From the Division of Cell and Molecular Biology, Toronto General
Research Institute, University Health Network and the § Department of
Medicine and Department of Laboratory Medicine and Pathobiology,
University of Toronto, Toronto, Ontario M5G 2M1, Canada
Received for publication, September 10, 2000, and in revised form, February 5, 2001
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ABSTRACT |
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The caudal homeobox gene Cdx-2 is a
transcriptional activator for approximately a dozen genes specifically
expressed in pancreatic islets and intestinal cells. It is also
involved in preventing the development of colorectal tumors. Studies
using "knockout" approaches demonstrated that Cdx-2 is
haplo-insufficient in certain tissues including the intestines but not
the pancreatic islets. The mechanisms, especially transcription
factors, which regulate Cdx-2 expression, are virtually unknown. We
found previously that Cdx-2 expression could be autoregulated in a cell
type-specific manner. In this study, we located an octamer (OCT)
binding site within the mouse Cdx-2 gene promoter. This site,
designated as Cdx-2POCT, is involved in the
expression of the Cdx-2 promoter. Both pancreatic and intestinal cell
lines were found to express a number of POU (OCT binding)
homeodomain proteins examined by electrophoretic mobility shift
assay. However, it appears that Cdx-2POCT interacts only
with OCT1 in the nuclear extracts of the intestinal cell lines
examined, although it interacts with OCT1 and at least two other POU
proteins that are to be identified in the pancreatic InR1-G9 cell
nuclear extract. Co-transfecting OCT1 cDNA but not five other POU
gene cDNAs activates the Cdx-2 promoter in the pancreatic InR1-G9
and the intestinal Caco-2 cell lines. In contrast,
Cdx-2POCT cannot act as an enhancer element if it is fused
to a thymidine kinase promoter. Furthermore,
Cdx-2POCT-thymidine kinase fusion promoters cannot be
activated by OCT1 co-transfection. Cell type-specific expression, cell
type-specific binding affinity of POU proteins to the
cis-element Cdx-2POCT, and the DNA
content-dependent activation of Cdx-2 promoter via
Cdx-2POCT by OCT1 suggest that POU proteins play important
and complicated roles in modulating Cdx-2 expression in cell
type-specific manners.
Transcription factors are almost never expressed as specific as
their downstream target genes (for review see Ref. 1). A given
transcription factor is always able to exert multiple biological
functions via regulating the expression of different downstream target
genes in tissue/cell type-specific manners. This is mainly attributed
to the temporal and spatial expression property of a given
transcription factor, its ability to bind to
cis-elements within different downstream target promoters, and more importantly, its ability to recruit other transcription factors or nuclear co-activators and/or repressors (1-3).
Cdx-2 (CDX2 for humans) is one of the three caudal-related homeodomain
(HD)1 proteins identified in
mammals (4-7). Post-embryogenesis, Cdx-2 is expressed in pancreatic
islets, differentiated intestinal epithelia including the
proglucagon-producing endocrine L cells (6-9). Cdx-2 mull
mutant mice have been generated recently (10, 11). Cdx-2 Although the physiological and pathological significance of Cdx-2
has been well recognized, the mechanisms, especially transcription factors, which regulate the expression of Cdx-2, remain virtually unknown. We have isolated the mouse Cdx-2 promoter and examined the
transcriptional properties of this promoter in pancreatic and
intestinal cell lines. We have found that Cdx-2 activates the
expression of its own promoter in a cell type-specific manner (12). We
have dissected the Cdx-2 gene promoter and demonstrated that Cdx-2
binds to two separated AT-rich motifs, namely the TATA and DBS sites,
within a proximal region of the Cdx-2 promoter. The DBS site is
critical for autoactivation, whereas the TATA site may serve as an
attenuating component for the autoregulatory loop (12).
Members of the POU HD protein family share a common feature of
harboring a bipartite DNA binding domain consisting of a POU-specific domain and a POU homeodomain joined by a short variable linker (13).
Many POU proteins act as transcriptional regulators via recognizing a
conserved 8-base pair octamer (OCT) binding element (13). POU proteins
are expressed in early embryogenesis and in diverse organs and/or
systems and play fundamentally important roles in the organ
development, cell type specification, and growth and differentiation of
cells (14, 15).
