From the
The LIM domain homeobox gene islet 1 (isl-1) is
expressed in the embryonic nervous system and may be an early marker of
motor neuron specification. isl-1 is expressed in all 4 islet
cell types, but a role for isl-1 in the regulation of insulin
gene expression has not been demonstrated, and the genetic targets for
isl-1 in the pancreas remain unknown. We show here that the
proximal rat proglucagon gene promoter binds an amino-terminally
truncated Trp-E-isl-1 fusion protein that lacks the LIM
domains. The proglucagon gene promoter also binds full-length in
vitro translated isl-1 containing the intact LIM domains.
isl-1 antisera detects binding of proglucagon gene sequences
to isl-1 present in a slowly-migrating complex in nuclear
extracts from InR1-G9 islet cells. The transcriptional properties of
the proglucagon gene promoter sequences that bind isl-1 (designated Ga, Gb, and Gc) were assessed after transfection of
reporter genes into wild type and isl-1-antisense
(isl-1(AS)) InR1-G9 islet cells. The proximal proglucagon gene
(Ga) promoter sequence reduced TK-CAT activity by
Glucagon and the glucagon-like peptides are encoded within a
common precursor, proglucagon, that is expressed in a highly
tissue-specific manner in the brain, intestine, and endocrine pancreas
(1). Glucagon, secreted from the pancreatic A cell, is a key regulator
of carbohydrate, lipid, and protein metabolism, and excess glucagon
secretion contributes to the metabolic derangements characteristic of
diabetes mellitus. Recent studies have implicated glucagon-like
peptide-1 as an important mediator of glucose-dependent insulin
secretion from the pancreatic islet
(2) . Accordingly,
understanding the factors important for the regulation of proglucagon
gene expression is of considerable interest and importance.
The
isolation of the genes and cDNAs encoding proglucagon has demonstrated
that proglucagon gene transcription originates from a single
transcription start site and gives rise to an identical proglucagon
mRNA transcript in each tissue
(3) . Nevertheless, gene transfer
and transgenic experiments have demonstrated that the control of
proglucagon gene transcription is highly tissue-specific, with
different regions of the proglucagon gene promoter identified as
necessary for transcriptional activation in the pancreas and intestine
(4-7). A series of deletional and mutational analyses have
identified several cis-acting DNA sequences in the proximal proglucagon
gene promoter that appear to be important for islet cell-specific gene
transcription
(8) . Two of these regions, originally designated
G2 and G3, have been shown to act as islet cell-specific enhancers,
whereas a more proximal region, G1, displays islet cell-specific
promoter activity
(8, 9) . These DNA sequences have been
shown to bind distinct proteins, including several that may be unique
to islet cell lines
(10, 11) . Although these proteins
are candidate transcriptional regulators that likely contribute to
islet cell-specific proglucagon gene transcription, the identify of
these factors has not yet been elucidated.
Recent studies suggest a
role for homeobox genes in the control of islet cell-specific gene
transcription. Two novel homeobox proteins isolated from islet cell
cDNA libraries by expression cloning, Lmx-1 and Cdx-3, bind to
sequences in the rat insulin gene promoter and activate insulin gene
transcription
(12) . Degenerate oligonucleotides complementary to
conserved sequences of the homeobox were used in polymerase chain
reaction experiments to isolate over a dozen distinct transcripts
encoding homeobox sequences from islet RNA
(13, 14) ;
however, the functional importance of these homeobox proteins in the
regulation of islet gene expression has not been established.
A cDNA
encoding the homeobox gene isl-1 was originally isolated by
screening a rat islet cell (RIN cell) library with a probe from the
insulin gene enhancer
(15) . The homeobox gene isl-1 is
expressed in all four principal cell types of the endocrine pancreas
(16, 17), however a role for isl-1 in the regulation of
insulin gene transcription has not been established
(12) .
Subsequent experiments localized isl-1 to neurons in the
central and peripheral nervous system
(16, 17) , and
developmental analysis of isl-1 in the embryonic chick spinal
cord has shown that isl-1 is regulated by inductive signals
and may serve as a useful early marker of motor neuron
differentiation
(18) . Additional evidence for the biological
importance of isl-1 derives from analyses of isl-1 genes in different species that demonstrate 100% conservation of
the isl-1 amino acid sequence in the human, hamster, and rat
isl-1 genes
(19) .
