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J. Biol. Chem., Vol. 276, Issue 40, 37011-37019, October 5, 2001
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From the
Received for publication, May 9, 2001, and in revised form, June 27, 2001
The transcription of type I collagen genes is
tightly regulated, but few cis-acting elements have been identified
that can modulate the levels of expression of these genes. Generation
of transgenic mice harboring various segments of the mouse pro- Type I collagen is a fibrillar collagen composed of two Different positive regulatory sequences have been identified in the
pro- Besides cis-acting elements able to modulate the level of
transcription of the pro- In order to identify new cis-regulatory elements within the
mouse pro- Plasmid Constructions--
We used two previously published
plasmids containing segments of the mouse pro-
pDR583 contains a segment of the Hoxb-7 promoter, extending
from
pGL3 control vector, pSV
pCMVX- Generation and Analysis of Transgenic Mice--
Transgenes and
transgenic embryos were generated using standard procedures (20, 21).
Cell Lines--
ROS17/2.8 cells are rat osteoblastic cells,
which produce type I collagen. They were cultured in 50% Dulbecco's
modified Eagle's medium, 50% HAMF12 (Life Technologies, Inc.),
supplemented with 10% fetal calf serum (Life Technologies).
RC.SVtsA58 cell line is a rabbit cortical collecting duct cell line
that was obtained in our laboratory (23) and does not produce type I
collagen. These cells were cultured in 50% Dulbecco's modified
Eagle's medium, 50% HAMF12 supplemented with 2 mM
glutamine, 5 mg/liter insulin, 50 nM dexamethasone, 5 mM transferrin, 30 nM selenium, 20 mM Hepes, and 2% fetal calf serum.
Transfection Experiments--
For transient transfection
experiments, cells were plated at 400,000 cells/well in six-well plates
(Nunc, Kamstrup, Denmark) and transfected using LipofectAMINE (Life
Technologies) following the manufacturer's instructions. In each well,
0.25 pmol of lacZ-containing plasmids were co-transfected
with 0.10 pmol of pGL3 control vector. In some experiments, pCMVX and
pCMVX-
For stable transfection experiments, linearized
lacZ-containing plasmids were mixed with linearized pSVneo
in a 10:1 molar ratio. They were transfected as described above in
10-cm diameter Petri dishes (Nunc). Seventy-two hours after the start
of transfection, cells were incubated in medium supplemented with 100 µg/ml G418 (Life Technologies). Under these conditions, untransfected
cells died within 10 days. Experiments were done in triplicate. In each triplicate, the transfected clones were pooled to eliminate an integration site effect.
In Vitro DNase I Digestion and Southern Blotting--
Cells were plated
at a density of 500,000 cells/10-cm Petri dish (Nunc) and grown to
confluence. Nuclei were isolated as previously described (25).
Approximately 107 nuclei were resuspended in 90 µl of a
buffer containing 15 mM Tris-HCl (pH 7.5), 15 mM NaCl, 60 mM KCl, 0.5 mM
spermidine, 0.5 mM spermine, 0.34 M sucrose, 1 mM dithiothreitol. Then 10 µl of assay buffer containing
0-60 IU/reaction of DNase I (Roche Molecular Biochemicals) were added
to the nuclei. The mixture was incubated at 37 °C for 15 min, and
the reaction was stopped by adding 200 µl of a buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM EDTA, 100 mM NaCl, 1% SDS, 800 µg of proteinase K. Proteins were
then digested overnight at 55 °C. The DNA was purified and digested with appropriate restriction enzymes with standard techniques (20). It
was then electrophoresed on a 0.8% agarose gel and blotted on a
Zeta-Probe membrane (Bio-Rad). This membrane was then processed
following the manufacturer's recommendations and hybridized with a
825-bp lacZ probe.
Electrophoretic Mobility Shift Assay--
Nuclear extracts from
ROS 17/2.8 cells were prepared as previously described (26). The probes
were end-labeled by filling in with [ Northern Blot Analysis--
Northern analysis of pro- Statistical Analysis--
To compare different groups of
transgenic embryos, we performed statistical analysis according to
analysis of variance followed by the Fisher's protected least
significant difference test (Statview).
Analysis of Transgenic Mice Suggests the Existence of a Repressor
Element in the Pro-
We first generated transgenic mice using pC3.112. This construct
contains 2.3 kb of the pro-
We then used pC15.56 to generate transgenic mice (Fig. 1A).
Three out of seven transgenic embryos expressed lacZ in
ossification and tendons at high levels, one expressed it at low
levels, and three did not show any X-gal staining (Fig.
1B).
To test the role of sequences located upstream of
Taken together, these data showed that the percentage of founder
embryos expressing the reporter gene at high levels was significantly lower with the two constructs containing the 6.5-kb pro-
Furthermore, we performed histological analysis of transgenic embryos
sections to precisely map the pattern of expression of the transgenes
out of ossification centers and tendons (data not shown). In
particular, transgenic embryos, at 15.5 days postconception never
disclosed X-gal staining in organ capsules, the lung, the trachea, the
pericardial membranes, the aortic trunk, the cardiac valves, the
digestive tract, the metanephros, the muscles, and the soft connective
tissues. These data suggested that neither the pro- The Repressor Activity of the 6.5-kb Fragment Is Not Mediated by
Modification of Chromatin Conformation--
Since chromatin structure
appears to play an important role in regulating gene expression, and in
particular the pro-
First, nuclei isolated from cells stably transfected with pC6.267 were
incubated with increasing amounts of DNase I, and the subsequently
extracted DNA was digested with NarI (Fig. 2B).
