THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 279, No. 45, Issue of November 5, pp. 46373–46383, 2004
Printed in U.S.A.

The Transcription Factor CCAAT-binding Factor CBF/NF-Y and Two
Repressors Regulate the Core Promoter of the Human Pro-␣3(V)S
Collagen Gene (COL5A3)*□
Received for publication, June 1, 2004, and in revised form, August 16, 2004
Published, JBC Papers in Press, August 16, 2004, DOI 10.1074/jbc.M406069200

Hitoshi Nagato‡§, Noritaka Matsuo‡, Hideaki Sumiyoshi‡, Keiko Sakata-Takatani‡,
Masaru Nasu§, and Hidekatsu Yoshioka‡¶
From the ‡Department of Anatomy, Biology and Medicine, and §Department of Immunology and Allergy, Faculty of
Medicine, Oita University, 1-1 Hasama-machi, Oita 879-5593, JapanTo elucidate the mechanisms underlining ␣3(V) collagen chain expression, we performed an initial analysisof the structure and function of the core promoter of thehuman COL5A3 gene. The core promoter, which lacks atypical TATA motif and has a high GC content, wasdefined within the ⴚ129 bp immediately upstream fromthe major transcription start site by transient transfection experiments. In this region, we identified four DNAprotein complexes, named A, B, C, and D, by a combination of DNase I footprinting and electrophoretic
mobility shift assays. Electrophoretic mobility shift assays using mutant oligonucleotide revealed that thecomplexes A, B, C, and D bind to ⴚ122 to ⴚ117, the ⴚ101
to ⴚ96, the ⴚ83 to ⴚ78, and the ⴚ68 to ⴚ57 bp, respectively. The competition assays using consensus oligonucleotides and supershift assays with specific antibodies
showed that complex A consists of CBF/NF-Y. In a chromatin immunoprecipitation assay, CBF/NF-Y protein directly bound to this region, in vivo. Functional analysis
showed that CBF/NF-Y activated the gene, whereas theproteins of complexes B and C repressed its activity.
Furthermore, overexpression of a mutant form of the
CBF-B/NF-YA subunit, which forms CBF/NF-Y with CBFA/NF-YB and CBF-C/NF-YC subunits, inhibited promoter activity.

Vertebrate collagens, a large family of extracellular proteins,are critically important for the formation and function of virtually every organ system (1). Among them, fibrillar collagen,which includes five different molecular types I, II, III, V, and
XI, participates in the formation of fibrils with molecules
packed in quarter-staggered arrays (2, 3). The fibrillar collagens are divided into major types (I–III) and minor types (Vand XI) based on their relative expression levels. Minor fibrillar collagen types V and XI are incorporated into the fibrils of
the much more abundant collagen types I and II, respectively,
and act as regulators of the sizes and shapes of the resultant
heterotypic fibrils (4 –7). The collagen molecules are either* 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 and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
□
S The on-line version of this article (available at http://www.jbc.org)
contains Table S1.
¶ To whom correspondence should be addressed: Dept. of Anatomy,
Biology and Medicine, Faculty of Medicine, Oita University, 1-1
Hasama-machi, Oita 879-5593, Japan. Tel.: 81-97-586-5670; Fax: 8197-549-6302; E-mail: hidey@med.oita-u.ac.jp.
This paper is available on line at http://www.jbc.org

homotrimers with ␣ chains or heterotrimers with two or threedifferent ␣ chains. The predominant molecular form of type V isthe heterotrimer [␣1(V)]2␣2(V) and is expressed in most tissues(8). Other forms of type V collagen include the [␣1(V)]3 homotrimer that is synthesized in cultures of hamster lung cells (9) andthe ␣1(V)␣2(V)␣3(V) heterotrimer that can be extracted fromthe placenta (10 –12). Cross-type heterotrimer composed of
␣2(V) and ␣1(XI) chains is present in the rhabdomyosarcoma
cell line A204 and bovine vitreous (13, 14). These findings
suggest that type V and type XI chains constitute a single
collagen type in which different combinations of chains associate in a tissue-specific manner. Recently, a fourth chain, ␣4(V),expressed in rat Schwann cells was reported (15). This ␣ chain,
which can form molecules with ␣1(V) and ␣2(V) chains, seems
to be the counterpart of the mouse and human ␣3(V) chain (16).
Type V collagen, which is widely expressed spatially andtemporally, is expressed in the mouse embryo as early as 11days post-coitum (17). Altered production of type V collagen isassociated with some connective tissue pathology, such as inflammation, some forms of cancer, and atherosclerosis (18 –20).
Characterization of the cis-acting elements and trans-acting
nuclear factors that modulate correct patterns of gene expression is necessary for understanding physiological and pathological conditions. Among the collagen genes, the transcriptional regulation of type I collagen has been most extensively
studied, showing common features of the proximal promoter
and tissue-specific enhancer (21, 22). With regard to minor
collagen, Penkov et al. (23) identified the nuclear factors binding to the proximal promoter of COL5A2. Their results indicatethat members of the homeotic complex and TALE class of
homeoproteins, PBX1/2, PREP1, and HOXB1, were involved inregulation of COL5A2. B-myb, a member of the myb genefamily, indirectly repressed the COL5A2 gene promoter byinteracting with the positive factor that binds to the first exon
(24). Lee and Greenspan (25) suggested that the GAGA boxesin the promoter and the first exon of the COL5A1 gene affectthe transcription. We have recently characterized the proximal
promoter of the COL11A1 (26) and Col5a1 genes (27). In thesestudies, we demonstrated that the CCAAT binding factor, CBF/
NF-Y, bound to the CCAAT motif in the proximal promoter andis involved in activation of the genes.
Recently, human and mouse full-length pro-␣3(V) sequenceswere provided (16). These were shown to be closely related tothe pro-␣1(V) chain. In situ hybridization of mouse embryo
detected its expression in epimysial sheaths of developing muscles and within nascent ligaments adjacent to forming bones
and in joints, where it probably forms molecules with ␣1(V) and␣2(V). In the present study, we cloned the human promoter
DNA fragment and characterized the core promoter of the46373

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Human Pro-␣3(V) Collagen Core Promoter

␣3(V) collagen gene. DNase I footprinting and DNA binding
assays have demonstrated four nuclear protein binding sites in
the promoter region. Cell transfection experiments showedthat one works as an activator and two as repressors. Finally,functional analysis combined with oligonucleotide competitionand supershift assays showed that CBF/NF-Y acts as a transcriptional activator of COL5A3.
MATERIALS AND METHODS

