THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 278, No. 35, Issue of August 29, pp. 32763–32770, 2003
Printed in U.S.A.

The Transcription Factor CCAAT-binding Factor CBF/NF-Y
Regulates the Proximal Promoter Activity in the Human ␣1(XI)
Collagen Gene (COL11A1)*
Received for publication, May 28, 2003, and in revised form, June 11, 2003
Published, JBC Papers in Press, June 11, 2003, DOI 10.1074/jbc.M305599200

Noritaka Matsuo‡, Wang Yu-Hua‡, Hideaki Sumiyoshi‡, Keiko Sakata-Takatani‡, Hitoshi Nagato,
Kumiko Sakai§, Mami Sakurai§, and Hidekatsu Yoshioka‡¶
From the ‡Department of Anatomy, Biology, and Medicine, and §Research Laboratory Center, Oita Medical University,
Hasama-machi, Oita 879-5593, Japan

We have characterized the proximal promoter regionof the human COL11A1 gene. Transient transfection assays indicate that the segment from ⴚ199 to ⴙ1 is necessary for the activation of basal transcription. Electrophoretic mobility shift assays (EMSAs) demonstrated
that the ATTGG sequence, within the ⴚ147 to ⴚ121 fragment, is critical to bind nuclear proteins in the proximal
COL11A1 promoter. We demonstrated that the CCAAT
binding factor (CBF/NF-Y) bound to this region using aninterference assay with consensus oligonucleotides anda supershift assay with specific antibodies in an EMSA.
In a chromatin immunoprecipitation assay and EMSA
using DNA-affinity-purified proteins, CBF/NF-Y proteins directly bound this region in vitro and in vivo. We
also showed that four tandem copies of the CBF/NF-Ybinding fragment produced higher transcriptional activity than one or two copies, whereas the absence of a
CBF/NF-Y-binding fragment suppressed the COL11A1
promoter activity. Furthermore, overexpression of adominant-negative CBF-B/NF-YA subunit significantly
inhibited promoter activity in both transient and stable
cells. These results indicate that the CBF/NF-Y proteinsregulate the transcription of COL11A1 by directly binding to the ATTGG sequence in the proximal promoter
region.

The collagen superfamily, one of the extracellular matrix
proteins, plays an important role, not only in stabilizing thetissues as structural components but also in regulating a variety of biological functions, such as development, differentiation, proliferation, and morphogenesis (1–3). Among them,types I, II, III, V, and XI collagens are included in the group of
fibril-forming collagens, based on their structural and functional features, and are divided into two subgroups, major (I, II
and III) and minor (V and XI) fibrillar collagen (4) on the basisof their contents in tissues.
Type XI collagen is a component of the collagen fibrillar
network found in cartilage (5), and consists of three geneticallydistinct polypeptide chains: ␣1(XI), ␣2(XI), and ␣3(XI); the lastof these is thought to be overglycosylated ␣1(II) chains (6).

* 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, and The Nakatomi
Foundation (to H. Y.). The costs of publication of this article weredefrayed 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.
¶ To whom correspondence should be addressed. Tel.: 81-97-5865670; Fax: 81-97-549-6302; E-mail: hidey@oita-med.ac.jp.
This paper is available on line at http://www.jbc.org

Although type XI collagen is a relatively minor collagen and isburied within the major collagen fibrils, it is important for the
regulation of fibril diameter (7). Chondrodysplasia mice (cho),which do not synthesize ␣1(XI) chains, show irregular collagen
fibrils in their cartilage (8). Contrary to the previous findings,
the ␣1(XI) chain is not restricted to cartilage (9 –14), and it wasdemonstrated that the ␣1(XI) chain could form a heterotrimer
with the ␣2(V) chain in a 2:1 ratio in non-cartilaginous cells
and tissues (15, 16). Although the precise function of this crosstype trimer remains unclear, the ␣1(XI) collagen gene is more
broadly expressed than other collagen genes, showing that the␣1(XI) collagen gene is the sole collagen gene to be expressed inboth cartilaginous and non-cartilaginous tissues. This implies
that the regulation of the ␣1(XI) collagen gene might be more
complex than expected.
We have previously reported the structural and functionalfeatures of the human ␣1(XI) collagen gene (COL11A1) promoter (17). DNase I footprinting has mapped nine areas, FP1 to
FP9, covering the region ⫺541 to ⫹1, where nuclear proteinsprobably bind. In characterizing the ⫺541 to ⫺199 segment,
FP9-protein, which has homology to the GATA consensus motif
and binds at ⫺531 to ⫺487, was recognized to be a distinct⬃100-kDa polypeptide (18). It was expressed in mesenchymal
cells, and its level of binding activity was positively correlatedwith the degree of cell differentiation in osteoblastic and skeletal muscle cell lines, in vitro. At ⫺395 to ⫺379, the coresequence of FP7 exhibits some homology with that of FPB1 in
COL5A2, and FP7-protein complexes can be competed with anexcess of FPB oligonucleotides in an EMSA using nuclear extracts from the 1120 cell line (17, 19). Further studies usingtransfection experiments and EMSAs in A204 cells, however,indicated that the FPB-protein complexes identified were PBX,PREP and HOX proteins and therefore different from the FP7binding proteins (20).
In this study, we have characterized the region downstreamof the ⫺541 to ⫺199 region in the human COL11A1 promoter.
Transient transfection assays indicate that the segment from⫺199 to ⫹1 is necessary for the activation of basal transcription, and EMSAs demonstrate that the ATTGG sequence
within the ⫺147 to ⫺121 fragment is critical for binding nuclear proteins. Furthermore, we identify the CCAAT binding
factor CBF/NF-Y as binding to this region.

