From the Department of Molecular Pharmacology, University of Göttingen, D-37070 Göttingen, Germany
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ABSTRACT |
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Cyclic AMP stimulates insulin gene transcription through a cAMP response element (CRE). In the present study the insulin CRE-binding proteins and their functions were investigated. A mutational analysis of nuclear protein binding in electrophoretic mobility shift assays in combination with specific antisera showed that in the CRE of the rat insulin I gene the imperfect CRE octamer-like sequence TGACGTCC interacts weakly with CREB and overlaps with two sequence motifs (TTGTTGAC and CCAAT) that bind winged helix-like proteins and the transcription factor NF-Y, respectively. Transient transfection of wild-type and mutant insulin CRE-reporter fusion genes and the inactivation of cellular CREB or NF-Y by overexpression of the dominant negative mutants KCREB or NF-YA29, respectively, indicate that cAMP inducibility of the insulin CRE is mediated by CREB or closely related proteins; however, NF-Y binding to the insulin CRE confers constitutive, basal activity and decreases the ability of CREB to mediate cAMP-stimulated transcription and calcium responsiveness. Results from these studies demonstrate that NF-Y binds to the insulin CRE and modulates the function of CREB. Together with the nonpalindromic sequence of the CRE octamer motif, the interaction of NF-Y with CREB may be responsible for the gene-specific transcriptional activity of the insulin CRE and explain why it has considerable basal activity but is less responsive to cAMP stimulation than others.
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INTRODUCTION |
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The second messenger cAMP regulates the transcription of many genes by a mechanism involving the activation of protein kinase A and subsequent phosphorylation of the nuclear transcription factor CREB1 (1-3). In addition to protein kinase A, CREB is phosphorylated on serine 119 (in CREB-327) by other kinases including calcium/calmodulin-dependent protein kinases, Ras-dependent RSK2, and a p38/HOG-1-dependent protein kinase (1-7). CREB can thus confer cAMP, calcium, and growth factor responsiveness to genes carrying CREB-binding sites called cAMP response elements (CRE) and typified by the consensus palindromic octamer sequence 5'-TGACGTCA-3'. CREB is expressed ubiquitously and many genes whose transcription is regulated by cAMP contain a CRE, or variants of it, in their 5'-flanking regions. CREB/CRE-directed transcription is thus of general importance in virtually all cells (1-7). Cyclic AMP responsiveness has been shown in individual cases to be conferred by transcription factors other than CREB such as AP-1 (8), AP-2 (9), Pit-1 (10), and SF-1 (11).
Although CREB is a ubiquitous protein, gene-specific differences exist
in the responsiveness of different CREs to transcriptional activation
(1-3, 12-15). In addition to CREB and the closely related proteins
ATF-1 and CREM, the CRE motif is recognized by a number of proteins
of the CREB/ATF family of transcription factors that are not activated
by cAMP but may compete with CREB for binding to the CRE (1-3). CREs
in which the DNA binding or function of CREB is modulated by the
presence of another protein at adjacent or overlapping sites have been
described (1-3, 13) and in some cases the accessory proteins have been
identified including the glucocorticoid receptor (14, 16), YY1 (17),
and C/EBP proteins (15, 18-21). Therefore, a given CRE may have unique
functional properties depending on its specific DNA sequence both
within the core octamer and nucleotides flanking this motif.
Gene-specific transcriptional activities have been reported also for
the CRE of the rat insulin I gene (22), although the molecular basis
for its unique functional properties has remained unclear. The peptide
hormone insulin is synthesized in cells of the islets of
Langerhans. It is a key regulator of blood glucose concentration, and
inappropriate regulation of insulin production and secretion causes
diabetes mellitus. Cyclic AMP stimulates insulin gene transcription
(22, 23). In the absence of other signals, cAMP inducibility of the
insulin gene is modest (22-24). However, cAMP stimulates insulin gene
transcription synergistically with glucose (25-28). Potent stimulators
of
cell cAMP levels are hormones including glucagon-like peptide-1
(29), which appears to be a physiologically important hormonal mediator
of the "incretin effect" on insulin secretion and has been proposed
as a new therapeutic agent for the treatment of
non-insulin-dependent diabetes mellitus (29). Therefore,
similar to the glucose competence concept of insulin secretion (29), a
synergistic interaction between the hormonally regulated
cAMP-dependent signaling system and the glucose-regulated signaling system may give
cells the ability to match the ambient concentration of glucose to an appropriate transcriptional response of
the insulin gene (24). Despite its suggested physiological significance, the molecular mechanism of cAMP-induced insulin gene
transcription is poorly understood.