In this study, we located an OCT binding site in the mouse, Cdx-2
promoter, designated as Cdx-2POCT. We demonstrate here that Cdx-2POCT is critical for Cdx-2 promoter expression. OCT1
and other to-be-identified POU proteins are expressed in pancreatic islet, and intestinal cell lines examined. It appears that
Cdx-2POCT physically interacts with only OCT1 expressed in
intestinal cell lines, whereas Cdx-2POCT interacts with
OCT1 and at least two other yet to-be-identified POU proteins expressed
in a pancreatic A cell line. Although OCT1 was found to bind to and
activate Cdx-2 promoter in pancreatic and intestinal cell lines,
Cdx-2POCT itself cannot act as an enhancer element when it
is fused to a heterologous promoter. In addition, the fusion promoters
cannot be activated by OCT1 co-transfection. We conclude that OCT1 and
other to-be-identified POU proteins play important and complicated
roles in modulating Cdx-2 expression in cell type-specific manners.
Materials--
Radioisotopes were obtained from Amersham
Pharmacia Biotech. Oligonucleotides were provided by ACGT Co. (Toronto,
Canada). Restriction enzymes and DNA modification enzymes were
of molecular biology grade and were purchased from several sources.
Plasmids--
Construction of the mouse Cdx-2 promoter and the
luciferase fusion gene plasmid and its deletion constructs have been
described previously (12). Cdx-2-LUC ( Cell Culture, Transient Transfection, and Luciferase Reporter
Gene Analysis--
Human colon cancer cell lines HT-29, COLO-205,
SW480, and Caco-2 were obtained from the American Type Culture
Collection (ATCC, Manassas, VA) and cultured according to
instruction. Cultivation of the baby hamster kidney (BHK) fibroblast,
pancreatic islet cell lines InR1-G9, In111, RIN-56, and HIT, and the
intestinal endocrine L cell lines GLUTag and STC-1 have been described
previously (8, 12, 19, 20). BHK, InR1-G9, and Caco-2 cell lines were
transfected by a calcium phosphate precipitation method (12). The cells
were harvested for the luciferase (LUC) reporter gene analysis 16 h after the transfection.
Nuclear Protein Extraction and Electrophoretic Mobility Shift
Assay (EMSA)--
Nuclear proteins from the cultivated cell lines were
prepared as described by Schreiber et al. (21). EMSA and
supershifting analysis were performed as described previously (19).
Contribution of Cdx-2POCT to the Expression of the
Cdx-2 Promoter--
We located an OCT binding site within the proximal
portion of the mouse Cdx-2 promoter, which we designated as
Cdx-2POCT (Fig. 1A). To examine the
contribution of this OCT binding site to the expression and
autoregulation of the Cdx-2 promoter, we generated a pair of Cdx-2-LUC
fusion gene constructs, Cdx-2-LUC ( Examination of OCT-binding Protein Expression by EMSA--
We then
conducted an examination of the expression of nuclear proteins that are
able to physically interact with Cdx-2POCT in pancreatic
and intestinal cell lines by EMSA. When the pancreatic islet A cell
line InR1-G9 was examined, four DNA protein complexes were obtained
designated as C1-C4 (Fig. 3, lanes
2 and 10). The formation of these four complexes could
be effectively competed by unlabeled native Cdx-2POCT probe
(lanes 3 and 4) but not by an unlabeled
nonspecific probe (lanes 5 and 6). Therefore,
these complexes all represent specific DNA binding events. Complexes C1, C2, and C3 could be effectively competed by another OCT binding probe (22) (lanes 7 and 8, see below), indicating
the likelihood that these three complexes all represent POU protein
binding events. When the mouse large intestinal endocrine L cell
line GLUTag was examined, only one complex, C3, was clearly observable
(lane 11). When the human colon cancer cell line Caco-2 was
examined, the only observable complex was C1 (lane 16). The
C1 complex was determined to represent the ubiquitously expressed POU
protein OCT1 by a supershifting analysis (compare lane 14 with 15 and lane 16 with 17).
