From the Sekiguchi Biomatrix Signaling Project,
ERATO, Japan Science and Technology Corporation, Karimata, Yazako,
Nagakute, Aichi 480-1195, Japan and the ¶ Division of Protein
Chemistry, Institute for Protein Research, Osaka University,
Yamadaoka, Suita, Osaka 565-0871, Japan
Received for publication, December 10, 2002
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ABSTRACT |
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Laminin-1 is the major component of the embryonic
basement membrane and consists of Laminins are the major basement membrane glycoproteins regulating
tissue morphogenesis through their effects on the proliferation, migration, and differentiation of various types of cells (1-3). Laminins consist of three subunit chains, Mouse F9 embryonal carcinoma cells, a cell culture model of early
mammalian embryogenesis, can be induced to differentiate into primitive
endoderm- and parietal endoderm-like cells upon treatment with retinoic
acid and dibutyryl cAMP with concomitant transcriptional activation of
the genes encoding the laminin The In this study, we isolated the 5'-flanking region of the mouse
LAMA1 gene. Using reporter gene assays and deletion
analyses, we identified an enhancer in the promoter sequence
responsible for laminin Cell Culture--
F9 and NIH/3T3 cells were obtained from Health
Science Research Resources Bank (Osaka, Japan). PYS-2 cells were kindly
provided by Dr. Atsuhiko Oohira (Institute for Developmental Research, Aichi Human Service Center, Aichi, Japan). These cells were cultured in
high glucose Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal calf serum in an atmosphere of 95% air, 5%
CO2, and 100% humidity. The differentiation of F9 cells
was induced by adding 0.1 µM
all-trans-retinoic acid (Sigma) and 1 mM
dibutyryl cAMP (Sigma) into the medium. EHS tumor-derived cells were
prepared cultured as described previously (26) with minor
modifications.2
Isolation of the Mouse LAMA1 Genomic Clones--
A mouse RPCI-23
bacterial artificial chromosome (BAC) library was screened using a
BAC/PAC library screening kit (GenoTechs, Tsukuba, Japan). The
oligonucleotide primers used for screening were:
5'-GAGTGTGCTCTTCCCAGCTC-3' and 5'-CCCCTGGAGGACAGACCT-3'. Genomic DNA
fragments containing exon 1 of the mouse LAMA1 gene were
digested with restriction enzymes, subcloned into pBluescript II
(Stratagene, La Jolla, CA), and sequenced. All DNA sequencing was
carried out using an ABI Prism dye terminator cycle sequencing kit and
a model 3100 DNA sequencer (PE Applied Biosystems, Foster City, CA).
Luciferase Reporter Plasmid Construction and Site-directed
Mutagenesis--
A 9.4-kb XbaI fragment containing 2.5 kb
of the 5'-flanking sequences of the mouse LAMA1 genomic DNA
was subcloned into the XbaI site of pBluescript II. An
XbaI and blunted EcoNI fragment then was inserted
into the pGL3-Basic vector (Promega, Madison WI) to generate a
To identify the enhancer element, the AflII fragment (
Site-directed mutagenesis of four potential protein binding sites was
carried out in the Transfection and Reporter Gene Assays--
Cells in 24-well
plates at 50-70% confluency were transfected using the Effectene
transfection reagent (Qiagen) with 200 ng of reporter plasmid and 20 ng
of the Renilla luciferase expression vector phRL-null
(Promega) as an internal control. 48 h later, the cells were
harvested in Passive lysis buffer (Promega), and the lysates were
assayed for luciferase activity using the dual-luciferase reporter
assay system (Promega). Firefly luciferase activities of various mouse
LAMA1 promoter constructs were normalized to that of the
Renilla luciferase and expressed based on the activity of
the pGL3-Basic or pGL3-Promoter plasmid as 1. The data are expressed as
the mean values ± S.E. of at least three experiments (duplicate
samples). The p values were obtained using Student's t test.
Electrophoretic Mobility Shift Assays (EMSA)--
Nuclear
extracts of various cells were prepared as described previously (27).