Studies of the transcriptional activity of reporter fusion genes in
insulinoma cells demonstrated that a CRE in the 5'-flanking region of
the rat insulin I gene is required and sufficient for cAMP induction
(22, 23). However, the insulin CRE shows unique functional properties;
it (i) has considerable basal transcriptional activity, (ii) gives a
weak cAMP response, and (iii) is not responsive to membrane
depolarization and calcium influx (22). These activities distinguish
the insulin CRE from typical CREs that share a perfect 5'-TGACGTCA-3'
octamer, bind CREB, and fully respond to cAMP and depolarization-induced calcium influx in insulinoma cells (22, 30-32).
The special properties of the insulin CRE may be explained by its
particular DNA sequence and distinct pattern of nuclear protein binding
(22). The insulin CRE octamer, 5'-TGACGTCC-3' (between 178 and
185
relative to the transcriptional start site), differs in one position
from the CRE octamer consensus motif. Cellular CREB binds weakly to the
insulin CRE (22), such that CREB cannot be detected among the nuclear
proteins binding to the insulin CRE in an electrophoretic mobility
shift assay with labeled insulin CRE used as probe (22). Furthermore,
the insulin CRE contains a CCAAT box motif that in some other genes has
been shown to confer cAMP inducibility (33-36). Therefore, the role of
CREB in the cAMP responsiveness of the insulin CRE was unclear, and the
identities of the other insulin CRE-binding proteins as well as their
functional significance were unknown. The present study addressed these
questions.
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EXPERIMENTAL PROCEDURES |
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Plasmid Construction--
The plasmid 85InsLuc was prepared by
cloning a fragment of the rat insulin I gene promoter (from
85 to
+49) with 5'-XhoI and 3'-BglII ends into the
XhoI-BglII sites of pXP2 (37). For 4xInsCRE(
85Ins)Luc, 4xm1-InsCRE(
85Ins)Luc,
4xm2-InsCRE (
85Ins)Luc, 4xm3-InsCRE(
85Ins)Luc,
4xm4-InsCRE(
85Ins)Luc, 4xE
-CAAT(
85Ins)Luc, and
4xG3B(
85Ins)Luc, four copies of the synthetic oligonucleotides with
5'-GATC overhangs (for sequences see Fig. 1 or further below) were
cloned in the forward orientation into the BamHI site of
85InsLuc. The plasmid
410InsLuc was prepared by cloning a fragment of the rat insulin I gene promoter (from
410 to +49) with
5'-XhoI and 3'-BglII ends into the
XhoI-BglII sites of pXP2 (37). Four bases in the
CRE octamer-like sequence (from
183 to
180) were selectively
deleted inside the insulin promoter with the restriction enzyme
AatII and T4 DNA polymerase, yielding the construct
410(
183/
180)InsLuc. Subcloning and plasmid isolation were
performed by standard procedures. All constructs were sequenced by the
enzymatic method to confirm the identity and the orientation of the
inserts.
Cell Culture and Transfection of DNA-- The pancreatic islet cell line HIT-T15 (38) was grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 5% horse serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were trypsinized and transfected in suspension by the DEAE-dextran method as described (30) with 2 µg of indicator plasmid per 6-cm dish. Rous sarcoma virus-chloramphenicol acetyltransferase plasmid (0.4 µg/6-cm dish) was added as a second reporter to check for transfection efficiency. When indicated, 2 µg of RSV-KCREB (39) or, unless indicated otherwise, 3 µg of an expression vector encoding the dominant negative mutant of NF-Y, NF-YA29 (40), were co-transfected per 6-cm dish. These co-transfections were done with a constant DNA concentration, which was maintained by adding Bluescript (Stratagene, La Jolla, CA). Cells were stimulated with forskolin (10 µM) or high KCl (45 mM final concentration) for 6 h before harvest. Cell extracts (30) were prepared 48 h after transfection. A chromatographic chloramphenicol acetyltransferase assay (41) and the luciferase assay (30) were performed as described previously. Thin layer chromatography plates were analyzed with a Fuji PhosphorImager.
