Matrix Biology 23 (2004) 87–99

Identification of a functional CBF-binding CCAAT-like motif in the core
promoter of the mouse pro-a1(V) collagen gene (Col5a1)
Keiko Sakata-Takatania,b, Noritaka Matsuoa, Hideaki Sumiyoshia, Tomiyasu Tsudab,
Hidekatsu Yoshiokaa,*aDepartment of Anatomy, Biology and Medicine, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Oita 879-5593, Japanb
Department of Immunology and Allergy, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Oita 879-5593, Japan
Received 11 August 2003; received in revised form 26 March 2004; accepted 26 March 2004

Abstract
We used structure-function analysis of the core promoter region to elucidate the transcriptional features of the mouse a1(V)collagen gene (Col5a1). The core promoter, which lacks a typical TATA motif and has a high GC content, was defined within
the 231 bp immediately upstream from the major transcription start site by transient transfection experiments. In this region, we
identified three nuclear-factor binding sites by electrophoretic mobility shift assay: BS1 (y195 to y167), BS2 (y134 to y106),
and BS3 (y110 to y80). Oligonucleotide competition and supershift assays revealed that Sp1, CBF, and Sp1-related protein
specifically bind to BS1, BS2, and BS3, respectively. The CCAAT-like motif, CAAAT, and flanking sequences are conserved
between the mouse and human gene. CBF, which recognizes this motif, activated the Col5a1 promoter, as previously reported for
Col1a1 and Col1a2. Furthermore, overexpression of a wild-type and mutant forms of CBF-B subunit altered this activity. These
results suggest that CBF is a key factor in the coordinated expression of type I and V collagen genes.
䊚 2004 Elsevier B.V./International Society of Matrix Biology. All rights reserved.
Keywords: a1(V) collagen promoter; CCAAT-binding factor CBF; Sp1; Gene regulation

1. Introduction
Fibril-forming collagens represent a structurally related group of molecules within the large family ofcollagens. This class of collagens includes nine distinct
polypeptides that associate into five types of collagen:types I, II, III, V and XI (Vuorio and de Crombrugghe,
1990; van der Rest and Garrone, 1991; Brown andTimpl, 1995). They are essential for the integrity of theextracellular matrix scaffold. Type V collagen, which isa minor component, co-polymerizes with the majorcollagen, type I, and regulates the diameter of collagenfibers (Adachi and Hayashi, 1986; Linsenmayer et al.,
1993; Andrikopoulos et al., 1995). Collagens are heterotrimer andyor homotrimer molecules made of three achain. The predominant molecular form of type V
Abbreviations: Sp1, specificity protein 1; CBF, CCAAT-binding
factor; RACE, rapid amplification of cDNA ends; EMSA, electrophoretic mobility shift assay.
*Corresponding author. Tel.: q81-97-586-5670; fax: q81-97-5496302.
E-mail address: hidey@med.oita-u.ac.jp (H. Yoshioka).

collagen is the heterotrimer wa1(V)x2a2(V), although
the a1(V)a2(V)a3(V) molecule has been extracted
from human placenta and the homotrimer wa1(V)x3 also
occurs in cultures of hamster lung cells (Burgeson etal., 1976; Sage and Bornstein, 1979; Haralson et al.,1980). Type V collagen production is elevated in many
pathological conditions, such as inflammation (Narayanan et al., 1983), some forms of cancer (Barsky et al.,1982), and atherosclerosis (Ooshima, 1981).
Numerous studies have identified the cis-acting regulatory elements and the trans-acting factors that bindto the regulatory regions in mammalian collagen genes(Ramirez and Di Liberto, 1990). The transcriptional
regulation of the a1(I) and a2(I) collagen genes has
been most extensively studied (Slack et al., 1993). A
common feature in the proximal regions of these promoters is the involvement of the ubiquitously expressedtranscriptional factors Sp1, Sp3 (Inagaki et al., 1994;
Tamaki et al., 1995; Ihn et al., 1996), and CBF (Maity
et al., 1988; Coustry et al., 1995; Hatamochi et al.,
1988; Maity and de Crombrugghe, 1998; Mantovani,1998). Sp1 is a well-characterized zinc-finger transcrip-

0945-053X/04/$30.00 䊚 2004 Elsevier B.V./International Society of Matrix Biology. All rights reserved.
doi:10.1016/j.matbio.2004.03.003