Single-stranded oligonucleotides were annealed at a concentration of 10 µM in annealing buffer (1 mM Tris-HCl (pH
7.5), 1 mM MgCl2, and 5 mM NaCl) at
95 °C for 5 min and then cooled to room temperature. Double-stranded
DNA was end-labeled with [
Nuclear extracts (5 µg) and, when indicated, unlabeled
oligonucleotide competitors were preincubated in 23 µl of the gel
mobility shift assay buffer (10 mM HEPES-KOH (pH 7.9), 50 mM KCl, 0.6 mM EDTA, 5 mM
MgCl2, 10% glycerol, 5 mM dithiothreitol, 0.7 mM phenylmethylsulfonyl fluoride, 2 µg/µl
pepstatin A, 2 µg/µl leupeptin, and 87 ng/µl poly(dI-dC)
(Amersham Biosciences)) for 10 min on ice. An oligonucleotide probe
(1 × 105 cpm) was added to the mixture and incubated
for an additional 30 min at room temperature. For antibody supershift
analyses, 1 µl of antibody was added and the incubation was continued
for an additional hour. The antibodies used for the supershift analyses were raised against Sp1 (PEP 2, Santa Cruz Biotechnology, Santa Cruz,
CA), Sp2 (K-20, Santa Cruz Biotechnology), Sp3 (D-20, Santa Cruz Biotechnology), and YY1 (H-414, Santa Cruz Biotechnology). DNA-protein complexes were separated from the free probe by 5% non-denaturing polyacrylamide gel electrophoresis. After
electrophoresis, the gel was blotted onto Whatman 3MM paper, dried, and
analyzed using a BAS2000 Image Analyzer (Fuji film, Tokyo, Japan).
The competitors used in EMSA were obtained by annealing of the
following oligonucleotides: wild-type Sp1, 5'-ATTGGATCGGGGCGGG GCGAGC-3' (forward) and 5'-GCTCGCCCCGCCCCGATCCAAT-3' (reverse); mutated
Sp1, 5'-ATTGGATCGGTTCGGGGCGAGC-3' (forward) and
5'-GCTCGCCCCGAACCGATCCAAT-3' (reverse); wild-type YY1,
5'-CGCTCCGCGGCCATCTTGGCGGCTGGT-3' (forward) and
5'-ACCAGCCGCCAAGATGGCCGCGGAGCG-3' (reverse); and mutated YY1, 5'-CGCTCCGCGATTATCTTGGCGGCTGGT-3' (forward) and
5'-ACCAGCCGCCAAGATAATCGCGGAGCG-3' (reverse).
Promoter Activity of the 5'-Flanking Sequence of the Mouse LAMA1
Gene--
A LAMA1 genomic clone was isolated from a mouse
BAC genomic library, and a 6.2-kb DNA fragment containing the
5'-flanking region of the mouse LAMA1 gene was subcloned and
fully sequenced. This sequence is available through the
GenBankTM data base (GenBankTM accession number
AB097426). Previously, Sasaki et al. (28) estimated the
5'-untranslated region of the mouse laminin
To identify the cis-regulatory elements controlling the
mouse LAMA1 gene transcription, a series of reporter
plasmids driven by the 5'-flanking region of the LAMA1 gene
of different lengths were constructed and transfected into mouse F9
cells before and after induction of differentiation by retinoic acid
and dibutyryl cAMP (Figs.
1A-C). When compared with the
Characterization of a Cell Type-specific Enhancer--
To further
localize the region critical for the enhancer activity, an 800-bp
AflII fragment from
To verify the activity of the 435-bp region as a cell type-specific
enhancer, this fragment was cloned 5' to the SV40 promoter in both the
forward and reverse orientation or as two copies in tandem and their
enhancer activities were examined in F9-stem cells, F9-PE cells, PYS-2
cells, EHS tumor-derived cells, and NIH/3T3 fibroblasts (Fig.
