Transcription Factor Occupancy of the Insulin Gene in
Vivo
EVIDENCE FOR DIRECT REGULATION BY Nkx2.2*
Michelle A.
Cissell
,
Li
Zhao
,
Lori
Sussel§,
Eva
Henderson
, and
Roland
Stein
¶
From the
Department of Molecular Physiology and
Biophysics, Vanderbilt University Medical School, Nashville,
Tennessee 37232 and the § Barbara Davis Center for Childhood
Diabetes, University of Colorado Health Sciences Center, Denver,
Colorado 80262
Received for publication, June 13, 2002, and in revised form, November 7, 2002
 |
ABSTRACT |
Consensus-binding sites for many transcription
factors are relatively non-selective and found at high frequency within
the genome. This raises the possibility that factors that are
capable of binding to a cis-acting element in
vitro and regulating transcription from a transiently transfected
plasmid, which would not have higher order chromatin structure, may not
occupy this site within the endogenous gene. Closed chromatin structure
and competition from another DNA-binding protein with similar
nucleotide specificity are two possible mechanisms by which a
transcription factor may be excluded from a potential binding site
in vivo. Multiple transcription factors, including Pdx-1,
BETA-2, and Pax6, have been implicated in expression of the insulin
gene in pancreatic
cells. In this study, the chromatin
immunoprecipitation assay has been used to show that these
factors do, in fact, bind to insulin control region sequences in intact
cells. In addition, another key islet-enriched transcription
factor, Nkx2.2, was found to occupy this region using the chromatin
immunoprecipitation assay. In vitro DNA-binding and
transient transfection assays defined how Nkx2.2 affected insulin gene
expression. Pdx-1 was also shown to bind within a region of the
endogenous islet amyloid polypeptide, pax-4, and glucokinase genes that were associated with control in
vitro. Because Pdx-1 does not regulate gene transcription in
isolation, these sequences were examined for occupancy by the other
insulin transcriptional regulators. BETA-2, Pax6, and Nkx2.2 were also found to bind to amyloid polypeptide, glucokinase, and
pax-4 control sequences in vivo. These
studies reveal the broad application of the Pdx-1, BETA-2, Pax6, and
Nkx2.2 transcription factors in regulating expression of genes
selectively expressed in islet
cells.
 |
INTRODUCTION |
Transcription of the insulin
(INS)1 gene is restricted to
pancreatic islet
cells. The 5'-flanking sequences within 350 base pairs of the transcription start site contain the binding sites for the
factors that control cell type-specific expression (1-4). An extensive
series of in vitro gel shift and cell transfection experiments indicates that regulation is mediated by islet-enriched DNA-binding transcription factors, including Pdx-1 (5-7), BETA-2 (8),
and Pax6 (9). Strikingly, each of these factors is also a key regulator
of pancreas development. Thus, homozygous loss of Pdx-1 leads to
pancreatic agenesis in mice (10, 11) and humans (12), whereas there are
severe defects in islet cell formation in BETA-2 (i.e.
,
, and
cells; Ref. 13) and Pax6 (i.e.
cells; Ref.
14) null mice.
Islet cell development and function is also affected by other
islet-enriched transcription factors (15, 16). For example, insulin is
not made in
cells of mice lacking Nkx2.2 (17). Interestingly, this
appears to be a rather specific effect as other
cell-enriched
markers continue to be expressed (e.g. Pdx-1 and islet
amyloid polypeptide (IAPP). Nkx2.2 and insulin are also co-expressed
during embryogenesis, although Nkx2.2 expression is not restricted to
cells in adult islets (17). The mechanism by which Nkx2.2 regulates
insulin gene transcription has not been resolved. Indeed, no direct
gene targets have yet been reported for Nkx2.2 in the pancreas.
Assays such as in vitro DNA binding, co-transfection of
reporter constructs, and transgenic knock-out animals provide evidence for the involvement of a factor in transcriptional control. However, such experiments are indirect measures of regulation and do not account
for conditions like competition with other binding proteins in
vivo, chromatin structure of the endogenous gene, and cascades of
regulatory interactions. In contrast, the chromatin immunoprecipitation (ChIP) assay (18-20) is a powerful tool for directly analyzing transcription factor site occupancy in vivo. Importantly,
one can determine the physical association of a specific DNA-binding factor with potential control sequences in intact cells with this assay. This technique has been applied to many experimental systems, including in
cells to examine the in vivo occupancy of
control sequences within the pdx-1 gene by Pax6 and HNF3
(21), within the glut2 glucose transporter gene by HNF1
(22), and within several
cell genes by Pdx-1 (23, 24).
