From the Department of Molecular Physiology and
Biophysics and the § Department of Surgical Oncology,
Vanderbilt University Medical Center, Nashville, Tennessee 37232
Received for publication, October 22, 2002, and in revised form, January 21, 2003
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ABSTRACT |
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Pancreatic duodenal homeobox factor-1, PDX-1, is
required for pancreas development, islet cell differentiation, and the
maintenance of Targeting of the pancreatic duodenal homeobox factor-1
(pdx-1)1 gene in
mice has established that expression in a common progenitor cell
population is essential for the development of both the endocrine and
exocrine compartments of the pancreas. PDX-1 acts by stimulating proliferation, branching, and differentiation of the pancreatic epithelium (1-3). In contrast, all other characterized islet endocrine- (e.g. PAX6 (4, 5), Ngn3 (6), BETA2 (7), and
exocrine (PTF1-p48 (8, 9)-enriched transcription factors act downstream
of PDX-1 and are principally involved in islet or exocrine cell
differentiation. Selective elimination of PDX-1 in mouse The recent success in reversing type 1 diabetes by islet
transplantation has led to renewed optimism for this form of treatment (16). However, the availability of human islets is limited and will
never be sufficient to treat all patients. Because islet-enriched transcription factors are essential for islet cell development, information valuable for generating transplantable cells will likely be
gained by understanding how their expression is regulated. Therefore,
efforts have recently focused on characterizing the transcriptional
control regions in genes necessary for islet cell formation, including
pdx-1 (17-23), BETA2 (24), pax6 (25, 26), pax4 (27), and ngn3 (28). In specific regards to
pdx-1, expression will likely be mediated by factors
involved in both the differentiation and maintenance of functional Experiments performed in transgenic animals have established that Mutational analysis of 17 conserved sequence blocks within Area II
revealed sites for both positive- and negative-acting regulatory factors (22). Gel shift analysis performed on the activating B8
( In the present study, we show that the B4/5 activator of Area II is
RIPE3b1/Maf, a 46-kDa islet Transfection Constructs--
The Area II and PstBst reporter
constructs were made using human ( Cell Transfections--
Monolayer cultures of pancreatic islet
Electrophoretic Mobility Shift Assays--
Double-stranded Area
II block 4 (B4, agcttTCTTTTTGCAAAGCACAGCAt), B5
(agcttAAAGCACAGCAAAAATATTAt), and B4/5
(agcttCTTTTTGCAAAGCACAGCAAAAAt) sequences, in which the
lowercase lettering corresponds to linker sequences, were excised from
pBluescriptKS2+ and Klenow-labeled with [ SDS-PAGE Fractionation--
Phosphatase Treatment--
Min6 or Anti-phosphotyrosine
Immunoprecipitation--
Immunoprecipitations using anti-Tyr(P) (4G10,
Upstate Biotechnology, Lake Placid, NY) were performed as described
previously (40). Briefly, SDS was added to a final concentration of
0.5% (w/v) to Immunohistochemistry--
Pancreata from 6-8-week-old mice were
fixed 4-5 h in 4% paraformaldehyde at 4 °C, washed, dehydrated,
embedded in paraffin, and then 5-µm sections cut and mounted on glass
slides. Double immunofluorescence was performed using guinea pig
Chromatin Immunoprecipitation Assay--
Chromatin
immunoprecipitation assays were performed with the following
modifications of a described method (19, 22). The B4 and B5 Affect Area II Activity--
Block mutations within
conserved B2 ( B4 and B5 Represent a Single cis-Element That Interacts with a
To determine the distribution of the cellular factor(s) forming complex
A, binding reactions were conducted with nuclear extracts from various
islet ( Complex A Contains an Approximately 46-kDa Protein(s)--
To
estimate the size of the protein(s) in complex A, Min6 nuclear extracts
were separated by SDS-PAGE and transferred to a PVDF membrane that was
cut into slices to represent distinct molecular masses. The
separated proteins were eluted from the membrane slices, renatured, and
tested for binding to the B4/5 probe. The binding specificity of
fraction 8 was identical to complex A found in unfractionated Min6
extracts (Fig. 3; data no shown). The
molecular mass range of the proteins in fraction 8 was 44-47 kDa.
These results indicate that complex A is composed of one or more
proteins of ~46 kDa.