3). The 435-bp fragment conferred high luciferase activity independent of its orientation in F9-PE, PYS-2, and
EHS tumor-derived cells but not in F9-stem and NIH/3T3 fibroblasts. The
tandem repeat of the 435-bp fragment was more potent than a single copy
in the enhancer activity. Given that the enhancer activity was only
detected in cells with parietal endoderm phenotypes, we concluded that
the 435-bp SacI-BglII ( Characterization of Nuclear Protein Binding by EMSA--
To
determine the regions in the 435-bp enhancer that interact with
DNA-binding proteins, we prepared a series of overlapping double-stranded oligonucleotides (data not shown) altogether covering the whole segment and used them as probes for EMSA analyses. Among 24 sets of oligonucleotides, four oligonucleotides designated protein
binding sites (PBS) 1-4 formed DNA-protein complexes with nuclear
extracts from EHS tumor-derived cells (Fig.
4). All of the four DNA-protein complexes
were detected not only with nuclear extracts from F9-PE and PYS-2 cells
but also with those from F9-stem and NIH/3T3 cells, implying that the
binding proteins are not unique to parietal endoderm cells.
To narrow down the enhancer activity within these four DNA segments, a
series of mutant double-stranded oligonucleotides with 6-bp
substitutions were used as competitors for the complex formation of a
32P-labeled probe and nuclear proteins (Fig.
5). For PBS1, an excess amount of
unlabeled oligonucleotides mut1-2 and mut1-3 competed with the protein
binding, whereas mut1-1 failed to compete. These results indicate that
a substituted sequence in mut1-1 (ATTAAG) is critical for the
DNA-protein complex formation. Similarly, the nucleotide sequences
substituted in mut2-3 (TAGGTG), mut3-1 (CCATCC), and mut4 Contribution of Individual Elements to Enhancer
Activity--
We next examined the contribution of these putative
enhancer elements to the overall enhancer activity of the 435-bp
fragment by introducing mutations in the 6-bp core sequences of the
PBS1, PBS2, PBS3, and PBS4 segments (Fig.
6). Mutation at PBS1 had no significant
effects on the 435-bp enhancer activity, although mutation in PBS2,
PBS3, and PBS4 reduced the enhancer activity by 72%, 93%, and 48%,
respectively. Double mutations in these three elements resulted in
further reduction of the enhancer activity to 2-5% of the control,
and mutations of all three sites completely abolished the enhancer
activity. Similar results were observed in PYS-2 cells, but not in
NIH/3T3 cells (data not shown). Mutation in the PBS3 element alone
eliminated more than 90% of the enhancer activity, suggesting that
PBS3 is the most critical for the enhancer activity. In contrast,
mutation in the PBS4 element had only a modest effect. These data are
consistent with the results that the XmnI-BglII
fragment (
Computer analyses using the TFSEARCH program (30) suggested that PBS2
and PBS4 contained putative binding motifs for Sp1-like (GTGTGG) and
YY1 (TAATGG) transcription factors, respectively. To test whether these
factors were responsible for the observed protein binding to PBS2 and
PBS4, we performed competition and supershift assays. Competitor
oligonucleotides with an authentic Sp1 (GGGGCGGGGC) or YY1 (GCGGCCATCT)
binding motif abolished protein binding to PBS2 and PBS4, respectively,
although those with mutations in the Sp1 or YY1 motif failed to compete
(Fig. 7, A and B).
Furthermore, antibodies to Sp1 and Sp3 produced supershifted complexes,
whereas antibodies to YY1 inhibited PBS4-protein complex formation.
Therefore, it seems probable that Sp1/Sp3 and YY1 bind to the PBS2 or
PBS4 sequences, respectively.
Parietal endoderm derives from the primitive endoderm, which in
turn derives from the inner cell mass of the blastocyst at 4.0-4.5
days post coitum in the mouse (31). Parietal endoderm cells are the
major fetal components of the yolk sac, synthesizing large amounts of
laminin and collagen IV, which are incorporated into Reichert's
membrane (32). Reichert's membrane plays a critical role in the
maternofetal exchange of nutrients (33) and is important for the
postgastrulation development of the murine embryo. Because parietal
endoderm cells continually secrete large amounts of Reichert's membrane components during development, they may be regarded as an
active in vivo protein biosynthetic system. However, the
regulatory mechanisms of genes encoding Reichert's membrane components
remain poorly understood. Elucidation of such mechanisms could have a significant impact on developing a system for the large scale biosynthesis of basement membrane components in vitro.