In this study, the ChIP assay was used to assess the in vivo
occupancy of insulin 5'-flanking sequences by Pdx-1, BETA-2, Pax6, and
Nkx2.2. In addition to directly demonstrating binding of Pdx-1, BETA-2,
and Pax6 to these sequences in intact
cells, Nkx2.2 was also found
to interact. Analysis of the insulin 5'-flanking region target
sequences by gel shift and transfected insulin-driven reporter assays
suggests that Nkx2.2 reduces transcription upon binding at
140/
119
bp in
cell lines. Our findings that BETA-2, Pax6, and Nkx2.2 bind
within the transcription control region of other islet-enriched genes
also highlights how the ChIP assay can be used to identify targeted genes.
 |
MATERIALS AND METHODS |
Cell Culture and Transfection Analysis--
Monolayer cultures
of insulin-producing pancreatic
cell lines (
TC-3, HIT T-15 2.2, and MIN6) were maintained as described previously (25). The
238 WT
CAT (chloramphenicol acetyltransferase) plasmid contains sequences from
238 to +2 bp of the rat insulin II enhancer/promoter region linked to
the CAT reporter gene (26). Construction of the insulin mutant (MT)
expression vector,
238 CAT
108,107 MT, has been described (25). The
Nkx2.2-binding element mutant was generated in
238WT CAT using the
QuikChange mutagenesis kit (Stratagene, La Jolla, CA) with an
oligonucleotide containing the
133,
132 mutation
(5'-CCTAGCACCAGGCccGTGTTTGGAAACTGCAGC-3'; mutated
nucleotides are underlined). The LipofectAMINE reagent (Invitrogen) was used to transfect 1 µg of the
238 CAT and
Rous sarcoma virus (RSV):luciferase (LUC) plasmids. The RSV
enhancer-driven LUC expression plasmid, pRSVLUC, was used as a recovery
marker. Extracts were prepared 40-48 h after transfection and assayed for LUC (27) and CAT (28) enzymatic activity. The CAT activity from the
test construct was normalized to RSV-driven LUC activity. Each
experiment was carried out at least three times.
ChIP Assay--
ChIP assays were performed on
TC-3 cells as
described (24) with the following modifications. Protein A-Sepharose
was replaced with protein A/G-Agarose (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA), and the following antibodies were used: 1 µl of
N-terminal (amino acids 1-75) or C-terminal (amino acids 271-283)
Pdx-1 whole rabbit polyclonal antiserum; 20 µl of rabbit polyclonal
Pax6 IgG (PRB-278P; Covance, Richmond, CA); 5 µl goat polyclonal
BETA2 IgG (sc-1084; Santa Cruz Biotechnology, Inc.); 25 µl mouse
monoclonal Nkx2.2 antibody (74.5A5; Developmental Studies Hybridoma
Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of
Iowa, Department of Biological Sciences, Iowa City, IA). Normal rabbit
(sc-2027), goat (sc-2028), and mouse (sc-2025) IgG preparations were
obtained from Santa Cruz Biotechnology, Inc.; 10 µg of normal IgG
from the same species of the experimental antiserum was used as a
control. The PCR oligonucleotides used to detect mouse gene control
sequences were as follows: INS, (
378) 5'-GGAACTGTGAAACAGTCCAAGG-3' and (
46) 5'-CCCCCTGGACTTTGCTGTTTG-3' (these primers amplify both mouse insulin I and II gene sequences); phosphoenolpyruvate
carboxykinase (PCK), (
434) 5'-GAGTGACACCTCACAGCTGTGG-3' and (
96)
5'-GGCAGGCCTTTGGATCATAGCC-3'; IAPP, (
290)
5'-AAGTCACTTCCTTACTGTCAGAC-3' and (
36)
5'-CAACTTGCTTAGCTCTGTCACCAG-3'; pax-4, (
2015)
5'-GCTGGAATGGCCTTGGCTGGCC-3' and (
1628)
5'-CCTGGCAGATGGTGGTGGAAGCG-3'; and glucokinase (GK), (
258)
5'-GTGATAGGCACCAAGGCACTGAC-3' and (
2)
5'-CGGTGCTTCTGTTCCAACCAGG-3'. The PCR cycling parameters were:
INS, PCK, IAPP, and GK, 1 cycle of 95 °C/2 min and 28 cycles of
95 °C/30 s, 61 °C/30 s, and 72 °C/30 s; pax-4, 1 cycle of 95 °C/2 min and 30 cycles of 95 °C/30 s, 68 °C/30 s,
and 72 °C/30 s.
