this gastrin AT-rich (gAT)
element and adjacent Sp1 consensus site as important regulatory
elements.
Addition of Sp1
antisera to 38A nuclear extracts in gel mobility shift assays
supershifted the slower migrating complex Ia (Fig. 2 B, lanes
2-4), confirming that the Sp1 transcription factor does bind
to this sequence. A control antibody did not supershift any complex
(Fig. 2 B, lane 5). Since Sp1 binding is dependent upon
its zinc finger domains, chelation of zinc abolishes Sp1 binding to DNA
(26) . Accordingly, addition of the chelator EGTA to the islet
nuclear extracts quantitatively reduced complex Ia and Ib formation but
not complex II (Fig. 2 C). Significantly, abolition of
Sp1 binding by EGTA enhanced complex II formation, suggesting that
there was competition between Sp1 binding and the binding of the gAT
factor to the adjacent AT-rich sequence.
The
factors binding in complex I and II could be separated physically by
ion-exchange chromatography. Crude nuclear preparations from 38A islet
cells were first applied to a cation exchange phosphocellulose column.
Subsequent elution with 0.5
M KCl yielded extract B,
a fraction enriched for Sp1 (complex I) without contamination by the
factor in complex II which binds the AT-rich sequence
(Fig. 2 D, lane 1). The gAT factor was enriched by
passage of the crude nuclear extract over an anion exchange
DEAE-Sepharose column. The subsequent 0.25
M KCl eluate
(extract C) contained only the AT-binding factor (Fig. 2 D,
lane 7). Reconstitution experiments were performed to test whether
various stoichiometric mixtures of the two enriched fractions showed
any evidence of cooperative interactions (Fig. 2 D, lanes
2-6). In no combination did a new binding complex appear in
gel mobility shift assays. While this is suggestive of mutually
exclusive binding by the two factors, it is possible that other
complexes form which are not stable during electrophoresis.
The nucleotides necessary for binding of
the gAT factor were also defined by competition with mutant
oligonucleotides. The gAT factor binding site lay adjacent to the Sp1
site but did not overlap with nucleotides necessary for Sp1 binding
since oligo M2 failed to compete for gAT factor binding (Fig. 4 A,
lane 4) but effectively competed Sp1 binding (Fig. 3 A,
lane 4). Conversely, oligos M3, M4, and M5 bound the gAT factor
but not Sp1 (Fig. 4 A, lanes 5-7). Furthermore, an
oligonucleotide containing a consensus Sp1 sequence did not displace
gAT factor binding (Fig. 4 A, lane 8). Oligo M1 competed
as well as the wild-type (Fig. 4 A, lane 2), indicating
that the critical residues for gAT factor binding were mutated in the
M2 oligo. Methylation interference assays were unsuccessful probably
due to the relative paucity of G residues in the gAT factor binding
domain. DNase I footprinting was therefore performed, which
demonstrated footprinting of the gAT factor over the AT-rich sequence
(Fig. 4 B, lane 1). The close agreement between the
nucleotides for binding shown in the competition assays and the
residues footprinted (Fig. 4 C) indicate that the AT-rich
element is directly adjacent to the GC-rich sequence necessary for Sp1
binding. Such proximity of these two sites makes steric interference
the likely mechanism which prevents simultaneous binding of the two
factors to the adjacent sequences.
Prior
studies defined the
This study has identified additional cis-regulatory elements
within the gastrin promoter lying between -163 and -140,
upstream of previously characterized regulatory sequences
(11, 14, 25) . The proximal 200 base pairs of
the gastrin promoter contain multiple positive and negative
cis-regulatory sequences, and scanning mutagenesis studies indicate
that no single element is essential for transcription but rather that
transcriptional activity is dependent upon the combined action of the
CACC, E-box, and proximal and distal Sp1 positive cis-regulatory
elements. Adjacent to each of these positive sites are negative
cis-regulatory elements which modulate the activity of these activating
elements. Transactivation by the CACC and E-box binding factor is
antagonized by a factor binding to the GNE
(11) . Sp1
transactivation via the proximal Sp1 site (-68) is inhibited by
factors interacting with the adjacent epidermal growth factor-response
element
(36) . Similarly, the present study demonstrated that
Sp1 transactivation at the distal Sp1 site (-150) is inhibited by
a competing factor binding to an adjacent AT-rich site.