The proglucagon gene proximal
promoter contains several AT-rich sequences that are candidate binding
sites for homeobox transcription factors
(8) . Furthermore, the
highly restricted cell-specific expression of the proglucagon gene
suggests that a combination of transcriptional regulators expressed in
a tissue-specific manner likely contributes to islet cell-specific
expression of the proglucagon gene in the islet A cell. To ascertain
whether isl-1 may be a candidate transcription factor that
controls proglucagon gene expression in the islets, we have examined
the importance of isl-1 in the regulation of proglucagon gene
transcription.
Analysis of the sequence of the rat proglucagon gene promoter
(8) identified 2 AT-rich sequences containing TAAT motifs in the
first 100 bp upstream of the transcription start site. To determine if
isl-1 binds to the proglucagon gene TAAT motifs, we prepared a
TrpE-isl fusion protein lacking the isl-1 LIM domains, since
previous studies have shown that the LIM domains inhibit homeodomain
binding of DNA sequences in vitro(24, 25) . The
amino-terminally truncated isl-1 fusion protein was incubated
with synthetic oligonucleotides containing TAAT motifs from the
proximal (first 100 bp) rat proglucagon gene promoter ().
All 3 proglucagon gene sequences (Ga/Gb/Gc) bound the TrpE-isl-1 fusion protein in an electrophoretic mobility shift assay
(Fig. 1). Competition experiments were carried out using the
Ga/Gb/Gc sequences as well as oligonucleotides containing TAAT
sequences from the insulin and amylin promoters (E2, P1, and AMY)
(). A 200-fold molar excess of unlabeled Ga markedly
diminished the binding of isl-1 to the Ga probe
(Fig. 1). In contrast, 1000-fold molar excess Gb was much less
effective in competing for Ga binding, suggesting that the core TAAT
motif alone (present in Ga/Gb/Gc) is not the only determinant for
isl-1 binding. Furthermore, the Gc sequence, which contains
the same TAAT motif as Ga but different 5`- and 3`-flanking nucleotide
sequences was much less effective in displacing Ga binding, providing
further evidence that the nucleotide sequences surrounding the TAAT
motif determine binding affinity (Fig. 1). In contrast, no
displacement of Ga binding was detected with a 1000-fold molar excess
of the TAAT-containing amylin or insulin E2 and P1 elements
(Fig. 1). Although unlabeled excess Gb effectively competed for
isl-1 binding to Gb, both Ga and Gc also diminished specific
isl-1 binding to Gb, although not as effectively as Gb alone
(Fig. 1). In contrast, a 1000-fold molar excess of unlabeled E2,
P1, and AMY probes was necessary to achieve a reduction of specific
isl-1 binding to the Gb probe. The proglucagon gene Gc
sequence was also effective in binding isl-1, and isl-1 binding to Gc was displaced by unlabeled Gc as well as by a
200-fold molar excess of Ga. Competition for isl-1 binding to
Gc was also observed with a 1000-fold molar excess of Gb and P1
sequences. In contrast, no displacement of isl-1 binding to Gc
was detected with a 1000-fold molar excess of the E2, AMY, or GATA
oligonucleotides (Fig. 1). The isl-1 homeodomain also
bound the E2 insulin gene probe, but the binding to E2 was more
effectively diminished in competition experiments using the proglucagon
gene Ga/Gb/Gc sequences as competitors, compared with the competition
observed with unlabeled E2 alone (Fig. 1).
A supershift of the high molecular weight isl-1 complex in
InR1-G9 extract was also seen with the Gb and Gc probes (data not
shown). The high molecular weight isl-1 complex was markedly
diminished after incubation with a 500-fold molar excess of unlabeled
competitor sequences from islet hormone gene promoters, including the
Ga, Gb, Gc, E2, AMY, and P1 sequences (Fig. 2B). In
contrast, no significant diminution of complex formation was detected
after incubation with excess GATA oligonucleotide. Addition of
isl-1 antisera also resulted in increased intensity of complex
A. The formation of this complex was attenuated in the presence of
excess competitor Ga sequences but was not comparably diminished after
competition with the other TAAT-containing oligonucleotides
(Fig. 2B).