Electrophoretic Mobility Shift Assay--
Nuclear extracts were
prepared from
TC-3 and MIN6 cells as described previously by Zhao
et al. (25). The TNT-coupled reticulocyte lysate
system (Promega, Madison, WI) was used to in vitro
transcribe and translate the Nkx2.2 encoding plasmid, pNkx2.2 (17). Ten µg of nuclear extract protein or 2 µl of in
vitro-translated protein was added to the binding buffer
containing 10 mM HEPES (pH 7.4), 1 mM EDTA,
10% (v/v) glycerol, 100 mM NaCl, 2 mM
dithiothreitol, and 1 µg poly(dI-dC). The double-stranded
oligonucleotide probes were labeled with [
-32P]ATP
with T4 polynucleotide kinase. Binding reactions, containing 40 fmol of
radiolabeled probe, were incubated at 4 °C for 30 min. The
competitor DNA was added at 10-50-fold molar excess prior to the
addition of probe. Anti-Nkx2.2 antibody was pre-incubated with protein
at 4 °C for 20 min before adding probe to the binding reaction. The
protein-DNA complexes were resolved on a 6% non-denaturing polyacrylamide gel, which was then dried and exposed to
autoradiographic film.
 |
RESULTS |
Transcription Factor Occupancy of Insulin Control Region Sequences
in Vivo--
The ChIP assay was first used to investigate binding of
Pdx-1 to 5'-flanking control sequences of the insulin gene in an islet
cell line,
TC-3. Two distinct antibodies to Pdx-1 were used to
immunoprecipitate formaldehyde cross-linked chromatin. Insulin 5'-flanking sequences (INS; nucleotides
378/
46) were selectively amplified by PCR from chromatin precipitated by anti-Pdx-1 antisera made to either the N- or C-terminal region, but not from chromatin treated with normal rabbit IgG or in the absence of antiserum (Fig.
1A). In contrast, Pdx-1
antisera did not immunoprecipitate control sequences from the PCK gene
(nucleotides
434/
96), which is not expressed in
cells (Fig.
1A). Significantly, the amplified region of the mouse PCK
promoter contains two TAAT motifs that can compete for Pdx-1 binding
in vitro (data not shown). Thus, although Pdx-1 can bind to
both the insulin and PCK control sequences in vitro, the
ChIP assay demonstrates that only Pdx-1 interacts with the endogenous
insulin gene in
cells.

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Fig. 1.
Pdx-1, BETA2, Pax6, and Nkx2.2 bind to
insulin gene control sequences in vivo.
Formaldehyde cross-linked chromatin from TC-3 cells was incubated
with antibodies raised to the N terminus or C terminus of Pdx-1
(panel A, lanes 3 and 4), BETA2
(panel B, lane 3), Pax6 (panel C,
lane 3), or Nkx2.2 (panel D, lane 3).
Immunoprecipitated DNA was analyzed by PCR with primers specific to
transcriptional regulatory sequences of the mouse insulin
(INS) and phosphoenolpyruvate carboxykinase (PCK)
genes. As controls, PCR reactions were performed with no DNA (all
panels, lane 1), input DNA (1:100 dilution; all
panels, lane 2), DNA immunoprecipitated by normal
immune sera that were species-matched to the source of the test
antibodies (panel A, lane 5; panels
B-D, lane 4), and DNA that was
immunoprecipitated in the absence of antiserum (panel A,
lane 6; panels B-D, lane 5).