Although the
gAT factor has not been fully characterized, it binds a TAAT motif
recognized by many homeodomain transcription factors
(37, 38) . Homeodomain factors often regulate
tissue-specific gene expression and in islet cells, isl-1 and
STF-1/idx-1 are well characterized pancreatic endocrine homeodomain
factors which regulate insulin and somatostatin gene expression,
respectively
(32, 33, 34) . However, the gAT
factor displayed minimal affinity for these homeodomain factor binding
sites in gel mobility shift assays. Rather, the gAT factor recognized a
sequence bound by the islet cell nuclear protein
Transactivation of gastrin transcription by Sp1 appears to be
attenuated by binding of the gAT factor to the adjacent AT-rich
sequence. Down-regulation of Sp1 transactivation by steric effects of
neighboring transcription factors has been shown to be important in
regulating transcription of other genes. In the collagen
Activation of gastrin
transcription in islet cells is strongly dependent upon the ubiquitous
transactivator Sp1 even though gastrin gene expression is highly
cell-specific and temporally restricted to the neonatal period.
Although negative regulation is probably a highly important determinant
of this tissue and temporal specificity, Sp1 levels can vary
dramatically in different developmental stages
(45) . In the
mouse, it is expressed at highest levels in tissues undergoing final
differentiation, such as in spermatids or hematopoietic cells. Lower
levels are seen in terminally differentiated cells. Within the
gastrointestinal tract, expression in the stomach is high at the time
of weaning and persists throughout adult life
(45) . Since there
is continuous differentiation of gut endocrine cells from the
population of endodermal stem cells throughout adult life, the
concurrent high level of Sp1 expression may be relevant. The
developmental expression of Sp1 within the pancreas has not been
examined but such studies would be of great interest. The developmental
regulation of Sp1 may thus influence the developmental expression of
Sp1-regulated genes, such as gastrin.
Thus far, two Sp1 binding
sites have been identified in the gastrin promoter. Sp1 binding to a
proximal G-rich element (-68 to -53) is an important
component of epidermal growth factor-stimulated gastrin gene expression
in rat pituitary GH4 cells, as described previously
(25) . As
the current report demonstrates, an additional upstream Sp1 binding
site is strongly activating in gastrin-expressing islet cell lines.
Mutations in either Sp1 site (Fig. 1, constructs 1 and 10) are
associated with diminished transcriptional activity compared to the
wild type. Prior studies have established that Sp1 binding at multiple
elements has a synergistic effect on transcriptional activation
(46) . One proposed mechanism for such long range interactions
involves DNA looping mediated by tetramers of Sp1 binding to proximal
and distal enhancers simultaneously
(47) . Such cooperative
interactions between Sp1 elements may similarly play a role in
activating gastrin transcription during islet cell differentiation.
Inhibiting binding at just one of these Sp1 sites by an islet cell
factor binding to the adjacent gAT site may be a critical determinant
in switching off gastrin transcription. Such an interaction may explain
why repression of gastrin expression coincides with full
differentiation of the pancreatic islets, when many pancreas-specific
transcription factors are maximally expressed. Future studies will need
to focus on identifying the relative levels of these different factors
at different times in pancreatic development. In addition to
elucidating the molecular mechanisms controlling pancreatic
development, these studies may provide insight into the reactivation of
gastrin expression with the neoplastic transformation of pancreatic
islets.
We thank Drs. Timothy C. Wang and David A. Brenner for
critical review of the manuscript and helpful advice, Philip J. Davis
for technical assistance, and Dr. Anil K. Rustgi for antisera.
Oligonucleotides were provided by the Center for the Study of
Inflammatory Bowel Disease at the Massachusetts General Hospital.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
ACACTAAATGAAAGGGCGGGGCAG
)
which bound two islet nuclear proteins in a mutually exclusive manner,
as defined by gel shift competition, methylation interference, and
DNase I footprinting assays. The general transactivator Sp1 recognized
the downstream GGGCGGGG sequence, but Sp1 binding was prevented when
another islet factor bound to the adjacent AT-rich sequence (CTAAATGA).
This gastrin AT-rich element is nearly identical to the binding site
(ATAAATGA) for the islet-specific transcription factor
TF-1.