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tion factor that binds to the GC box or a similar motif.
Sp1 and the related protein, Sp3, work as activators of
the a1(I) and a2(I) collagen genes under basal or TGFb-inducible conditions. CBF binds to the CCAAT motif,
which is one of the common elements present in theproximal promoter. CBF, also called NF-Y or CP1,
consists of three different subunits, CBF-A, CBF-B, and
CBF-C, all of which are necessary to form the CBFDNA complex (Maity et al., 1992). CBF acts as atranscriptional activator for the a1(I) and a2(I) collagengenes. The CCAAT element is located between nucleotides y84 and y80 in the mouse a2(I) gene, andbetween nucleotides y100 and y96 in the mouse a1(I)gene in a reverse orientation. Mutation of the CCAAT
motif decreases basal promoter activity in both genes(Karsenty et al., 1988; Karsenty and de Crombrugghe1990).
Lee and Greenspan (1995) characterized the promoterof the human a1(V) collagen gene. A minimal promoter
region within 212 bp immediately upstream of the majortranscription start site contains the region where Sp1binds. Furthermore, they suggested that GAGA boxesin the promoter and the first exon may be implicated inaffecting levels of transcription. In the present study, wecharacterized the core promoter of the mouse a1(V)collagen gene. Cell transfection experiments and DNAbinding assays have demonstrated that the factors whichbind to the core promoter region are the same as those
responsible for the expression of type I collagen genes.
In particular, CBF binds to the CAAAT sequence, but
not to the CCAAT sequence, and activates the gene.
2. Results

the 59 region of mgKS1. We thus isolated another clone,
mgKS3, encoding a further 13 kb upstream from the 59
end of mgKS1. (The nucleotide sequence has been
submitted to the DDBJyEMBLyGenBank under accession number, AB098608.)
The location of the transcription start site was identified by 59 RACE using different combinations ofprimer sets and a nuclease protection assay (Yoshioka
et al., 1995). We performed 59 RACE with two primer
sets, RACE1 and RACE3 primers, and RACE2 andRACE4 primers. After subcloning, 14 positive RACE
clones were selected and sequenced. As shown in Fig.
1b, the ten 59 RACE products extended to the same
upstream site were isolated from three independent
PCRs. This information strongly suggested that this
upstream site is the major start site of Col5a1 transcription. We prepared a 360-nucleotide (nt) riboprobe containing this putative start site for use in a nuclease
protection experiment. After RNase digestion, we
observed one major resistant product of approximately
170 nt, and several minor products (Fig. 1c). The major
protected band has a size consistent with protection
starting at the major transcriptional start site identified
by 59 RACE. We concluded that the Col5a1 genecontains multiple transcription start sites, with a major
one located 361 bp upstream from the ATG codon. The
nucleotide sequence of around mouse promoter is similar
to that of human counterpart except BS3 region, whichis missing in human gene (Fig. 1b and Fig. 3a). The
similarity between both species from y231 to q70 is
85%. The Col5a1 promoter lacks typical TATA andCCAAT boxes, and has a high GC content as shown inhuman gene (Lee and Greenspan, 1995).

2.1. Structural analysis of the 59 portion of Col5a1

2.2. Functional analysis of the Col5a1 promoter region

To isolate the 59 part of Col5a1, we screened a mouse
genomic library using mHY217, a most 59 clone ofmouse a1(V) cDNA, which was isolated previously(Wu et al., 1998). Comparison of the genomic sequencesof isolated clones with that of mHY217 revealed thatthe genomic clones did not contain the 59 sequences of
mHY217, but sequences downstream from the ATG
codon, presumably due to a short portion of 59 UTR of
cDNA. Therefore, we decided to isolate a cDNA clonethat included the 59 region further upstream by using
the 59 RACE procedure. We performed 59 RACE with
two specific primers, RACE1 and RACE2. A 322-bp
RACE product overlapped with mHY217, and contained
230 bp in the 59 region. We re-screened the genomic
library using this cDNA as a probe, and isolated a 15kb genomic clone, mgKS1 (Fig. 1a). Southern blot andsequencing analyses revealed that mgKS1 contained
only 682 bp upstream from the ATG codon. To isolate
a clone that contained more of the 59 region, we rescreened with a 0.55-kb fragment containing most of

To determine the smallest region required or sufficient
to encompass the basal promoter of the Col5a1 gene, aseries of genomic fragments fused to a firefly luciferase
reporter gene, pGL3-basic vector, were generated (Fig.
2a). These were transiently transfected into a1(V)producing cells, NIH3T3 and A204 (Lee and Greenspan,
1995). Seven constructs containing progressive 59 deletions of a 1.7-kb fragment were used to identify moreclosely the probable active promoter region (Fig. 2a).
As shown in Fig. 2b, in the constructs from y1700yq
39-Luc to y231yq39-Luc, luciferase activity was significantly higher than that of the pGL3-basic vector.
Removal of the region between y231 and y84 reduced
transcriptional activity to near basal levels. Confirming
these results, the y1700yy232-Luc and y468yy232Luc plasmids, which lack the y231 to q39 region,
showed no transcriptional activity, suggesting that thisregion contains positive cis-acting elements (Fig. 2c).
The plasmids containing a GAGA box in the 59 untranslated region, y1700yq156-Luc and y231yq156-