Cells and Cell Culture Conditions—Most of the cell lines used in thisstudy were purchased from the American Type Culture Collection(ATCC) and the Japanese Collection of Research Bioresources (JCRB)
cell banks. A204 cells (human embryonal rhabdomyosarcoma cell line,
ATCC HTB-82), HT-1080 cells (human fibrosarcoma cell line, ATCC
CCL-121), U251MG cells (human glioblastoma cell line, JCRB,IFO50288), IMR32 cells (human neuroblastoma cell line, ATCC CCL1277), and A498 cells (human epithelial cell line, ATCC HB-44) were
cultured in Dulbecco’s modified Eagle’s medium containing 10% heatinactivated fetal bovine serum (Sanko Junyaku, Tokyo, Japan) at 37 °C
in a 5% CO2/air environment. Jurkat cells (human T cell line, ATCC
TIB-152) and YST-1 cells, a primary cell line of human Schwannoma
cells (kindly provided by Dr. Y. Nagashima, Yokohama City University
School of Medicine), were cultured in RPMI 1640 medium containing
10% fetal bovine serum in the same environment.
Isolation of RNA—Total RNA was isolated from cultured cells using
Isogen (Nippon Gene Co. Ltd., Tokyo, Japan) according to the manufacturer’s instructions. RNA was quantified by optical density (A260)
and stored at ⫺80 °C until used.
Reverse Transcription-Polymerase Chain Reaction (RT1-PCR)—The
expression of COL5A3 mRNA in various cell lines was investigated by
RT-PCR. Three micrograms of total RNA was reverse transcribed byrandom hexamer priming using SuperScript II reverse transcriptase(Invitrogen). The single-stranded cDNA was amplified by PCR using
COL5A3-specific primer pairs and glyceraldehide-3-phosphate dehydrogenase primers. The primers are listed in Supplemental Materials
Table S1.
PCR was carried out for 30 cycles using a step cycle of 94 °C for 30 s,
55 °C for 20 s, 72 °C for 30 s, followed by 72 °C for 7 min. The PCR
products were analyzed by electrophoresis on a 4% ethidium bromidestained agarose gel. The amplified fragment was eluted from the gel
and subcloned into the pGEM-T Easy vector (Promega) for sequencing.
Screening Human BAC Library and Cloning of Genomic DNA Fragment—A human genomic BAC library was screened to obtain the
COL5A3 genomic fragment using the PCR protocol supplied by the
manufacturer (Genome System, Inc., St. Louis, MO). The primers specific for the COL5A3 used in the PCR-based screening are listed in
Supplemental Materials Table S1.
The DNA from the PCR-positive BAC clone was purified and thesequences at both ends were determined by a direct sequencing method
using Sp6 and T7 primers specific for the BAC cloning vector arms. The
sequences were compared with those in the EMBO/GenBankTM database. To obtain a smaller DNA fragment that contains the promoter
region of COL5A3, the DNA was digested with ApaI because thisrestriction site was located just upstream of the initiating ATG codon.
The resulting fragments were subcloned into the pBluescript (SK⫹)
vector. The sublibrary was screened by colony hybridization.
Rapid Amplification of 5⬘-cDNA Ends (5⬘-RACE)—The 5⬘-RACE experiment was carried out according to the method described by Frohman et al. (28). Briefly, 10 ␮g of total RNA from A204 or YST-1 cells
was reverse transcribed using the COL5A3-specific primer, RACE1 orRACE3 (Fig. 2C). Homopolymeric dC was added to the 3⬘-end of the firststrand cDNA using terminal deoxynucleotidyl transferase in the presence of dCTP. The dC-tailed cDNA was amplified with PCR using apoly(dG) containing the 5⬘ adapter primer and a 3⬘ COL5A3-specificprimer, RACE2 or RACE4, which was located just upstream of RACE1
or RACE3, respectively (Fig. 2C). PCR was carried out for 35 cycles
using a step cycle of 94 °C for 1 min, 55 °C for 1 min, 72 °C for 2 min,followed by 72 °C for 8 min. The 5⬘-RACE products were purified byelectrophoresis on a 4% ethidium bromide-stained agarose gel, and thensubcloned into the pGEM-T Easy vector for sequencing.

1
The abbreviations used are: RT, reverse transcription; CBF/NF-Y,
CCAAT-binding factor; RACE, rapid amplification of cDNA ends;
EMSA, electrophoretic mobility shift assay; CHIP, chromatin immunoprecipitation assay; PBS, phosphate-buffered saline; ss, single-stranded; FP, footprint; wt, wild-type; CEBP, cAMP element-binding protein.