The above results would suggest that either the intestinal cell lines
express fewer number of POU proteins or most POU proteins expressed in
intestinal cell lines cannot bind to Cdx-2pOCT. To examine
these two possibilities, we then conducted EMSA to examine four human
colon cancer cell lines using a synthetic OCT probe (22). DNA sequence
of the synthetic OCT probe is shown in Fig. 1B. This
artificial probe has been used successfully previously by us to examine
the expression of POU proteins in normal and malignant human mammary
gland epithelial cell lines (22). When this probe was used, nuclear
extracts from four intestinal cell lines were found to form a number of
complexes (Fig. 4A). Among these complexes, C1, C2, and C4 were observable for all four colon cancer cell lines examined (lanes 1-4). The formation of
C1, C2, and C3 in the Caco-2 cell line was completely competed by the unlabeled OCT probe (lanes 7 and 8) but not by
the mutant OCT probe (lanes 9 and 10), indicating
that they represent POU protein binding. The native
Cdx-2pOCT probe was found to inhibit the formation of C1
and C4 but not C2 and C3 (lane 6). This result would suggest that the POU proteins in C2 and C3, expressed by the Caco-2 cell line,
are unable to bind to Cdx-2pOCT. To further examine this observation, we conducted EMSA using the native Cdx-2POCT
probe against four colon cancer cell lines. As shown in Fig.
4B, the nuclear extracts from all these cell lines examined
were found to form only one major complex, C1 (Fig. 4B).
Taken together, our results suggest that with the exception of OCT1,
the other to-be-identified POU proteins are also expressed in the
intestinal cell lines examined in this study. However, they are not
capable of binding to Cdx-2POCT.
When the synthetic OCT probe was applied to the pancreatic islet cell
line InR1-G9, three complexes from C1 to C3 were obtained (Fig.
5). Supershifting analyses further
revealed that C1 represents OCT1 (lane 2). The identities of
C2 and C3 cannot be determined using antibodies against two other POU
proteins, namely OCT2 and OCT11 (Skn-1a/Skn-1i) (lanes
3-5). We also cannot detect OCT2 and OCT11 expression in the
pancreatic and intestinal cell lines used in this study by Western blot
analysis (data not shown).
OCT1 Activates Cdx-2 Promoter in Pancreatic and Intestinal Cell
Lines--
Our observations indicated that OCT1 is expressed in most
pancreatic and intestinal Cdx-2-expressing cell lines, and OCT1 physically interacts with the Cdx-2 promoter. We then examined if
co-transfection of OCT1 and other POU gene cDNAs would activate Cdx-2 promoter. We found that OCT1 activated Cdx-2 promoter nearly 6-fold in the pancreatic InR1-G9 cell line (Figs. 2A and
6A) and ~4-fold in the
intestinal Caco-2 cell line (Figs. 2B and 6B). Among the other five POU genes examined, only OCT2A showed a moderate activation on the Cdx-2 promoter. Because OCT2 is not expressed in any
of the Cdx-2-expressing cell lines examined, such a moderate activation
may have no biological significance.
Cdx-2POCT Cannot Act As an Enhancer Element When It Is
Fused to a Heterologous Promoter--
To examine whether the 31-bp
native Cdx-2POCT itself could act as an enhancer element,
we fused this element with the thymidine kinase (TK) promoter (19) in
either 5' to 3' (forward) or 3' to 5' (reverse) orientations. The
Cdx-2POCT-TK-LUC fusion genes together with the wild type
TK-LUC were then transfected into the pancreatic InR1-G9 and the BHK
fibroblast cell lines. As shown in Fig.
7A, these fusion promoters
show a 40-70% activity compared with that of wild type TK promoter
when transfected into the InR1-G9 cell line. Therefore, the 31-bp
Cdx-2POCT itself cannot act as an enhancer element for this
Cdx-2-producing cell line. In contrast, these two fusion promoters are
10-20-fold more active compared with the parental TK promoter when
they were transfected into the BHK fibroblasts (Fig. 7B).
Furthermore, these two fusion promoters showed a negative response to
OCT1 co-transfection in both InR1-G9 and BHK cell lines examined. These
results suggest that OCT1 activates Cdx-2 promoter through binding to
Cdx-2POCT in a DNA content-dependent manner.
In 1991, James and Kazenwadel (4) examined the expression of Hox
and related homeobox genes in mouse intestines by reverse transcription-polymerase chain reaction using degenerate primers. With
the exception of the detection of Cdx-1 (23), they also obtained a
portion of a novel caudal cDNA, which they named Cdx-2 (4).
Complete cDNA sequences for mouse Cdx-2 were reported in 1994 (6,
7). However, in 1992 German et al. (5) isolated a complete
caudal cDNA from a hamster insulinoma cell line, HIT. This cDNA
was named Cdx-3. Cdx-2 and Cdx-3 are now considered to be the same gene
in different rodents. We and others (8, 9, 12) have used the term
Cdx-2/3 previously. To follow the nomenclature regulation for HOX/Hox
genes (24), we now use the term CDX2 for this gene in humans and the
term Cdx-2 in rodent species.