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89

Fig. 1. Structural analysis of the 59 region of Col5a1. (a) Schematic representation of the structure of the Col5a1 gene. Top: Location of the
genomic clones, mgKS1 and mgKS3. Bottom: Partial restriction map of a fragment from the mgKS3 clone. P, Pst I; S, Sac I; A, Apa I; H, Hind
III; E, EcoR I; K, Kpn I; Sm, Sma I. Open and closed boxes represent untranslated and coding regions, respectively. The major start site oftranscription is marked with an arrow. (b) Alignment of the sequences of mouse Col5a1 (upper) and human COL5A1 (lower) (Lee and Greenspan,
1995) promoter regions and parts of exon 1. Only the nucleotides of the human that differ from the mouse are shown. The small bars and asterisks
indicate identical and missing nucleotides, respectively, in the corresponding regions of both species. The start site determined by 59 RACE isdenoted by black dots. The approximate 59 ends of the major and minor RNase-protected bands are indicated by a black and striped bars,
respectively. Nucleotide numbering of both species start with q1, which correspond to the major transcription start sites indicating with small
arrows. The primers used for 59 RACE and the RNase protection assay, the ATG codon of the translation start site, and the 59 end of mgHY217(Wu et al., 1998) are indicated by arrows, a box, and an open dot, respectively. The binding sites for nuclear factors, BS1–BS3, and the GAGAbox identified in the human promoter are indicated with horizontal lines and open bars, respectively. The critical nucleotides in BS1-BS3 areemphasized with black squares. (c) RNase protection assay of the 360-nt riboprobe using RNA from mouse lung (lane 2) and yeast (lane 3).
The undigested riboprobe is shown in lane 1, and size makers (in bp) are indicated in lane M. The major protected products is indicated by anarrow and, the minor products by open and closed arrowheads, respectively.

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Fig. 2. Luciferase assay of the Col5a1 promoter. (a) Schema of luciferase reporter constructs. The assay was performed using 59 (b) and 39 (c)stepwise-deleted constructs. A firefly luciferase reporter construct was cotransfected with the Renilla vector, pRL-TK, used as an internal control
for transfection efficiency. The histograms indicate percentage activity normalized to the internal control and shown relative to that of the y
1700yq39-Luc construct transfected into NIH3T3 cells. The data represent the mean of three independent tests "S.D. Two different cell lines,
NIH3T3 (white histogram) and A204 (black histogram), were used for transfection studies.

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91

Luc, did not elevate promoter activity, as was reported
for the human gene (data not shown) (Lee and Greenspan, 1995).
2.3. Identification of nuclear factor binding sites in thebasal Col5a1 promoter
On the basis of the above data, we concentrated on
the y231 to y84 region to characterize the minimum
promoter for Col5a1. EMSA were performed to examine
the transcription factors that bind in this region, using
nuclear extracts from NIH3T3 cells. First, we used two
oligonucleotides, corresponding to the sequences fromy250 to y167 and from y176 to y80, as probes(Fig. 3a). The y250yy167 probe produced one major
protein–DNA complex and the y176yy80 probe produced two major protein–DNA complexes (Fig. 3b).
Each band was competitively inhibited by corresponding
unlabeled excess oligonucleotide. Furthermore, the bandin the y250 to y167 region and the upper band in they176 to y80 were competitively inhibited by the
y176yy80 and the y250yy167 probes, respectively.
Because both bands migrated at the same position, theresults suggest that the DNA-binding proteins might beidentical or similar. To identify the DNA-binding sitesof these proteins more closely, we prepared shorter
oligonucleotides (Fig. 3a). The protein–DNA complex
in the y250 to y167 region was detected by the
y216yy167 and y195yy167 probes, but not by the
y250yy207 probe, indicating that the binding site is
located in the y195 to y167 region (binding site 1:
BS1) (Fig. 3a,c lanes 1–3). The band detected by the
y195yy167 probe migrated more slowly than that
detected by the y216yy167 probe, and became adoublet (Fig. 3c lane 3Fig. 5c lane 2). This may reflect
a slight difference in the conformation of the DNA–
protein complex induced by each probe. In the y166
to y80 region, the lower band was detected by they166yy93 probe, the y166yy106 probe, and they134yy106 probe, indicating that the protein–DNA
complex corresponding to the lower band is located in
the y134 to y106 region (BS 2) (Fig. 3a,c lanes 5, 6
and 8). However, the band was detected by neither they166yy121 nor the y120yy80 probe (Fig. 3c lanes
7 and 9). This result suggests that the region aroundy120 is critical for the formation of the protein–DNA
complex. However, the upper band was detected by they120yy80 and y110yy80 probes, indicating that thebinding site is located in the y110 to y80 region (BS3) (Fig. 3a,c lanes 9 and 10). No upper band was
detected by the y166yy93 probe (Fig. 3c lane 5),
implying that the y93 to y80 region is critical in theformation of the protein–DNA complex corresponding
to the upper band.
The two sequences, 59-CCCCACCCCC-39 in they195 to y167 region (BS 1), and 59-GGGGGTGGGG-

Fig. 3. Binding of nuclear factors to the Col5a1 proximal promoter.
(a) Positions of the designed oligonucleotides corresponding toCol5a1 nucleotides y250 to y80 for EMSA. BS1, BS2, and BS3
indicate nuclear-factor binding sites. (b) Detection of protein–DNA
complexes with two oligonucleotides covering the sequence fromy250 to y80. (c) Determination of the binding sites of nuclear factors to the Col5a1 proximal promoter. The arrows indicate the bindingof specific complexes in (b) and (c). Note that three specific bands,
indicated by arrowheads, can be seen in lane 3. Compare with lanes
2 and 7 in Fig. 5c.