Nuclear Extracts-- Nuclear extracts were prepared from HIT cells by the method of Dignam et al. (42) with the modification described (22).
Electrophoretic Mobility Shift Assay--
Using 15 µg of
protein from nuclear extracts, the electrophoretic mobility shift assay
was performed as described (41). In some binding reactions 2.5 µl of
an antiserum directed against the B subunit of NF-Y, -NF-YB (43), 2 µl of a specific anti-CREB antiserum (R1090) (44), or an equal volume
of preimmune serum was added to the binding reaction, and the assay was
then performed as described (22).
Oligonucleotides--
The sequences of the CRE oligonucleotides
of the rat insulin I gene (wild type and mutants 1 to 4) and the rat
somatostatin gene are shown in Fig. 1. The sequences of other
oligonucleotides were as described previously or read as follows (only
one strand with the 5'-GATC overhang is shown): TTR-HNF-3, containing a
fragment of the transthyretin promoter from 111 to
85 that includes
an HNF-3-binding site (45); TTR-HNF-4, containing a fragment of the
transthyretin promoter that includes an HNF-4-binding site (46),
5'-GATCCGGCAAGGTTCATA-3'; G3B, containing domain B of the rat glucagon
G3 enhancer-like element from
247 to
234 (41, 45); Glu-C/EBP-site,
containing the binding site of C/EBP
in the rat glucagon gene from
241 to
212 (47); CG
-CAAT, containing a CCAAT sequence motif from
the proximal promoter of the human gene encoding the
subunit of
glycoprotein hormones (45); CTF/NF-I consensus, consensus site for
CTF/NF-I (48, 49), 5'-GATCCTTTTGGCTTGAAGCCAATATGAGA-3'; MSV-CAAT,
containing the NF-Y-binding CAAT box from the long terminal repeat of
the Moloney murine sarcoma virus (50, 51),
5'-GATCCAGCGAACTGATTGGTTAGTTCA-3'; E
-CAAT, containing the
NF-Y-binding CAAT box from the murine class II genes of the major
histocompatibility complex (51), 5'-GATCCATTTTTCTGATTGGTTAAAAGTA-3'.
Materials-- A stock solution of forskolin (100 mM) was prepared in dimethyl sulfoxide and further diluted in cell culture medium. Controls received the solvent only.
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RESULTS |
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Mutational Analysis of Nuclear Protein Binding to the Insulin
CRE--
Using an electrophoretic mobility shift assay, it has been
shown previously that three unidentified nuclear proteins bind to the
rat insulin I gene CRE (complexes 1, 2, and 3) (Ref. 22; see also Fig.
2A, lane 3). As a first approach to characterize these
insulin CRE-binding proteins a mutational analysis was performed. The
sequences of the oligonucleotides used are shown in Fig.
1. Oligonucleotides containing the
insulin CRE wild-type sequence or mutants 1 to 4 were labeled and
incubated with nuclear extracts from the insulin-producing -cell
line HIT. The labeled CRE of the rat somatostatin gene was used for
comparison. As shown in Fig.
2A, complex 1 binding to the
insulin CRE was also detected using mutant 2 as probe, whereas
complexes 2 and 3 were no longer formed. In contrast, when mutants 3 or
4 were used as probes, complex 1 was not detectable, whereas complexes
2 and 3 persisted (Fig. 2A). It has been shown previously
(22) and was confirmed in the present study (not shown) that complexes
1, 2, and 3 are not formed with labeled mutant 1. Note that a new
protein complex was detected with labeled mutant 3 that comigrated with
complex A on the labeled somatostatin CRE (Fig. 2A, compare
lanes 2 and 1). A band of similar mobility but
lower intensity was detected with labeled mutant 4 (Fig. 2A, lane
5). Labeled mutant 4 showed also an additional band migrating more
slowly than complex 1 (Fig. 2A, lane 5); this band was not
further investigated. The results of this mutational analysis are
summarized in Fig. 2B, which indicates the mutations that
abolish the binding of complex 1 or complexes 2 and 3, respectively.