In Vitro Transcription and RNase Protection Assay—RNase protection assays were carried out to confirm the transcription initiation sites.
A 252-bp genomic fragment, containing putative transcription initiation sites from the data of the 5⬘-RACE experiment, was generated by
PCR. This PCR product was subcloned into pGEM-T Easy vector, followed by digestion with EcoRI, and subcloned into the EcoRI site ofpBluescript (SK⫹) vector. After linearization with BamHI, the antisense riboprobe was synthesized using the in vitro transcription kit,
MAXIscript (Ambion, Austin, TX), according to the manufacturer’s
instructions. This riboprobe was examined by 5% polyacrylamide gel
containing 8 M urea, and the full-length riboprobe was recovered fromthe gel. RNase protection analysis was performed using a ribonuclease
protection assay kit, RPAIII (Ambion), following the manufacturer’s
protocol. Briefly, 20 ␮g of total RNA isolated from A204 or YST-1 cells
and 8 ⫻ 104 cpm of riboprobe were dissolved in 10 ␮l of hybridizationbuffer and incubated overnight at 42 °C. After digestion with the RNase
A/T1 mixture, protected RNA fragments were separated on a 6% polyacrylamide gel containing 8 M urea. As a size marker, 32P-end-labeled
␾X174 DNA/HaeIII marker was loaded onto the same gel. After the run
had finished, the gel was transferred onto 3MM paper (Whatman) anddried under vacuum. The dried gel was visualized by autoradiographyusing a Bio Imaging Analyzer FLA-5000 (Fuji Film, Tokyo, Japan).
Construction of Chimeric Plasmids—The ApaI genomic fragment
corresponding to the region from ⫺1785 to ⫹112 bp of the COL5A3
was blunt-ended with T4 DNA polymerase and subcloned into the
SmaI site of pGL3-Basic vector (Promega). To generate the 5⬘ stepwise deletion constructs, PCR procedures were applied. PCR was
performed using sets of oligonucleotide primers that are SacI sitelinked 5⬘ and HindIII site-linked 3⬘ primers specific for the COL5A3
sequence and the pGL3⫺1785/⫹109 plasmid as a template. These
PCR products were subcloned into the pGEM-T Easy vector, followedby digestion with SacI and HindIII, and subcloned into the SacI/
HindIII site of pGL3-Basic vector.
Internal deletion and substitution mutation constructs were generated by site-directed mutagenesis using the pGL3⫺310/⫹109 plasmid
as a template. The primers used in the PCR amplification are listed in
Supplemental Materials Table S1. The PCR products were digested
with endonuclease, followed by self-ligation. All mutagenesis plasmids
were digested with SacI and HindIII and re-cloned into the SacI/
HindIII site of pGL3-Basic vector. Construction of the dominant negative CBF-B/NF-YA was generated by RT-PCR as previously described(26, 29).
Transient Transfection and Luciferase Assays—Transient transfection experiments were carried out using A204 and YST-1 cells. The
cells were plated at a density of 2 ⫻ 105 per 35-mm dish ⬃18 h before
transfection. For transient transfection, 1 ␮g of plasmid DNA wastransfected into these cells by using the LipofectAMINE Plus reagent
system (Invitrogen). Plasmid pRL-TK vector (Promega) was always
cotransfected as an internal control for transfection efficiency. Afteran additional cultivation for 48 h, the transfected cells were harvested, lysed, centrifuged to pellet the debris, and subjected to luciferase assay. Luciferase activities were measured as chemiluminescence in a luminometer (Lumat LB 9507, PerkinElmer Life Sciences)using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol. The cotransfection experimentswith a mutant type of CBF-B/NF-YA subunit expression vector were
performed as previously described (26, 27). All transfections wererepeated in triplicate and the results were expressed as the mean ⫾S.D. of three independent experiments.
Preparation of Nuclear Extracts—Nuclear extracts from A204 andYST-1 cells were prepared according to the method of Dignam et al. (30).
Briefly, cells, 1 ⫻ 108, were scraped in ice-cold phosphate-bufferedsaline (PBS), centrifuged for 5 min at 1,500 ⫻ g, and washed with PBS
before being recentrifuged. The pellets were resuspended in buffer (10mM HEPES, pH 7.8, 10 mM KCl, 0.1 mM EDTA, 0.1% Nonidet P-40),
incubated on ice for 10 min, and homogenized. Nuclei were pelleted bycentrifugation at 3,000 ⫻ g for 10 min at 4 °C, followed by resuspension
in buffer (50 mM HEPES, pH 7.8, 420 mM KCl, 5 mM MgCl2, 0.1 mM
EDTA, 20% glycerol) and mixed by rotation at 4 °C for 1 h. Aftercentrifugation at 24,000 ⫻ g for 30 min at 4 °C, the supernatants werecollected and stored at ⫺80 °C until used. The protein concentration of
the nuclear extracts was determined by Bradford assay (Bio-Rad) using
bovine serum albumin as a standard.
DNase I Footprinting Assay—Probes for DNase I footprinting weregenerated by PCR amplification using sets of oligonucleotides that are
SacI site-linked 5⬘ and HindIII site-linked 3⬘ primers specific for
COL5A3, and all PCR products were subcloned into the pGEM-T Easy
vector. These plasmids were digested with HindIII and radiolabeledHuman Pro-␣3(V) Collagen Core Promoter

46375

FIG. 1. RNA expression of the
COL5A3 in various human cell lines.
RT-PCR analysis on total RNA from different cell lines. Lane 1, 100-bp laddermarker; lane 2, A204; lane 3, YST-1; lane
4, HT1080; lane 5, Jurkat; lane 6,
U251MG; lane 7, IMR32; lane 8, A498.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was included as a positive
control for the PCR.