To determine the DNA sequence requirements
for isl-1 binding to the proglucagon Ga sequence, we
synthesized three Ga mutants () and tested their
isl-1-binding properties in the EMSA experiment using InR1-G9
nuclear extracts. Mutant GaM1, which contains an intact TAAT motif but
two nucleotide substitutions in the nucleotides 12 and 13 bp 3` to the
TAAT site also formed a high molecular weight complex that was
supershifted with isl-1 antisera (Fig. 2C). In
contrast, the GaM2 sequence with a disrupted TAAT motif did not form
the high molecular weight isl-containing complex with InR1-G9 extract
(Fig. 2C). Furthermore, mutation GaM3 (that contains an
intact TAAT sequence but an AT-GC mutation of the two nucleotides
immediately 3` to the TAAT site) also failed to form the high molecular
weight isl-1 complex. These observations suggest that although
the TAAT site appears to be important for isl-1 binding to Ga,
nucleotides that flank the TAAT motif are also critical for the
formation of the proglucagon gene-isl-1 complex. Moreover,
although the specific mutations in GaM2 and GaM3 eliminated isl-1 binding (Fig. 2C), no effect on the formation of
complex A (which is specifically competed by excess Ga;
Fig. 2B) was seen in the same experiment
(Fig. 2C). These data suggest that the nucleotide
determinants of isl-1 binding to Ga are highly specific, since
the same Ga mutations did not affect protein binding and complex A
formation.
To ascertain whether the proglucagon gene sequences that
bind isl-1 are functionally important for proglucagon gene
transcription, we ligated the Ga/Gb/Gc sequences adjacent to the
thymidine kinase promoter, and the transcriptional activity of the
different TK-CAT plasmids was assessed following transfection of
InR1-G9 cells. The Ga sequence repressed the basal activity of the TK
promoter to 45% of control values (Fig. 3). The GaM1 mutation,
which displayed intact isl-1 binding in EMSA experiments
(Fig. 2C), also repressed transcriptional activity;
however, no repression was seen with either the GaM2 or GaM3 mutants
that failed to bind isl-1 (Fig. 3). In contrast (to the
repression of transcriptional activity observed with the wild type Ga
sequence) the Gb and Gc proglucagon gene sequences strongly activated
transcription (2-3-fold) from the TK promoter in InR1-G9 cells
(Fig. 3A) but not in BHK fibroblasts (Fig. 3),
NIH3T3 fibroblasts, or JEG choriocarcinoma cells (data not shown).
isl-1 was originally thought to be an insulin gene
transcription factor. In vitro translated isl-1 was
shown to bind to the TAAT motifs present in the insulin gene FLAT
element, and mutation of the TAAT motifs in the insulin gene eliminated
the ability of isl-1 to bind the insulin FLAT element
enhancer
(15) . Nevertheless, although isl-1 antisera
prevented in vitro translated isl-1 from binding to
the insulin gene E2 and P1 elements, isl-1 could not be
identified in DNA-protein complexes using nuclear extracts from islet
cell lines and the E2 or P1 probes
(27) . Taken together, these
observations suggest that isl-1 may not form a complex on the
insulin gene promoter in cells that actively transcribe the insulin
gene in vivo. Although isl-1 also binds sequences in
the rat somatostatin promoter
(28) , recent studies have
suggested that the major homeobox protein binding to the somatostatin
gene TAAT sites is IDX-1/STF-1
(13, 29) .
The
subsequent isolation of novel homeobox genes sequences from an
insulinoma cDNA library suggests that multiple homeobox transcription
factors likely compete for binding to the proglucagon gene promoter
AT-rich sequences, since Northern blot analysis demonstrated that
several homeobox genes are expressed in InR1-G9 cells and
Analysis of the DNA-binding properties of the TrpE-isl-1 fusion protein suggests that the isl-1 binding sites in
the proglucagon gene promoter display a higher affinity for isl-1 than the TAAT sites in the insulin gene FLAT or P1 elements.
Competition experiments using excess unlabeled oligonucleotides
demonstrated that the E2/P1 sequences were not very effective
competitors for isl-1 binding when using the proglucagon gene
Ga/Gb/Gc sequences as probes. In contrast, the proglucagon gene
sequences effectively displaced isl-1 from an E2 probe in EMSA
experiments. Taken together, these observations support the hypothesis
that the AT-rich sequences in the proximal proglucagon promoter may be
target sites for isl-1 action.