The bases that are required for the binding of bands 2 and 3 fall
within a sequence that shows high similarity with a consensus motif for
binding sites of HNF-3 proteins and related members of the winged helix
family of transcription factors (match of 10 bases out of 12) (52)
(Fig. 2B). The bases that are critical for the binding of
complex 1 are very similar to a consensus NF-Y-binding site (match of 8 bases out of 10) (53) (Fig. 2B).
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Evidence That Complexes 2 and 3 Represent Winged Helix-like
Proteins--
The significance of these sequence similarities was
further investigated. As shown in Fig.
3A, nuclear protein binding to labeled insulin CRE forming complexes 2 and 3 was selectively competed
away by an oligonucleotide (TTR-HNF-3) that contained a well
characterized binding site for HNF-3 proteins in the transthyretin promoter (46, 52). It was, however, not competed away by an oligonucleotide (TTR-HNF-4) that contained another fragment from the
transthyretin promoter with an HNF-4 binding site (Fig. 3A). TTR-HNF-3 did not compete for protein binding to labeled m2-InsCRE (which lacks complex 2 and 3 binding) but did selectively compete for
protein binding to labeled m4-InsCRE that comigrated with complexes 2 and 3 of labeled InsCRE (Fig. 3A). TTR-HNF-3 also competed
for protein binding to labeled m3-InsCRE which comigrated with complex
3 of labeled InsCRE (Fig. 3A); at the same time the intensity of the band comigrating with complex 2 was only somewhat reduced by TTR-HNF-3 (Fig. 3A), revealing that the mutation
in mutant 3 allowed the binding of two new proteins, one comigrating with complex A of labeled SomCRE as mentioned earlier (Fig.
2A) and one that, although it does comigrate with, is
distinct from complex 2; noteworthy, this complex does comigrate with
complex B of labeled SomCRE (see Fig. 2A). The sequence
similarity of their binding sites within the insulin CRE to an HNF-3
consensus site and the competition by TTR-HNF-3 suggest that the
binding specificity of the proteins forming complex 2 and 3 is related to that of winged helix proteins. Winged helix proteins share a
conserved DNA-binding domain and thus exhibit related but distinct DNA-binding specificities (52, 54, 55). The winged helix protein
HNF-3 is expressed in islets and also in HIT cells (56-58). However, the addition of antisera directed against HNF-3
, -3
, or
-3
to the binding reaction did not affect nuclear protein binding to
the insulin CRE (data not shown). Evidence has been presented recently
that another, not yet identified member of the winged helix family of
transcription factors binds to the glucagon and somatostatin genes
(45); it interacts with domain B of the glucagon G3 element and the
somatostatin upstream element (45). When an oligonucleotide containing
domain B of the glucagon G3 element (G3B) was used as probe, protein
complexes were formed that comigrated with complexes 2 and 3 of the
insulin CRE (Fig. 3B). Cross-competition of the insulin CRE
and G3B for binding to these complexes was observed using a similar
molar excess of the competitors (Fig. 3B). Thus, based on
their binding specificity, the proteins forming complexes 2 and 3 on
the insulin CRE could be members of the winged helix family of
transcription factors distinct from HNF-3
but the same as those
binding to the glucagon G3 element.
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Complex 1 Represents Binding of NF-Y--
The binding site of
complex 1 within the insulin CRE contains a CAAT motif with strong
similarity to an NF-Y consensus site (Fig. 2B). Sequences
related to "CAAT boxes" can, however, be recognized by diverse
transcription factors with distinct DNA-binding domains including C/EBP
(59) and CTF/NF-I (48). As shown in Fig.