The DNA fragments were then separated on an agarose gel and blotted onto a nylon membrane, which was hybridized with a probe corresponding to lacZ. The probe hybridized with four fragments of
different size (Fig. 2, B and D). The high
molecular weight one was generated by NarI digestion of the
DNA extracted from nuclei untreated with DNase I. The 8- and 7-kb ones
were generated by NarI digestion and DNase I digestion at
two hypersensitive sites located in the pro-
Nuclei isolated from ROS17/2.8 cells stably transfected with pC6.273
were also used to identify DNase I-hypersensitive sites within the
transgene. They were incubated with increasing amounts of DNase I, and
the extracted DNA was digested with HindIII (Fig. 2C). The lacZ probe hybridized with a 10.5-kb
HindIII restriction fragment when DNA was extracted from
DNase I-untreated nuclei. In nuclei treated with increasing amounts of
DNase I, the lacZ probe hybridized with additional DNA
fragments migrating at about 7, 6.5, and 4 kb (Fig. 2, C and
E). These fragments correspond to HindIII
digestion and DNase I digestion at hypersensitive sites located in the
proximal promoter, the first intron, and the fifth intron, respectively.
The DNase I-hypersensitive sites in the two transgenes were identical
to those present in the endogenous gene, in type I collagen-producing cells, suggesting that the repressor effect was not mediated by modifications of the chromatin structure.
A 6.5-kb Fragment of the Pro-
To test the ability of the repressor element to inhibit the activity of
an heterologous promoter, the pro- Identification of a 123-bp Repressor Element Using Deletion
Analyses of the Pro-
We then tested whether the inhibitory sequence was still active when it
was cloned far from the transcription start site, as in the endogenous
gene. For this purpose, we used a construct, named pC15.56.2XN, in
which the 2-kb inhibitory sequence was located 13 kb upstream of the
transcription start site (Fig. 4).
To identify more precisely the inhibitory element, transient
transfection experiments were performed using subsegments of the 2-kb
element, cloned upstream of 3.2 kb of the pro-
Sequencing of the 312-bp segment showed the existence of a 123-bp
sequence that is an almost perfect three-time repeat of a 41-bp motif
(Fig. 5A). This 123-bp
decreased the activity of the reporter gene by 37%, suggesting that it
corresponded to the repressor element (Fig. 5B). This
sequence was named COIN-1 (for collagen-inhibitory element-1).
Each of the three 41-bp motifs contains a CACCTG sequence, known as an
E2-box. To test whether the E2-boxes were important in mediating the
inhibitory effect of COIN-1, we compared Repression Correlates with the Binding of
Three retarded complexes (complexes B, C, E, in Fig.
6) were seen when WT1 was used as a
probe. Complexes B and C, but not complex E, were competed by a
100-fold molar excess of the corresponding unlabeled probe (Fig. 6).
One additional complex (complex A) was seen when WT2 was used as a
probe (Fig. 6). In contrast, complex A was not seen when MUT2 or DEL2
were used as probes (Fig. 6). Complex A disappeared when a
30-fold or a 100-fold molar excess of the unlabeled wild type probe
(WT2) was added to the binding reaction (Fig. 6). In contrast, complex
A was not competed by a 30- or 100-fold molar excess of the unlabeled
mutated probe (MUT2). Taken together with the results of transfection
experiments, these data suggested that complex A corresponded to the
binding of a repressor factor.
Computer analysis of COIN-1 suggested that the E2-boxes could be
involved in the binding of a transcription factor named Overexpression of
To study the effect of Molecular mechanisms governing type I collagen gene expression are
still quite elusive, and in particular the elements that down-regulate
the highly active pro- Electromobility shift assays showed that COIN-1 was able to bind a
transcription factor named The fact that COIN-1 is located far upstream of the transcriptional
start site and that its inhibitory effect is
orientation-dependent raises the question of its mode of
action. In eukaryotes, transcription can be repressed through different
mechanisms, including modifications of chromatin structure,
interference with the binding of activators, and interactions with
components of the general transcription machinery (reviewed in Ref.
38). The binding of Analysis of the transgenic embryos confirmed that an
osteoblast-specific element is located within 2.3 kb of the pro- In conclusion, we have identified a 123-bp repressor element located at
about 14 kb upstream of the transcription start site in the mouse
pro- We thank B. de Crombrugghe for the generous
gift of ROS 17/2.8 cells and H. Kondoh for the generous gift of the
pCMVX- *
This work was supported by grants from the Association pour
la Recherche sur le Cancer (to J. R.) and from the University of Paris
(to J. R.).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.
§
Recipient of a fellowship from the Ministère de l'Education,
de la recherche, et de la Technologie.
Published, JBC Papers in Press, July 25, 2001, DOI 10.1074/jbc.M104185200
The abbreviations used are:
bp, base pair(s);
kb, kilobase pair(s);
X-gal, 5-bromo-4-chloro-3-indolyl-
EF1 Binds to a Far Upstream Sequence of the Mouse Pro-
1(I)
Collagen Gene and Represses Its Expression in Osteoblasts*
§,,, and
INSERM U489 and Université Paris VI,
Paris, France and ¶ Cohesion Technologies,
Palo Alto, California 94303
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1(I) collagen promoter led us to suspect that a repressor element was located between 
10.5 and 17 kilobase pairs. Stable and
transient transfection experiments in ROS17/2.8 osteoblastic cells
confirmed the existence of such a repressor element at about 14
kilobase pairs and showed that it consisted in an almost perfect
three-time repeat of a 41-base pair sequence. This element, which we
named COIN-1, contains three E2-boxes, and a point mutation in at least two of them completely abolished its repressor effect. In gel shift
assays, COIN-1 bound a DNA-binding protein named 
EF1/ZEB-1, and
mutations that abolished the repressor effect of COIN-1 also suppressed
the binding of EF1. We also showed that the repressor effect of
COIN-1 was not mediated by chromatin compaction. Furthermore, overexpression of EF1 in ROS17/2.8 osteoblastic cells enhanced the
inhibitory effect of COIN-1 in a dose-dependent manner and repressed the expression of the pro-1(I) collagen gene. Thus, EF1
appears to repress the expression of the mouse pro-1(I) collagen
gene, through its binding to COIN-1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
chains and one 2 chain coiled around each others in a triple helix. It is the most abundant protein of mammalian bodies, and a major component of most extracellular matrices. In the extracellular space,
type I collagen molecules self-assemble into highly organized fibrils
and then fibers, which largely contribute to the high tensile strength
of the structural framework supporting body structures (reviewed in
Ref. 1). Nevertheless, an abnormal accumulation of type I collagen,
along with other components of the extracellular matrix, can greatly
and irreversibly impair functions of various organs including lung,
kidney, liver, or skin. Thus, the production of type I collagen needs
to be tightly regulated, and this regulation appears to occur mostly at
a transcriptional level (reviewed in Ref. 2). It involves a control of
the levels of expression of type I collagen genes as well as a control
of their coordinate expression and their cell-specific expression.