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Fig. 4. Mutation analysis of the binding of nuclear factors to the Col5a1 promoter. (a) Schematic representation of the locations and nucleotide
substitutions of mutant oligonucleotides, m1–m4. The binding of nuclear factors was examined in the y216 to y167 (b), the y166 to y93
region (c), and y110 to y60 regions (d), with labeled wild-type (WT) or mutant (m1–m4) nucleotides in the presence or absence of excess
unlabeled WT or mutant competitor. The specific binding of nuclear factors is indicated with arrowheads. BS1*, BS2* and BS3* indicate theregions containing BS1, BS2 and BS3, respectively.

39 in the y110 to y80 region (BS 3), are inverted
complementary motifs (Fig. 1b and Fig. 4a). Thissuggests that identical or closely related factors are
involved in both regions. On the basis of these data, we
generated mutant oligonucleotides substituted at critical
positions, m1–m3, for a DNA-binding assay to assess
the three BS regions (Fig. 4a). In the regions containing
BS1 and BS3, the mutant oligonucleotides, m1 and m3,
failed to form protein–DNA complexes (Fig. 4b, lane 4
and Fig. 4d, lane 4). Moreover, these mutant oligonucleotides did not compete with isotope-labeled wild-type
oligonucleotides at the level at which the wild-type
oligonucleotides inhibited their binding (Fig. 4b, lanes
5 and 6 and Fig. 4d, lanes 5 and 6). In the region
containing BS2, the mutant oligonucleotide, m2, also
failed to form a protein–DNA complex (Fig. 4c, lane4), but the wild-type sequence and m4, which is mutatedaround y132, formed these complexes (Fig. 4c, lanes
2 and 8). A competition assay confirmed that m2 did
not inhibit the binding of the normal protein–DNA
complex (Fig. 4c lane 6).
2.4. Identification of nuclear factors in the basal Col5a1
promoter
To identify the nuclear factors associated with the
Col5a1 promoter, competition experiments were carriedout by adding oligonucleotides containing the bindingsites for several known transcription factors that occur
in BS1, BS2, and BS3. Protein–DNA complexes in they216 to y167 region (BS1) competed against excess
unlabeled Sp1- and CBF-binding oligonucleotides (Fig.
5a, lanes 5 and 6). In the y166 to y80 region, thefaster migrating complex in BS2 was competitively

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93

Fig. 5. Identification of nuclear factors binding to the Col5a1 promoter. The competition assay with labeled probes corresponding to the regions
y216 to y167 (a) and y166 to y80 (b) using oligonucleotides containing consensus binding sites for known nuclear factors, Oct-1 (lane 3),
NF-1 (lane 4), Sp1 (lane 5), CBF (lane 6), and mutant CBF (lane 7), as competitors. Control assays were performed without nuclear extract orcompetitor (lane 1), and with nuclear extract but without competitor (lane 2). BS2*BS3* indicates the region containing BS2 and BS3. For thesupershift assay, nuclear extract was incubated with labeled probes, y195yy167 and Sp1 consensus oligonucleotides (c) and y166yy93
oligonucleotide (d) after the addition of antibodies against CBF-A (lanes 3C and 3D), CBF-B (lanes 4C and 4D), CBF-C (lanes 5C and 5D),
Sp1 (lanes 8C, 11C, and 6D), and Oct-1 (lane 7D). Control assays were performed without nuclear extract or antibody (lanes 1C, 6C, 9C, and1D), with nuclear extract but without competitor (lanes 2C, 7C, 10C, and 2D), and with nuclear extract and normal goat IgG (lane 8D). Specificprotein–DNA complexes and supershifted bands are indicated by arrows and arrowheads, respectively.

inhibited by CBF, whereas the slower one was competitively inhibited by both Sp1 and CBF in BS3 (Fig. 5b,lanes 5 and 6). We then evaluated supershift assays with
antibodies against known factors (Fig. 5c,d). The complex in BS1 was supershifted with anti-Sp1 antibodies,but not with anti-CBFs antibodies (Fig. 5c). The complexes in BS3 were not markedly supershifted with antiSp1 antibodies (data not shown). The discrepancy
between the oligonucleotide competition and supershift
assays regarding the involvement of CBF in BS1 andBS3 is due to a cross-reaction between the consensus
CBF-binding oligonucleotides and the Sp1 DNA-binding
domain (data not shown). The complex in BS2 wassupershifted by anti-CBF-A, -B, and -C antibodies, but
by neither anti-Sp1 nor anti-Oct-1 antibodies, which isconsistent with the data from the oligonucleotide competition assay (Fig. 5d).
To determine whether the Sp1 family or CBF activate
the basal promoter of Col5a1, we examined the BS1,
BS2, and BS3 regions using a luciferase assay, by
introducing mutations into these regions (Fig. 6a). The
BS2 mutation, m2-Luc, caused a decrease in promoter
activity in NIH3T3 and A204 cells, as shown in Fig.
6b. However, the BS1 and the BS3 mutations, m1-Luc
and m3-Luc, respectively, produced different results inthe two cell lines. Overexpression of Sp1 also did notaffect on wild, m1-Luc and m3-Luc promoter (data notshown).
Because the above experiment showed that CBF binds
in BS2 where the CCAAT-like motif, 59-ATTTG-39