The 46-kDa Complex A Protein(s) Corresponds to the InsC1 Activator,
RIPE3b1--
Because the RIPE3b1 protein(s) that binds to and
activates the InsC1 control element has the same cell-restricted
distribution (38) and molecular size (see Fig. 3 and Ref. 38), we
compared the binding properties of B4/5 to InsC1 (Fig.
4). Both InsC1 and B4/5 competed
effectively for complex A binding when either B4/5 or InsC1 were used
as probes (Fig. 4B). In addition, RIPE3b1/complex A activity
was affected in the same manner by B4/5 or InsC1 mutations that either
modestly (e.g. InsC1mt1, Ref. 38) or profoundly (e.g. InsC1mt3, Ref. 38) (Fig. 1B,
B4/5MT) affected activity. The resulting competition
patterns were consistent with each element binding the same factor(s)
(Fig. 4B).
RIPE3b1 binding activity is inhibited by the actions of a tyrosine
phosphatase (40). To test whether complex A formation on B4/5 is also
regulated in this manner, Min6 nuclear extracts were incubated in the
presence or absence of CIAP and a general (sodium pyrophosphate, NaPPi)
or phosphotyrosine-specific (sodium orthovanadate,
Na3VO4) phosphatase inhibitor. B4/5 and InsC1
binding activities were monitored in the treated extracts. The binding characteristics of complex A were affected in exactly the same manner
with both probes (Fig. 5A).
Complex A mobility was shifted upon incubating the InsC1 Can Substitute for B4/5 to Drive Area II
Activation in A Large Maf Transcription Factor within
To directly determine whether RIPE3b1/Maf binds within Area II of the
endogenous pdx-1 gene, a chromatin immunoprecipitation assay
was performed using formaldehyde cross-linked chromatin from Area II, when compared with Area I and III, was the only
pdx-1 control region capable of independently directing
pancreatic Mutation of either B4 or B5 reduced activation of the PstBst-driven
fragment, and all of our subsequent analysis focused on characterizing
their activator(s). Using B4, B5, or B4/5 in gel shift assays revealed
that a specific, The RIPE3b1 activator was recently independently isolated from Importantly, the large Maf-recognizing antiserum demonstrated that a
protein(s) in this family binds to Area II control region sequences in
intact Collectively, the data presented here and elsewhere demonstrate that
insulin and pdx-1 are bona fide transcriptional
targets for RIPE3b1/Maf control. Inspection of the consensus large Maf binding motif (TGC (N)6-7 GCA, Ref. 48) also revealed a
greater similarity of B4/5 to InsC1 than was initially apparent (Fig.
6). Interestingly, the presence of this consensus binding site in the
transcription control region of other selectively expressed genes
suggests a general, but significant, role in controlling The functional cooperativity observed between the large Mafs and Pax6
in activating lens gene expression may also be of relevance to both
insulin and pdx-1 in the cell function. Selective expression in the pancreas
appears to be principally regulated by Area II, one of four conserved regulatory sequence domains found within the 5'-flanking region of the
pdx-1 gene. Detailed mutagenesis studies have identified potential sites of interaction for both positive- and negative-acting factors within the conserved sequence blocks of Area II. The islet
cell-enriched RIPE3b1 transcription factor, the activator of insulin C1
element-driven expression, was shown here to also stimulate Area II by
binding to sequence blocks 4 and 5 (termed B4/5). Accordingly, B4/5
DNA-binding protein's molecular mass (i.e. 46 kDa),
binding specificity, and islet
cell-enriched distribution were
identical to RIPE3b1. Area II-mediated activation was also unaffected
upon replacing B4/5 with the insulin C1/RIPE3b1 binding site. In
addition, the chromatin immunoprecipitation assay showed that the Area
II region of the endogenous pdx-1 gene was precipitated by
an antiserum that recognizes the large Maf protein that
comprises the RIPE3b1 transcription factor. These results
strongly suggest that RIPE3b1/Maf has an important role in generating
and maintaining physiologically functional
cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
cells
in vivo also results in a reduction in both insulin secretion and islet
cell numbers (1). These animals become glucose
intolerant and diabetic, largely because of their inability to
synthesize appropriate amounts of PDX-1-regulated gene products that
are involved in maintaining glucose homeostasis (1) (e.g. insulin (10, 11), GLUT2 (12), and glucokinase (13)). Moreover, mutations in pdx-1 cause pancreatic agenesis (2, 3) and a
form of maturity onset diabetes of the young in humans (14, 15). These
data have established an essential role for PDX-1 in islet
cell
development and function.