4A, complex 1 with the insulin
CRE is not competed away by oligonucleotides containing a C/EBP-binding
site (Glu-C/EBP) (47) or an CTF/NF-I consensus site (49). Complex 1 binding is, however, competed away by CAAT boxes from the subunit
of the choriogonadotropin gene (CG
-CAAT) (60), the long terminal
repeat of the Moloney murine sarcoma virus (MSV-CAAT) (50, 51), and the
E
gene from the murine class II genes of the major
histocompatibility complex (E
-CAAT) (51) (Fig. 4A), all
of which are recognized by NF-Y (50, 51, 60). CG
-CAAT competed also
for complex 2 and 3 binding (Fig. 4A), consistent with the
fact that CG
-CAAT is recognized by the winged helix-like proteins
binding to G3B (45). Cross-competition was observed between InsCRE
and E
-CAAT as increasing amounts of InsCRE and E
-CAAT competed
for complex 1 with labeled InsCRE (Fig. 4B) and protein
binding to labeled E
-CAAT (Fig. 4C). Irrespective of
whether InsCRE or E
-CAAT were used as probe, E
-CAAT was a
stronger competitor than InsCRE (Fig. 4, B and
C), suggesting that E
-CAAT binds with somewhat higher affinity. Competition for protein binding to E
-CAAT was also seen
with m2-InsCRE, whereas m4-InsCRE (NF-Y site mutated) did not compete
(Fig. 4C), indicating that the competition by InsCRE was
specific. The addition of an antiserum directed against NF-Y to the
binding reaction abolished complex 1 of labeled InsCRE, whereas
complexes 2 and 3 remained unaffected (Fig. 4D). Protein binding to labeled E
-CAAT comigrated with complex 1 binding to the
insulin CRE and was markedly reduced by the anti-NF-Y antiserum (Fig.
4D). These results, when taken together, strongly suggest that complex 1 represents the binding of NF-Y or a closely related protein to the insulin CRE.
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Mutational Analysis of Sequences within the Insulin CRE That Are
Required for Function--
As a first approach to study the functional
significance of the proteins binding to the insulin CRE, the
transcriptional activity of the insulin CRE and the mutants 1 to 4 was
investigated in transient transfection experiments. Four copies of the
insulin CRE wild-type sequence or mutants 1 to 4 were cloned in front of the truncated insulin promoter (from 85 to +49) fused to the luciferase reporter gene. These fusion genes were transiently transfected into HIT cells. As has been shown before (22), the insulin
CRE conferred basal activity (12.6-fold increase) as well as modest
cAMP responsiveness to the minimal promoter (2-3-fold stimulation)
(Fig. 5; see also Fig.
6). Mutant 1 did not show any
transcriptional activity (Fig. 5). Mutant 2 conferred basal activity as
did the wild-type sequence but did not respond to cAMP stimulation
(Fig. 5). The mutations in mutants 4 and 3 reduced and abolished,
respectively, basal activity, whereas they enhanced cAMP responsiveness
(Fig. 5). Forskolin stimulated transcription through m4-InsCRE 12-fold
and through m3-InsCRE 148-fold (Fig. 5).
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Transcriptional Activity of Binding Sites for NF-Y and Winged
Helix-like Proteins in Islet Cells--
The above results obtained
with mutant 2, which lacks complex 2 and 3 binding, and with mutants 3 and 4, which lack NF-Y binding, suggest that basal activity may be
conferred to the insulin CRE by NF-Y; NF-Y may not, however, mediate
cAMP responsiveness. Consistent with this assumption the NF-Y-binding
site E-CAAT (binding complex 1) conferred basal activity to the
minimal promoter but not cAMP responsiveness (Fig. 6). Basal activity
of E
-CAAT was less than that of InsCRE (Fig. 6), which may be
explained by a different spacing of the NF-Y-binding sites in the
oligomerized E
-CAAT construct which allows functional synergism in a
somewhat less efficient way. G3B, which binds the winged helix-like
proteins (complexes 2 and 3), did not show any transcriptional activity (Fig. 6). These data suggest that proteins others than NF-Y and the
winged helix-like proteins of complexes 2 and 3 confer cAMP responsiveness to the insulin CRE.