The expression of multiple
homeobox genes in a given cell type, taken together with the
demonstration that most homeobox genes can bind the core TAAT sequence,
makes it difficult to ascertain precisely which homeobox genes exert
transcriptionally important effects on a given promoter. The results of
our experiments using isl-1-depleted antisense InR1-G9 islet
cells demonstrate that although the proximal proglucagon gene Ga
sequence clearly binds isl-1 and Ga sequences mediate
inhibition of a heterologous promoter, this transcriptional property is
not affected by the marked reduction of isl-1 in the antisense
InR1-G9 cells. In contrast, the transcriptional activation mediated by
the Gb/Gc sequences was eliminated following transfection of isl-1 antisense InR1-G9 cell lines, strongly suggesting that isl-1 mediates the Gb/Gc-dependent activation of the proglucagon gene
promoter.
The observation that isl-1 is detected as part of
a high molecular weight complex in InR1-G9 nuclear extracts by EMSA
experiments suggests that protein-protein interactions may be important
for the formation of a transcriptionally active isl-1 complex
on the proglucagon gene promoter. Increasing evidence implicates the
LIM domains as important mediators of protein-protein interaction.
Truncation of the LIM domains from the Lmx-1 protein abrogates the
synergistic activation of the insulin gene minienhancer in transfected
BHK cells
(12) . The LIM protein RBTN2 and the basic
helix-loop-helix protein TAL1 have been shown to form a complex in
erythroid cell nuclei
(30, 31) , and the LIM domains in
the human cysteine-rich protein appear to mediate dimerization even in
the absence of DNA
(32) . Mutational analyses have recently shown
that the LIM domains in the homeodomain Xlim-1 protein function as
negative regulatory elements that constrain Xlim-1
activity
(25) . Accordingly, it seems reasonable to postulate
that the LIM domains of isl-1 may be important for
protein-protein interaction that contributes to complex formation in A
cells of the islets. Alternatively, the heterodimer formation observed
between the POU protein UNC-86 and the LIM homeobox protein Mec-3 is
not dependent on the LIM repeats
(33) , suggesting that LIM
homeobox proteins may contain multiple distinct domains capable of
mediating protein-protein interaction in vitro. Future
experiments should address the identity of the specific partners that
complex with isl-1 that likely contribute to the regulation of
proglucagon gene transcription in the endocrine pancreas.
The location (relative to
the transcription start site) and surrounding nucleotide sequences of
the TAAT motifs (underlined) from the rat insulin (rlns I),
human amylin (hAmylin), and rat proglucagon (rGlu)
gene promoters are shown. Mutations in the nucleotide sequences are
designated M, and the specific mutated nucleotides are shown in
boldface.
50%, but no
change in the activity of the Ga-TK-CAT plasmid was seen after
transfection of isl-1(AS) InR1-G9 cells. In contrast, the
Gb/Gc sites activated transcription 2-3-fold in wild type InR1-G9
cells, and the isl-1-dependent activation of gene
transcription through the Gb/Gc element was eliminated following
transfection of isl-1(AS) InR1-G9 cells. These data
demonstrate that the LIM domain homeobox gene isl-1 1) is not
constrained from DNA binding by its LIM domains and 2) functions as a
positive regulator of proglucagon gene transcription in the endocrine
pancreas.
EMSA
A 702-base pair polymerase chain reaction
fragment of isl-1 cDNA (hamster, rat, and human isl-1 amino acid sequences are 100% identical
(19) ) containing
the complete homeodomain and 3`-sequences from amino acid 118 to the
stop codon was sequenced and then inserted in the TrpE expression
vector path 11 to generate the plasmid TrpE-isl-1. The
standard binding reaction contained the following components in a final
volume of 18 µl: 10 mM HEPES (pH 7.8), 75 mM KCl,
2.5 mM MgClExperiments Using a TrpE-isl-1
Fusion Protein
, 0.1 mM EDTA, 1 mM
dithiothreitol, 3% Ficoll, 1 mg/ml bovine serum albumin, 500 ng (when
using nuclear extract, see Fig. 2-4) or 1 µg (for
TrpE-isl-1 fusion protein) of poly(dI-dC), 10,000 cpm of
end-labeled, double-stranded oligonucleotide, and 12 µg of crude
nuclear extract (Fig. 2-4) or 2 µg of TrpE-isl-1 fusion protein extract. The mixture was incubated for 20 min at 30
°C and loaded directly onto a 5% nondenaturing polyacrylamide gel.