cells.
cell-selective expression of the pdx-1 gene is regulated by
sequences 5' to the transcription start site (18, 23). Control also
appears to be largely mediated by those conserved between the
vertebrate pdx-1 genes (19, 20, 22). Thus,
cell-specific
reporter gene expression was driven in transfection assays by areas of
sequence identity shared between the chicken, mouse, and human genes
(i.e. Area I,
2839/
2520 base pair (bp) (19), Area III,
1879/
1799 bp (19), Area IV,
6047/
6529 bp),2 or only the mouse and
human genes (Area II,
2141/
1961 bp (19, 22)). In contrast,
the 5'-non-conserved sequences were inactive (18, 23). A
pdx-1 gene fragment spanning Areas I and II also directed
transgene expression to islet
cells in vivo (termed PstBst,
2917/
1918 bp (18, 23)), although only Area II, and not
Areas I (22) or III (18), functioned independently in these in
vivo assays. Collectively, these data strongly suggest that Area
II represents the core of the mammalian pdx-1 transcription control region.
2068/
2060-bp) and B14 (
2006/
1996-bp) elements demonstrated specific binding to Pax6 and Foxa2 (formerly termed HNF3
),
respectively. Mutation of the Foxa2 binding site in Area II limited
expression of the PstBst transgene to a subset of the islet
cells
in vivo (22). In addition, conditional deletion of Foxa2
specifically from
cells decreased pdx-1 mRNA and
protein expression in mice (29). The ability of Pax6 and Foxa2 to bind
within Area II of the endogenous pdx-1 gene also strongly
supports a direct role in mediating transcription in
cells
(22).
cell-enriched protein(s) essential for
both cell type-specific (30, 31) and glucose-inducible (32, 33)
transcription of the insulin gene. Moreover, an antiserum that
recognizes the recently isolated large Maf protein(s) of the insulin
C1/RIPE3b1 activator revealed binding to Area II of the endogenous
pdx-1 gene in
cells. We propose that RIPE3b1/Maf is
required for transcription of genes critical to
cell function.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2141/
1890-bp) and mouse
(Pst/
2917bp:Bst/
1890-bp) pdx-1 sequences (23), which
were cloned directly upstream of the herpes simplex thymidine kinase
(TK) promoter in a chloramphenicol acetyltransferase (CAT) expression
vector, pTK(An) (34). The block transversion and insulin C1 (InsC1)
substitution mutants in B4/5 were constructed in Area II:pTK and
PstBst:pTK using the QuikChange mutagenesis kit (Stratagene). Each
construct was determined to be correct by DNA sequencing.
(
TC-3, HIT-T15, and Min6) and non-
(NIH3T3) cell lines were
maintained as described previously (35). The LipofectAMINE reagent
(Invitrogen) was used to introduce 1 µg each of Area II:pTK or
PstBst:pTK and 0.5 µg of pRSVLUC. The activity from the Rous sarcoma
virus enhancer-driven luciferase plasmid served as an internal
transfection control for the pdx-1:pTK constructs.
Luciferase (36) and CAT (37) enzymatic assays were performed 40-48 h
after transfection. Each experiment was carried out more than three
times with at least two independently isolated DNA preparations.
32P]dATP. The
InsC1 probe spans nucleotides
126 to
101 of the rat insulin II gene
and was labeled as described (38). Nuclear extracts were prepared as
described previously (39). Binding reactions (20 µl total volume)
were conducted with 5-10 µg of extract protein and labeled probe
(8 × 104 cpm) in binding buffer containing 10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM dithiothreitol, 1 mM EDTA, 10% glycerol,
and 1 µg of poly(dGdC) (final concentrations). The conditions for the
competition analyses were the same, except that excess (see figures for
amounts) of the specific competitor DNA was included in the mixture
prior to the addition of probe. Anti-c-Maf antiserum (10 µg, M-153, Santa Cruz Biotechnology) was added to binding reactions 10 min prior
to addition of the probe for supershift analysis. This antiserum, referred to in the text as
c-Maf M-153, was made to an N-terminal region of c-Maf that is common to the other large Mafs and cross-reacts with each (i.e. MafA, NRL, and MafB). The samples were
resolved on a 6% nondenaturing polyacrylamide gel
(acrylamide:bisacrylamide ratio 29:1) and run in TGE buffer (50 mM Tris, 380 mM glycine, 2 mM EDTA,
pH 8.5). The gel was dried and subjected to autoradiography.