Cdx-2 is expressed in intestinal epithelia along the entire
crypt-villus axis of the small intestine and in crypts of the colon
(7). It is also expressed in pancreatic A and B cell lines (5, 8, 9)
and in the large and small intestinal endocrine L cell lines (8). More
recently, the Cdx-2 expression in human gastric cancer cell lines has
been reported (25). CDX2 expression was found to be ceased in
the colon cancer cells of patients at later stages, and mutations in
the CDX2-coding region has been found in both colon cancer patients and
colon cancer cell lines
(26-28).2 More importantly,
Cdx-2+/ Indeed, about a dozen potential downstream target genes of Cdx-2 have
been identified including insulin (5), proglucagon (8, 9),
sucrase-isomaltase (7), phospholipase A/lysophospholipase (29),
lactase-phlorizin hydrolase (30), carbonic anhydrase 1 (31),
calbindin-D9K (32), vitamin D receptor (33), and the
homeobox gene Hoxc-8 (34). Cdx-2, therefore, provides a very good model
system for addressing a fundamentally important biological question:
How can a single transcription factor exert its multiple biological
functions in tissue/cell type-specific manners?
One of the essential steps for understanding tissue-specific biological
functions of Cdx-2 is to investigate mechanisms controlling its
expression in different types of cells. For this purpose, we isolated
the mouse Cdx-2 promoter and examined the transcriptional properties of
this promoter in pancreatic islet, intestinal endocrine, and human
colon cancer cell lines. We demonstrated that Cdx-2 is able to activate
it own promoter in pancreatic and intestinal cell lines that express
CDX2/Cdx-2, but repress its own promoter in fibroblasts (12). This
observation, on one hand, provided an explanation for why one wild type
Cdx-2 allele is sufficient for certain tissue such as pancreas. On the
other hand, it raised a question why one functional allele is
insufficient for other tissues including intestines. We proposed that,
except for Cdx-2 itself, other transcription factors and signals might
determine whether Cdx-2 expression in a given cell reaches the
threshold for triggering the autoregulatory loop. Some of these factors or signals may function only, or more effectively, in one type of cells
such as pancreatic cells. If this hypothesis is correct, it would
provide an explanation as to why various abnormalities observed among
Cdx-2+/ In this study, we assessed the contribution of Cdx-2POCT in
Cdx-2 expression. Because the mutant promoter shows ~30%
activity compared with that of the wild type, we conclude that this
site is important for Cdx-2 expression. In addition, because the
mutant promoter loses more than a 2-fold response to Cdx-2 activation, we suggest that this binding site might be also involved in the autoregulation process.
To examine POU proteins that are expressed and capable of binding to
Cdx-2POCT, we conducted EMSA using both the typical
synthetic OCT binding probe (22) and the native 31-bp
Cdx-2POCT probe. Our results indicated that although
Cdx-2POCT forms four specific DNA-protein complexes with
the pancreatic InR1-G9 cell line, it forms only one complex, C3, with
the intestinal endocrine cell line GLUTag and one complex, C1, with the
colon cancer cell line Caco-2. Because Cdx-2 haplo-insufficient occurs
in intestines but not pancreata, our results would suggest that
pancreatic islet cells might express more POU proteins as
transcriptional activators for Cdx-2. To further examine this, we need
to identify each individual complex formed between the InR1-G9 cell
line and the Cdx-2POCT probe.
The C1 complex was found to represent the ubiquitously expressed POU
protein OCT1. However, four colon cancer cell lines examined do express
OCT-binding proteins other than OCT1 detected by EMSA using the
synthetic OCT binding probe. These observations would suggest that,
except for OCT1, other POU proteins expressed in pancreatic and
intestinal cell lines have different binding affinity to the 31-bp
cis-element Cdx-2POCT. This could be due to the
expression of different profiles of POU proteins by pancreatic islets
and intestine cells. Alternatively, the same POU protein(s) expressed in pancreatic and intestine cells may have different binding
affinities. The latter hypothesis is plausible because the binding
ability of POU proteins could be influenced by posttranslational events including phosphorylation status (35-38). Grenfell et al.