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To further characterize the in vivo occupancy of the INS
transcription unit, ChIP analysis was performed with antibodies to other potential transcriptional regulators, specifically BETA2 (8) and
Pax6 (9). Both anti-BETA2 (Fig. 1B) and anti-Pax6 (Fig.
1C) antibodies effectively immunoprecipitated INS control sequences from formaldehyde cross-linked
TC-3 chromatin, but not
PCK. Neither the INS nor PCK regulatory region was amplified from
chromatin isolated in control immunoprecipitations.
Finally, we tested if the endogenous INS promoter is occupied by
Nkx2.2, a factor that was proposed to be involved in expression as a
result of its dramatic and selective effect on insulin levels in Nkx2.2
null mice (17). Anti-Nkx2.2 antibodies, but not control reactions,
immunoprecipitated INS sequences from
TC-3 cells (Fig. 1D), whereas the PCK promoter was not immunoprecipitated by
anti-Nkx2.2. The same patterns were observed with antisera to Nkx2.2,
BETA2, Pdx-1, and Pax6 with MIN6
cells (data not shown). These
results demonstrate that Nkx2.2, as well as Pdx-1, BETA2, and Pax6, is physically associated with endogenous INS control region sequences in
cell lines. The binding detected in vivo for Pax6
(
317/
311 bp, Ref. 9), Pdx-1 (
201/
196 bp, Ref. 6,
85/
70 bp,
Ref. 5), and BETA2 (
100/
91bp, Ref. 8) within the insulin gene presumably involves the control sites detected in DNA element gel shift assays.
Identification of an Nkx2.2-binding Site between Nucleotides
140
and
119 in the Insulin Gene--
The insulin 5'-flanking region
(
378/
46 bp) analyzed in the ChIP studies was found to contain
potential Nkx2.2-binding elements at nucleotides
140 to
119 (site
1) and
193 to
172 (site 2) upon comparison to the Nkx2.2
consensus-binding site (29) (Fig. 2A). To determine whether
Nkx2.2 binds to these INS elements, gel mobility shift assays were
performed with in vitro-translated Nkx2.2 and probes to site
1, site 2, and the consensus element (Fig. 2B). Two major
protein-DNA complexes were commonly formed with each element in the
Nkx2.2 and control in vitro-translated reactions (see
asterisk-labeled complexes in Fig. 2A).
Significantly, a unique faster mobility complex was detected in the
Nkx2.2 reaction with the INS site 1 and consensus element probes,
although not with INS site 2 (Fig. 2B). To determine whether
Nkx2.2 was involved in formation of this faster mobility complex,
antiserum specifically raised against this protein was added to the
binding reactions. This complex was selectively super-shifted in the
reactions performed in the presence of Nkx2.2 with the consensus and
site 1 probe (Fig. 2B). A complex of identical mobility was
formed with the INS site 1 probe in
TC-3 and MIN6
nuclear
extracts and was also super-shifted with the anti-Nkx2.2 antibody (Fig.
2C). In addition, competition assays demonstrated that
Nkx2.2 bound effectively to the consensus-binding element and INS site
1 and not to site 2 or a site 1 element with a two base pair mutation
in the core-binding sequence (INS site 1
133,
132 MT) (Fig.
2D). Collectively, these data indicated that Nkx2.2 binds to
site 1 at
140 to
119 bp of the insulin gene in
cells.

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Fig. 2.
Nkx2.2 binds to a site between nucleotides
140 and 119 in the insulin gene. A, sequences of the
oligonucleotides used in the gel shift assays are shown in comparison
with the Nkx2.2 consensus-binding element (29). The core-binding
site nucleotides are underlined. B, gel shift
assays were performed with radiolabeled probes corresponding to the
Nkx2.2 consensus-binding site (lanes 1-3), INS site 1 (lanes 4-6), and INS site 2 (lanes 7-9).