The Mutations in Mutants 1 to 4 Alter CREB Binding-- It has been shown before that the transcription factor CREB is not detected among the nuclear proteins that bind to the labeled insulin CRE in the electrophoretic mobility shift assay (22). This is confirmed in Fig. 7A showing that the addition of an antiserum directed against CREB to the binding reaction had no effect on nuclear protein binding to the insulin CRE (compare lanes 5 and 6). The anti-CREB antiserum did, however, abolish the binding of complexes A and B to the labeled somatostatin CRE used as a positive control (Fig. 7A, compare lanes 1 and 2). The somatostatin CRE is a well characterized high affinity CREB-binding site (1-3). The base substitution in mutant 3 converts the CRE octamer-like sequence of the insulin CRE (TGACGTCC) into a perfect CRE octamer consensus sequence (TGACGTCA) (Fig. 1). It was noted above that two new protein complexes were detected that bind to labeled m3-InsCRE but not to labeled InsCRE and that comigrate with complexes A and B of the labeled somatostatin CRE (see Figs. 2A, 3A, and also 7A). As shown in Fig. 7A, binding of these complexes to m3-InsCRE was abolished by the addition of anti-CREB antiserum (compare lanes 3 and 4). This shows that the mutation in m3-InsCRE increases the affinity of CREB such that the binding of proteins with CREB-like immunoreactivity becomes detectable in the electrophoretic mobility shift assay with labeled m3-InsCRE. This offers an explanation for the marked cAMP responsiveness of m3-InsCRE (Fig. 5).
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Functional Significance of CREB and NF-Y Binding to the Insulin CRE as Assessed by Using Dominant Negative Mutants-- The binding of CREB or closely related proteins to the insulin CRE, although weak, could be functionally significant. To further explore this possibility co-transfection experiments were performed using an expression vector encoding a dominant negative mutant of CREB, KCREB (39). Whereas forskolin stimulated insulin CRE-mediated transcription in controls, forskolin had no stimulatory effect when in addition KCREB was overexpressed (Fig. 8A), indicating that CREB or closely related proteins are required for cAMP responsiveness of the insulin CRE. Forskolin slightly decreased activity in the presence of KCREB (Fig. 8A), which is similar to what was found using m1- and m2-InsCRE (Fig. 5) and remains unexplained. When taken together with the results from the mutational analysis of the insulin CRE showing that mutations to the insulin CRE that increase CREB binding enhance cAMP responsiveness, whereas mutations to the insulin CRE that decrease CREB binding prevent cAMP responsiveness (see above), the present data strongly suggest that cAMP responsiveness is conferred to the insulin CRE by CREB or closely related proteins.
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Role of CREB and NF-Y in the Regulation of the Intact Insulin
Promoter--
Within the insulin promoter (from 410 to +49) 4 bases
of the insulin CRE octamer-like sequence were selectively deleted
(construct
410(
183/
180)InsLuc, Fig.
9A) which abolishes CREB and
NF-Y binding (see above). This internal promoter deletion decreased basal activity by 40% and abolished transcriptional activation by cAMP
(Fig. 9A) indicating that insulin CRE-binding proteins contribute to basal activity and are required for the stimulation by
cAMP of insulin promoter activity. These results extend previous mutational analyses (23, 26). Co-transfection of expression vectors
encoding the dominant negative mutants KCREB or NF-YA29 together with
the wild-type insulin promoter construct (
410InsLuc) inhibited or
enhanced, respectively, cAMP-induced insulin gene transcription (Fig.
9B), suggesting a role of CREB and NF-Y in the regulation
also of the intact insulin promoter.
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DISCUSSION |
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In the present study the insulin CRE-binding proteins and their function were characterized. It is shown that in addition to CREB, which interacts with low affinity with the CRE octamer-like sequence, the transcription factor NF-Y and winged helix-like proteins bind to overlapping sites within the insulin CRE. This combination of CRE-binding proteins forms a composite CRE with gene-specific properties. Cyclic AMP responsiveness is mediated by CREB; however, NF-Y binding to the insulin CRE confers basal activity and modulates the function of CREB. The data offer an explanation why the insulin CRE has considerable basal activity but is less responsive to cAMP stimulation than others.
The winged helix protein HNF-3 is expressed in insulin- (56) and
glucagon-producing islet cell lines (57, 58, 61) and regulates glucagon
gene transcription through a binding site within the G2 element of the
glucagon gene (57, 61). The winged helix-like proteins binding to the
insulin CRE are distinct from HNF-3
. Although it is not excluded
that they enhance responsiveness of the insulin CRE, the present study
does not establish a role for these proteins and, thus, their identity
and functional significance remains to be shown. Islet cells express
multiple winged helix proteins as has been suggested based on indirect
evidence (45) and is confirmed by cDNA
cloning.2 The
cross-competition between the insulin CRE and G3B indicates that the
winged helix-like proteins binding to the insulin CRE are the same as
those binding to domain B of the G3 enhancer element of the glucagon
gene. On this element the winged helix-like proteins synergize with the
paired domain transcription factor Pax6 binding to an adjacent site and
confer in this combination cell-specific transcriptional activity (41,
45, 62-64).