(35) have demonstrated that Oct-1 and Oct-2 bind differentially to three octamer binding sequences, the one from the H2B promoter (ATGCTAATAA, similar to the synthetic OCT probe utilized in this study), a TAATGARAT motif, and a perfect consensus overlapping octamer/TAATGARAT motif (ATGCTAATGAGAT). The binding activity of these
octamer probes could be modulated by PKA, PKC, and casein kinase 2 (35). Inamoto et al. (36) found that MAT1 is able to target
cyclin-dependent kinase-activating kinase to Oct factors and lead to their phosphorylation. This may play a role in the recruitment of transcription factor IIH to the preinitiation complex or
in subsequent initiation and elongation reactions (36). Zhang et
al. (37), however, shown that Oct-1 is a repressor for
HLA-DRA. Expression of the tumor suppressor gene Rb leads to the
phosphorylation of Oct-1 relieving its repressive effect on HLA-DRA. To
assess the overall contribution of POU proteins in regulating Cdx-2
expression in different types of cells, it is necessary to examine the
phosphorylation status of these POU proteins in each type of cells in
both physiological and pathological conditions.
The expression of POU genes in the gastrointestinal tract has not been
actively examined. Hussain et al. (39), however, demonstrated that both InR1-G9 and GLUTag cell lines do express Brn-4,
a class III POU gene that acts as a regulator for proglucagon gene
expression. Because the C3 complex is observed for both InR1-G9 and
GLUTag cell lines (Fig. 3, lanes 10 and 11), it
is possible that this complex represents Brn-4. This complex was
not observed for the four colon cancer cell lines examined. It will be
interesting to examine whether Brn-4 or the protein component for the
complex C3 is a tissue-specific transcriptional activator for Cdx-2 in proglucagon producing cells.
Another interesting observation is that although OCT1 activates Cdx-2
promoter via binding to Cdx-2POCT, this 31-bp
cis-element itself cannot act as an enhancer element for the
heterologous TK promoter. Furthermore, the Cdx-2POCT-TK
fusion promoters do not respond to OCT1 activation. Therefore, OCT1
activates Cdx-2 promoter in a DNA content-dependent manner.
Because OCT1 activates both the In summary, we demonstrated in this study the complexity of the role of
the 31-bp cis-element Cdx-2POCT in regulating
Cdx-2 expression and autoregulation. The complexity has been shown at three different levels: 1) expression of POU proteins may occur cell
type specifically, 2) binding affinity of POU proteins to Cdx-2POCT also shows cell type specificity, 3) activation
of Cdx-2 promoter via Cdx-2POCT by OCT1 depends upon its
native DNA content. We suggest that POU proteins play important and
sophisticated roles in modulating Cdx-2 expression in a cell
type-specific manner.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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/
mutants die between 3.5 and 5.5 days
postcoitum, whereas Cdx-2+/
mutants show
multiple malfunctions including the formation of multiple intestinal
adenomatous polyps. However, malfunctions in the pancreata have
not been detected in the Cdx-2 heterozygotic mice. Therefore, Cdx-2 is
haplo-insufficient for selected tissues including the intestines but
not the pancreas.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
138 to +137) and its mutant
were constructed by subcloning the correspondent polymerase chain
reaction products into a promoter-less luciferase reporter gene
plasmid. The forward primers used in polymerase chain reaction are
Cdx-2pOCT and Cdx-2pOCT(M) as indicated in Fig. 1B. The
reverse primer represents the mouse Cdx-2 DNA sequence from +119 to
+137 (GAGCTTCTGCCTCCCGGG). OCT1, OCT2A, and OCT2B expression plasmids
were kindly provided by Dr. Winship Herr (16), OCT3A was a gift
from Dr. Graeme Bell (17), and the Skn-1a and Skn-Ii were gifts of Dr.
Bogi Andersen (18). Construction of Cdx-2 expression plasmid and its
antisense counterpart (Cdx-2(AS)) has been described previously (19). OCT1(AS) was generated by flipping the 1.1-kb EcoRI fragment
in the pCG-OCT1 expression vector (16).
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ABSTRACT
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138 to +137 wild type) and
Cdx-2-LUC (
138 to +137 mutant). These two fusion genes are identical
with the exception that Cdx-2-LUC (
138 to +137 mutant) carries a
mutated OCT binding site (Fig. 1B). We then transfected
these two fusion genes into the pancreatic InR1-G9 cell line (Fig.