For experiments with antisera (Fig. 2-4), 5 µl
isl-1-specific
(17) or preimmune antisera was mixed
with the reticulocyte lysate or nuclear extract immediately before
adding the premixture. Nuclear extracts from InR1-G9, BHK, and COS7
cells were prepared as described
(20) . The EMSA was performed
essentially as in Ref. 21. The EMSA experiments employed
double-stranded synthetic oligonucleotides as probes, corresponding to
the sequences shown in , with an additional 5 bases
comprising a 5`-BamHI overhang (top strand) and a 5 base
5`-BglII overhang (bottom strand) for end-labeling. The
nonspecific competitor DNA was a double-stranded oligonucleotide
containing a GATA binding site from the human cardiac a-myosin heavy
chain gene (5`-TTCAACGTGCAGCCGGAGATAAGGCCAGGCCCGAA-3`).
Figure 2:
The proglucagon gene sequences bind
full-length in vitro translated isl-1 as well as an
isl-1-immunoreactive protein present in InR1-G9 islet cell
nuclear extracts. PanelA, lysate products (from
5-10% of each reaction) were used for EMSA reactions in the
absence or presence of nuclear extracts, and isl-1 antisera
(antibody). The incubations were carried out at either 30 °C or 0
°C as shown. A and B designate as yet
unidentified DNA-protein complexes. PanelB,
competition for isl-1/proglucagon gene-Ga complex formation in
InR1-G9 extract in the presence of specific and nonspecific (500-fold
molar excess) competitor sequences. The competitor oligonucleotides are
shown in Table I. Ab + isl-1 designates the supershifted
complex seen with immune but not preimmune isl-1 antisera.
A refers to a DNA-protein complex not yet definitively
identified. PanelC, effect of mutations in the
proglucagon gene Ga sequence on isl-1 binding and complex
formation in InR1-G9 nuclear extract. The oligonucleotides containing
specific mutations, shown in Table I, were used in EMSA analyses with
InR1-G9 nuclear extract, in the presence (+) and absence (-)
of isl-1-specific antisera. A denotes a specific
DNA-protein complex, as seen in panels A and
B.
In Vitro Translation
The full-length rat isl-1 cDNA was transcribed from linearized plasmid templates with T7 RNA
polymerase, Riboprobe II Core kit, and 5 mM
mG(5`)ppp(5`)G for capping synthesized transcripts in a
final volume of 50 µl. RNase-free DNase I was added to the solution
to remove the DNA template for 15 min at 37 °C. The quality of
transcript synthesis was checked on a 1% agarose gel. Approximately 5
µg of RNA were added to a 50-µl in vitro translation
mixture including nuclease-treated rabbit reticulocyte lysate in
accordance with the manufacturer's instructions. A control
translation mix with no added RNA was treated identically. Identical
translation reactions using [
S]-labeled
methionine were performed in parallel with the isl-1 template
to verify that equivalent amounts of protein were synthesized in each
experiment. The labeled translation reactions were analyzed on a 10%
acrylamide gel.
Analysis and Generation of Antisense Cell
Lines
Isolation of RNA and Northern blot analysis was carried
out as described. Four different isl-1 antisense expression
plasmids isl-1(AS)A-D contained various lengths of
isl-1 5`-untranslated, intron and translated sequences.
Plasmid AS-isl-1A contained 440-bp rat isl-1 genomic
sequences 5` to the translation start site and 30-bp 5`-coding
sequences. This 470-bp isl-1 fragment was subcloned into the
EcoRI and HindIII sites of plasmid pSR1neo in the
antisense orientation. Plasmid AS-isl-1B contained 100-bp rat
isl-1 genomic sequences 5` to the translation start site and
30-bp 5`-coding sequences (total 130 bp) subcloned into the same
restriction sites in pSR1neo. Plasmid AS-isl-1D contained
hamster full-length isl-1 coding sequences
(19) . This
1060-bp fragment was subcloned into the HindIII and
BamHI sites of the mammalian expression vector pSR1neo in the
antisense orientation. Plasmid AS-isl-1C was produced by
ligating a 360-bp EcoRI fragment of AS-isl-1D
(containing sequences from the translation start site to the
EcoRI site of the isl-1 cDNA) into
pSR1neo
(22) . All of the antisense constructs were confirmed by
DNA sequencing. G418-resistant InR1-G9 clones transfected with the
isl-1 antisense expression plasmids were individually
expanded, and the levels of isl-1 RNA and protein were
assessed by Northern and Western blotting as described
previously
(19) .