Several lines of evidence strongly suggest that NF-Y binds to the
insulin CRE. First, the insulin CRE contains a CCAAT box motif with
high similarity to an NF-Y consensus site. Second, this CCAAT box motif
is recognized by a nuclear protein (complex 1). Third, complex 1 binding is not competed for by binding sites of the CAAT box-binding
proteins CTF/NF-I or C/EBP, but is competed for by several NF-Y binding
sites. Fourth, there is cross-competition for comigrating complexes
between the insulin CRE and a well characterized NF-Y-binding site,
E-CAAT. Finally, complex 1 is recognized by an antiserum directed
against NF-YB. NF-Y (also called CBF and CP1) is a ubiquitous CCAAT
box-binding protein and extremely conserved in evolution (40, 53, 65,
66). Like the TATA box, CCAAT boxes are widespread promoter elements
typically located between
60 and
100 base pairs 5' to the start of
transcription, suggesting a role in basal transcription (67). Most
CCAAT boxes located at this position are NF-Y-binding sites (67). NF-Y
has been shown to interact with the TATA box-binding protein (68), to be important for transcription re-initiation (43), and to facilitate in vivo recruitment of upstream DNA binding transcription
factors (69). Thus, NF-Y may have a general role in the assembly of proximal promoter complexes. Although, in general, NF-Y is
constitutively active, it has been implicated directly or indirectly in
the regulation of some promoters by, for example, heme (70), sterol
(71, 72), calcium depletion (73), and the viral Tax protein (74). In
the insulin promoter the NF-Y-binding CCAAT box is located further
upstream at
179 and falls within an element that confers cAMP
responsiveness. The CCAAT box motif has been implicated in the cAMP
inducibility of several genes (33-36). Some of these CCAAT boxes have
been shown to bind NF-Y. Boularand et al. (36) reported that
an inverted CCAAT box motif at
67 of the human tryptophan hydroxlase
gene promoter was required and sufficient for cAMP induciblity in
pinealocytes. This inverted CCAAT box in the mouse gene has been shown
to bind NF-Y (75). In addition, it has been demonstrated that a CCAAT
box that binds NF-Y can mediate induction by cAMP of the rat hexokinase
II gene in L6 myotubes (35). However, the present study clearly
demonstrates that NF-Y does not confer cAMP responsiveness to the
insulin CRE in islet cells.
Instead, cAMP responsiveness seems to be conferred by CREB. In the absence of other proteins, CREB binds with low affinity to the insulin CRE (Ref. 22 and this study). This may largely be due to the deviation in position 8 of the core octamer-like motif of the insulin CRE (TGACGTCc) from the consensus sequence (TGACGTCA) rather than to the bases flanking the octamer in the insulin CRE. This is suggested by mutant 3 which bound CREB much more strongly than the wild-type or mutant 4. The x-ray crystal structure of the AP-1·DNA complex indicates that the corresponding position in the AP-1 site is recognized on the opposite strand by alanine and cysteine residues of Fos and Jun that are conserved in CREB (76). Mutations of the insulin CRE that altered CREB binding changed cAMP responsiveness correspondingly. In addition a dominant negative mutant of CREB abolished cAMP inducibility of the insulin CRE. Thus, although CREB binds weakly to the insulin CRE, and although the insulin CRE contains a CCAAT box motif that has been shown to mediate cAMP inducibility in other genes (see above), the results of the present study, when taken together, strongly suggest that the cAMP responsiveness of the insulin CRE is conferred by CREB or a closely related protein.
Whereas NF-Y does not confer cAMP responsiveness, the present results
define a role for NF-Y in insulin CRE-mediated transcription. Insulin
CRE mutants that lack NF-Y binding still responded to cAMP but had lost
basal activity. An NF-Y-binding site alone was not cAMP responsive but
did confer basal activity in islet cells. NF-Y is a heterotrimeric
protein and the assembly of the NF-Y subunits follows a specific
pathway. NF-YB and NF-YC associate with each other to form a binary
complex which then interacts with NF-YA. Formation of this ternary
complex is required for binding to DNA (40, 53, 65, 66, 77). Mutants of
NF-Y subunits, such as NF-YA29 (40), that still interact with the other
subunits but do not bind DNA act as dominant negative mutants (40, 66).