2A) and the intestinal Caco-2
cell line (Fig. 2B). The mutated promoter was found to
activate the reporter gene in both cell lines ~30% compared with the
wild type promoter indicating that this OCT binding site is critical
for Cdx-2 expression. The wild type promoter could be activated
>10-fold by Cdx-2 cDNA co-transfection, whereas the mutant
promoter could be activated <5-fold. This result suggests that
Cdx-2POCT could also be implicated in Cdx-2 autoregulation
(12). In addition, the wild type Cdx-2 promoter could be activated
approximately 5-fold by OCT1 cDNA co-transfection, whereas the
mutant promoter has no response to OCT1 co-transfection (Fig. 2). Cdx-2
(AS) and OCT1 (AS), which contain Cdx-2 or OCT1 cDNA, respectively,
in the antisense orientation, cannot activate Cdx-2 promoter (Fig.
2).
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Fig. 1.
Schematic presentation of the mouse Cdx-2
promoter and the DNA probes/primers utilized. A,
CRE, cAMP response element; E box, the consensus
sequence CANNTG; TATA and DBS, two Cdx-2
binding sites identified previously (12). The ATGCAAAT motif is located
between 125 to
118 bp relative to the transcription start site.
B, * indicates that this synthetic OCT binding probe and its
mutant have been previously utilized for detection of OCT-binding
proteins in mammary glands (22).
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Fig. 2.
Assessment of the role of
Cdx-2POCT in Cdx-2 promoter expression.
SK-LUC, a promoter-less luciferase reporter gene construct;
Cdx-LUC(WT) and Cdx-LUC(M), luciferase reporter
genes driven by the wild type or the OCT binding site mutated Cdx-2
promoter (-138 to +137), respectively; Cdx-2 and
OCT1, cDNAs of these two transcription factors driven by
a CMV promoter; Cdx-2(AS) and OCT1(AS), Cdx-2 and
OCT1 cDNAs inserted into the expression vector pBAT7 in the
antisense orientation (8). Pancreatic InR1-G9 cells (A) or
intestinal Caco-2 cells (B) were transfected by the
combination of the indicated plasmids (5 µg each). Luciferase
activity was measured 16 h after transfection. The data are
expressed as mean relative luciferase activity (n = 3) ± S.E. normalized to: 1) the activity obtained after
transfection of the promoter-less SK-LUC in the same experiment, and 2)
total amount of protein utilized in each assay.
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Fig. 3.
Detection of nuclear proteins that bind to
Cdx-2POCT by EMSA. NE, nuclear extracts;
Comp./S, unlabeled Cdx-2POCT probe;
Comp./NS, an unrelated unlabeled 30-bp probe;
Comp./OCT, unlabeled OCT probe (Fig. 1B);
Ab/OCT1, anti-OCT1 antibody (2 µl); OCT1, the
supershifted complex by anti-OCT1 antibody; FP, free probe.
Approximately 5 µg of nuclear proteins were utilized in each
reaction.
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Fig. 4.
Examination of the expression of OCT-binding
proteins in colon cancer cell lines by EMSA using both the OCT
(A) and the Cdx-2POCT (B)
probes. NE, nuclear extracts. Competitors:
Cdx, Cdx-2pOCT; OCT, a synthetic OCT
probe (Fig. 1B); OCT(m), OCT mutant probe (Fig.
1B). FP, free probe. Approximately 5 µg of
nuclear proteins were utilized for each reaction.
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Fig. 5.
Examination of the identities of DNA-Protein
complexes by supershifting analysis. NE, nuclear
extracts. Ab (OCT), anti-POU protein antibodies:
1, OCT1; 2, OCT2; 11, OCT11
(Skn-1a/Skn-1i); and G4, an unrelated (anti GATA-4)
antibody. FP, free probe; OCT1, the supershifted
complex by anti-OCT1 antibody.
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Fig. 6.
Co-transfection of OCT1 cDNA activates
Cdx-2 promoter in pancreatic and intestinal cell lines.
SK-LUC, a promoter-less luciferase reporter gene construct.
Five µg of indicated luciferase reporter gene construct was
transfected into the pancreatic InR1-G9 (A) and intestinal
Caco-2 (B) cell lines with 5 µg of indicated cDNA.
Luciferase activity was measured 16 h after transfection. The data
are expressed as mean relative luciferase activity (n = 3) ± S.E. normalized to: 1) the activity obtained after
transfection of the promoter-less SK-LUC in the same experiment, and 2)
total amount of protein utilized in each assay.