In this study, we have cloned the promoter region of the mouse
LAMA1 gene and identified the distal enhancer ( Sp1/Sp3 and YY1 have broad tissue distribution and have been implicated
in the regulation of several tissue-specific genes as well as
housekeeping genes (34-37). Sp1 binding sites have also been
identified in heat shock protein 47 (38) and the laminin YY1 is also known to act as a transcriptional activator or repressor
depending on its promoter context. The transcriptional activity of YY1
appears to be regulated at the posttranslational level, possibly
through interaction with other proteins. In fact, a wide variety of
transcription factors including Sp1 and nuclear receptor co-activators
have been shown to associate with YY1 (34, 36, 42-44). Considering
these findings, the parietal endoderm-specific activation of the
LAMA1 gene may be controlled by complex transcriptional pathways involving interactions among three ubiquitous factors (Sp1/Sp3, YY1, and an unidentified factor), tissue-specific co-factors, and posttranslational modification such as phosphorylation and acetylation. Recently, it was demonstrated that Akt/protein kinase B
activates the transcription of all three chains of laminin-1 as well as
type IV collagen (45). It has also been shown that the DNA binding and
transcriptional activities of YY1 and Sp1/Sp3 are regulated by
acetylation and deacetylation (44, 46, 47). It remains to be explored
whether Sp1/Sp3, YY1, and an unidentified factor binding to PBS3 are
targets of such modifications.
Although parietal endoderm-specific enhancer elements have been
identified in the In conclusion, we have identified a parietal endoderm-specific enhancer
of the mouse LAMA1 gene, which could explain the increased mRNA levels of laminin-1 during early mouse development. Further characterization of this enhancer, i.e. identification of
the nuclear protein(s) binding to PBS3 and/or other factors interacting with Sp1/Sp3 and YY1, will clarify the novel mechanism(s) operating in
the regulation of parietal endoderm-specific gene expression. This
435-bp enhancer system may also provide a clue to understanding the
molecular basis of the large amount of production of basement membrane
components in EHS tumor and parietal endoderm cells.
1,
1, and
1 chains.
The expression of laminin-1 is induced in mouse F9 embryonal carcinoma
cells upon differentiation into parietal endoderm through
transcriptional up-regulation of the genes encoding these subunits.
Here, we identified a 435-bp enhancer in the 5'-flanking region of the
mouse laminin
1 (LAMA1) gene that activated its
transcription in a differentiation-dependent manner. This
enhancer was also active in PYS-2 parietal yolk sac-derived cells but not in NIH/3T3 fibroblasts, indicating that it was a parietal
endoderm-specific enhancer. This enhancer was also active in
Engelbreth-Holm-Swarm (EHS) tumor-derived cells characterized by
excessive production of laminin-1 and other basement membrane components, suggesting that EHS tumors have a transcriptional control
mechanism similar to that of parietal endoderm cells. Electrophoretic
mobility shift analyses revealed four protein binding sites (PBS1-PBS4)
in the 435-bp region. However, these DNA-binding proteins were detected
not only in parietal endoderm cells (i.e. differentiated F9
cells, PYS-2 cells, and EHS tumor-derived cells) but also in
undifferentiated F9 cells and NIH/3T3 cells. Mutational analyses
revealed that three of these binding sites (PBS2, PBS3, and PBS4)
function synergistically to confer the parietal endoderm-specific
enhancer activity. The proteins binding to PBS2 and PBS4 were
identified as the Sp1/Sp3 family of transcription factors and YY1, respectively.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
and
, which are assembled and disulfide-bonded in a cross-shaped structure with three
short arms and one long rodlike arm. To date, five
chains (
1-5), three
chains (
1-3), and three
chains (
1-3)
have been identified, and these assemble into at least 12 distinct laminin isoforms (4-7). Among these isoforms, laminin-1 is the major
component of the early embryonic basement membrane and has been shown
to be required for normal development (8, 9).