TC-3 and Min6 nuclear extracts
(30 µg) were separated on a 10% SDS-polyacrylamide gel (SDS-PAGE)
and then electro-transferred onto an Immobilon polyvinylidene
difluoride (PVDF) membrane (Millipore). The extract lanes were cut
horizontally into 3-mm slices. The molecular mass range of each
lane fraction was determined by comparison with colored Rainbow protein
markers (Amersham Biosciences). The proteins from each fraction were
eluted as previously described (38) and analyzed for B4/5 and InsC1
binding activity in electrophoretic mobility shift assays.
TC-3 nuclear extract (3-5
µg) was incubated for 10 min at 4 °C or 30 °C with and without
0.5 units of calf intestinal alkaline phosphatase (CIAP, Promega) in
the presence or absence of sodium orthovanadate
(Na3VO4, 10 mM) or sodium
pyrophosphate (NaPPi, 10 mM) in phosphatase buffer (20 mM Tris-HCl, pH 7.4, 1 mM dithiothreitol, 0.1 mM EGTA, 2 mM MgCl2, 1× protease
inhibitor mixture (CØmplete, Roche Diagnostics)) (10 µl total
volume). The samples were analyzed for InsC1 and B4/5 binding after
addition of 10 µl of 2× gel shift binding buffer.
TC-3 nuclear extract (100 µg protein) in a buffer
containing 10 mM Tris-HCl, pH 7.4, 1 mM EDTA,
10% glycerol, 1 mM Na3VO4, and 2 mM dithiothreitol (final concentrations) and then heated to
65 °C. After diluting the SDS to 0.05%, anti-Tyr(P) or control mouse IgG was added along with protein A-Sepharose beads. The washed
beads were then resuspended in 1× SDS-PAGE loading buffer and the
immunoprecipitated proteins separated on a 10% SDS-polyacrylamide gel.
After transfer to an Immobilon PVDF membrane, the 44-47-kDa eluted
proteins were assayed for B4/5 and InsC1 binding activity.
-human insulin (Linco) and rabbit
-mouse c-Maf (
c-Maf M-153)
as primary antibodies at dilutions of 1:2000 and 1:100, respectively.
Secondary antibodies were Cy3- or Cy5-labeled donkey anti-guinea pig
and anti-rabbit IgG diluted to 1:500 (Jackson ImmunoResearch
Laboratories). Fluorescent images were captured on a Zeiss LSM510
confocal microscope at an optical depth of 1 µm, false colors were
assigned, and the images merged in Photoshop 5 (Adobe).
Immunoperoxidase staining was performed with Vectastain Elite
kits (Vector Labs) and with 3,3'-diaminobenzidine tetrahydrochloride
substrate (Zymed Laboratories Inc.) according to the
manufacturer's recommendations. Rabbit anti-c-maf antibody (M-153) was
diluted 1:1000.
c-Maf M-153
antiserum (10 µg) was incubated with sonicated formaldehyde cross-linked
TC3 chromatin. Normal rabbit IgG (10 µg, sc-2027, Santa Cruz Biotechnology) was used as a control. The protein-DNA complexes were isolated with A/G-agarose beads (Santa Cruz
Biotechnology). The PCR oligonucleotides used to detect mouse control
sequences were: pdx-1 Area II,
2208
5'-GGTGGGAAATCCTTCCCTCAAG-3' and
1927 5'-CCTTAGGGATAGACCCCCTGC-3',
and phosphoenolpyruvate carboxykinase (PEPCK),
434
5'-GAGTGACACCTCACAGCTGTGG-3' and
96 5'-GGCAGGCCTTTGGATCATAGCC-3'. The
PCR cycling parameters were 1 cycle of 95 °C/2 min and 28 cycles of
95 °C/30 s, 61 °C/30 s, 72 °C/30 s for PEPCK and 1 cycle of
95 °C/2 min and 28 cycles of 95 °C/30 s, 57.5 °C/30 s,
72 °C/30 s for Area II.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2131/
2115 bp), B3 (
2110/
2102 bp), B4
(
2100/
2093 bp), and B5 (
2089/
2086 bp) reduce Area II:pTK
activity in
cell lines (Fig.