However, the gastrin AT-binding factor appeared to differ from
TF-1 in its gel mobility shift pattern. Transfections of rat
insulinoma cells revealed that mutations which blocked binding to the
AT-rich element but allowed Sp1 binding up-regulated transcriptional
activity. These results suggest that the gastrin AT-binding factor
blocks transactivation by Sp1 and may have a role in the repression of
gastrin transcription seen at the end of islet differentiation.
(
)
of 5`-flanking DNA of the human gastrin gene
appropriately regulates the transient expression of gastrin in fetal
islets
(9) . Detailed promoter analysis utilizing cultured
gastrin-expressing rat insulinoma cell lines has identified regulatory
elements that lie within 200 bp of the transcriptional start site
(10, 11, 12) . These include a sequence between
-109 and -75 of the gastrin promoter that binds three
transcription factors. A transcriptional activator binds to a CACC
element at the 5` end of this domain
(12) . However, a repressor
binds to an immediately adjacent 3` sequence, the Gastrin Negative
Element (GNE), which is similar to a repressor binding sequence in the
-interferon promoter
(11) . Immediately 3` to the GNE
sequence is another positive element containing an E-box motif
(CANNTG), which has been shown to bind multiple helix-loop-helix
transcription factors
(13, 14) .
Plasmid Constructions
Linker scanning
mutagenesis was performed utilizing a promoter construct containing 200
bp of 5`-flanking DNA of the human gastrin gene subcloned into the
promoterless luciferase vector pXP2, as described previously
(12) . 11 linker scanning mutant constructs were generated by
substituting a 10-bp cassette specifying adjacent XhoI and
ScaI restriction sites (CTCGAGTACT) in sequential 10-bp blocks
from -170 to -60 bp utilizing the unique site elimination
method
(15) . The mutagenic primers contained 13 complementary
nucleotides flanking both ends of the 10-bp substitution. The second
primer set mutated a unique BglII site in the pXP2 vector.
After heat-denaturing at 100 °C and snap-cooling to 0 °C, the
plasmid vector with a 1000-fold molar excess of both primers was
incubated with T4 DNA polymerase, T4 DNA ligase (both New England
Biolabs), and all 4 dNTPs. Newly synthesized double-stranded plasmid
constructs were digested with BglII (New England Biolabs) and
transformed into the mismatch repair deficient
mutSEscherichia coli strain
(16) , miniprep DNA was prepared via a modified alkaline lysis
procedure (Qiagen), and mutants were selected by digestion again with
BglII and re-transformation into E. coli DH5-
cells. All mutant constructs were confirmed by sequencing.
Figure 3:
Characterization of the Sp1 binding domain
by mutational analysis and methylation interference assays. A,
gel mobility shift assays were performed with Sp1-enriched 38A fraction
B and the WT full-length probe in the presence of a 25-fold
excess of Sp1 consensus, wild-type, and mutant competitor
oligonucleotides. The nucleotide sequences for the plus strand are
listed below and the underlined residues indicate the mutated base
pairs. B, methylation interference assay performed as
described under ``Materials and Methods.'' Lanes 1 and 3 represent free probe and lane 2 represents
probe bound to Sp1. The bar at the right represents methylated
G and A residues that interfere with Sp1 binding. C, schematic
representation of residues important for Sp1 binding to the gastrin
promoter ( underlined) as defined by mutational gel-shift
analysis ( X) and methylation interference ( O); WT,
gatccACAC-T AAA TGA AAG GGC GGG GC AGa ; M 1 ,
gatccAACAGAAATGAAAGGGCGGGGC-AGa; M2, gatccACACTCCCGGAAAGGG-CG GGG CA Ga
; M 3 , ga tc cAC ACT AA AT-TCCCGGGCGGGGCAGa; M4, gatccACACTAAATGAAA
TTTCGGGGCAGa; M5, ga tcc ACA CT AA AT GA AA GC GA GTG GC-AGa; Sp1,
ATTCGATCGGGGCGGGGC-GAGC.
Cells
RIN 1046-38A
(10, 18) ,
AR4-2J
(19) , Rat 2 (ATCC CRL 1764), Panc-1 (ATCC CRL
1469), and HepG2 (ATCC HB 8065) cells were cultured as described in
Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum and 2% penicillin-streptomycin (all Bio-Whittaker).