1(I) collagen gene (reviewed in Ref. 2). The 220-bp pro-1(I)
proximal promoter is extremely active in transfection experiments and
in vitro transcription assays, and it has been described as
one of the most potent eukaryotic promoters (3, 4). It contains
enhancers such as a CCAAT-box, Sp1-binding sites, and other GC-rich
sequences (4, 5). The first intron of the pro-1(I) collagen gene
also contains positive regulatory elements such as an AP-1 binding site
(6) or an Sp1-binding site that is involved in maintaining bone density
(7). The role of this site in maintaining normal levels of expression
of the pro-1(I) collagen gene throughout life has been shown by knock-in experiments (8). A cis-acting element located in
the 3'-flanking region of the pro-1(I) collagen gene has been shown to drive high levels of reporter gene expression in transiently transfected fibroblastic cells (9). Contrasting with the existence of
these enhancers, type I collagen turnover appears to be a slow process,
which suggests that inhibitory factors are essential to control the
overall level of expression of the pro-1(I) collagen gene.
Nevertheless, few repressor elements have been described in this gene.
An inhibitory element located between 361 and 339 bp1 has been identified in
the mouse pro-1(I) collagen promoter, in transient transfection
experiments (10). A GC-rich repressor element has also been described
in the first intron of the human gene (11). It had a repressor effect
in transient transfection experiments, but sequences adjacent to this
element completely abolished its repressor effect (11). Moreover,
deletion of most of the first intron did not increase the levels of
expression of the pro-1(I) collagen gene in vivo (8). A
member of the Krüppel-like family of transcription factors named
cKrox is the only transcription factor that has been reported as being
able to down-regulate the level of expression of the pro-1(I)
collagen gene (12). Nevertheless, its role in modulating the
transcription of this gene remains controversial (12, 13). In transient transfection experiments, overexpression of the mouse cKrox gene enhanced the transcription of a reporter gene cloned downstream of a
promoter containing three copies of a cKrox binding site (13), while
overexpression of a truncated form of the human cKrox gene had an
inhibitory effect on the expression of the pro-1(I) collagen gene
(12).
1(I) collagen gene, regulatory elements responsible for its cell-specific expression have been identified. Only
a discrete subset of cells of mesenchymal origin synthesize type I
collagen. These cells are mostly fibroblasts, osteoblasts, and
odontoblasts. Studies performed using transgenic mice harboring various
fragments of the mouse, rat, or human pro-1(I) collagen gene have
shown that there is a modular arrangement of separate cell-specific
cis-acing elements responsible for the expression of the
pro-1(I) collagen gene in different type I collagen-producing cells
(14-16). So far, three cell-specific elements have been
identified within the mouse gene: an element located within 900 bp of
the proximal promoter induced reporter gene expression in some skin fibroblasts; a second element located between 1656 and 1570 bp
conferred high levels of reporter gene expression in osteoblasts and odontoblasts; and a third element located between 2300 and 3200 bp conferred reporter gene expression in tendon and fascia fibroblasts (14, 17). The cis-acting element(s) responsible for the expression of the pro-1(I) collagen gene in fibroblasts other than those present in fascia, tendons and skin remain(s) to be identified.
1(I) collagen gene, we have generated transgenic mice harboring segments of the corresponding promoter extending up to 17
kb and containing or not containing the first five introns of the gene.
Analysis of these mice led to the identification of a repressor element
that we named COIN-1 (for collagen-inhibitory element-1). COIN-1 is a three-time repeat of a 41-bp motif
containing an E2-box and is located 14 kb upstream of the
transcriptional start site. It binds a widely expressed transcription
factor called EF1/ZEB-1, which appears to be responsible for the
repressor effect of COIN-1, and is able to down-regulate the level of
expression of the endogenous gene independently on chromatin compaction.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1(I) collagen promoter
cloned upstream of the lacZ reporter gene in the placH
expression vector (14). pJ251 contains a segment of the pro-1(I)
promoter extending from 2310 to +110 bp. pJ320 contains a sequence of
this promoter extending from 3150 to +110 bp. Plasmids containing
segments of the mouse pro-1(I) promoter extending upstream of 3150
bp were constructed using placH and pDT816 (18). pDT816 is a cosmid
clone that contains all of the mouse pro-1(I) exons and introns and
17 kb of the 5'-flanking region. pC15.56 was generated by cloning an
SpeI/XbaI fragment extending from 15 kb to +110
bp in placH. pC3.112 was obtained by cloning an
XbaI/EcoRV fragment extending from +192 to +3196
bp in pJ251, immediately upstream of the lacZ gene, and in
frame with it. pC3.112 is thus coding for a fusion protein that
contains the segment of the pro-1(I) collagen chain encoded by the
first six exons of the pro-1(I) collagen gene (minus the signal
peptide), and lacZ. PC6.267 and pC6.273 were generated by
cloning an EcoRI/BamHI segment extending from
17 to 10.5 kb in pJ320 and in pC3.112, respectively. pC320.4 was
obtained by cloning an EcoRI/SpeI fragment
extending from 17 to 12.5 kb of the pro-1(I) promoter in pJ320.