with [␣-32P]dCTP using Klenow fragment to fill in the HindIII overhanging sites. After digestion with SacI, these probes were examined by
a 4.5% non-denaturing polyacrylamide gel and 3⬘ end-labeled probes
were recovered from the gel. DNase I footprinting was performed according to the protocol described by Galas and Schmitz (31) with somemodifications. Briefly, the 3⬘ end-labeled probe (50,000 cpm) was incubated at 25 °C for 30 min with 50 ␮g of nuclear extracts (or bovine
serum albumin as control) in a 100-␮l reaction mixture containing 20
mM Tris-HCl (pH 7.9), 3 mM MgCl2, 100 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol, 5 ␮g of bovine serum albumin, and 4 ␮g of poly(dI-dC).
After the addition of 5 units of DNase I (or 0.1 unit with bovine serum
albumin as control) to the mixture, the incubation was continued for 5
min at 25 °C. The reaction was stopped by adding 100 ␮l of a stop buffer(200 mM sodium acetate, 20 mM EDTA, 1% SDS, 10 ␮g of yeast tRNA),and DNA fragments were subsequently extracted with phenol/chloroform and precipitated with ethanol before being loaded onto a 6%
polyacrylamide gel containing 8 M urea. After electrophoresis, the gel
was transferred onto 3 MM paper, dried, and exposed to a BioImaging
Analyzer FLA-5000.
Electrophoretic Mobility Shift Assay (EMSA)—Wild-type and mutant
probes used for EMSA were generated by PCR using each set of HindIIIsite-linked primers, and all PCR products were subcloned into thepGEM-T Easy vector. All plasmids were digested with HindIII and thedigested fragments were radiolabeled with [␣-32P]dCTP using Klenow
fragment to fill in the HindIII overhanging sites. The binding reaction
was carried out for 30 min at 25 °C in 25 ␮l of binding buffer (50 mM
HEPES, pH 7.8, 250 mM KCl, 25 mM MgCl2, 5 mM EDTA, 50% glycerol)containing 20,000 –30,000 cpm of labeled probe, 3 ␮g of poly(dI-dC), and5–15 ␮g of nuclear extracts. For the competition assays, double strand
oligonucleotides containing a consensus CBF, CBF mutant, NF-1,
CEBP, GATA-1, c-Myb, and MEF-1 binding site were generated byannealing equimolar complementary oligonucleotides. The consensus
sequences are shown in Fig. 7A. For the supershift assay, anti-CBF-A/
NF-YB, anti-CBF-B/NF-YA, anti-CBF-C/NF-YC, anti-NF-1, antiCEBP, and anti-GATA-1 polyclonal antibodies and preimmune goat
IgG (Santa Cruz Biotechnology, Santa Cruz, CA) were purchased. For
these interference assays, 5–100-fold molar excess of unlabeled competitor or 2 ␮g of antibody was added to the reaction mixture for 1 h at 4 °C
before the addition of radiolabeled probe. The DNA-protein complexes
were separated on a 4.5% non-denaturing polyacrylamide gel in a 0.25⫻
Tris borate electrophoresis buffer at 200 V. Following completion ofrunning, the gel was transferred onto 3MM paper and dried undervacuum. The dried gel was visualized by autoradiography using a
BioImaging Analyzer FLA-5000.
Chromatin Immunoprecipitation (CHIP) Assay—CHIP assays were
performed using a chromatin immunoprecipitation assay kit (Upstate
Biotechnology, Lake Placid, NY) according to the manufacturer’s protocol. Briefly, A204 and YST-1 cells were inoculated at a density of 5 ⫻106 per 100-mm dish. The cells were fixed in a final concentration of 1%
formaldehyde for 10 min at room temperature. After washing twicewith PBS, cells were removed from dishes in PBS containing 1 mM
EDTA, and harvested by centrifugation at 1,000 ⫻ g for 1 min at 4 °C.
The cell pellets were resuspended in SDS lysis buffer, incubated for 10
min at 4 °C, and sonicated four times for 10 s yielding DNA fragments
200 –1000 bp in size. After centrifugation, the supernatant was dilutedin CHIP dilution buffer, precleared with salmon sperm DNA/protein
A-agarose slurry, and immunoprecipitated with the indicated antibodies overnight at 4 °C. Immunocomplexes were captured on the ssDNA/
protein A-agarose slurry, and washed with low salt wash buffer, highsalt wash buffer, and LiCl wash buffer and finally washed twice with
TE buffer. The immunocomplexes were eluted by incubation for 15 min
at 25 °C with 200 ␮l of elution buffer (1% SDS, 100 mM NaHCO3, 1 mM
dithiothreitol). To reverse the cross-linking of DNA, the elutes weretreated with 8 ␮l of 5 M NaCl and incubated for 4 h at 65 °C, followed bytreatment with proteinase K for 1 h at 45 °C. The DNA fragments wereextracted with phenol/chloroform and precipitated with ethanol. PCR
were carried out for 40 cycles using a step cycle of 94 °C for 30 s, 55 °C
for 20 s, 72 °C for 30 s, followed by 72 °C for 8 min. The PCR products
were analyzed by electrophoresis on a 4% ethidium bromide-stained
agarose gel.
DNA Sequencing—Nucleotide sequences were determined by automated DNA sequencing (ABI PRISM 310 Genetic Analyzer, Applied
Biosystems, Foster, CA) using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems).
RESULTSRNA Expression of COL5A3 in Cultured Cells—Initially, we
examined the expression of the COL5A3 in various culture cells
using the RT-PCR technique. As shown in Fig. 1, the predictedband of 295 bp was observed in YST-1 (strong) and A204 (weak)cells. The PCR products were verified by nucleotide sequence toconfirm the specificity of the amplified COL5A3 sequence. By
contrast, the band was not detected in HT1080, Jurkat,
U251MG, IMR32, and A498 cells. From these results, we decided to use A204 and YST-1 cell lines for the analysis of the
COL5A3 promoter.
Structural Analysis of the 5⬘ Portion of COL5A3—To obtain
the COL5A3 genomic fragment, we screened a human genomic
BAC library by PCR, and isolated one positive clone, 218M16.
Compared with the EMBO/GenBankTM data base, DNA sequences of both ends of the clone were identical with those of
two clones, AC008742 and AC020931, in chromosome 19 of
Homo sapiens. From the data base, this BAC genomic fragment, which was ⬃141 kb in size, contained the promoter
region and both exons and introns 1–32 of COL5A3 (Fig. 2A).
To subclone a DNA fragment containing the promoter region,
the BAC genomic fragment was digested with ApaI endonuclease and subcloned into the pBluescript (SK⫹) vector. The sublibrary was screened by colony hybridization and the insert
DNA of the positive clone was sequenced using COL5A3-specific or vector-specific primers. As a result, a 1897-bp genomic
fragment containing the 5⬘-flanking region of COL5A3 wasisolated (Fig. 2B).
To determine the transcription initiation sites of COL5A3,
5⬘-RACE experiments and RNase protection assays were performed. The 5⬘-RACE was carried out using total RNA derived
from A204 and YST-1 cells. Sixteen independent clones, eightclones from A204 cells and eight clones from YST-1 cells, wereselected and sequenced. As shown in Fig. 2C, the 5⬘-ends of the
COL5A3 cDNA were located at 113 to 119 bp upstream fromthe initiating ATG codon, and 13 of the 16 RACE products weregenerated from the same position, which suggested that the
major transcription initiation site is located 119 bp upstream
from the initiating ATG codon. On the basis of these data, we
also performed RNase protection assays to confirm the transcription initiation sites. In this experiment, we used a 357nucleotide riboprobe spanning nucleotides containing the putative transcription initiation site, and at least two specificprotected bands were observed in both A204 and YST-1 cells

46376

Human Pro-␣3(V) Collagen Core Promoter

FIG. 2. Structural analysis of the 5ⴕ region of the COL5A3. A, schematic representation of BAC clone containing COL5A3. AC020931 andAC008742 clones, which are assigned on human chromosome 19, are only shown with the portions that overlap with the BAC218M16 clone. E1,
E2, E3, E31, and E32, indicated with filled boxes, are the exons of COL5A3. SP6 and T7 indicate primers on the vector arms. B, partial restriction
map of COL5A3. Open and closed boxes represent untranslated and coding regions of exon 1, respectively. The transcription initiation site isrepresented by an arrow. Restriction endonuclease sites are shown as follows: P, PstI; B, BamHI; A, ApaI; K, KpnI; N, NotI. C, nucleotide sequenceof the proximal promoter region and part of exon 1 of COL5A3. The 5⬘ ends of the RACE products are shown by the black dots (A204) and the blackboxes (YST-1). RACE 1– 4, indicated with horizontal arrows, are sites of the primers used for 5⬘-RACE experiments. The transcription initiationsite and the translation initiator ATG codon are shown with a short arrow and a box, respectively. FP1 and FP2, indicated with striped bars, are
the DNase I-footprinted sites. RPA1 and RPA2, indicated under bars, are the approximate 5⬘ ends of the RNase-protected bands. The putative sites
where the factors bind are underlined. D, RNase protection analysis. Lane 1, undigested riboprobe; lane 2, RNA derived from A204 cells; lane 3,
RNA derived from YST-1 cells; lane 4, yeast RNA (negative control); lane 5, 32P-end-labeled ␾X174 DNA/HaeIII marker. Arrows indicate specificprotected bands. The ladders seen in the lower portion of lanes 2– 4 are nonspecific.