The RIN 38A cell line expresses gastrin, as determined by Northern blot
hybridization with a rat gastrin Exon III riboprobe (data not shown).
Transfections
RIN 38A cells were transiently
transfected with a modified DEAE-dextran protocol followed by a 10%
dimethyl sulfoxide shock, as described previously
(12) .
Luciferase activity was assayed 48 h after transfection with luciferin,
ATP, and coenzyme A (Promega) utilizing a monolight luminometer
(Analytical Luminescence Laboratory). Transfections were performed at
least 3 times and variation between experiments was not greater than
15%.
Nuclear Extracts and Gel Mobility Shift
Assays
Nuclear extracts were prepared by the method of Dignam
(20) and quantitated utilizing a Bradford protein assay
(21) . Partial purification of crude RIN 38A extracts was
performed by passage over a phosphocellulose column (Whatman) or a
DEAE-Sepharose column (Pharmacia Biotech Inc.) as described previously
(22) to partially purify Sp1 and the gastrin AT-binding factor
(gAT factor), respectively. Proteins adsorbed to the phosphocellulose
or DEAE-Sepharose column were eluted with a 0.5 or 0.25
M KCl
solution, respectively. The trace amount of gAT factor that was eluted
off the phosphocellulose column was removed by incubation with the
AT-rich oligo (gatccCTGACACTAAATGAAAGGGa) at a final concentration of
0.15 µ
M prior to passage over the phosphocellulose column
again. All samples were then dialyzed against buffer A (20 m
M HEPES (pH 8), 10% glycerol, 1.5 m
M MgCl, 75
m
M KCl, 1 m
M dithiothreitol, 0.2 m
M phenylmethylsulfonyl fluoride, and a 0.5 µg/ml mixture of
aprotinin, antipain, pepstatin A, and leupeptin).
-
P]ATP (DuPont NEN) by T4 polynucleotide
kinase (New England Biolabs) in a 20-µl binding reaction containing
25 m
M HEPES (pH 7.9), 100 m
M KCl, 5 m
M MgCl
, 1 m
M EDTA, 1 m
M dithiothreitol, 10% glycerol, and 0.5-1.0 µg of
poly(dA-dT)/poly(dA-dT) (Pharmacia) as a nonspecific competitor. The
30-bp wild-type synthetic probe used in this study
(gatccACACTAAATGAAAGGGCGGGGCAGa) spans the gastrin promoter from
-163 to -140 bp and was polyacrylamide gel-purified.
Competition experiments were carried out by preincubating the nuclear
extract with a 25-125-fold excess of cold competitor
oligonucleotides prior to the addition of probe. For Sp1 supershift
assays, extracts were preincubated with varying amounts of a polyclonal
Sp1 antibody (Santa Cruz) or control rabbit antiserum (12CA5 polyclonal
antisera, gift from Anil K. Rustgi) for 10 min. Zinc chelation assays
were performed by preincubating the nuclear extracts in buffer
containing either 10 or 25 m
M EGTA prior to the addition of
probe. EGTA was selected for its ability to efficiently chelate
Zn
but not Mg
cations
(23) ,
the latter being a component of the gel shift assay buffer. All samples
were then incubated for 10 min at room temperature prior to loading
onto a 0.5
TBE, 4% nondenaturing polyacrylamide gel run at 10
V/cm. For oligonucleotide sequences, upper case letters represent
gastrin gene sequence and lower case letters represent BamHI
or BglII cloning sites added onto the native sequence.
Methylation Interference and DNase I Footprinting
Assays
Methylation interference assays were performed as
described previously
(24) . The P singly
end-labeled double-stranded 30-bp WT oligonucleotide was partially
methylated with dimethyl sulfate (Aldrich) and then purified with two
rounds of ethanol precipitation. 20,000 cpm of probe was incubated with
nuclear extracts in a typical gel shift reaction and run on a 4%
nondenaturing polyacrylamide gel as described earlier. An autoradiogram
of the gel revealed bands corresponding to Sp1 and gAT binding
complexes as well as unbound probe, all of which were excised and
electroeluted. After piperidine cleavage, the samples representing
bound and free probe were resolved on an 8% sequencing gel.