pC320.1 was obtained by cloning an EcoRI/PstI
fragment extending from 17 to 16 kb in pJ320. pC320.2XN was
obtained by cloning a PstI/PstI fragment extending from 16 to 14 kb in pJ320, in the 5'-3' orientation. pC320.312, pC320.400, and pC320.1.3 were obtained by cloning
subfragments of this 2-kb segment: a SpeI/StuI
fragment, a StyI/SpeI fragment, and a
StyI/StyI fragment in pJ320, respectively.
pC320.1.5 was obtained by cloning a PstI/SpeI
fragment extending from 14 to 12.5 kb in pJ320. pC15.56.2XN was
obtained by cloning the PstI/PstI fragment
extending from 16 to 14 kb in pC15.56 in the 5'-3' orientation.
pC123, pC123.2.1m, pC123.2m, pC123.3m, and pC123.3d were obtained by
cloning double-stranded oligonucleotides in pJ320.
583 to +81 bp. It is cloned upstream of the firefly luciferase reporter gene, of an SV40 splice site and polyadenylation
signal, and downstream of a polyadenylation cassette which prevents
read-through transcription. In pDR6, the 6.5-kb pro-1(I) promoter
fragment extending from 17 to 10.5 kb was inserted upstream of the
Hoxb-7 promoter in pDR583.
-gal control vector, and pSVneo
contain the luciferase reporter gene, the -galactosidase reporter gene, and the neomycin resistance gene, respectively, cloned downstream of the SV40 promoter and enhancer (Promega).
EF1 contains the cDNA encoding EF1, cloned in the
pCMVX expression vector (19).
-Galactosidase activity was assessed on 15.5-day postconception
embryos as previously described (14). For each embryos, staining was
scored semiquantitatively, using a 0-2 scale, by an investigator not
aware of the construct harbored by the embryo. It was scored 0 for no
expression, 1 for low levels of expression, and 2 for high levels of
expression. To screen for transgenic mice, genomic DNA was extracted
from the embryo's yolk sacs with the DNeasy Tissue Kit (Qiagen,
Hilden, Germany) following the manufacturer's instructions, and a
sequence of the lacZ gene was amplified by PCR, as
previously described (22).
EF1 were also co-transfected in increasing concentrations
(0.05, 0.1, and 0.2 pmol). Reporter gene expression was measured
72 h after the start of transfection. All transfection experiments
were done in triplicate and repeated at least three times. Results are
expressed as mean ± S.E.
-Galactosidase Assay and Luciferase Assay--
Cell
extracts were prepared as previously described (24). -Galactosidase
activity was measured with the luminescent -galactosidase detection
kit (Roche Molecular Biochemicals) following the manufacturer's instructions in a luminometer (EG&G, Bad Wilbad, Germany). Luciferase activity was also assayed by using a luminometer as previously described (24).
-32P]dCTP using
the Klenow fragment of the E. coli DNA polymerase I. Then
0.3 ng of each probe was incubated with 10 µg of nuclear extracts at
room temperature for 30 min, in 20 µl of EF1 binding buffer (27).
Competition experiments were performed using a 30-100-fold molar
excess of nonlabeled competitor. Supershift experiments were performed
by adding an anti-EF1 antibody in the binding reaction. The
complexes were resolved by electrophoresis through 4% polyacrylamide
gels containing 22 mM Tris borate (pH 8.0) and 0.5 mM EDTA.
1(I)
collagen transcription was conducted with total RNA from ROS17/2.8
cells transfected with 0.1 pmol of pCMVX or 0.1 pmol of pCMVX-EF1.
Total RNAs were prepared with RNAwiz (Ambion, Austin, TX) following the
manufacturer's instructions. They were fractionated and blotted
onto nylon membrane (Amersham Pharmacia Biotech) using standard
techniques (20). Hybridization was performed using a random
prime-labeled cDNA probe for the pro-1(I) RNA (28). An 18 S RNA
probe was also used as a probe to control for differences in the total
amount of RNA loaded. The signals were quantitated using a STORM 860 PhosphorImager (Amersham Pharmacia Biotech) and the ImageQuant software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1(I) Promoter, and This Is Confirmed Using Stable
Transfection Experiments in ROS17/2.8--
In order to identify new
cis-acting regulatory elements within the mouse pro-1(I) collagen
gene, we generated transgenic mice harboring segments of the promoter
located upstream of 4 kb and/or the first five introns.
lacZ was used as a reporter gene, since X-gal staining
allows us to easily detect tissues and cells expressing the transgene
during embryonic development. Foster mothers were sacrificed at 15.5 days postconception, because at this time, the endogenous gene is
expressed in most type I collagen-containing tissues as well as in
ossification centers (28).
1(I) proximal promoter, a sequence extending from the 3'-end of exon 1 (and thus lacking the sequence coding for the signal peptide) to the 5'-end of exon 6 and
lacZ cloned in frame with the sixth exon (Fig.
1A). Out of 12 transgenic embryos, seven expressed lacZ in ossification centers at
high levels, one at low levels and four showed no staining after
overnight incubation with X-gal (Fig. 1B).
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Fig. 1.
Analysis of the levels of expression of the
lacZ reporter gene in transgenic mice and in stable
transfection experiments. A, partial map of pDT816 and
schematic representation of the constructs used to generate transgenic
mice. B, BamHI; E, EcoRI;
Sp, SpeI; A, Asp718I;
H, HindIII; X, XbaI;
N, NcoI. The black boxes
correspond to the first six exons. B, semiquantitative
analysis of the levels of expression of the lacZ gene in
ossification centers for each construct. Results are expressed as
percentages, and the corresponding absolute number of transgenic
embryos are in parentheses. C, results of stable
transfection experiments in ROS17/2.8 cells. -Galactosidase activity
was measured in pools of stably transfected clones. Results are
expressed per 100 µg of proteins, and the activity of pC3.112 was
considered as 100%. Values represent the mean ± S.E. from three
separate experiments.