1,
1, and
1 chains (10, 11).
The coordinate expression of these subunit genes during F9 cell
differentiation suggests that a common mechanism is operating in their
transcriptional regulation. Several studies on the transcriptional
regulation of laminin subunit expression during the differentiation of
F9 cells have been reported previously (12). In the laminin
1
(LAMB1) gene promoter, a retinoic acid-responsive element
has been identified previously (13-16), whereas
differentiation-dependent elements in the first intron have
been identified in the laminin
1 (LAMC1) gene (17).
However, the molecular mechanisms mediating the coordinate activation
of these genes are poorly understood, and the function of the laminin
1 (LAMA1) gene promoter has not been studied in any species.
1 and
1 chains are common components of several laminin
isoforms (laminin-1, -2, -6, -8, and -10) and have a wide distribution in basement membranes. In contrast, the laminin
1 chain has a restricted tissue distribution and is predominantly expressed in the
epithelial basement membrane during embryonic development (5, 18-21).
Moreover, the laminin
1 chain expression is thought to be the
limiting factor in the secretion of laminin-1, because the
1 and
1 chains, which are preassembled into a disulfide-linked
1-
1
dimer, cannot be secreted without the trimeric assembly with the
1
chain (22). These findings prompted us to investigate the mechanism
restricting the laminin
1 chain expression in a differentiation-dependent and cell type-specific manner.
1 expression during F9 cell differentiation.
This enhancer was also active in the PYS-2 mouse teratocarcinoma cell line that exhibits parietal endoderm phenotypes (23) but not in NIH/3T3
fibroblasts, suggesting that this enhancer functions in a parietal
endoderm-specific manner. Interestingly, this enhancer was also active
in Engelbreth-Holm-Swarm
(EHS)1 tumor-derived cells,
which are characterized by an excessive secretion of laminin-1 and
other basement membrane components (24, 25). We further demonstrated
that the synergy of three cis-elements was required for the
enhancer activity. DNA-binding proteins interacting with two of these
cis-elements were identified as Sp1/Sp3 and YY1, zinc finger
transcription factors widely expressed in many tissues, suggesting that
posttranslational modifications of these factors and/or cooperative
interactions with other proteins are important for parietal
endoderm-specific enhancer activity.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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2527/
30 (relative to the initiation codon) plasmid. To generate the
longest promoter construct,
6198/
30, a blunted NsiI and
SpeI fragment was inserted into the
2527/
30 plasmid (see
Fig. 1). All of the 5'-deletion constructs were generated in these
plasmids by using the endogenous restriction sites and the appropriate
restriction sites in the polylinker.
3684
to
2892) from the mouse LAMA1 genomic DNA was inserted
into the AflII site of the pcDNA3.1(+) vector
(Invitrogen). The fragments with appropriate restriction sites at both
ends were inserted into the pGL3-Promoter vector (Promega) to generate
3684/
2892,
3684/
3516,
3082/
2892,
3516/
3082,
3516/
3214, and
3214/
3082 plasmids. Using a similar approach,
plasmids carrying a double copy and the reverse direction of the
3684/
2892 enhancer,
3516/
3082(+)x2, and 3516/
3082(
),
respectively, were constructed.
3516/
3082 plasmid using the
GeneEditorTM in vitro site-directed mutagenesis
system (Promega) and/or the Gene- TailorTM site-directed
mutagenesis system (Invitrogen). The sequences of the mutagenic primers
are available upon request. All of the mutants were verified by sequencing.
-32P]dCTP and the DNA
polymerase Klenow fragment (Invitrogen). Labeled DNA was separated from
free dCTP by filtration through a ProbeQuantTM G-50 Micro
Column (Amersham Biosciences).
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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1 transcript to be
128-bp long. According to this finding, neither a TATA box nor a CCAAT
box was found proximal to the putative transcription start site. Mouse
and human (GenBankTM accession number AC021879)
LAMA1 genes display a high degree of sequence conservation
in the proximal promoter regions (
200 ~+1), suggesting that the
LAMA1 proximal promoter regions contain binding sites for
the transcription factors necessary for basal expression in rodents and humans.