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Fig. 7.
Cdx-2POCT cannot act as an
enhancer element when fused to a heterologous promoter.
TK-LUC, a reporter gene construct in which the luciferase
reporter gene expression is driven by a thymidine kinase gene promoter;
OCT-TK-LUC(F) and OCT-TK-LUC(R), the 31-bp
Cdx-2POCT was inserted in front of the TK promoter in 5'-3' (forward)
or 3' to 5' (reverse) orientation. Pancreatic InR1-G9 cells
(A) or BHK fibroblasts (B) were transfected by
the combination of the indicated reporter gene and the cDNA
plasmids (5 µg). Luciferase activity was measured 16 h after
transfection. The data are expressed as mean relative luciferase
activity (n = 3) ± S.E. normalized to: 1) the
activity obtained after transfection of the promoter-less SK-LUC in the
same experiment, and 2) total amount of protein utilized in each
assay.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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mice were found to develop multiple
malfunctions including a homeotic shift on vertebrae, stunted growth,
abnormalities on tails, and the development of multiple intestinal
neoplasia within three months of their birth. However, no observable
malfunction has been found in the pancreata of these heterozygotic mice
(10), and proglucagon gene expression appears normal in the
Cdx-2+/
mice.3 Such extraordinary
multiple malfunctions observed in the heterozygotic null mutants
suggest that 1) Cdx-2 is haplo-insufficient for selected tissues, and normal cellular phenotype in these tissues requires the expression of both alleles, 2) Cdx-2 has a broad spectrum of
downstream target genes, and 3) Cdx-2 requires co-factors to achieve
its diverse and cell type-specific biological functions.
mice vary, in degree, time, and
location, from one mouse to another.
769 to + 137 and the
138 to +137
Cdx-2 gene promoters, the cis-element(s) critical for this
content-dependent activation should be downstream of the
Cdx-2POCT itself. The to-be-defined cis-element(s) may overlap with Cdx-2 binding sites and the
area with which components for general transcription machinery might interact. It is possible that OCT1 and other to-be-identified POU
proteins may interact with other transcription factors that bind to
yet-to-be-defined cis-element(s) downstream of
Cdx-2POCT for activating Cdx-2 expression. This contact may
promote further protein-protein interactions and recruitment events
that interface more effectively with the transcriptional machinery.
Indeed, it has been shown previously that OCT1 requires different
co-factors such as OCA-B (40), proximal sequence element-binding
transcription factor (41), and VP-16 (42) for its diverse biological
functions. OCT1 is also able to interact with MAT1, a subunit of
cyclin-dependent kinase-activating kinase (36).
Furthermore, it has been shown that OCT1 and the glucocorticoid
receptor interact synergistically to activate the transcription of
mouse mammary tumor virus and many other cellular genes (43-45). Other
steroid hormone receptors including progesterone receptor and androgen
receptor, were also found to be able to efficiently recruit POU
proteins to adjacent octamer motifs in the cell (45) or to interact
with OCT1 independently of DNA (46). To identify co-factor(s) that are
essential for OCT1 to activate Cdx-2 expression will add more to our
understanding of cell type-specific autoregulation and cell
type-specific functions of Cdx-2.
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ACKNOWLEDGEMENT |
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We thank Drs. Winship Herr, Graeme Bell, and Bogi Andersen for providing cDNAs of OCT1, OCT2A, OCT2B, OCT3A, Skn-1a, and Skn-1I and Dr. Vincent Giguere for the TK-LUC luciferase reporter gene.
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FOOTNOTES |
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* This work is supported by Operating Grant MOP36398 from the Medical Research Council of Canada (to T. J.).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: Div. of Cell and Molecular Biology, Toronto General Research Inst. University Health Network, Univ. of Toronto, Rm. 421, 67 College St., Toronto, Ontario, M5G 2M1, Canada. Tel.: 416-340-4800, ext. 4768; Fax: 416-340-3453; E-mail: tianru.jin@utoront.ca.
Published, JBC Papers in Press, February 6, 2001, DOI 10.1074/jbc.M008277200
2 T. Jin and H. Li, unpublished data.
3 D. Drucker, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: HD, homeodomain; PCR, polymerase chain reaction; AS, antisense; BHK, baby hamster kidney; LUC, luciferase; EMSA, electrophoretic mobility shift assay; bp(s), base pair(s); TK, thymidine kinase.
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