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Fig. 6. Functional analysis of the Col5a1 promoter. (a) Schematic representation of the mutant firefly luciferase reporter constructs, m1-Luc-m3Luc. (b) Transient transfections with the wild-type construct from y231 to q39 and the mutant construct were carried out in NIH3T3 (white
histogram) and A204 (black histogram) cells. A firefly luciferase reporter construct was cotransfected with the Renilla vector, pRL-TK, used as
an internal control for transfection efficiency. The histograms indicate the percentage activity normalized to the internal control and shown relative
to the activity of the wild-type construct transfected into NIH3T3 cells. The data represent the mean of three independent tests"S.D.

(reverse CAAAT), is located, we examined the binding
affinity of the CCAAT-like motif, including the prototype ATTGG (reversed CCAAT), using EMSA. DNA
containing ATTTG in the Col5a1 collagen promoter, or
the mutant ATTCG, bound to CBF to similar degrees,
but with a slightly weaker affinity than that of the
CCAAT sequence. However, DNA with the TTTGG
mutation completely failed to bind to CBF (Fig. 7).
Finally, we tested the effect using a wild-type andmutant forms of CBF-B subunit, which interacts with
the CBF-A and CBF-C subunits to form CBF as previously described (Mantovani et al., 1994). Overexpression of wild type significantly increased promoter
activity using y231yq39-Luc in NIH3T3 and A204
cells. However, in cotransfection with the mutant type,the promoter activity decreased approximately 20% and
30% in NIH3T3 and A204 cells, respectively (Fig. 8).

3. Discussion
The cloned 1.7-kb genomic fragment from the 59
flanking region of Col5a1 had strong transcriptional
activity. The promoter lacks the canonical TATA andCCAAT boxes, but has a high GC content; these arefeatures of housekeeping genes. Because deletion analysis showed that the promoter activity is located within
the y231 to q39 region and that removal of thesequences between y231 and y84 reduced transcriptional activity to near basal levels, we concentrated on
characterizing the y231 to y84 region. Using EMSA,
we identified three specific regions that clearly bind to
nuclear proteins. EMSA with unlabelled competitor
oligonucleotides and antibodies showed that Sp1 bindto the y195 to y167 region, where the CACCC
sequence is critical. Although its complementary

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Fig. 7. Comparison of the binding affinity of CBF to different oligonucleotides containing CCAAT-like motifs. Electrophoretic mobility shift assays were performed using labeled y134yy106
oligonucleotides containing ATTTG (wild-type, lane 1), ATTGG(prototype, lane 2), ATTCG (lane 3), and TTTGG (lane 4).

sequence, GGGTG, in the y110 to y80 region, showed
the same results as the CACCC sequence by in competition assays, we could not confirm the presence ofsupershifted bands in this region using Sp1 antibodies.
A factor related to Sp1 might be involved in this region.
However, this region is lacking in the human gene (Fig.
1b). Taken together with the data of the luciferase assay
using mutations (Fig. 6b), these results suggest that
Sp1ySp3 or related factors do not greatly affect basic
promoter function, or that their involvement is dependent
upon cell type, wherein their contributions may differ.

95

In contrast, despite the lack of a canonical CCAAT siteor the reversed ATTGG site, CBF bound to the CCAATlike motif (reversed ATTTG) in the y134 to y106
region, and activated the Col5a1 promoter. Moreover,overexpression of a wild and mutant types of CBF-B
subunit altered this promoter activity.
The CCAAT box is the most common element ineukaryotic promoters, found between 50 to 110 bp
upstream from the start of transcription in both orientations. The mammalian promoters containing the CCAAT
box are divided into three groups based on the effect on
promoter activity of mutations to the motif (Maity andde Crombrugghe, 1998). In group I, mutation of the
CCAAT motif in the promoter decreases basal promoter
activity. In groups II and III, on the other hand, the
CCAAT motif is necessary for the induction of promoter
activity by various agents, and the alteration of promoter
activity during cell growth, respectively. In the collagengene family, CBF binds to the CCAAT motif in thea1(I) and a2(I) collagen promoters, resulting in anelevation of basal promoter activity. Similarly, we foundthat CBF activates basal promoter activity in the mousea1(V) collagen gene. CBF requires the perfect pentanucleotide, CCAAT, except in a few genes (Mantovani,1998). The globin r39E gene and the ApoA-I gene
(Wandersee et al., 1996; Papazafiri et al., 1991) containa CAAAT sequence to which CBF can bind. For CBF
to bind to the CCAAT motif, three 59 (C, Pu, Pu) andfive 39 (CyG, AyG, G, AyC, G) flanking nucleotides
also appear to be important (Fig. 9a) (Mantovani, 1998).
These 59 and 39 flanking sequences of Col5a1 perfectly
match those of Col1a1.
Lee and Greenspan reported that the human COL5A1
minimal promoter region contains GAGA boxes, whichhave been found in the TATA-less promoter regions ofFig. 8. Overexpression of a wild-type (WT) and mutant (DN) forms of CBF-B. NIH3T3 and A204 cells were transfected with the y231yq39Luc construct together with plasmid DNA expressing a wild-type, or mutant form of CBF-B, or control vector with no insert (pCXN2). Transfections were normalized by cotransfection with the Renilla vector, pRL-TK. The data represent an average of five independent tests "S.D.
Asterisks indicate statistically significant compared to control vector (P-0.05).