(Fig. 2D). The protected bands were about 115 and 100 nucleotides in size, and the larger protected band corresponded to
the results of the 5⬘-RACE. Therefore, we concluded that
COL5A3 contains at least two transcription initiation sites andthat the major transcription initiation site is located 119 bp
upstream from the initiating ATG codon. The COL5A3 promoter also lacks a canonical TATA box and has a highGC content.
Functional Analysis of the COL5A3 Promoter Region—To
define the proximal regulatory regions in the COL5A3 promoter, a series of chimeric constructs containing progressive 5⬘
end and internal deletions linked to the luciferase gene weretransfected into A204 and YST-1 cells (Fig. 3A), and luciferase
assays were carried out. The longest construct, pGL3⫺1785/⫹109, derived from a 1.9-kb genomic fragment of the 5⬘-flanking region of COL5A3, had strong transcriptional activity. The
activity of each construct was compared with this construct. As
shown in Fig. 3B, deletion from ⫺1785 to ⫺129 bp had no
significant change on luciferase activity; however, deletion to⫺40 bp resulted in a significantly lower luciferase activity
compared with that of pGL3⫺1785/⫹109, and deletion to the⫹3 bp showed almost complete loss of luciferase activity in both
A204 and YST-1 cells. In addition, the deletion mutant of
pGL3⫺310/⫹109(del⫺129/⫹2), which is deleted between ⫺129
and ⫹2, also showed a significantly lower luciferase activity inboth cells. These results indicate that the region between the⫺129 and ⫹2 bp is important for basal transcriptional activityof the COL5A3 promoter.

Human Pro-␣3(V) Collagen Core Promoter

FIG. 3. Luciferase assay of the COL5A3 promoter. A, schematic
illustration of the 5⬘-deletion and internal deletion constructs of the
COL5A3 promoter. B, luciferase activity in A204 (black histogram) andYST-1 cells (dotted histogram). All of the constructs were cotransfectedwith the pRL-TK vector as an internal control for transfection efficiency. Relative luciferase activities (%) were normalized against theactivity of pGL3-control vector. Data are the mean ⫾ S.D. of three
independent experiments.

Identification of Nuclear Factor Binding Sites in the Core
Promoter of the COL5A3—To identify transcription factor binding sites on the proximal promoter region of COL5A3, we initially performed DNase I footprinting. Two different probes,
which contain the region from the ⫺160 to ⫺51 bp (Fig. 4A) andfrom the ⫺96 to ⫹30 bp (Fig. 4B), were used in this experiment.
As shown in Fig. 4, A and B, two protected regions, FP (footprint) 1 in the ⫺124 to ⫺117 bp, and FP2 in the ⫺69 to ⫺63 bp
regions, were observed in the COL5A3 promoter.
To further investigate the binding of nuclear proteins to theproximal COL5A3 promoter, we carried out EMSA experiments. From the footprinting data, we prepared three overlapping oligonucleotides covering the region between ⫺160 and⫺51 bp (Fig. 5). The ⫺160/⫺101wt probe, covering the ⫺160 to⫺101-bp region, which contains the FP1 site, could bind nuclear protein extracted from A204 and YST-1 cells in a dosedependent manner (Fig. 6A). The band was competitively inhibited by corresponding excess unlabeled oligonucleotide (Fig.
6B). We prepared two substitution oligonucleotides, ⫺160/⫺101m2 and ⫺160/⫺101m1 (Fig. 5). The former was mutatedat the FP1 site and the latter at a site unrelated to FP1 region.
As shown in Fig. 6A, the ⫺160/⫺101m2 probe could not form a
DNA-protein complex, whereas the ⫺160/⫺101m1 probe couldin A204 and YST-1. Furthermore, this DNA-protein complex A
disappeared completely in the presence of 100-fold excess of
unlabeled ⫺160/⫺101m1 probe, but not in the presence ofexcess unlabeled ⫺160/⫺101m2 probe (Fig. 6B).
Although a protected region was not observed in DNase I
footprinting between positions ⫺115 and ⫺71 bp, the ⫺115/⫺71wt probe could bind nuclear proteins extracted from A204

46377

FIG. 4. DNase I footprinting analysis of the proximal promoter
region of the COL5A3. A 3⬘-end labeled probe of ⫺160/⫺51 (A) and⫺96/⫹30 (B) were used. These probes were incubated with bovine
serum albumin (lanes 1 and 5), A204 nuclear extracts (lanes 2 and 6),
and YST-1 nuclear extracts (lanes 3 and 7). Undigested 3⬘-end labeled
nucleotides of ⫺160/⫺51, ⫺129/⫺51, ⫺96/⫺51, and ⫺80/⫺51 (lane 4),and ⫺96/⫹30, ⫺80/⫹30, ⫺60/⫹30, ⫺40/⫹30, and ⫹3/⫹30 (lane 8) were
loaded on the same gel as a size marker. The protected regions, FP1 andFP2, are indicated by vertical bars.

FIG. 5. The probes used for EMSA experiments. Oligonucleotide
sequences of wild-type and mutant probes. FP1 and FP2 indicate the
DNase I footprinted sites. Bars in the mutant probes show the same
nucleotide sequences as those of wild-type.

and YST-1 cells, and three and two DNA-protein complexes
were observed in A204 cells and YST-1 cells, respectively (Fig.
6C, lanes 1 and 10). To clarify whether these complexes were
specific or not, we carried out EMSA experiments using unlabeled ⫺115/⫺71wt probe and salmon sperm DNA (ssDNA) as
competitors. The three DNA-protein complexes in A204 cells
were competed out in the presence of the unlabeled ⫺115/⫺71wt probe in a dose-dependent manner (Fig. 6C, lanes 2–5),
whereas the upper DNA-protein complex was abolished in thepresence of only a 5-fold excess ssDNA (Fig. 6C, lane 6). The