13 kb, we generated
transgenic embryos using pC6.267 (Fig. 1A). Only four out of
11 transgenic embryos harboring pC6.267 expressed the reporter gene. It
was expressed in ossification centers and tendons in all cases
but at high levels only in two cases (Fig. 1B). We then generated transgenic embryos harboring pC6.273. (Fig. 1A).
Only two out of six transgenic embryos harboring pC6.273 expressed the
lacZ reporter gene, and in both cases it was expressed in ossification centers at low levels (Fig. 1B). In these two
cases, X-gal staining was restricted to ossification centers.
1(I) promoter sequence, extending from 17 to 10.5 kb, than with the corresponding constructs lacking this upstream sequence (2 out of 17 versus 10 out of 19, respectively, p < 0.05). Hence, these data suggested that the 6.5-kb fragment of the
pro-1(I) promoter, extending from 17 to 10.5 kb contains a
repressor element. To confirm this, we stably transfected pC3.112 and
pC6.273 in ROS17/2.8 osteoblastic cells. -Galactosidase activity was
68% lower in ROS17/2.8 cells stably transfected with pC6.273 than in
those stably transfected with pC3.112 (Fig. 1C). Besides,
-galactosidase activity was similar when ROS17/2.8 cells were stably
transfected with pC6.273 and pC6.267, suggesting that the first five
introns do not contribute to the inhibitory effect (data not shown).
1(I) promoter
sequence located between 17 kb and 3.2 kb nor the first five
introns contain new tissue-specific cis-acting elements active during
embryonic development.
2(I) collagen gene expression (25), and since the
DNase I-hypersensitive sites present within the mouse pro-1(I)
collagen gene have been precisely mapped (29), we studied whether DNase
I-hypersensitive sites present within the endogenous gene were also
present within constructs containing the repressor element and stably
integrated in the genome of ROS17/2.8 cells. The pro-1(I) collagen
gene contains two DNase I-hypersensitive sites between 17 kb and 11 kb, another one immediately upstream of the transcription start site,
one within the first intron, and one within the fifth intron (Fig.
2A).
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Fig. 2.
Study of DNase I-hypersensitive sites in two
constructs (pC6.267 and pC6.273) stably transfected in ROS17/2.8
cells. A, representation of some of the hypersensitive
sites (arrows) present in the endogenous mouse pro- 1(I)
collagen gene in type I collagen-producing cells (29). The
black boxes represent exons 1-6. B
and C, schematic representation of the analysis of DNase
I-hypersensitive sites present in pC6.267 and pC6.273, respectively.
The location of the lacZ probe used for hybridization is
indicated under the lacZ box.
Open bars show the products obtained after
digestion with DNase I and NarI (B) or
HindIII (C). The arrows indicate the
hypersensitive sites. D and E, study of DNase
I-hypersensitive sites in pC6.267 and pC6.273, respectively.
D, DNA from ROS17/2.8 cells stably transfected with pC6.267
was digested with NarI and increasing amounts of DNase I and
hybridized with a lacZ probe. The arrows indicate
the position of the resulting DNA fragments. E, DNA from
ROS17/2.8 cells stably transfected with pC6.273 was digested with
HindIII and increasing amounts of DNase I and hybridized
with a lacZ probe. The arrows indicate the
position of the resulting DNA fragments.
1(1) promoter sequence
extending from 17 to 10.5 kb. The 4-kb band was generated by
NarI digestion and DNase I digestion at a hypersensitive
site lying in the proximal promoter (Fig. 2, B and
D).
1(I) Promoter Represses Reporter
Gene Expression in Transiently Transfected ROS17/2.8 Cells--
To
confirm that the inhibitory effect of the fragment located between 17
and 10.5 kb was not mediated by modifications of chromatin
conformation, we performed transient transfection experiments with
constructs containing (pC6.267, pC6.273) or not containing (pJ320,
pC3.112) the repressor element. Relative -galactosidase activities
in ROS17/2.8 cells transfected with pC6.267 and pC6.273 were 2 times
lower than -galactosidase activities in cells transfected with pJ320
and pC3.112, respectively (Fig.
3A).
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Fig. 3.
Transient transfection experiments.
A, schematic representation of pJ320, pC6.267, pC3.112, and
pC6.273, and results of the transient transfection experiments
performed using these constructs. Each construct was co-transfected in
ROS17/2.8 cells with the pGL3 control vector to correct for
transfection efficiency. The activity of a construct containing the
17 to 10.5 kb segment was compared with the activity of a similar
construct lacking this fragment and was considered as 100%. Values
represent the mean ± S.E. from at least three separated
experiments. B, schematic representation of pDR583 and pDR6
and results of transient transfection experiments performed using these
constructs. Each construct was co-transfected in RC.SVtsA58 renal
collecting duct cells with the pSV-gal control vector to correct for
transfection efficiency. The activity of pDR6 was compared with the
activity of pDR583, considered as 100%. Values represent the mean ± S.E. from at least three separate experiments.
1(I) promoter fragment extending
from 17 to 10.5 kb was cloned upstream of a 664-bp segment of the
Hoxb-7 proximal promoter and of the luciferase reporter gene
(pDR6) and transiently transfected in RC.SVtsA58 tubular epithelial
cells. Luciferase activity was more than 2 times lower in cells
transfected with pDR6 than in cells transfected with pDR583, a
construct lacking the 6.5-kb fragment (Fig. 3B).
1(I) Promoter--
To delineate more precisely
the boundaries of the inhibitory element, transient transfection
experiments were performed using subsegments of this element, cloned
upstream of 3.2 kb of the pro-1(I) proximal promoter and of
lacZ. Reporter gene activity in cells transfected with these
constructs was compared with the activity of pJ320, which contains only
3.2 kb of the pro-1(I) collagen promoter cloned upstream of
lacZ. A 35-45% decrease in the levels of reporter gene
expression was observed with pC6.267, pC320.4, or pC320.2XN (Fig.