-
P]dNTPs (DuPont NEN)
after restriction digestion with BamHI. After complete
excision of the labeled fragment by digestion with SalI,
20,000 cpm of gel-purified probe was incubated with gAT-factor-enriched
nuclear extract C for 10 min at room temperature in a
100-µl binding reaction containing 10 m
M Tris (pH 8.0), 5
m
M MgCl
, 1 m
M CaCl
, 2 m
M dithiothreitol, 100 m
M KCl, and 3 µg of
poly(dA-dT)/poly(dA-dT). The samples were digested with varying amounts
of deoxyribonuclease I (Promega) for 2 min at 20 °C and then
immediately ethanol precipitated with 5
M ammonium acetate and
2.5 µg of yeast tRNA (Sigma). The digested samples were resolved on
an 8% sequencing gel.
Sp1 and a Factor Recognizing the AT-rich Sequence Bind to These
Adjacent Gastrin Promoter Elements Independently
Gel mobility
shift assays were performed by incubating crude nuclear extracts
prepared from RIN 38A insulinoma cells with a 30-bp double-stranded
wild-type probe encompassing both new elements
(gatccACACTAAATGAAAGGGCGGGGCAGa) in order to characterize the islet
nuclear proteins binding to the -160 to -141 sequence. Fig.
2 A shows the presence of 3 binding complexes, labeled Ia, Ib,
and II ( lane 1). Complexes Ia and Ib were competed by a
125-fold excess of a consensus Sp1 oligonucleotide, suggesting that
they represent Sp1 factor binding to the downstream GC-rich element
( lane 2). Complex II was not affected by competition with the
GC-rich Sp1 sequence, indicating that it represents binding by a
distinct factor to another sequence. However, complex II was competed
away by an oligonucleotide containing only the upstream AT-rich
sequence ( lane 3). This AT-rich sequence did not compete for
binding of the Sp1-like factor found in complex Ia and Ib, again
suggesting the factors recognize discrete sites.
Figure 2:
Gel mobility shift assays with 38A islet
cell extracts. A, crude 38A nuclear extract was incubated with
4 fmol of double-stranded P-end-labeled WT probe
(gatccACACTAAATGAAAGGGCGGGGCAGa) with or without a 125-fold excess of
cold oligonucleotide corresponding to the consensus Sp1 binding site
(ATTCGATCGGGGCGGGGCGAGC) in lane 2 or to the AT-rich domain
(gatccCTGACACTAAATGAAAGGGa) in lane 3 prior to electrophoresis
on a 4% nondenaturing polyacrylamide gel as described under
``Materials and Methods.'' In the oligonucleotide sequences,
the lower case letters represent BamHI or
BglII restriction sites. B, Sp1 supershift assay was
performed with crude 38A nuclear extract preincubated with 2.0, 1.0, or
0.1 µl of a rabbit polyclonal Sp1 antisera ( lanes
2-4) prior to the addition of WT probe. Lane 5 represents the reaction with 1.0 µl of a control rabbit
antisera. C, gel shift assay performed in
Zn
-deficient conditions. Divalent zinc cations were
chelated by preincubating crude 38A nuclear extracts with either 10 or
25 m
M EGTA prior to the addition of WT probe. D, gel
shift assay with varying titrations of the Sp1-enriched
phosphocellulose eluate B and gAT factor-enriched
DEAE-Sepharose eluate C using full-length WT probe. The amount
of protein in each reaction are: Extract B ( lanes
1-4, 6.7 µg; lane 5, 4.5 µg; lane
6, 2.2 µg; lane 7, 0 µg); Extract C ( lane 1, 0 µg; lane 2, 1.3 µg; lane
3, 2.7 µg; and lanes 4-7, 4.0
µg).
Complex Ib may also
represent binding by Sp1 or an Sp1-like factor because of the identical
sequence specificity, sensitivity to zinc chelation, and
chromatographic co-purification with Sp1. However, the anti-Sp1
antisera does not apparently supershift the Ib complex. This may be a
consequence of the species difference between the 38A extract (rat) and
the antigenic specificity of the antibody (human Sp1), or that complex
Ib may represent binding by an immunologically distinct member of the
Sp1 family
(27, 28) . Nevertheless, complex Ib does not
represent simultaneous binding of both Sp1 and gAT factors.