In vitro-translated control (C) or Nkx2.2
(NK) protein and Nkx2.2-specific antiserum (+) were added to
the binding reactions. The positions of the Nkx2.2-binding complex and
the antibody super-shifted (SS) Nkx2.2 complex are marked
with an arrow; nonspecific binding complexes are marked with
asterisks. C, TC-3 (lanes 1-2) and
MIN6 (lanes 3-4) cell nuclear extracts were analyzed for
INS site 1 binding in the presence (+) or absence ( ) of
Nkx2.2-specific antiserum. The positions of the Nkx2.2-binding complex
and the antibody super-shifted (SS) Nkx2.2 complex are
marked with an arrow. D, in
vitro-translated Nkx2.2 binding to the INS site 1 (lanes
1-9) and the Nkx2.2 consensus-binding site (Con,
lane 10) probe was conducted in absence ( ) or presence of
a 10-50-fold molar excess of the consensus Nkx2.2-binding site
(lanes 2-3), INS site 1 (lanes 4-5), INS site 2 (lanes 6-7), and the INS site 1 133,132 mutant
(lanes 8-9) competitor.
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|
Nkx2.2 Appears to Inhibit Insulin Gene Expression--
To
understand how Nkx2.2 influences insulin transcription, the
133/
132
mutant of site 1 that disrupted binding was introduced into an insulin
enhancer/promoter-driven CAT reporter. The effect on activation was
compared with a mutant in the RIPE3b1/C1 activator site at
108/
107
bp and the wildtype
238/+2bp CAT construct in transfected HIT T-15,
TC-3, and MIN6
cell lines. Regulatory patterns for both the INS
site 1 and RIPE3b1/C1 mutated constructs were similar in all three
cell lines. Thus, activation was stimulated 1.8- (HIT T-15), 3.3- (
TC-3), and 1.8- (MIN6) fold in the site 1 mutant and, as expected,
reduced in the RIPE3b1 activator-binding site mutant (25) (Fig.
3). These results suggest that Nkx2.2 functions as a negative regulator of insulin expression in
cell lines.

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Fig. 3.
Nkx2.2-binding site 1 inhibits insulin-driven
reporter activity. HIT T-15, TC-3, and MIN6 cells were
transfected with the wildtype (WT) ( 238 WT CAT,
white bars), Nkx2.2 ( 238 CAT 133,132 MT; dark
bars), or RIPE3b1/C1 ( 238 CAT 108,107 MT, grey
bars) mutant versions of the rat insulin II 238/+2 bp CAT
expression plasmid. The rat insulin I and II genes share five of six
nucleotide identity with the mouse insulin II genes in the core
sequence of the Nkx2.2-binding site 1 and retain gel shift binding
(data not shown). CAT results are normalized to the LUC activity
from cotransfected pRSVLUC and are reported relative to 238 WT CAT
activity. The asterisk denotes that there was a
statistically significant increase between 238 CAT 133, 132 MT and
the WT activity in HIT T-15 (p < 0.0001, Student's
t test), TC-3 (p < 0.001), and MIN6
cells (p < 0.05). The data was compiled from at least
six independently performed transfections.
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Pdx-1, Nkx2.2, BETA2, and Pax6 Also Bind in Vivo within
the Control Region of Other
Cell-enriched Gene
Products--
Because
cell-specific expression of the insulin gene
appears to be controlled by the combinatorial actions of Pdx-1, BETA2, and Pax6 (30-32), the ChIP assay was used with
TC-3 cells to
determine whether these factors also bound within control region
sequences of other islet-enriched gene products, specifically IAPP, GK, and pax-4. The Pdx-1-specific antibodies were found to
selectively immunoprecipitate the control sequences of the IAPP
(33-35), GK (36), and pax-4 (37) genes that bound Pdx-1
in vitro and were mutationally sensitive in transfection
analyses (Fig. 4A).

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Fig. 4.
Pdx-1, BETA2, Pax6, and Nkx2.2 occupy the
control region in vivo of many genes selectively
expressed in cells. Formaldehyde
cross-linked chromatin from TC-3 cells was incubated with antibodies
raised to the N terminus or C terminus of Pdx-1 (panel A,
lanes 3 and 4), BETA2 (panel B,
lane 3), Pax6 (panel C, lane 3), or
Nkx2.2 (panel D, lane 3). Immunoprecipitated DNA
was analyzed by PCR with control region-specific primers to the mouse
IAPP, pax-4, and GK genes. Control reactions were performed
as described in legend to Fig. 1.