4). In contrast, no inhibitory activity was observed with pC320.1 or pC320.1.5 (Fig. 4). Taken together, these
experiments showed that the repressor element was located between 16
and 14 kb. Besides, transient transfection experiments also showed
that the inhibitory effect of the 2-kb repressor element was completely
abolished when it was cloned in the reverse orientation (data not
shown) and hence that the repressor effect of this sequence was
orientation-dependent.
View larger version (15K):
[in a new window]
Fig. 4.
Transient transfection experiments. A
schematic representation is shown of the constructs used to perform
transient transfections experiments and results of these experiments.
Each construct was co-transfected in ROS17/2.8 cells with PGL3 control
vector to correct for transfection efficiency. Values are
expressed as relative -galactosidase activity, the activity of pJ320
being considered as 100%. All values represent the mean ± S.E.
from at least three separate experiments.
-Galactosidase activity was 2 times lower in cells transfected with pC15.56.2XN than in cells
transfected with pC15.56 (Fig. 4), confirming that the activity of the
repressor element was not position-dependent.
1(I) proximal promoter
and of lacZ. As previously, -galactosidase activity observed with these constructs was compared with the one of pJ320. A
40% decrease in the levels of reporter gene expression was observed with a plasmid containing a 312-bp segment located at ~14 kb upstream of the transcriptional start site (pC320.312), as shown in Fig. 4. In
contrast, no repressor effect was observed with pC320.400 and
pC320.1.3, which contain a 400-bp and a 1.3-kb subsegment of the 2-kb
repressor sequence, respectively (data not shown).
-galactosidase activities
in ROS17/2.8 cells transiently transfected with pJ320, with pC123, and
with plasmids harboring mutations or deletions in the three E2-boxes (Fig. 5B). Mutations or deletions of the three E2-boxes
completely abolished the repressor effect of COIN-1, confirming that
the E2-boxes played a key role in mediating the repressor effect of COIN-1. Mutations in the two most 3' E2-boxes (pC123.2m) had the same
effect. In contrast, a mutation in the most 5' E2-box only (pC123.1m)
did not modify the inhibitory effect of COIN-1 (Fig. 5B).
View larger version (20K):
[in a new window]
Fig. 5.
Analysis of COIN-1. A,
sequence of COIN-1. This sequence is an almost perfect three-time
repeat of a 41-bp motif containing an E2-box. The nonidentical
nucleotides are underlined. The E2-boxes are in
boldface type. B, schematic
representation of constructs used to perform transient transfection
experiments and results of these assays. A star indicates a
point mutation in an E2-box (CACCT 
CATCT). An
open box indicates a deleted E2-box. Each
construct was co-transfected in ROS17/2.8 cells with PGL3 control
vector to correct for transfection efficiency. Values are expressed as
relative -galactosidase activity, the activity of pJ320 being
considered as 100%. All values represent the mean ± S.E. from at
least three separate experiments.
EF1--
To study the
proteins able to bind to COIN-1, we performed electrophoretic mobility
shift assays using nuclear extracts from ROS17/2.8 cells and five
probes. WT1 corresponds to the most 3' 41-bp motif; DEL1 is similar to
WT1, except for a deletion of the E2-box; WT2 corresponds to the two 3'
motifs; MUT2 is similar to WT2 except for a point mutation in the two
E2-boxes (CACCTG CATCTG); and DEL2 is similar to
WT2 except for deletions of the two E2-boxes.
View larger version (78K):
[in a new window]
Fig. 6.
Electrophoretic mobility shift analysis of
the proteins binding to COIN-1. Lane 1 shows
the proteins binding to WT1. Lanes 4 and
11 show the proteins binding to WT2. Complex A was only seen
with the WT2 probe, which contains two E2-boxes. Lanes
2, 5, and 6 show the proteins binding
to DEL1, MUT2, and DEL2. Complex A was not seen with MUT2 and DEL2 that
contain mutated or deleted E2-boxes, respectively. In lane
3, a competition assay was performed using a 100-fold
molar excess of the WT1 unlabeled probe. In lanes
7-10, competition assays were done using a 30-100-fold
molar excess of the WT1 and the MUT2 unlabeled probes. Complex A was
not eliminated in competition experiments with MUT2, which harbors a
point mutation in the E2-boxes, while it was eliminated in competition
experiments with WT2. In lane 12, incubation of
the nuclear extracts with an anti- EF1 antibody eliminated the
formation of complex A and produced a slower migrating complex
(star).
EF1 (30).
Since EF1 has been reported to inhibit transcription (27, 31, 32),
it appeared as a good candidate for mediating the inhibitory effect of
COIN-1. To test whether EF1 was present in complex A, we added an
anti-EF1 antibody in the binding reaction. Complex A disappeared and
was partially supershifted in the presence of the antibody, confirming
that complex A contained EF1 (Fig. 6).
EF1 Represses Reporter Gene and Pro-1(I)
Collagen Gene Expression in Transfection Experiments--
To test
whether overexpression of EF1 enhances the inhibitory effect of
COIN-1, we co-transfected ROS17/2.8 cells with pC123 and either with an
expression vector containing the cDNA encoding EF1
(pCMVX-EF1) or with an empty expression vector (pCMVX). Transfection
experiments with increasing amounts of pCMVX-EF1 induced a
dose-dependent inhibition (up to 51%) of the activity of
pC123 (Fig. 7A).
View larger version (14K):
[in a new window]
Fig. 7.
Effects of EF1
overexpression. A, transient transfection experiments
in ROS17/2.8 cells were performed using pC123, together with increasing
amounts of pCMVX and pCMVX-EF1. PGL3 control vector was used to
correct for transfection efficiency. Values are expressed as percentage
of inhibition by pCMVX-EF1 when compared with pCMVX. Values
represent the mean ± S.E. from three separate experiments.