Using this technique, the present study shows that inactivation of NF-Y
in islet cells decreases basal activity of the insulin CRE while it
enhances cAMP responsiveness. Thus, these data indicate that NF-Y
modulates the function of CREB. NF-Y decreases the ability of CREB to
mediate cAMP-induced gene transcription. It also inhibits
depolarization responsiveness, and through NF-Y the insulin CRE gains
constitutive basal activity that is conferred to the insulin promoter
(22, 23, 26). The interaction of CREB with NF-Y at the insulin CRE
could include direct protein-protein interaction. The transcription
factor YY1, that binds immediately downstream of the CRE of the mouse
c-fos promoter, interacts directly with CREB, and this
interaction has been suggested to lead to repression of
CRE-dependent transcription (17). A direct interaction between CREB and NF-Y remains to be shown. However, as the basis of
functional synergism, evidence has already been presented for complex
formation between NF-Y and other transcription factors such as C/EBP
proteins on the serum albumin promoter (78) and ATF-2 on the
fibronectin promoter (79). Alternatively, CREB and NF-Y could compete
for binding to the insulin CRE as their binding sites overlap. This is
reminiscent of CREB-binding sites which overlap with binding sites for
the glucocorticoid receptor in the human glycoprotein hormone
-subunit gene (14) or for C/EBP
and related proteins in the
phosphoenolpyruvate carboxykinase gene (15, 18-21). In these cases a
specific negative regulation by glucocorticoids (14) and a
tissue-dependent activation by cAMP (15, 18-21),
respectively, is assumed to be achieved through these overlapping
sites.
To maintain glucose homeostasis, insulin gene transcription appears to be regulated synergistically by glucose metabolism and the hormonally regulated cAMP pathway (24, 25). Studies on many genes have taught that promoter activity depends on a synergistic interaction between multiple promoter-binding proteins. Transcriptional activation in response to extracellular signals thus involves the assembly or multiprotein complexes on enhancers and promoters induced by regulated transcription factors through interaction with other proteins. Consistent with this view, several specific cooperative effects of CREB have been shown including synergistic interactions with HNF-4 in the tyrosine aminotransferase gene (80), liver-enriched transcription factors in the phosphoenolpyruvate carboxykinase gene (15), and steroidogenic factor-1 in the aromatase CYP19 gene (81). Some of the insulin CRE-binding proteins identified in the present study are therefore likely to interact with constitutive or glucose-regulated transcription factors of the insulin promoter. In the absence of glucose, stimulation of insulin gene transcription by cAMP is modest. Together with the nonpalindromic sequence of the CRE octamer motif, the binding of NF-Y to the insulin CRE that inhibits the ability of CREB to mediate cAMP-induced transcription reported in our studies may be wholly or partially responsible for this modest response.
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ACKNOWLEDGEMENTS |
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We greatly appreciate the generous gift of
unique reagents from the following investigators: J. F. Habener,
Boston, MA, for anti-CREB antiserum; R. H. Costa, Chicago, IL,
anti-HNF-3 and -3
antibodies; E. Lai, New York, anti-HNF-3
antibody; R. H. Goodman, Portland, OR, plasmid RSV-KCREB; D. Mathis, C. Benoist, Illkirch, France, and R. Mantovani, Milano, Italy,
-NF-YB, NF-YA29 expression vector. We are grateful to M. Schwaninger
for helpful discussions and preparing one of the constructs, E. Oetjen
for critical reading of the manuscript and help in preparing some of
the figures, and C. Spinhoff for typing the manuscript.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grants SFB236/A25 and SFB402/A3.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
Pharmacology, University of Göttingen, Robert-Koch-Str. 40, D-37070 Göttingen, Germany. Tel.: 49-551-395787; Fax:
49-551-399652; E-mail: wknepel{at}med.uni-goettingen.de.
1 The abbreviations used are: CRE, cAMP response element; CREB, CRE-binding protein; HNF, hepatocyte nuclear factor.
2 S. Herzig and W. Knepel, unpublished observation.
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REFERENCES |
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