103/
30 plasmid, the
178/
30 plasmid showed significantly higher
activities in both undifferentiated and differentiated F9 cells
(designated F9-stem and F9-PE cells, respectively), indicating that the
basal promoter activity is localized within the
103 to
178 region
(FspI to SfoI). Six other reporter plasmids with
longer 5' sequences (i.e.
237/
30,
676/
30,
1036/
30,
2046/
30,
2527/
30, and
2888/
30) showed
transcriptional activity similar to
178/
30 in both F9-stem and
F9-PE cells. Intriguingly, the transcriptional activity in F9-PE cells
was dramatically elevated when the 5'-flanking region was extended to
3516, although such potentiation in transcriptional activity was not
observed in F9-stem cells. These results indicate that the 630-bp
region encompassing
3516 to
2888 contains an enhancer that is only
effective in F9 cells in the differentiated state. Because
differentiated F9 cells exhibit a parietal endoderm-like phenotype, we
examined the transcriptional activity of these deletion constructs in
the PYS-2 parietal yolk sac-derived cells as well as EHS tumor-derived
cells that secrete a large amount of laminin-1 (Fig. 1, D
and E). A dramatic increase in the transcriptional activity
was also detected with the
3516/
30 construct, but not with the
2888/
30 construct, in both PYS-2 and EHS tumor-derived cells,
whereas a basal promoter activity was also detectable within the
103
to
178 region. These results suggest that the 630-bp region from
3516 to
2888 harbors an enhancer activity closely associated with
parietal endoderm cells. Although the exact origin of the EHS tumor has
not been determined, overproduction of extracellular matrix proteins
similar to those of Reichert's membrane (29) as well as gene
expression profiles determined by microarray
analysis3 indicates that EHS
tumor cells are also parietal endoderm-like cells, lending support for
the parietal endoderm-specific enhancer activity within the
2888/
3516 630-bp region.
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Fig. 1.
LAMA1 promoter reporter gene
constructs and cell type-specific promoter activity in transient
transfection assays. A, a schematic representation of
the region around exon 1 of the LAMA1 gene. A series of
LAMA1 promoter reporter genes were constructed as described
under "Experimental Procedures." These constructs were transfected
into F9-stem (panel B), F9-PE (panel C), PYS-2
(panel D), and EHS tumor-derived cells (panel E)
together with phRL-null as an internal control. 48 h after
transfection, the cells were harvested for the luciferase assay. The
relative luciferase activities are shown as the means ± S.E. of
at least three experiments (duplicate samples).
3684 to
2892 and its 5'- and
3'-deletion constructs were tested directly for their enhancer activity
using the heterologous SV40 promoter. The 800-bp AflII fragment showed high enhancer activity (i.e. a 100-fold
increase relative to the basal promoter activity) in EHS tumor-derived cells (Fig. 2) as well as in F9-PE and
PYS-2 cells (data not shown). Deletion from the 3'-end to
3516
(AflII-SacI fragment) and from the 5'-end to
3082 (BglII-AflII fragment) abolished the
enhancer activity. In contrast, a 435-bp
SacI-BglII fragment covering nucleotides
3516
to
3082 retained the full enhancer activity, although further deletion constructs (
3516/
3214 and
3214/
3082) did not. These results indicate that both regions (SacI-XmnI and
XmnI-BglII) contain the regulatory element
required for the high expression of LAMA1 in EHS
tumor-derived cells, making it likely that the 435-bp region from
nucleotides
3516 to
3082 is sufficient for the enhancer activity in
EHS tumor-derived cells. Similar results were also obtained with F9-PE
and PYS-2 cells (data not shown).
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Fig. 2.
Identification of a minimal enhancer region
( 3516/
3082) by deletion analysis. The AflII
fragment (
3684 to
2892) subcloned into the pGL3-Promoter vector
showed full enhancer activity in EHS tumor-derived cells. As shown
schematically on the left, several deletion mutants were
constructed from this AflII fragment and tested for their
luciferase activity. The data are the means ± S.E. of at least
three experiments (duplicate samples).