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Fig. 9. The flanking sequence of CCAAT motif in the collagen promoters and schema of the core promoter region of Col5a1. (a) The 59 and 39
flanking sequences of the CCAAT motif in a1(V), a1(I), and a2(I) collagen promoters with reverse orientation. The nucleotides that differ fromthe consensus sequence are indicated with underlining. (b) Schematic summary of the binding of nuclear factors to the core promoter region of
Col5a1. Sp1 bind to a CACCC box in the y195 to y167 region. CBF binds to a CCAAT-like motif in the y134 to y106 region. The Sp1family may bind in the y110 to y80 region.

a number of extracellular matrix genes, immediately
upstream and downstream from the major transcription
start site (Lee and Greenspan, 1995). The region containing the downstream 56-bp GAGA box is sensitive
to cleavage with S1 nuclease and has promoter activity
in COL5A1, although the upstream region does not.
They speculated that the binding factor at the GAGAbox affects chromatin structure. However, we observed
no promoter activity in the corresponding region of themouse gene. Because the sequence is not similar in both
species, it may only influence the human gene, and not
the mouse gene. Our preliminary experiment showed afaint band in the upstream GAGA box of the mousepromoter by EMSA (data not shown). Although we
could not identify a factor that binds to the upstream
GAGA boxes, one may contribute to promoter activity(Fig. 1b and Fig. 9b).
Type V collagen is found in most connective tissue
matrices, usually co-expressed with type I collagen. In
the proximal promoter of a1(I) and a2(I) collagengenes, Sp1, which binds at several sites, and CBF areresponsible for basal promoter activity. CBF is especiallyrequired for the coordinated expression of both type I
collagen genes. Type III collagen is also co-expressedwith type I collagen, but CBF cannot bind to its proximal
promoter, suggesting that other factors are involved in
the mechanism underlying the co-expression of type I
and III collagen genes (Ruteshouser and de Crombrugghe, 1989). Since CBF activates the basal promoter of
the a1(V) collagen gene, as shown in our experiment,
CBF may be a key factor in the co-expression of type I
and type V collagens. For collagen fibril formation, twocollagen molecules must be produced simultaneously,
even if the ratio of their production is different spatiallyand temporally. Sox9 and related factors are necessary
for the specific production of type II and XI collagens,which form collagen fibrils in cartilaginous tissue(Lefebvre et al., 1997; Bridgewater et al., 1998). Similarly, type I and V collagens co-polymerize to form
fibrils in non-cartilaginous tissues. Because CBF itself
is the ubiquitous factor, it may be utilized for the broad
expression of type I and V collagens. A specific factor,
such as cbfa1, which is critical for osteoblast differentiation (Ducy et al., 1997), probably binds to someregion of the a1(V) gene. The identification of factors
other than CBF that affect cell-specific expression
should provide further information on the expression of
the type V collagen gene.
4. Materials and methods
4.1. Isolation and characterization of genomic clones
To isolate the 59 flanking region of the mouse a1(V)collagen gene, a partially Sau3A1-restricted genomic
library constructed in the Lambda FIX II vector (Stratagene, La Jolla, CA) was screened using the productof a rapid amplification of cDNA ends (RACE) as aprobe (Yoshioka et al., 1995). RACE was carried out
according to the method described by Frohman et al.
(1988). Briefly, 20 mg of total RNA from 18-day-old
mouse lung was reverse-transcribed using a Col5a1specific primer, RACE1 (Fig. 1b). After tailing, thecDNA molecules were amplified by the polymerase
chain reaction (PCR) using a 59 adaptor primer and a
39 Col5a1-specific primer, RACE2. Amplifications werecarried out for 35 cycles of 94 8C for 1 min, 55 8C for
1 min, and 72 8C for 2 min, followed by a final