1B, HIT-T15,
MIN6, and
TC-3) (22). To further examine the
significance of these elements in Area II activation, each was mutated
within the mouse pdx-1 `PstBst' region that spans Area I
and Area II (Fig. 1A). In the context of the more active
PstBst:pTK expression construct, the B4 and B5 mutants reduced activity
to a greater extent than in Area II:pTK (Fig. 1B). Combining
the B4 with B5 mutations in PstBst:pTK reduced activity further than
either individual mutation (Fig. 1B). In contrast, the B2
and B3 mutants had less effect on PstBst:pTK activation (data not
shown). The following experiments were designed to characterize the B4
and B5 activators in
cells.
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Fig. 1.
The conserved B4 and B5 sequence blocks
regulate PstBst-mediated activation in cells. A, a schematic diagram illustrating the
position of the
2560/
1880-bp PstBst region in the mouse
pdx-1 gene. The location of the Area I and Area II control
regions and the characterized conserved block mutants within
Area II are also shown. The Foxa2 (19, 23) and Pax6 (22) control
elements were characterized previously. B, the normalized
activity of the transfected mutant (MT) Area II:pTKCAT and
PstBst:pTKCAT constructs is expressed as a percent activity of the wild
type Area II and PstBst reporter ± S.E. of the mean.
PstBst:pTKCAT is ~2- to 4-fold more active than Area II:pTKCAT in
transfected
cell lines.
Cell-enriched Protein(s)--
To define the factors associated with
B4- and B5-mediated regulation, gel shift experiments were performed
with probes spanning B4, B5, and B4+B5 (B4/5), and
TC-3 or MIN6 cell
nuclear extracts (Fig. 2). Identical
results were obtained with MIN6 and
TC-3 cells, and they were used
interchangeably in these analyses. Two common protein-DNA complexes
were detected with the B4 and B4/5 probes (labeled as A and
B in Fig. 2B), whereas no binding was found with
B5 (data not shown). The binding affinity of B4 and B4/5 for these
complexes was determined with the wild type and B4/5 double mutant site
(B4/5 MT) competitors. As expected, both B4 and B4/5 reduced the levels
of these complexes, although B4/5 was roughly 20-fold more effective
(Fig. 2B). In contrast, B5 did not compete for binding (data
not shown), whereas the B4/5 MT only competed away complex B,
consistent with the conclusion that it is unrelated to activation (Fig.
2B). These results suggested that B4 and B5 define a single
activator-binding site, which is regulated by the factor(s) found
within the slower mobility complex A.
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Fig. 2.
B4 and B5 comprise a single element that
binds a cell-enriched factor.
A, the B4, B5, and B4/5 probe sequences are shown. The
conserved B4 and B5 block sequences are in bold. The mutant
B4/5 competitor contains transversion mutations of the conserved bolded
sequences. B, gel shift binding reactions were conducted
with the B4, B5, or B4/5 probe using
TC-3 and Min6 nuclear extracts
in the presence of a -fold molar excess of unlabeled wild type
(WT) or mutant (MT) competitor to probe. The
complexes labeled A and B are discussed under "Results."
C, nuclear extracts from
(
TC-3,
Ins-1, Min6, HIT-T15) and non-
(
TC-6, RC2-E10, NCB20,
MDCK, BHK, NIH-3T3, and
H4IIE) cell lines as well as rat liver (rliver)
were analyzed for B4/5 binding activity.
: Ins-1, Min6, HIT-T15;
,
TC-6) and non-islet cell
types (neuronal, RC2.E10 (41, 42), NCB20 (43, 44); liver, H4IIE (45),
normal rat liver; kidney, MDCK, BHK; fibroblast, NIH 3T3). Complex A
was uniquely detected in the
cell extracts (Fig. 2C).
These results suggest that the factor(s) in activator complex A is
enriched in
cells.
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Fig. 3.
The RIPE3b1 complex contains a protein(s) of
~46 kDa. Min6 nuclear extracts were electro-transferred onto an
Immobilon PVDF membrane after SDS-PAGE. The proteins were eluted from
membrane slices and assayed for B4/5 and InsC1 binding activity.