46378Human Pro-␣3(V) Collagen Core Promoter

FIG. 6. EMSA analysis of the COL5A3 promoter. A, the 32P-labeled ⫺160/⫺101wt (lanes 2–7), ⫺160/⫺101m1 (lanes 9 –14) and ⫺160/⫺101m2(lanes 16 –21) probes were incubated with nuclear extracts from A204 and YST-1 cells. The DNA-protein complex is indicated by A with an arrow.
Lanes 1, 8, and 15 were performed without nuclear extracts. B, competition analysis using an excess of unlabeled oligonucleotides. The 32P-labeled⫺160/⫺101wt probe was incubated with nuclear extracts from A204 and YST-1 cells in the presence of 100-fold excess unlabeled ⫺160/⫺101wt(lanes 3 and 7), ⫺160/⫺101m1 (lanes 4 and 8), and ⫺160/⫺101m2 (lanes 5 and 9) probes. Control assays were performed without nuclear extract

Human Pro-␣3(V) Collagen Core Promoter
middle and lower complexes remained even in the presence of
100-fold excess ssDNA (Fig. 6C, lane 9). We concluded that the
middle and lower DNA-protein complexes are specific but theupper complex is nonspecific in A204 cells. On the other hand,
the two DNA-protein complexes of YST-1 cells did not disappear in the presence of ssDNA (Fig. 6C, lanes 15-18). These
results showed two nuclear protein binding sites between the⫺115 and ⫺71 bp of the COL5A3 promoter. To verify these
nuclear protein binding sites, we prepared substitution mutation probes, ⫺115/⫺71m3, -m4, -m5, and -m6 (Fig. 5), andcarried out EMSA experiments. As shown in Fig. 6D, the ⫺115/⫺71m3 and ⫺115/⫺71m6 probes could not form the DNAprotein complexes B and C, respectively.
The position from ⫺80 to ⫺51 bp contained the FP2-protected site. As shown in Fig. 6E, the ⫺80/⫺51wt probe could
bind nuclear proteins extracted from A204 and YST-1 cells(lanes 1 and 10), and two and one DNA-protein complexes wereobserved in A204 cells and YST-1 cells, respectively. From thecompetition analysis, the upper DNA-protein complex observedin A204 cells was abolished in the presence of a 10-fold excess
of ssDNA (lane 7), although the lower complex was not competed out in the presence of 100-fold excess ssDNA (lane 9). We
concluded that the lower band, DNA-protein complex D, existed between the ⫺80 and ⫺51 bp. As shown in Fig. 6F,⫺80/⫺51 m7 mutation probe (Fig. 5) could form the DNAprotein complex D, but ⫺80/⫺51m8 and -m9 probes could not.
These results were consistent with the result of the DNase I
footprinting analysis.
Identification of Nuclear Factors in the Core Promoter of
COL5A3—To identify the nuclear binding proteins in the core
promoter of the COL5A3, we performed a computer analysis(transfac.gbf.de/TRANSFAC/). The FP1 site, where a protein
binds to form complex A, has the CCAAT motif that is recognized by transcription factors such as CBF/NF-Y, NF-1, CEBP,
and GATA-1 (Figs. 2C and 7A). The binding site for DNAprotein complex B contained putative transcription factor-binding sites for c-Myb and CEBP and that of complex C wasconsistent with E-box (Figs. 2C and 7A). However, at the FP2
site, where a protein binds to form complex D, a known factor
could not be identified. As shown in Fig. 7B, an excess of theconsensus oligonucleotides of CBFwt inhibited the binding ofthe ⫺160/⫺101wt probe, whereas NF-1, CEBP, GATA-1 consensus, and CBFmt oligonucleotides could not abolish the binding activity. On the other hand, c-Myb and CEBP, and MEF-1
consensus oligonucleotides could not compete out complexes B
and C, respectively (Fig. 7C).
To confirm the binding of CBF/NF-Y to the CCAAT motif, we
performed an interference assay using specific antibodies. As
shown in Fig. 8, DNA-protein complex A was only supershiftedby anti-CBF/NF-Y antibodies, namely anti-CBF-A/NF-YB,
CBF-B/NF-YA, and CBF-C/NF-YC, but not by other specificantibodies against NF-1, CEBP, and GATA-1, and control IgG.
To examine whether CBF/NF-Y binds directly to COL5A3, we
performed a CHIP assay. DNA-protein complexes were immunoprecipitated with antibodies, the cross-links reversed, andrecovered DNA fragments were monitored by PCR using prim-

46379

ers for the ⫺200 to ⫹109 bp region of the COL5A3 gene. As
shown in Fig. 9, DNA fragments immunoprecipitated withpolyclonal antibodies against CBF/NF-Y could be amplified by
PCR using the indicated primers (lane 5) as well as the positive
control (lane 2), whereas those immunoprecipitated with normal IgG (lane 4) or without antibody (lane 3) could not. These
results indicate that CBF/NF-Y binds specifically to the
CCAAT motif in the COL5A3 core promoter, in vivo.
The CBF/NF-Y and Two Repressors Regulate the Core Promoter of COL5A3—From the DNase I footprinting and EMSA
analysis, four nuclear protein binding sites were identified
between ⫺160 and ⫺51 bp. To clarify whether these nuclear
protein binding sites were functional, a series of substituted
mutations of a luciferase construct (Fig. 10A) were transfected
into A204 and YST-1 cells and luciferase assays were carriedout. The activity of each construct was compared with that of
pGL3⫺310/⫹109wt. As shown in Fig. 10B, the activity of the
pGL3⫺310/⫹109m2 construct was suppressed, whereas thoseof pGL3⫺310/⫹109m3 and -m6 constructs increased in both
A204 and YST-1 cells. On the other hand, the activities of the
pGL3⫺310/⫹109 m8 and m9 constructs did not change compared with that of the pGL3⫺310/⫹109wt construct. These
results show that the nuclear protein binding factor of DNAprotein complex A, namely CBF/NF-Y, activates the COL5A3
promoter and those of complex B and C suppress. Consistent
with these data, overexpression of the dominant-negative CBFB/NF-YA subunit, which interacts with the CBF-A/NF-YB and
CBF-C/NF-YC subunits to form CBF/NF-Y (27), suppressed thepromoter activity in both A204 and YST-1 cells by ⬃30% (p ⬍0.05) (Fig. 11).
DISCUSSION