3516 to
3082) fragment acts as a parietal endoderm-specific enhancer.
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Fig. 3.
Parietal endoderm-specific enhancer activity
of the 435-bp fragment ( 3516/
3082). The 435-bp fragment
(
3516 to
3082) was inserted to pGL3-Promoter in the forward (+) or
reverse (
) orientation as well as in a tandem repeat ((+)
× 2). The constructs were introduced into non-parietal
endoderm cells (F9-stem and NIH/3T3) or parietal endoderm cells (F9-PE,
PYS-2, and EHS tumor-derived cells). The activities are shown as the
means ± S.E. of at least three experiments (duplicate
samples).
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Fig. 4.
Localization of protein binding sites within
the 435-bp enhancer. A, sequence of the mouse
LAMA1 435-bp enhancer ( 3516 to
3082). The regions
forming DNA-protein complexes in EMSA (from PBS1 to PBS4) are indicated
with lines with arrowheads at both ends. The
putative Sp1/Sp3 and YY1 binding sites are shown in bold and
italics. The restriction sites depicted in Fig. 2 are
boxed. B, DNA-protein complex formation on
PBS1-PBS4. Nuclear extracts prepared from F9-stem, F9-PE, PYS-2, EHS,
and NIH/3T3 cells were used with the indicated oligonucleotide as
probes.
2 (ATAATG)
were identified to be critical for protein binding in PBS2, PBS3, and
PBS4, respectively.
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Fig. 5.
Delineation of sequence motifs essential for
nuclear protein binding using mutated oligonucleotide competitors.
A, sequences of the oligonucleotides used as competitors in
EMSA. Wild-type and mutant oligonucleotide sequences containing 6-bp
substitutions are shown. B, the interaction of
32P-labeled oligonucleotide probes with DNA-binding
proteins were analyzed in the presence of a 100-fold excess of
unlabeled specific (S), nonspecific (NS), and
mutated competitors to delineate regions critical for DNA-protein
interaction. Nuclear extracts were prepared from EHS tumor-derived
cells.
3214/
3082) lacking PBS1 through PBS3 showed little, if
any, enhancer activity, whereas the SacI-XmnI fragment (
3516/
3214) lacking only PBS4 had significant enhancer activity (Fig. 2). Together, these results indicate that synergy of
three protein binding sites (PBS2, PBS3, and PBS4) accounts for the
bulk of the activity of the 435-bp enhancer.
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Fig. 6.
Functional analysis of the protein binding
sites on the 435-bp enhancer activity. pGL3-Reporter plasmids
containing the wild-type 435-bp enhancer ( 3516/
3082) and the
mutated enhancers (mut1-4) were transfected into EHS tumor-derived
cells and tested for luciferase activity. The regions altered by
site-specific mutagenesis are indicated by X. The values
represent the percentage of the luciferase activity (mean ± S.E.)
of three separate experiments (versus the activity of the
wild-type construct (
3516/
3082). **, p < 0.01; *, p < 0.05).
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Fig. 7.
Binding of Sp1/Sp3 and YY1 to the 435-bp
enhancer. The 32P-labeled PBS2 (panel A)
and PBS4 (panel B) oligonucleotides were incubated with
nuclear extracts from EHS tumor-derived cells. Competition assays were
performed with a 100-fold excess of unlabeled specific (S),
nonspecific (NS), wild-type consensus (Sp1,
YY1), or mutated (Sp1mut, YY1mut)
oligonucleotides. For the antibody supershift analysis, Sp1-, Sp2-,
Sp3-, or YY1-specific polyclonal antibodies were added to the reaction
mixture. The asterisk points to the supershifted band. Note
that the DNA-protein complex formation was completely abrogated in the
presence of the YY1 antibody.