K. Sakata-Takatani et al. / Matrix Biology 23 (2004) 87–99

extension at 72 8C for 8 min. The RACE products were
subcloned using the TA Cloning Kit (Promega, Madison,
WI). Positive genomic clones were characterized as
described previously (Yoshioka et al., 1995). Sequenceswere analyzed with an ABI 310 sequencer (Perkin–
Elmer Life Sciences and Applied Biosystems).
4.2. Determination of the transcription start site
For this purpose, RACE and ribonuclease protection
assays were used. In addition to the primers describedabove, another set of Col5a1-specific primers, RACE3
and RACE4, was used for the RACE procedure (Fig.
1b). Ribonuclease protection assays were performed
using an RPA III Kit (Ambion, Austin, TX) according
to the manufacturer’s instructions. The DNA templatefor generating the riboprobe was derived from mgKS3
genomic DNA (Fig. 1a), using PCR with the RNase2
and RACE3 primers. The product was subcloned into
the TA vector and linearized at the HindIII site. Total
RNA (10 mg) was hybridized overnight at 42 8C to105 counts per minute (cpm) of radiolabeled antisense
riboprobe produced with an in vitro transcription kit(Ambion). Following hybridization, the reactions wereincubated with single-strand-specific RNase, and theprotected fragments were analyzed on a 5% denaturing
polyacrylamide gel (Yoshioka et al., 1995).
4.3. Construction of chimeric plasmids for luciferase
assay
Luciferase reporter constructs were generated using
the promoterless firefly luciferase reporter vector, pGL3Basic (Promega, Madison, WI). SacI-site-linked 59 and
XhoI-site-linked 39 primers specific for the Col5a1
genomic sequence determined above were used to amplify the mgKS3 genomic fragment by PCR. Initially, they811yq39 fragment was amplified and cloned into thepGEM-T Easy vector. The y468yq39-Luc and y231y
q39-Luc constructs were produced by digesting thisfragment with SacIyXhoI and SmaIyXhoI, respectively,
and were ligated into the corresponding sites in pGL3.
The y84yq39-Luc, y1yq39-Luc, and y231yq156Luc fragments were generated by PCR amplification,cloned into the pGEM-T vector and ligated into the
SacIyXhoI sites of pGL3. To construct the fragments
y1700yq39-Luc, y1095yq39-Luc, and y811yq39Luc, the upstream fragments y1700yy469, y1095yy469, and y811yy469 were amplified by PCR andligated into the SacI site in y468yq39-Luc in thecorrect orientation. To generate the y1700yy232-Luc
fragment, the y1700yy469 fragment was ligated to
y468yy232-Luc. To generate the y468yy232-Luc
fragment, y468yq39-Luc was digested with SacIy
SmaI to remove the 39 section. For the y1700yq156Luc fragment, the y1700yq39-Luc fragment was cut

97with SmaIyXhoI and ligated to the y231yq156 fragment derived from the original genomic fragment. The
XhoI site, CTCGAG, was introduced into the y231yq
39-Luc fragment to generate the mutant constructs, m1Luc, m2-Luc, and m3-Luc. The upstream fragments
were amplified by PCR using SmaI-site-linked 59 and
XhoI-site-linked 39 primers, cloned into the pGEM-T
vector, transferred into the SmaIyXhoI sites of pGL3,
and ligated to the downstream fragments linked to XhoI
sites. All PCR-derived constructs were fully sequenced
and compared with the original genomic clone.
4.4. Construction of CBF-B mutant construct
The CBF-B construct was generated by RT-PCR using
RNA extracted from A204 cells. A set of primers weresense: 59-GTCGACGGAGGGACCATGGAGCAGTATA-39; andantisense: 59-TCTAGAGGGTTAGGACACTCGGATGATC-39.
(Underlining indicates tagged SalI and XbaI restriction sites for cloning). This PCR products was cloned
into the pGEM-T Easy vector and subcloned into the
SalIyXbaI site in the empty vector.
The CBF-B mutant construct was generated by replacing Arg-Gly-Glu (amino acids 312–314) with Ala-AlaAla (Mantovani et al., 1994). Two sets of primers forthe 59 and 39 CBF-B mutant fragments were:sense: 59-GTCGACGGAGGGACCATGGAGCAGTAATA-39;
antisense: 59-GCGGCCGCCTTCCGTGCCATGGCATGAC-39;
sense: 59-GCGGCCGCAGGTGGACGATTTTTCTCTC-39; andantisense: 59-TCTAGAGGGTTAGGACACTCGGATGATC-39.
(Underlining indicates tagged SalI and XbaI restriction sites for cloning, and italicized sequences are NotI
sites, which introduce mutation). These PCR products
were cloned into the pGEM-T Easy vector and subcloned into the SalIyNotI sites for the 59-CBF-B mutant,followed by the NotIyXbaI sites for the 39-CBF-B mutant
into the empty vector.
Finally, wild and mutant forms were cloned into themammalian expression vector, pCXN2.
4.5. Cells and culture conditions
The cell lines used in this study were NIH3T3 mouse
fibroblasts and human rhabdomyosarcoma A204
(HTB82) cells. These cells were grown and maintainedin Dulbecco’s modified Eagle’s medium supplemented
with 10% heat-inactivated fetal bovine serum (SankoJunyaku, Tokyo, Japan).