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IAPP and pax-4 control sequences were also
immunoprecipitated from
TC-3 cells by anti-BETA2 antibodies but not
by control antibodies (Fig. 4B). These results confirm the
in vitro studies that indicated that BETA2 was involved in
pax-4 expression (37). BETA2 has also recently been shown to
bind to the GK promoter in vivo and stimulate expression
(38). In addition, antisera to Pax6 (Fig. 4C) and Nkx2.2
(Fig. 4D) selectively immunoprecipitated IAPP, GK, and
pax-4 control sequences. Interestingly, the pax-4 promoter has been shown to contain a mutationally sensitive binding site that may be regulated by a factor in the Nkx2 family (39), although the precise binding protein had not been identified. In sum,
these results suggest that Pdx-1, BETA2, Pax6, and Nkx2.2 define the
core of a
cell-enriched transcription complex.
 |
DISCUSSION |
Though assays such as in vitro binding, co-transfection
with reporter constructs, and transgenic knock-out animals provide evidence for the involvement of a factor in transcriptional control, they do not prove this directly. This is especially problematic for
factors, such as Pdx-1, that recognize relatively non-selective binding
sites. The consensus Pdx-1-binding element, TAAT(T/G) (40), is expected
to occur once in every 512 bp. As control region sequences are often
several hundred bases in length, the presence of this motif is not
uncommon and would not unequivocally demonstrate Pdx-1 regulation even
if it were capable of binding to a cis-acting control
element in vitro. Likewise, transfection experiments do not
account for the native chromatin structure of endogenous genes, which
may preclude physiological regulation by a factor that, nonetheless,
can bind in vitro. The ChIP assay combines two highly
specific techniques, antibody-specific protein-DNA precipitation and
PCR amplification, to demonstrate a physical association between a
transcription factor and specific control region sequences in intact
cells. The ChIP protocol can establish direct interactions of a
transcription factor and a gene that are known to be functionally
related and, importantly, can also be used to identify interactions
with candidate target genes.
Using the ChIP assay, we observed occupancy of insulin
5'-flanking control region sequences in
TC-3 and MIN6 cells
by Pdx-1, BETA2, Pax6, and Nkx2.2 (Fig. 1). Our demonstration of Pdx-1
occupancy of insulin control sequences corroborates a recent study that was published while our work was in progress (23). Though there was
evidence to suggest a direct role for Pdx-1 (5-9, 13, 41), BETA2 (8,
13), and Pax6 (9) in insulin gene expression, it has been unclear
whether Nkx2.2 directly or indirectly contributed. Thus, the evidence
supporting an involvement was based upon the loss of insulin expression
in Nkx2.2 null mice, under circumstances where other
cell markers
continued to be expressed (17). However, a control site for Nkx2.2
within the insulin gene had not been characterized previously. Based on
the reported consensus Nkx2.2-binding element (29), we have localized
an Nkx2.2-binding site between
140 and
119 bp of the insulin gene
by in vitro gel shift analysis (Fig. 2). Importantly, this
site is mutationally sensitive in transfection assays, indicating that
Nkx2.2 binding can affect insulin gene activity (Fig. 3). Collectively,
our results demonstrate that Nkx2.2 can directly regulate insulin
transcription in
cells.
One would have expected that Nkx2.2 would have had a positive effect on
insulin transcription from the results of the Nkx2.2 gene ablation
studies in mice (17). However, Nkx2.2 appears to act as a weak
repressor in
cell transfection assays performed with either an
insulin enhancer (Fig. 3) or consensus site-driven reporter construct
(29). Nkx2.2 is also known to function as a transcriptional repressor
in the developing central nervous system, a function necessary in cell
fate specification in the ventral neural tube (42). Interestingly,
structure-function analysis of the Nkx2.2 protein has identified a
strong C-terminal transactivation domain whose function is masked by
the highly conserved NK2-specific domain (NK2-SD) in
TC-3 cells
(29). As a consequence, it is possible that a co-factor expressed
during pancreatic development or a stage-specific protein modification might interfere with the NK2-SD-mediated repressor function of Nkx2.2
and allow it to function as a transcriptional activator during
development. In contrast, Nkx2.2 repressor activity would result if
cell lines or mature
cells lack this effector activity. By
extension of this reasoning, we presume that Nkx2.2 also inhibits pax-4, GK, and IAPP gene expression upon binding in
cells. Although the Nkx.2.2 regulatory site(s) within these genes has
not been defined precisely, it is likely to be found among the
consensus-like binding sites within their control region sequences
(i.e. pax-4,
1940/
1933 bp,
1876/
1869 bp;
GK,
542/
536 bp,
486/
479 bp; IAPP,
325/
319 bp,
286/
280
bp,
267/
261 bp,
146/
140 bp).