Construction of Reporter Plasmids and Cell
Transfections
InR1-G9 cells were transfected in suspension by
the DEAE-dextran method
(6) . Minienhancer multimers were
constructed from the appropriate oligonucleotides ()
synthesized with restriction enzyme BamHI and BglII
recognition sites on opposite ends. The ligated oligonucleotides were
inserted into the BamHI site of the PUTKAT vector adjacent to
the thymidine kinase promoter
(23) . All plasmids were sequenced
to verify copy number and orientation. Cells were harvested 48 h after
transfection and analyzed for chloramphenicol acetyltransferase
activity as described (6). Transfection efficiencies were monitored by
cotransfecting the plasmid Rous Sarcoma virus -galactosidase and
measuring the
-galactosidase activity in each sample. CAT activity
was normalized for variations in protein concentration and transfection
efficiency.
Figure 1:
EMSA
analysis of isl-1 binding to the proglucagon gene Ga, Gb, Gc,
and insulin gene E2 sequences. Increasing amounts (20, 200, or
1000-fold molar excess) of designated unlabeled oligonucleotides (shown
at the top of each figure; see Table I) were used to
compete for isl-1 binding. COMP, competitor DNA
sequence; FP, free probe. 10,000 cpm of end-labeled,
double-stranded oligonucleotide and 2 µg of unpurified bacterial
extract containing the TrpE-isl-1 fusion protein were used in
each reaction.
The observation
that the isl-1 homeodomain (without the LIM domains) binds
sequences in the proximal proglucagon gene promoter suggested that
isl-1 is a candidate regulator of proglucagon gene
transcription. To determine if an isl-1 protein including the
LIM domains could also bind to the proglucagon gene promoter, we
prepared full-length in vitro translated isl-1 for
use in EMSA experiments. Contrary to previous reports (that the
isl-1 LIM domain sequences inhibit isl-1 binding
(24, 26) ), we detected a specific
DNA-protein complex using in vitro translated isl-1 and the Ga probe (Fig. 2A). Addition of isl-1 antisera to the EMSA reaction eliminated the formation of the
isl-1Ga complex. To detect evidence for isl-1 binding to the Ga sequence in islet cells, we incubated nuclear
extracts from the InR1-G9 glucagon-producing islet cell line with the
proglucagon gene Ga sequence, producing several distinct DNA-protein
complexes (Fig. 2A); addition of anti-isl-1 antisera to the EMSA reaction resulted in a clear supershift of a
high molecular weight complex (Ab + isl-1;
Fig. 2A, lane4). Addition of excess
in vitro translated isl-1 to the InR1-G9 extract
eliminated the formation of both the high molecular weight complex and
the complex designated B, and isl-1 antisera attenuated the
formation of the in vitro translated isl-1/Ga complex
in the presence of InR1-G9 extract, resulting in a slight supershift of
this lower band (isl-1 + Ab; Fig. 2A).
Figure 3:
Transcriptional properties of the
proglucagon gene enhancer sequences in InR1-G9 islet cells and BHK
fibroblasts. Minienhancer multimers were constructed from the
appropriate wild type (A) and mutant (B)
oligonucleotides (Table I), and ligated into the BamHI site of
the PUTKAT vector (23), immediately 5`-to the TK promoter. All
constructs were sequenced to verify the correct copy number and
orientation (arrows) of the inserted oligonucleotides. InR1-G9
(A and B) and BHK cell transfections (each plasmid in
triplicate) and CAT assays were carried out, as described previously
(6). The data shown represent the mean ± S.E. of three separate
experiments.
Since the EMSA experiments identified more than one DNA-protein
complex that formed with the Ga/Gb/Gc sequences, we wished to determine
the specific contribution of isl-1 to the control of
proglucagon transcription as mediated by the Ga/Gb/Gc elements.
isl-1 antisense expression vectors were constructed and
transfected into wild type InR1-G9 cells. After selection with G418,
surviving InR1-G9 clones were individually expanded, and isl-1 mRNA transcripts in each clone were analyzed by Northern blotting.