B, transient transfection experiments were performed in
ROS17/2.8 cells, using pCMVX and pCMVX-EF1. The amounts of
pro-1(I) collagen mRNA in cells transfected with pCMVX-EF1
decreased by 35% when compared with cells transfected with pCMVX
(considered as 100%). Values represent the mean ± S.E. of a
PhosphorImager quantitation of pro-1(I) mRNA normalized to
18 S RNA.
EF1 on the level of expression of the
endogenous gene, pCMVX-EF1 was transiently transfected in ROS17/2.8 cells. Pro-1(I) mRNA levels were decreased by 35% in ROS17/2.8 cells transfected with pCMVX-EF1, when compared with cells
transfected with pCMVX (Fig. 7B). Transfection efficiency
was approximated by X-gal staining of transiently transfected cells
with pSV-gal control vector. About one-third of the cells showed
X-gal staining, suggesting that the inhibitory effect of EF1 on the
level of expression of the pro-1(I) collagen gene is highly significant.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1(I) proximal promoter are unknown. By
generating transgenic mice and performing transfection experiments, we
have identified a 123-bp repressor element in the mouse pro-1(I)
collagen promoter. The existence of such a repressor element was first
suggested by comparing the levels of expression of the lacZ
reporter gene in 15.5-day postconception transgenic embryos harboring
various segments of the mouse pro-1(I) promoter. When a segment of
the promoter extending from 17 to 10.5 kb was cloned upstream of
2.3 kb of the pro-1(I) proximal promoter and of the first five
introns, the percentage of transgenic embryos expressing the reporter
gene at high levels dropped from 58 to 0% (compare the results
obtained with pC3.112 and pC6.273 in Fig. 1). Stable and transient
transfection experiments confirmed the presence of this element, since
the average levels of expression of the lacZ reporter gene
were decreased by 68 and 60%, respectively, when the 17 to 10.5 kb
fragment was cloned upstream of 2.3 kb of the pro-1(I) proximal
promoter and of the first five introns. A similar inhibitory effect was
observed in transient transfection experiments when the 17 to 10.5
kb segment was cloned upstream of an heterologous promoter. Using
transient transfection experiments in ROS17/2.8 cells, the repressor
element was progressively narrowed to a 123-bp sequence, located 14 kb
upstream of the transcription start site. Sequencing of this element,
that we named COIN-1, showed that it consists of an almost perfect
three-time repeat of a 41-bp motif containing a CACCTG E2-box. These
E2-boxes play a key role in mediating the inhibitory effect of COIN-1,
since it was completely abolished by a point mutation in at least two of them in transient transfection experiments.
EF1 and that point mutations in the
E2-boxes, which abolished the repressor effect of COIN-1, also
abolished the binding of EF1. Furthermore, overexpression of EF1
in transiently transfected ROS17/2.8 cells decreased the activity of a
construct containing COIN-1 in a dose-dependent manner and
down-regulated the expression of the endogenous pro-1(I) collagen
gene. Thus, it is very likely that EF1 binds to COIN-1 and mediates
its inhibitory effect. EF1 is a DNA-binding protein that belongs to
an emerging family of two-handed zinc finger transcription factors. It
is expressed in lens, central nervous system, neural crest derivatives,
and various mesodermal tissues (33). It contains two widely separated
clusters of C2H2 Krüppel-like zinc
fingers and a homeodomain-like segment, but only the two clusters of
zinc fingers seem to be involved in DNA binding (34). In
vitro studies have shown that EF1 binds cis-acting
elements containing two E2-boxes (34), which is in complete agreement
with our results. Furthermore, the affinity of a zinc finger cluster
for its binding site appears to be largely increased when a guanine
residue is located immediately downstream of the CACCTG
motif (34), which is the case for all three CACCTG motifs in
COIN-1. In previously described EF1-binding sites, only one E2-box
out of two contained such a guanine residue (34). COIN-1 is thus the
first EF1-binding element that contains a high affinity binding site
for each cluster of zinc finger, and it might bind EF1 with a
greater affinity than the other EF1-binding sites. Moreover, since
two E2-boxes are sufficient to allow the binding of EF1, it would be
of interest to examine in greater detail the respective role of each of
the three E2-boxes contained in COIN-1 and the number of EF1
molecules able to bind to COIN-1. The inhibitory effect of COIN-1 is in
agreement with results reported by other groups, who showed that EF1
had a repressor effect and was able to decrease the activity of the
1-crystallin promoter, of the pro-1(II) collagen promoter, and of
the 4-integrin promoter (27, 31, 32). The ability
of EF1 to down-regulate the pro-1(I) collagen gene in
osteoblastic cells may explain that EF1-null mice display a variety
of defects in bones, where large a amount of type I collagen is
produced during embryonic development (35). The repression of the
pro-1(I) promoter activity mediated by COIN-1 may not appear
dramatic, but it is likely to be physiologically relevant for at least
two reasons. First, the pro-1(I) proximal promoter being very
active, transcriptional repression is probably a key phenomenon in
controlling the level of expression of the corresponding gene. Second,
since type I collagen protein turnover is very slow (36), only a modest
increase in pro-1(I) mRNA participate in the onset of fibrosis
(37).