Sp1 and gAT Factor Binding Domains Are Directly
Contiguous
The nucleotides within the -163 to -140
sequence necessary for DNA binding of these two factors were then
localized more precisely. First, the nucleotides necessary for DNA
binding of the Sp1 factor were defined utilizing gel mobility shift
assays with mutant oligonucleotide competitors which sequentially
spanned the sequence from -163 to -140 bp (Fig.
3 A, legend). Utilizing the Sp1-enriched 38A nuclear fraction
B in a gel shift assay, specific binding residues were
identified (Fig. 3 A). Mutant oligonucleotides M1 and M2
competed as well as the wild-type oligonucleotide ( lanes
2-4). Mutant oligonucleotides M3, M4, and M5 were unable to
compete, however, indicating that those mutated base pairs interacted
with the Sp1 factor ( lanes 5-7). As expected, an Sp1
consensus oligonucleotide competed away all binding activity to the
gastrin probe ( lane 8). These binding interactions were
directly confirmed in a methylation interference assay by delineating
the specific G and A residues in this gastrin promoter sequence that
interact with Sp1 (Fig. 3 B). The nucleotides in the sequence
necessary for Sp1 binding are summarized in Fig. 3 C, as
defined first by competition analysis and then corroborated by
methylation interference.
Figure 4:
Characterization of gAT factor binding
domain by mutational analysis and DNase I footprinting. A, gel
mobility shift assays were performed with gAT-factor enriched extract
C and full-length WT probe in the presence of a 25-fold excess
of the cold mutant oligonucleotides listed in Fig. 3 B, DNase I
footprinting assay of gAT factor binding to a 52-bp
BamHI/ SalI fragment Klenow labeled on the minus
strand. The bar on the right indicates nucleotide residues
protected from digestion by DNase I. C, schematic
representation of residues critical for gAT factor binding to the
gastrin promoter ( underlined) as defined by mutational gel
shift analysis ( X) and DNase footprinting
( O).
Binding of the gAT Factor Blocks Transcriptional
Activation by Sp1
To determine the functional significance of
this mutually exclusive interaction between these 2 factors,
heterologous promoter pT81 reporter genes containing the WT and mutant
oligonucleotides listed in Fig. 3 A (WT, M1-M5) were
designed. These constructs were transfected into the gastrin-expressing
islet cell line RIN 38A and luciferase reporter gene activity was
measured. A single copy of the -163 to -140 WT gastrin
promoter sequence weakly activated transcription (2.3-fold above the
basal enhancerless pT81), indicating that the intact bipartite domain
functions as a weak activator in islet cells (Fig. 5). Construct M1,
which can bind both the gAT and Sp1 factors ( lane 3, Figs.
3 A and 4 A), behaved similarly to the wild-type,
weakly activating transcription. However, construct M2, which cannot
bind the gAT factor but can bind Sp1 ( lane 4, Figs. 3 A and 4 A), showed a 4.8-fold increase in activity above the
wild-type and an 11.0-fold increase over basal, indicating that the gAT
factor functions to inhibit the strong transcriptional activation
mediated by Sp1. Constructs M3, M4, and M5 which all bind the gAT
factor but not Sp1 ( lanes 5-7, Figs. 3 A and
4 A) only weakly activated transcription to levels comparable
to the wild-type construct. These findings suggest that in RIN 38A
insulinoma cells, the wild-type gastrin promoter preferentially binds
the gAT factor, which behaves as a weak activator. Binding of the gAT
factor prevents Sp1 binding to the adjacent sequence, blocking the
stronger transcriptional activation mediated by Sp1.
The Gastrin-AT (gAT) Factor Binds an AT-rich Motif
Recognized by the Islet Cell Transcription Factor
To
further characterize the gAT factor, competition studies were then
performed with AT-rich consensus motifs of known transcription factors
expressed in the pancreas and gastrointestinal tract. Fig. 6 indicates
that there was only partial competition for gAT factor binding with
binding sequences derived from the ubiquitous POU-domain factor Oct-1
(29) , the liver and intestinal homeodomain factors HNF-1 and
HNF-3
(30) , and the glucagon G2 element
(31) . There was
minimal competition with elements for the pancreatic islet homeodomain
factors isl-1
(32) and STF-1/idx-1
(33, 34) .