DISCUSSION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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3516 to
3082) responsible for the expression of the laminin
1 chain during the parietal endoderm differentiation of F9 cells. The enhancer was
also active in PYS-2 and EHS tumor-derived cells but not in NIH/3T3
cells, suggesting that the enhancer activity is parietal endoderm-specific. Consistent with the definition of an enhancer, the
435-bp sequence enhanced luciferase gene expression in either the
forward or reverse orientation from the heterologous SV40 promoter. By
EMSA analysis, four protein binding sites (PBS1-PBS4) were identified
in the 435-bp enhancer. Although the proteins binding to these elements
were detected not only in parietal endoderm cells but also in
undifferentiated F9 and NIH/3T3 cells, three of these elements (PBS2,
PBS3, and PBS4) appear to be essential for the parietal
endoderm-specific enhancer activity. The proteins binding to PBS2 were
identified as Sp1/Sp3, and the proteins binding to PBS4 were identified
as YY1.
1 genes
(39), both of which are highly expressed in F9 cells differentiated
into parietal endoderm cells. However, it remains to be determined
whether these sites are involved in their parietal endoderm-specific
expression. Supershift analyses with anti-Sp1, anti-Sp2, and anti-Sp3
antibodies revealed that either Sp1 or Sp3 could bind to PBS2. It has
been reported that Sp3 can function as a positive regulatory factor or
as a repressor of Sp1-mediated transcription depending on its
alternatively spliced isoforms (40, 41). Further studies are required
to determine which isoforms are involved in the 435-bp enhancer activity.
1(IV) and
2(IV) collagen genes (48, 49), the
proteins binding to these elements have not been identified. There is
no clear sequence similarity between the enhancer elements in the
collagen IV genes and the presently identified 435-bp enhancer. A
parietal endoderm-specific enhancer has also been identified in the
5'-flanking region of the platelet-derived growth factor
receptor
gene (50), the expression of which is also induced in F9 cells during
the differentiation into parietal endoderm cells. GATA-4, a member of
the GATA transcription factor family, is considered to be responsible
for the platelet-derived growth factor
receptor enhancer activity.
This is consistent with a recent report that GATA-4 and GATA-6 are key
regulators of differentiation of the extra-embryonic endoderm (51). The
435-bp enhancer has several GATA-like motifs, but it seems unlikely
that these motifs are involved in the DNA-protein complex formation,
because the double-stranded oligonucleotides containing the GATA-like
motifs did not produce any significantly shifted band in the EMSA
analysis and GATA-4 failed to activate the 435-bp enhancer (data not
shown). These observations indicate that the parietal endoderm-specific gene expression can be conferred by either GATA-dependent
or GATA-independent mechanisms. In search of the parietal
endoderm-specific enhancer of the LAMB1 and LAMC1
genes, we cloned ~4-kb and ~7-kb genomic DNA segments covering the
5'-flanking regions of the LAMB1 and LAMC1 genes
and examined their enhancer activity in PYS-2 cells. However, none of
these DNA segments showed as strong transcriptional activity as the
435-bp enhancer.4 Further
sequences upstream of these region or the introns of the mouse
LAMB1 and LAMC1 genes may contain a regulatory
element similar to the 435-bp enhancer.
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ACKNOWLEDGEMENTS |
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We thank Dr. Atsuhiko Oohira for providing the PYS-2 cell line, Dr. Koji Kimata for providing the EHS tumor, and Dr. Masakuni Okuhara for helpful discussions and critical review of the paper.
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FOOTNOTES |
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* 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: Sekiguchi Biomatrix Signaling Project, ERATO, Japan Science and Technology Corporation, 21, Karimata, Yazako, Nagakute, Aichi 480-1195, Japan. Tel.: 81-561-64-5020; Fax: 81-561-64-2773; E-mail: hayashiy@aichi-med-u.ac.jp.
Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M212578200
2 Y. Hayashi, unpublished observations.
3 S. Futaki and Y. Hayashi, unpublished observations.
4 T. Niimi, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: EHS tumor, Engelbreth-Holm-Swarm tumor; BAC, bacterial artificial chromosome; EMSA, electrophoretic mobility shift assay; PBS, protein binding site; kb, kilobase(s).
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