The cloned 1.9-kb genomic fragment from the 5⬘-flanking
region of COL5A3 had strong transcriptional activity. The promoter has multiple transcriptional start sites, lacks the canonical TATA box around ⫺30 bp from the major transcriptional
start site, and has a high GC content. These structural features
are characteristic for the promoter of housekeeping genes. To
define the proximal regulatory regions in the COL5A3 promoter, a series of chimeric constructs containing progressive 5⬘
end and internal deletions linked to the luciferase gene weretransfected into A204 and YST-1 cells. The region from ⫺129 to⫹2 bp was necessary for the basal transcriptional activity of
COL5A3. With the combination analysis using footprinting and
EMSA, we identified four nuclear protein binding sites locatedbetween the ⫺160 and ⫺51 bp. Among them, we demonstrated
that CBF/NF-Y bound to the CCAAT motif in the ⫺122 to⫺117-bp region and acts as an activator, and the factors locatedin the ⫺101 to ⫺96 bp and in the ⫺83 to ⫺78-bp regions act
as repressors.
Transcription of collagen genes is controlled by a series ofcomplex interactions of positive and negative transcription factors. A number of regulatory elements required for constitutive
and inducible expressions have been identified in the promoterof both type I collagen genes (21, 22). The previous studies haveidentified the ubiquitous factors, Sp1 and Sp3, and CBF/NF-Y

and competitor (lane 1), and with nuclear extracts but without competitor (lanes 2 and 6). C, competition analysis using an excess of unlabeledoligonucleotides. The 32P-labeled ⫺115/⫺71wt probe was incubated with nuclear extracts from A204 and YST-1 cells in the presence of 5-, 10-, 20-,and 100-fold excess of unlabeled ⫺115/⫺71wt oligonucleotide (lanes 2–5 and 11–14) and ssDNA (lanes 6 –9 and 15–18) as competitors. The specificDNA-protein complexes are indicated by B and C with arrows, and nonspecific complex (N.S.) is indicated by an asterisk. D, binding assay using
6-bp substituted mutation probes. The 32P-labeled ⫺115/⫺71wt, -m3, -m4, -m5, and -m6 probes were incubated with nuclear extracts from A204
(lanes 1–5) and YST-1 cells (lanes 6 –10). E, competition analysis using an excess of unlabeled oligonucleotides. 32P-Labeled ⫺80/⫺51wt probe wasincubated with nuclear extracts from A204 and YST-1 cells in the presence of 5-, 10-, 20-, and 100-fold excess of unlabeled ⫺80/⫺51wt probe (lanes
2–5 and 11–14) and ssDNA (lanes 6 –9 and 15–18) as competitors. The specific DNA-protein complex is indicated by D with an arrow, and thenonspecific complex (N.S.) is indicated by an asterisk. F, binding assay using 6-bp substituted mutation probes. The 32P-labeled ⫺80/⫺51wt, -m7,
-m8, and -m9 probes were incubated with nuclear extracts from A204 (lanes 1– 4) and YST-1 cells (lanes 5– 8).

46380

Human Pro-␣3(V) Collagen Core Promoter

FIG. 8. Supershift analysis using specific antibodies against
various transcription factors. The 32P-labeled ⫺160/⫺101wt probe(lanes 1–17) and CBFwt consensus oligonucleotide probe (lanes 18 –26)
were incubated with nuclear extracts from A204 and YST-1 cells in thepresence of various antibodies, anti-CBF-A/NF-YB (lanes 3, 11, 20, and24), anti-CBF-B/NF-YA (lanes 4, 12, 21, and 25), anti-CBF-C/NF-YC(lanes 5, 13, 22, and 26), anti-NF-1 (lanes 6 and 14), anti-CEBP (lanes
7 and 15), anti-GATA-1 (lanes 8 and 16), and control IgG (lanes 9 and
17). Control assays were performed without nuclear extract and antibody (lanes 1 and 18), and with nuclear extracts but without antibody(lanes 2, 10, 19, and 23).

FIG. 9. Chromatin immunoprecipitation analysis. A CHIP assay
was performed to confirm the binding of CBF/NF-Y to the COL5A3
promoter, in vivo. Lane 1, 100-bp ladder marker; lane 2, input; lane 3;
immunoprecipitation without antibody; lane 4, immunoprecipitation
with normal IgG; lane 5, immunoprecipitation with polyclonal antibodies against CBF-A/NF-YB, CBF-B/NF-YA, and CBF-C/NF-YC. All immunoprecipitated DNA fragments were analyzed by PCR with theindicated primers.

binding factors for basal activity of these promoters (32–37).
Other putative transcription factors such as c-Krox, BFCOL1,
and IF-1 are also involved in the transcriptional regulation oftype I collagen genes (38 – 40). In COL5A3, we demonstrated
that CBF/NF-Y and two repressors are involved in the regulation of the core promoter. CBF/NF-Y was originally identified
as the proteins bound to the major histocompatibility complex
class II conserved Y box in Ea promoters and the yeast CYC1

FIG. 7. Competition analysis using various consensus oligonucleotides. A, consensus sequences of competitors. Bars in CBFmt show
the same nucleotide sequences with those of the wild-type. B, competition analysis using various consensus oligonucleotides as competitors.
The 32P-labeled ⫺160/⫺101wt probe was incubated with nuclear ex-

tracts from A204 and YST-1 cells in the presence of 100-fold excess
unlabeled CBFwt (lanes 2 and 8), CBFmt (lanes 3 and 9), NF-1 (lanes 4
and 10), CEBP (lanes 5 and 11), and GATA-1 (lanes 6 and 12). Lanes 1
and 7 were performed without competitor. C, competition analysis
using various consensus oligonucleotides as competitors. The 32P-labeled ⫺115/⫺71wt probe was incubated with nuclear extracts from
A204 and YST-1 cells in the presence of 100-fold excess competitors,
ssDNA (lanes 2 and 8), ⫺115/⫺71wt (lanes 3 and 9), c-Myb (lanes 4 and10), CEBP (lanes 5 and 11), and MEF-1 (lanes 6 and 12). Lanes 1 and 7
were performed without competitor.

Human Pro-␣3(V) Collagen Core Promoter

46381

FIG. 10. Functional analysis using
substitution mutation constructs of
the COL5A3 promoter activity. A,
schematic illustration of constructs thatare mutated in putative nuclear protein
binding sites in the COL5A3 proximal
promoter. B, luciferase activities in A204
and YST-1 cells transfected with the
pGL3⫺310/⫹109wt and substitution mutation constructs. All of the constructs
were cotransfected with pRL-TK vector as
an internal control for transfection efficiency. Relative luciferase activities (%)
were normalized against the activity ofthe pGL3⫺310/⫹109wt construct. Data
are the mean ⫾ S.D. of three independent
experiments. Asterisks indicate statistically significant results (p ⬍ 0.05).