Having used the ChIP assay to demonstrate binding of Nkx2.2 to several
genes expressed in an islet-enriched manner, we extended this study to
examine if other key
cell regulatory factors also bound within
their regulatory domains. Previously, Pdx-1 had been shown to bind to
its own promoter region in the ChIP assay (23, 24). Two distinct
antisera raised against Pdx-1 were found to immunoprecipitate the IAPP
and pax-4 control sequences. Each of these regions has been
reported to be a target of Pdx-1 function (IAPP, Refs. 34, 35, and 41;
pax-4, Ref. 37) and was independently shown to be
occupied in the ChIP assay (23). However, whereas we observed a clear
interaction of Pdx-1 and the GK gene using antisera to both the N and C
termini of Pdx-1, another group was unable to detect this association
using a different Pdx-1 antiserum (23). This difference highlights an
important limitation of the ChIP assay, which despite the power of this
technique to firmly establish a physical association between a
transcription factor and a control region with immunoprecipitating
antisera, a negative result does not provide definitive evidence for
the lack of such interactions. Another example involves the inability
to detect the occupancy of the GLUT2 promoter by Pdx-1 in the ChIP
assay (data not shown and Ref. 23), despite a great deal of evidence supporting a role in activation (15, 16). There are two likely explanations for these negative results. The microenvironment of the
GLUT2 control region might preclude the exposure of Pdx-1 antigenic
determinants required for immunoprecipitation, possibly due to
interactions with adjacent binding proteins. Alternatively, it is
possible that Pdx-1 does not directly regulate GLUT2 transcription or
does so through elements that are located outside of the analyzed region. Both studies focused on the proximal region of the GLUT2 promoter found to bind Pdx-1 in vitro (data not shown and
Ref. 23) and, as a result of methodological limitations, Pdx-1-GLUT2 interactions that lie at
1000 bp away would not have been detected.
Interestingly, the Pdx-1, Pax6, BETA2, and Nkx2.2 regulatory factors of
the insulin gene were all found to bind in intact
cells to
the regulatory control regions of other genes selectively transcribed
in islet cells. How each of these factors precisely mediates control
within the context of the insulin, pax-4, IAPP, and GK
transcription units is still unclear. As islet cell development was so
profoundly affected in the null mutants of these factors, the knock-out
studies in mice cannot be used to access how each factor controls islet
expression post-natally under normal or stressed states. Our data
strongly suggests that Pdx-1, Pax6, BETA2, and Nkx2.2 represent the
core components of a transcription complex of an islet-enriched gene,
and presumably also contribute in their expression during development.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Marc Daniels and David Duong
for advice and assistance with the ChIP assay protocol and John Lelay
in providing technical support. We thank Drs. Chris Wright and Joel
Habener for generously providing the N-terminal (amino acids 1-75) and C-terminal (amino acids 271-283) Pdx-1 antiserum, respectively.
 |
FOOTNOTES |
*
This work was supported by Grants NIH RO1 DK-50203 and
DK-55091 from the National Institutes of Health (to R. S.). Partial support was also provided by the Vanderbilt University Diabetes Research and Training Center Molecular Biology Core Laboratory (Public
Health Service Grant P60 DK20593 from the NIH).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, November 7, 2002, DOI 10.1074/jbc.M205905200
 |
ABBREVIATIONS |
The abbreviations used are:
INS, insulin;
IAPP, islet amyloid polypeptide;
ChIP, chromatin immunoprecipitation;
CAT, chloramphenicol acetyltransferase;
WT, wildtype;
MT, mutant;
RSV, Rous
sarcoma virus;
LUC, luciferase;
PCK, phosphoenolpyruvate carboxykinase;
GK, glucokinase.
 |
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