Only one competitor completely displaced binding, the ``B''
element found in the elastase I promoter which binds the islet
transcription factor TF-1
TF-1
(35) . The 8-bp core binding
sequence in the elastase promoter for
TF-1 (ATAAATGA) is nearly
identical to the gastrin AT-rich element (CTAAATGA) and both include
the important binding residues ( underlined) in the gastrin
promoter for the gAT factor (CTAAATGA). DNA sequence elements with this
core consensus motif are also found within the insulin promoter
(GAAATGA)
(35) , and an oligonucleotide competitor containing
this insulin sequence competed away gAT factor binding from the gastrin
probe as effectively as the elastase B oligo (data not shown).
TF-1 factor as islet-cell specific because it
was detected in 38A islet cells but not in acinar AR42J cells or
fibroblast Rat 2 cells
(35) . In order to characterize the
tissue distribution of the gAT factor, gel shift studies were performed
with nuclear extracts from these cell lines utilizing the wild-type
gastrin probe. Fig. 7 indicates that the identical gAT factor complex
is identifiable in both 38A and AR42J cells but not in Rat 2 cells,
suggesting that the gAT factor is therefore distinct from
TF-1.
These differences in electrophoretic patterns were also observed when
the elastase B element and gastrin wild-type probes were run in
parallel gel mobility shift assays with 38A and AR42J nuclear extracts
(data not shown). Furthermore, a survey of additional cell lines
revealed that binding activity was also detected in the ductular
pancreatic cell line Panc-1 and weaker activity was observed in the
hepatic cell line HepG2.
TF-1. Although
their sequence specificity is similar, the gAT binding factor differs
from
TF-1 in electrophoretic mobility and cell specificity in gel
shift assays. Interestingly,
TF-1 was first identified as a factor
which binds the B cis-regulatory element (ATAAATGA) in the enhancer of
the elastase I gene
(35) . Although the elastase gene is
specifically expressed in pancreatic exocrine cells, a transgene
comprising multiple copies of the isolated
TF-1 element is
expressed only in islet
-cells
(35) . The islet cell
factors binding to the
TF-1 sequence also activate insulin gene
transcription via a
TF-1-like element found within the insulin
enhancer
(35) . Multiple copies of both the isolated elastase
and insulin
TF-1 elements strongly activate islet-specific
transcription of a basal promoter reporter gene in RIN 38A cells
(35) . In contrast, 5 tandem copies of the gastrin-AT element
subcloned into the minimal promoter construct pT81-Luc activated
transcription only a modest 2-fold above basal levels in RIN 38A cells
(data not shown). Collectively, these results indicate that the gAT
factor is not
TF-1 even though both bind similar DNA sequences.
Further characterization of both
TF-1 and the gAT binding factor
will be necessary to more precisely define their relationship.
(I) gene
promoter, the GC-rich Sp1 binding site directly overlaps with the CCAAT
box binding site such that Sp1 and nuclear factor-1 interactions are
mutually exclusive
(39, 40) . Although each factor can
activate the collagen gene independently, nuclear factor-1 functions as
an inhibitor of Sp1-mediated transcriptional activation. Other examples
of transcriptional regulation mediated by the displacement of Sp1 by
competing factors include the ornithine decarboxylase gene
(41) and the
-globin gene
(42) . A more complex
interaction has been described between Sp1 and the pituitary-specific
homeodomain factor Pit-1 in the regulation of the growth hormone gene
(43, 44) . Binding studies indicate that their
interactions are also mutually exclusive. Although they do not bind
simultaneously, maximal growth hormone gene activity occurs when the
binding sites for both factors are intact, indicating that
transcriptional regulation is mediated by sequential rather than
simultaneous binding of the two factors.
Figure 1:
Linker
scanning mutagenesis of the human gastrin 5`-flanking region. 11 linker
scanning mutant gas200 pXP2-luciferase constructs as described under
``Materials and Methods'' were transfected with DEAE-dextran
into 38A islet cells and luciferase activity was assayed at 48 h. The
XX in the promoter map represents the 10-bp block
substitution. Luciferase activity is expressed as % activity of the
wild-type gas200 pXP2 construct (WT) and represents the mean ±
S.D. of 4-6 samples. Each transfection experiment was repeated at
least 3 times.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.