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K. Sakata-Takatani et al. / Matrix Biology 23 (2004) 87–99

4.6. Transient transfections and luciferase assays
Approximately 2=105 cells were seeded in 20 mm
dishes 24 h before transfection using 5 mg of various
Col5a1 promoter reporter constructs co-transfected with
0.25 mg of Renilla pRL-TK plasmid. Transient transfections were performed using the calcium-phosphate precipitation method (Yoshioka et al., 1995; Graham andvan der Eb, 1973). After transfection, the cells wereincubated at 37 8C for 3 h, shocked for 1 min in a 15%(vyv) glycerol solution, washed with phosphate-bufferedsaline, and incubated with fresh medium. After 48 h,
the cells were washed with phosphate-buffered saline
and lysed in Passive Lysis Buffer (Promega) at room
temperature for 5 min. Firefly and Renilla luciferase
activities were determined using the Dual-Luciferase
Reporter Assay System (Promega), according to the
manufacturer’s instructions using a luminometer (Lumat
LB 9507, Perkin–Elmer Life Sciences). Transfection
experiments were independently repeated 3–5 times.
For co-transfection experiments with a wild andmutant types of CBF-B expression vector, 1.25 mg of
Col5a1 reporter plasmid DNA was transfected together
with 3.75 mg of wild or mutant types of CBF-B cDNA,
or pCXN2 vector DNA (Mantovani et al., 1994). Transfections were normalized by co-transfection with 0.25
mg of the Renilla vector pRL-TK.

oligonucleotides were generated using mutant luciferase
constructs as DNA templates. DNA fragments weredigested with HindIII and end-labeled with wa-32PxdCTP
using Klenow fragment (Amersham Biosciences).
EMSA was carried out by incubating 5–10 mg ofnuclear extract, 3 mg of poly(dI-dC), 5= reaction buffer
containing 50 mM HEPES-KOH (pH 7.8), 250 mM
KCl, 5 mM EDTA-2Na (pH 8.0), 50% glycerol, and 25
mM MgCl2 (Yoshioka et al., 1995). Approximately
30 000 cpm of radiolabeled probe was added to thepreincubation mixture. After 30 min incubation at 25
8C, DNA–protein complexes were resolved on nondenaturing 4.5% acrylamide gels in 0.25 TBEy2.5%
glycerol at 4 8C and visualized by autoradiography. For
the competition assays, double-stranded oligonucleotides
containing a consensus Sp1, Oct-1, NF-1, CBF, or CBF
mutant binding site were generated by annealing equimolar complementary oligonucleotides at 100 8C.
The consensus sequences are:Sp1:
Oct-1:NF-1:
CBF:

59-ATTCGATCGGGGCGGGGCGAGC-39,
59-TGTCGAATGCAAATCACTAGAA-39,
59-TTTTGGATTGAAGCCAATGATA-39,
59-AGACCGTACGTGATTGGTTAATCTCTT39,
CBF mutant: 59-AGACCGTACGAAATACGGGAATCTCTT-39.

4.7. Preparation of nuclear extracts
Nuclear extracts were prepared from 1=108 NIH3T3
cells (Yoshioka et al., 1995; Morris et al., 1986). Cells
were scraped in phosphate-buffered saline and centrifuged for 5 min at 1500 rev.ymin. The pellets were
resuspended in 10 ml hypotonic buffer (10 mM HEPES
wpH 7.8x, 10 mM KCl, 0.1 mM EDTA-2Na, 0.1% NP40)
and homogenized for 10 min on ice. The homogenate
was centrifuged for 10 min at 3000 rev.ymin at 4 8C.
The supernatant was removed, and the nuclei in thepellet were resuspended in 400 ml of low-salt buffer (20mM HEPES wpH 7.8x, 420 mM KCl, 0.1 mM EDTA2Na, 20% glycerol, 5 mM MgCl2) by stirring with apipette tip. Nuclear proteins were extracted for 30 min
at 4 8C with continuous gentle mixing with a rotator,and centrifuged for 15 min at 15 000 rev.ymin. The
supernatants were subsequently centrifuged for 30 min
at 24 000=g at 4 8C. The protein concentration of theextracts was determined using Bio-Rad reagent. Solutions containing the nuclear proteins were frozen immediately at y80 8C. The samples were thawed gently on
ice just before electrophoretic mobility-shift assays were
performed.
4.8. Electrophoretic mobility-shift assay (EMSA)
Probes for EMSA were generated by PCR using
HindIII-site-linked Col5a1-specific primers. The mutant

For the supershift assay, 2 mg of polyclonal antibody
directed against Sp1, Oct-1, or CBF subunits A, B or
C, or preimmune goat polyclonal IgG (Santa Cruz
Biotechnology, CA) were added to the reactions andincubated at 4 8C for 1 h before the addition of probe.
Acknowledgments
We thank Dr F. Ramirez for reviewing the paper, Dr
T. Kumamoto and Dr K. Sugisaki for support during
this work, and the staff of Division of Biomolecular
Medicine and Medical Imaging, and Division of Radioisotope Research, Institute of Scientific Research, Oita
University where we performed some experiments. This
work was supported by Grants-in-Aid for Scientific
Research (11470312 and 14370468 to H.Y.) from the
Ministry of Education, Culture, Sports, Science andTechnology of Japan.
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