Binding specificity was determined by competition with a 10-fold excess
of unlabeled B4/5 or InsC1 (data not shown). Each fraction represents a
different molecular mass range. The position of complex A detected in
unfractionated Min6 nuclear extracts is indicated.
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Fig. 4.
B4/5 and InsC1 form similar
cell protein-DNA complexes. A, the
sequences of B4/5 and InsC1 probes. B4 and B5 sequences are contained
within the blocked region, and mutated sequences in InsC1mt1 and
InsC1mt3 are shown. B, binding reactions were conducted with
Min6 and
TC-3 nuclear extract. The molar ratio of the wild type or
mutant competitor to the labeled probe is shown. C, the
c-Maf M-153 polyclonal antiserum was incubated with MIN6 nuclear
extract and then analyzed for B4/5 binding. Arrows denote
the location of complex A and the antibody supershifted (SS)
complex.
cell nuclear
extract at 30 °C with both the B4/5 and InsC1 probes, presumably
because of the actions of an endogenous tyrosine phosphatase (40). CIAP
treatment reduced binding to each probe, an effect blocked by addition
of NaPPi or Na3VO4 (Fig. 5A). In
addition, B4/5 bound specifically to the 46-kDa fraction
immunoprecipitated from
TC-3 nuclear extracts with the
anti-phosphotyrosine immunospecific monoclonal antibody, 4G10 (Fig.
5B, compare 4G10 immunoprecipitate binding to InsC1 (40) and
B4/5). Collectively, these results strongly suggest that RIPE3b1 binds
to both the pdx-1 B4/5 and InsC1 elements.
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Fig. 5.
Complex A binding to B4/5 is sensitive to
tyrosine dephosphorylation. A, B4/5 and InsC1 binding
reactions with Min6 nuclear extracts were incubated at either 4 or
30 °C, either alone or in the presence of CIAP, CIAP + 10 mM Na3VO4, or CIAP + 10 mM NaPPi. B, TC-3 nuclear extract was
immunoprecipitated with either the anti-phosphotyrosine antibody 4G10
or normal mouse IgG. The immunoprecipitated proteins (labeled
4G10 and IgG) and whole nuclear extract were then
fractionated by SDS-PAGE and transferred to PVDF membranes (Immobilon).
Protein fractions 1 (53.7-62.7 kDa), 2 (41.7-53.6 kDa), and 3 (29.9-41.6 kDa) were eluted and used in B4/5 and InsC1 gel shift
assays along with unfractionated
TC-3 nuclear extract.
Cells--
Considering the interchangeability of
B4/5 and InsC1 in gel shift assays, it was surprising to find only
modest sequence identity between human (h) and mouse
(m) B4/5 and mouse InsC1 (Fig.
6A). However, methylation
interference assays over B4/5 suggested some similarity in contact
nucleotides for RIPE3b1 with InsC1 (Ref. 30 and Fig. 6A;
data not shown). Because of sequence dissimilarity between B4/5 and
InsC1, we tested whether InsC1 could substitute for B4/5 in the context
of the PstBst:pTK reporter. Replacement of B4/5 with InsC1 maintained
the same high level of activation found for wild type PstBst in Min6
cells (Fig. 6B). Furthermore, mutants in B4/5 (B4mt,
B5mt, B4/5mt) and InsC1 (mut3) that compromised complex A/RIPE3b1
binding also reduced PstBst activity only in Min6 cells. These data
strongly suggest that the
cell-enriched RIPE3b1 transcription
factor activates the B4/5 control element in Area II.
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Fig. 6.
InsC1 can substitute for B4/5 in driving Area
II activation. A, the sequence of human (h)
and mouse (m) B4/5 is compared with mInsC1. The
non-conserved bases in the human and mouse B4/5 are in lowercase
letters. The bases shown to be important for complex A and RIPE3b1
binding in methylation interference assays are indicated with
asterisks (InsC1 (30), B4/5, data not shown). B,
mInsC1 ( 124/
105) was inserted into PstBst (PB):pTKCAT in place of
B4/5 (
2100/
2082), and the activity was compared with other
PstBst:pTKCAT constructs in transfected Min6 and NIH-3T3 cells. The
normalized activity of the mutant PstBst:pTKCAT is expressed as the
percentage of the wild type ± S.E.