UAS CCAAT box (41, 42). It was purified independently using
affinity columns containing the Y box, the ␣2(I) collagen
CCAAT, and the ␣-globin CCAAT sequence (36, 43, 44). CBF/
NF-Y is a ubiquitous heterotrimer composed of three subunits,
CBF-A/NF-YB, CBF-B/NF-YA, and CBF-C/NF-YC, all necessary for DNA binding (45). CBF/NF-Y requires the perfectpentanucleotide, CCAAT, except in a few genes (46). For CBF/
NF-Y to bind to the CCAAT motif, three 5⬘ (C, Pu, Pu) and five3⬘ (C/G, A/G, G, A/C, G) flanking nucleotides also appear to beimportant (Fig. 12A). These 5⬘- and 3⬘-flanking sequences areconserved in COL5A3 except one for nucleotide at the 3⬘-flanking region.
One of the repressors might bind to the E-box, located at ⫺83
to ⫺78 bp, in the core promoter region of COL5A3. The factor
binding to the E-box usually works as an activator, such as
MyoD (47). One of the factors for the specific expression ofmouse Col1a1 in tendon fibroblasts binds to the E-box locatedbetween ⫺3.2 and ⫺2.3 kb (48). The E-box in the 3⬘-flanking
region is also involved in the stimulation of transcription of
Col1a1 (49). The E2-box, a subset of the E-box, has a ciselement containing both activator and repressor binding sites.
The ␦EF1, which is a zinc finger protein, bound to ⫺307 to⫺100 bp in the promoter of rat Col2a1 gene, and repressed its

transcription (50). Seki et al. (51) reported that, in the snail andslug, the C2H2 type zinc finger protein suppressed the promoter activity of Col2a1, and that four E-box up to the ⫺1,000
bp from the transcription start site were responsible with theadditive efficiency. Other putative suppressors bound to ⫺101
to ⫺96 bp were also examined with the competition assay using
the oligonucleotide motifs of known factors. The results showedneither c-Myb nor CEBP, which were candidate factors predicted from a computer search. c-Myb could transactivate the
COL1A2 gene by binding at around ⫺1,025 bp, whereas B-Myb,which has a similar structure, could not (52). B-Myb repressed
the transcription of both type I collagen genes (53) and COL5A2(24). The Myb family may have a role in the regulation of
COL5A3.
Heterotrimer ␣1(V)␣2(V)␣3(V) molecules were originally extracted from human placenta (10 –12). The ␣3(V) chain was
also isolated from synovial membrane, gingival, skin (11), anduterus (54). In situ hybridization of mouse embryo detects
␣3(V) expression in the epimysial sheaths of developing muscles and within nascent ligaments adjacent to forming bones
and in joints (16). Chernousov et al. (55) reported the isolationof heparin sulfate-binding collagenous protein, p200, which isexpressed by Schwann cells in developing peripheral nerves.

46382

Human Pro-␣3(V) Collagen Core Promoter

FIG. 11. Effect of overexpression of dominant negative CBF-B/
NF-YA on the COL5A3 promoter activity. Cells were cotransfectedwith 0.5 ␮g of pGL3⫺310/⫹109 construct and 1.5 ␮g of mutant CBF/
NF-Y expression vector (pCXN2-CBFmut) or empty vector (pCXN2).
Relative luciferase activities (%) were normalized against the activity ofoverexpressed pCXN2 empty vector and the results are the mean ⫾ S.D.
of three independent experiments. Asterisks indicate statistically significant results (p ⬍ 0.05).

obscure. Our previous study suggested that CBF/NF-Y is required for the coordinated expression of the ␣1(V) gene andboth type I collagen genes (26). In this study, we showed that it
binds to the COL5A3 promoter. Penkov et al. (23) identified
PBX1/2, PREP1, and HOXB1 proteins as an FPB-bound complex in the proximal COL5A2 promoter. Although they did notrecognize a cis-acting element that binds CBF/NF-Y, it may
exist in some region of the COL5A2 promoter.
In fibrillar collagen genes, the expression of the type II
collagen gene is clearly different from those of type I collagengenes. The promoter of the ␣1(II) gene has a weak activity,
and the tissue-specific expression is regulated by the enhancer region in the first intron, where the Sox family binds(56). Although type III collagen is coordinately expressedwith type I collagen in fetal tissues and blood vessels, themechanism of regulation seems to be different from those oftype I collagen genes. Two distinct positive factors, one ofwhich is a heat-resistant protein of ⬃95 kDa, bind in theproximal promoter of the Col3a1 gene (57). CBF/NF-Y itself
is a ubiquitous transactivating factor; therefore, it may beutilized for the broad expression in noncartilaginous tissue
such as type I and V collagen genes. However, it is difficult to
explain the decrease in promoter activity from ⫺129 to ⫺40
bp by the sole involvement of CBF/NF-Y because the dominant negative mutant of CBF-B/NF-YA was partially suppressed (Fig. 11). Other activators that could not be identified
under the conditions of our system might bind in this region.
On the contrary, we showed two repressors in this region
instead of activators (Fig. 12B). The identification of factors
other than CBF/NF-Y should provide further information on
the type V collagen expression, followed by characterizationof different roles for the isoforms of type V collagen.
Acknowledgments—We thank Dr. Y. Nagashima for the generous
gifts of YST-1 cells, Dr. K. Murakami for support during this work, andthe staffs of the Division of Biomolecular Medicine and Medical Imaging
and the Division of Radioisotope Research, Institute of Scientific Research, Oita University, where we performed some experiments.
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FIG. 12. The flanking sequence of the CCAAT motif in thecollagen promoter, and schematic of the core promoter of
COL5A3. A, the sequences are forward orientation in ␣3(V) and reverse
orientation in ␣1(V), ␣1(I), and ␣2(I) collagen. The nucleotides thatdiffer from the consensus sequence are indicated with underlines. B,
schematic illustration of four nuclear protein binding sites and transcriptional activities in the COL5A3 core promoter. The indicated sequences are critical for the binding of nuclear proteins.

They named the rat ␣4(V) chain that shows 82% amino acidsequence identity to human ␣3(V) chain (15). In our experiment, human ␣3(V) chain is expressed in the Schwann cell line,
YST-1. The expression of ␣3(V) chain is less abundant compared with ␣1(V) chain, but distributed in many more tissues
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has been extensively studied. It is usually co-expressed with
type I collagen and forms type I/V heterotypic fibrils. The
nature of the ␣1(V)␣2(V)␣3(V) form has remained relatively

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