EF1 to COIN-1 does not seem to modify chromatin
structure, since the DNase I-hypersensitive sites identified within the
mouse pro-1(I) collagen promoter or within the first and the fifth
introns, in type I collagen-producing cells (29), were still present in
constructs containing COIN-1 stably integrated into the genome of
ROS17/2.8 cells. Furthermore, COIN-1 was active not only in stable
transfection experiments but also in transient transfection
experiments. Thus, EF1 is likely to repress the pro-1(I) promoter
activity by interacting with other transcription factors, as suggested
for the 1-crystallin gene (27). The interactions between EF1 and
other components of the transcription machinery may be direct,
involving the repression domain of EF1, but they may also be
indirect, since recent studies reported that EF1 was able to recruit
co-repressors named C-terminal binding proteins (39). Quite
surprisingly, the inhibitory effect of COIN-1 was
orientation-dependent. Other cis-repressor
elements, such as a potent repressor located in the myelin basic
protein gene, have been reported to be active only in one orientation (40). This led to the assumption that the regulation of gene transcription can involve the formation of DNA-multiprotein complexes through distant regions of DNA and that those higher order
formations may require a correct three-dimensional structure given by
the binding of transcription factors in precise orientation (41).
1(I) proximal promoter (compare results obtained with pC3.112 and pC15.56) and that a tendon- and fascia-specific element is located between 3.2
and 2.3 kb (compare results obtained with pC6.267 and pC6.273). In
contrast, it did not disclose the existence of a new tissue-specific element, either upstream of 3.2 kb or within the first five introns. The absence of tissue-specific elements within the first intron extend
results obtained by Hormuzdi et al. (8). They also showed the absence of tissue-specific elements in this intron by generating knock-in mice lacking most of it (12). Analysis of heterozygous mice
showed that it was important for maintaining normal levels of
expression of the pro-1(I) collagen gene in lung and muscle during
adult life. Nevertheless, study of homozygous mice showed that this
intron was not necessary for inducing the expression of the pro-1(I)
collagen gene in type I collagen-producing cells. The absence of
tissue-specific elements within a fragment of promoter extending from
17 to 3.2 kb active during embryonic development extends results
obtained by Krempen et al. (43). They generated transgenic
mice harboring segments of the mouse pro-1(I) promoter extending up
to 19.5 kb that were cloned upstream of the green fluorescent protein
reporter gene (42). Analysis of these mice did not show new
tissue-specific elements, with the exception of an element located
at 8 to 7 kb that enhanced the expression of the reporter gene in
endometrial cells and in muscle cells of the uterus during the oestrous
cycle in adult females.
1(I) collagen gene. This element, which we named COIN-1, is a
three-time repeat of a 41-bp motif. Each repeat contains an E2-box, and
the repressor effect of COIN-1 appears to occur through the binding of
EF1 to these E2-boxes. COIN-1 was able to decrease the activity of
the pro-1(I) promoter not only in transient transfection experiments
but also in stable transfection experiments and in transgenic mice.
Furthermore, overexpression of EF1 enhanced the inhibitory effect of
COIN-1 and down-regulated the expression of the endogenous gene. These
data suggest that this inhibitory sequence could be an important player
in the regulation of the overall levels of expression of type I
collagen genes.
ACKNOWLEDGEMENTS
EF1 expression vector and the anti-EF1 antibody. We are
also grateful to J. Chambaz and C. Lasne for welcoming and helping
us in the IFR 58 transgenic facility. We thank A. Calmont and G. Bou-Gharios for carefully reading the manuscript.
FOOTNOTES
To whom correspondence should be addressed: INSERM U489,
Hôpital TENON, 4 rue de la Chine, 75020 Paris, France. Tel.:
33-1-56-01-69-93; Fax: 33-1-56-01-69-99; E-mail:
jerome.rossert@tnn.ap-hop-paris.fr.
ABBREVIATIONS
-D-galactopyranoside.
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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N. Matsuo, S. Tanaka, M. K. Gordon, M. Koch, H. Yoshioka, and F. Ramirez CREB-AP1 Protein Complexes Regulate Transcription of the Collagen XXIV Gene (Col24a1) in Osteoblasts J. Biol. Chem., March 3, 2006; 281(9): 5445 - 5452. [Abstract] [Full Text] [PDF]
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T. T. Antoniv, S. Tanaka, B. Sudan, S. De Val, K. Liu, L. Wang, D. J. Wells, G. Bou-Gharios, and F. Ramirez Identification of a Repressor in the First Intron of the Human {alpha}2(I) Collagen Gene (COL1A2) J. Biol. Chem., October 21, 2005; 280(42): 35417 - 35423. [Abstract] [Full Text] [PDF]
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 M. Ponticos, T. Partridge, C. M. Black, D. J. Abraham, and G. Bou-Gharios Regulation of Collagen Type I in Vascular Smooth Muscle Cells by Competition between Nkx2.5 and {delta}EF1/ZEB1 Mol. Cell. Biol., July 15, 2004; 24(14): 6151 - 6161. [Abstract] [Full Text] [PDF]
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K. N. Maclean, E. Kraus, and J. P. Kraus The Dominant Role of Sp1 in Regulating the Cystathionine {beta}-Synthase -1a and -1b Promoters Facilitates Potential Tissue-specific Regulation by Kruppel-like Factors J. Biol. Chem., March 5, 2004; 279(10): 8558 - 8566. [Abstract] [Full Text] [PDF]
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 O. Rahkonen, M. Su, H. Hakovirta, I. Koskivirta, S. G. Hormuzdi, E. Vuorio, P. Bornstein, and R. Penttinen Mice With a Deletion in the First Intron of the Col1a1 Gene Develop Age-Dependent Aortic Dissection and Rupture Circ. Res., January 9, 2004; 94(1): 83 - 90. [Abstract] [Full Text] [PDF]
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P. Christmas, N. Carlesso, H. Shang, S.-M. Cheng, B. M. Weber, F. I. Preffer, D. T. Scadden, and R. J. Soberman Myeloid Expression of Cytochrome P450 4F3 Is Determined by a Lineage-specific Alternative Promoter J. Biol. Chem., June 27, 2003; 278(27): 25133 - 25142. [Abstract] [Full Text] [PDF]
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 K. Sooy and M. B. Demay Transcriptional Repression of the Rat Osteocalcin Gene by {delta}EF1 Endocrinology, September 1, 2002; 143(9): 3370 - 3375. [Abstract] [Full Text] [PDF]
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