Cell Nuclei Binds to
Area II--
The RIPE3b1 transcription factor was recently isolated
and shown to be a member of the large Maf transcription factor family, most likely MafA (33, 46).3
To determine whether the B4/5 binding complex A contained a large Maf
protein, Min6 nuclear extract was preincubated with a polyclonal antiserum raised to N-terminal sequences of c-Maf shared with other
members of the large Maf family. This c-Maf antiserum, termed
c-Maf
M-153, cross-reacts with MafA, MafB, and NRL
(46).4 Complex A was
completely supershifted by
c-Maf M-153, whereas IgG had no effect
(Fig. 4C). These results strongly suggested that complex A
contains the RIPE3b1/Maf protein. Immunohistochemical analysis
performed with
c-Maf M-153 on adult mouse pancreas also showed that
the large Maf protein(s) of the RIPE3b1/Complex A activator was nuclear
and expressed almost exclusively in insulin-producing
cells (Fig.
7).
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Fig. 7.
RIPE3b1/Maf is found within the islet
nuclei. Large Maf expression was detected in
adult pancreas sections using
c-Maf M-153 antiserum. Note
that large Maf protein staining was (A) localized to the
nuclei of islet cells by immunoperoxidase and (B)
co-localized with insulin expression by immunofluorescence. The
blue-stained nuclei (A) reveal the absence of large Maf
expression in surrounding acinar cells.
TC-3
cells. The cross-linked DNA was precipitated with the Maf antiserum and
PCR-amplified with Area II and PEPCK promoter-specific primers. The Maf
antibody was capable of immunoprecipitating Area II sequences, whereas
the control IgG could not (Fig. 8).
However, the Maf antiserum did not immunoprecipitate transcription
control sequences from the PEPCK gene, which is not transcribed in
cells. These results demonstrate that RIPE3b1/Maf occupies the Area II region of the pdx-1 gene in
cells.
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Fig. 8.
RIPE3b1/Maf binds to the Area II region
in vivo. Cross-linked chromatin from TC-3
cells was incubated with
c-Maf M-153 antibody. The
immunoprecipitated DNA was analyzed by PCR for Area II and PEPCK
transcriptional regulatory sequences. As controls, PCR reactions were
run on input chromatin with no DNA and with DNA obtained after
precipitating with rabbit IgG.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
cell-selective transgene expression (18, 22). This
property and the uniqueness of this domain within the human and mouse
genes imply that Area II represents the core of the mammalian
pdx-1 transcription unit. Mutagenesis of conserved sequence
blocks within Area II revealed sites for both positive- and
negative-acting regulators, including the B8 and B14 control elements
that bind the Pax6 (22) and Foxa2 (20, 23) activators, respectively. However, the rather general distribution of these two developmental regulators suggests that a more
cell-restricted factor(s) likely contributes to the expression pattern observed for Area II-driven reporters. The objective of this study was to determine whether the B2,
B3, B4, and/or B5 activators had such properties. Our analysis revealed
that the B4 and B5 mutants reduced expression driven by the mouse
PstBst fragment spanning Areas I and II in all
cell lines tested.
The B2 and B3 mutants had a lesser affect and were not analyzed
further. B4 and B5 were found to comprise a single regulatory element
activated by RIPE3b1/Maf, a
cell-enriched nuclear factor known for
its important role in insulin gene transcription.
cell-enriched complex bound to these sequences
(Fig. 2). The results of the transfection studies performed with B4,
B5, and B4/5 mutants in PstBst were also consistent with this
conclusion (Fig. 1). Because the size and cellular distribution of the
specific B4/5 binding complex was similar to the InsC1/RIPE3b1 complex,
the binding and functional properties of InsC1 were compared with B4/5.
InsC1 not only specifically and effectively competed with B4/5 in
gel shift assays but also was functionally indistinguishable in
transfections performed with InsC1 substitution mutants in PstBst (Fig.
6). In addition, the apparent dependence upon tyrosine phosphorylation
for RIPE3b1 binding to InsC1 (40) was also found with B4/5 (Fig.
5).