Several InR1-G9 isl-1(AS) clones were obtained that contained
markedly reduced levels of isl-1 mRNA transcripts
(Fig. 4A). To ascertain whether these clones also
contained reduced levels of immunoreactive isl-1, nuclear
extracts were prepared and analyzed by Western blotting
(Fig. 4B). These experiments demonstrated that the
InR1-G9 isl-1(AS) clones also contained markedly reduced
levels of immunoreactive isl-1. We next transfected the TK-CAT
plasmids containing the Ga/Gb/Gc sequences into the InR1-G9
isl-1(AS) cells (Fig. 5). No change in the relative
transcriptional activity (compared with the results observed in wild
type cells) of the Ga-TK-CAT plasmids was detected following
transfection of the isl-1-depleted InR1-G9 isl-1(AS)
cells. In contrast, the isl-1-dependent transcriptional
activation conferred by the Gb and Gc elements was eliminated in
InR1-G9 isl-1(AS) cells. Furthermore, the relative levels of
proglucagon mRNA transcripts were also lower in the InR1-G9
isl-1(AS) cells (data not shown).
Figure 4:
A, inhibition of isl-1 gene expression in InR1-G9 isl-1(AS) cell lines. The
antisense clones shown in lanes2-6 were
derived from transfection with different isl-1 antisense
constructs A (lanes 2 and 3), B (lane 4), C (lane
5), and D (lane 6) that contained various amounts of
isl-1 sequences in the 3`-5` orientation. Control B9
represents a G418-resistant InR1-G9 clone transfected with the psR1neo
expression vector alone. The Northern blot shown in panel A was hybridized with an isl-1 DNA probe containing mostly
5`-untranslated sequences. The relative migration position of the 18 S
ribosomal RNA is indicated with an arrow. B, Western
blot analysis of immunoreactive isl-1 in wild type and
InR1-G9(AS) cells. 40 µg of nuclear protein from wild type and
antisense InR1-G9 cells was electrophoresed on a 10% SDS-PAGE gel and
transferred to a nitrocellulose membrane for hybridization with
isl-1-specific antisera (17). A 39-kDa
isl-1-immunoreactive band was detected in the wild type
InR1-G9 cells (and G418-resistant controls, not shown) but was greatly
reduced in InR1-G9(AS) cells. The relative migration positions of
molecular size markers are indicated. The blot was rehybridized with a
monoclonal antisera (AB-3) against the nuclear protein p53 (Oncogene
Science, Uniondale, NY).
Figure 5:
Transcriptional properties of the
proglucagon gene enhancer sequences in InR1-G9 isl-1(AS)
cells. The proglucagon gene Ga/Gb/Gc enhancer plasmids shown in Fig. 3
were transfected into InR1-G9 isl-1(AS) cells (data obtained
from transfection of several different InR1-G9 antisense clones were
comparable). InR1-G9 transfections were carried out on at least three
different occasions (each plasmid in triplicate), and CAT assays were
carried out, as described previously (6). The data shown represent the
mean ± S.E. of three separate
experiments.
TC1.9
glucagon-producing islet cell lines
(14) . Nevertheless, no
experiments to date have examined whether islet homeobox genes bind to
and regulate the proglucagon gene promoter. The results of our
experiments demonstrate that the isl-1 homeodomain as well as
full-length isl-1 (either translated in vitro or
present in nuclear extracts) binds to two specific sites in the first
100 bp of the rat proglucagon gene promoter. Deletional and mutational
analyses of transfected proglucagon-CAT fusion genes have previously
demonstrated that a specific region, designated G1, functions as an
islet cell-specific promoter in vitro(8) . G1 extends
from -65 to -100 and contains an inverted TAAT motif on the
opposite strand. A second TAAT motif (encompassed by the Ga/Gc sites)
is located immediately 3` to G1, approximately 25 bp upstream of the
TATA box. Our results demonstrate that the proglucagon gene Ga/Gb/Gc
sites are capable of binding the isl-1 homeodomain in
vitro. Furthermore, our experiments show that since full-length
in vitro translated isl-1 and isl-1 in islet
cell nuclear extracts can also bind to proglucagon gene sequences, the
isl-1 LIM domains are not necessarily inhibitory for isl-1 binding to the proglucagon gene promoter in vitro.
Table:
TAAT sequences in the proximal promoter regions
of the insulin, glucagon, and amylin genes
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.