TC-3
(data not shown) and HIT T-15 cells (46) using a biochemically based
InsC1 affinity matrix chromatography strategy. MafA, a member of the
large Maf family of basic leucine zipper proteins, was identified by
mass spectrophotometric analysis of the purified fractions. The
presence of Maf in the RIPE3b1 complex was further demonstrated using
an antiserum that cross-reacts with N-terminal epitopes common to all
the proteins in the large Maf family (Fig. 4C). These
results formed the primary basis for concluding that MafA is the
RIPE3b1 activator (46). This is likely to be correct for
cell
lines; however, we have used specific molecular and immunological
reagents to show that other members of the large Maf family are
expressed in the adult islet
cells, specifically c-Maf and MafB
(data not shown). Because MafA, c-Maf, and MafB are equally capable of
binding and activating B4/5-like sequences in vitro (Ref. 48
and data not shown), these results suggest that several members of the
large Maf family may control RIPE3b1-like-mediated transcription in the
cell. This broader composition of the RIPE3b1/Maf activator is also
consistent with the importance of the large Maf proteins in myeloid
(49, 50) and lens (51-53) cell differentiation.
cells (Fig. 8). In addition, this antiserum also
supershifted the
cell-enriched A/RIPE3b1 complex formed with the
B4/5 probe (Fig. 4C) and immunohistochemically localized Maf
protein in the nuclei of mouse islet
cells (Fig. 7). We and others
have also shown that the large Maf proteins can activate islet target
gene expression in transfection assays (33,
46).5 As a consequence, we
conclude that RIPE3b1/Maf binds to B4/5 and activates Area II-mediated
transcription in
cells. Furthermore, because only MafA was
identified during the biochemical isolation and characterization of the
RIPE3b1/InsC1 binding complex, it is most likely the principal large
Maf activator in
cells.
cell-specific expression (e.g. islet-specific
glucose-6-phosphatase catalytic subunit related protein (54),
183/
165; islet amyloid polypeptide (55-57),
540/
528 bp). It is
likely that the
-cell-enriched RIPE3b1/Maf activator acts in concert
with more widely distributed factors (e.g. Pax6 and Foxa2)
to mediate selective expression.
cell. Thus, Pax6 directly
regulates c-Maf gene expression during lens development (58, 59), and together they function cooperatively to stimulate crystalline gene
expression in differentiating lens fibers (58-61). It is also intriguing to consider that the loss in
cell function upon
eliminating insulin receptor (62) or insulin receptor substrate-2 (63, 64) signaling may be mediated, at least in part, by directly effecting
MafA activation. Thus, because signaling by all of these effectors in
cells is imparted by tyrosine phosphorylation, large Maf activation
would be directly influenced in the absence of insulin receptor and/or
insulin receptor substrate-2 function, resulting in reduced insulin and
pdx-1 transcription as seen in islets of
Irs2
/
mice (47). In support of this theory,
cell
mass and function are restored upon transgenic expression of PDX-1 in
Irs2
/
mice (47). Our studies are currently focused on
determining the nature of the interactions between RIPE3b1/Maf and
other factors that are required for assembly of the pdx-1
and insulin transcription complexes and, more broadly, the significance
of RIPE3b1/Maf in pancreatic development.
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ACKNOWLEDGEMENT |
---|
We thank members of the Stein laboratory for stimulating discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants RO1 DK50203 and P01 DK42502 (to R. S.) and Juvenile Diabetes Research Foundation Grants JDRF 398212 (to S. E. S.) and 32001678 (to T. M.). Partial support was provided by the Vanderbilt University Diabetes Research and Training Center, Molecular Biology Core Laboratory (Public Health Service Grant P60 DK20593).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. Tel.: 615-322-7026; Fax: 615-322-7236; E-mail: Roland.Stein@mcmail.vanderbilt.edu.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M210801200
2 R. Stein, unpublished observations.
3 T. Matsuoka, L. Zhao, and R. Stein, unpublished observations.
4 T. Matsuoka, L. Zhao, and R. Stein, unpublished observations.
5 T. Matsuoka, L. Zhao, and R. Stein, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PDX-1, pancreatic duodenal homeobox factor-1; CAT, chloramphenicol acetyltransferase; B4/5, sequence blocks 4 and 5; PEPCK, phosphoenolpyruvate carboxykinase; TK, thymidine kinase; InsC1, insulin C1; PVDF, polyvinylidene difluoride; CIAP, calf intestinal alkaline phosphatase; BHK, baby hamster kidney; MDCK, Madin-Darby canine kidney.
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