1
The abbreviations used are: FPB, Foot Printing B; EMSA, electrophoretic mobility shift assay; C/EBP, CCAAT/enhancer binding protein;
CBF, CCAAT binding factor; wt, wild type; CTF, CCAAT transcription
factor; NF-Y, nuclear factor for Y box.

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Human ␣1(XI) Collagen Proximal Promoter
MATERIALS AND METHODS

Cells and Cell Culture—Human rhabdomyosarcoma cell line A204
(17, 18) was used in this study. Cells were cultured in Dulbecco’s
modified Eagle’s medium supplemented with 10% heart-inactivatedfetal bovine serum (Sanko Junyaku, Tokyo) at 37 °C in humidified 5%
CO2/95% air.
Construction of Chimeric Plasmids—Essentially, all COL11A1 promoter-luciferase gene constructs were derived from ⫺1454 COL11A1/
CAT plasmid that fused to the CAT gene as described previously (17).
To obtain various 5⬘ deletion constructs, these fragments were generated by PCR using sets of oligonucleotide primers, which are SacI
site-linked 5⬘- and XhoI site-linked 3⬘-primers specific for the promoter
sequence. These PCR products were cloned into the pGEM-T Easy
vector (Promega, Madison, WI), followed by digestion with SacI and
XhoI, and subcloned into the SacI/XhoI site of pGL3-Basic vector.
The constructs that contained tandem fragments, PGL3(⫺199/⫺65)x2 and pGL3(⫺147/⫺121)x4, were chosen. Their DNA sizes werechecked on agarose gel, and the direction of the constructs was confirmed by sequence.
For internal deletion and mutation constructs, site-directed mutagenesis was apply (21). The constructs were derived from pGL3⫺541/⫹271 plasmid.
For 6 base-substitution, we introduced EcoRI sequence, GAATTC, forsubstitution sites. The primers for 6 base-substitution constructs,
pGL3⫺541E1, pGL3⫺541E2, pGL3⫺541E3, and pGL3⫺541E4, wereas follows: E1: sense, 5⬘-gaattcTGGGTCTGACCCTCAGCCTG-3⬘; antisense, 5⬘-gaattcCGACTCTGGGGCGGCCCCAA-3⬘; E2: sense, 5⬘-gaattcCTCAGCCTGCTTGTCAGTTT-3⬘; antisense, 5⬘-gaattcACCCAATCACACGACTCTGG-3⬘; E3: sense, 5⬘-gaattcTGTGATTGGGTCTGACCCTC3⬘; antisense, 5⬘-gaattcTGGGGCGGCCCCAAGCCCGC-3⬘; and E4:
sense, 5⬘-gaattcCTGACCCTCAGCCTGCTTGT-3⬘; antisense, 5⬘-gaattcTCACACGACTCTGGGGCGG-3⬘ (the EcoRI sites for mutation are indicated by lowercase letters). PCR products were digested with EcoRI,
followed by self-ligation. All mutagenesis plasmids were digested with
SacI and XhoI and re-cloned into the SacI/XhoI site of pGL3-Basic
vector.
The primers for pGL3⫺541CBFm, pGL3-del⫺147/⫺121, and pGL3del⫺199/⫺65 were as follows: for CBF mutant constructpGL3⫺541CBFm: sense, GaaATacGggCTGACCCTCAGCCTGCTT;
antisense, ccCgtATttCACGACTCTGGGGCGGCCC (mutated nucleotides are indicated by lowercase letters); for deletion construct pGL3del-147/⫺121: sense, 5⬘-AGCCTGCTTGTCAGTTTCGC-3⬘; antisense,
5⬘-GGGGCGGCCCCAAGCCCGCC-3⬘; for deletion construct pGL3del⫺199/⫺65: sense, 5⬘-GGCGGAGGAGGGGGCTGCCC-3⬘; antisense,
5⬘-GAGCAGGCCCAGCCCACCAA-3⬘.
Construct of the dominant-negative CBF-B/NF-YA was generated byreverse transcriptase PCR as described previously (22, 23). Two sets ofprimers were as follows: for 5⬘-CBF-B/NF-YAmut: sense, 5⬘-gtcgacGGAGGGACCATGGAGCAGTATA-3⬘; antisense, 5⬘-GCGGCCGCCTTCCGTGCCATGGCATGAC-3⬘, and for 3⬘-CBF-B/NF-YAmut: sense, 5⬘-GCGGCCGCAGGTGGACGATTTTTCTCTC-3⬘; antisense, 5⬘-tctagaGGGTTAGGACACTCGGATGATC-3⬘ (functional mutated nucleotides areindicated by lowercase letters.)
These PCR products were cloned into the pGEM-T Easy vector andsubcloned into the SalI/NotI site for 5⬘-CBF-B/NF-YA mut, followed by
NotI/XbaI site for 3⬘-CBF-B/NF-YAmut into the empty vector. Finally,
this dominant-negative form of CBF-B/NF-YA was cloned into pCXN2
mammalian expression vector (24). All constructs mentioned above
were sequenced on an ABI 310 sequencer (Applied Biosystems) according to the manufacturer’s protocol.
Transfection and Luciferase Assays—The cells were inoculated at adensity of 2 ⫻ 105 per 35-mm dish 24 h before transfection. For transient transfection, each of plasmid DNA was transfected into these cells
by using the calcium phosphate precipitation method followed by a 15%
glycerol shock for 60 s (25). After an additional cultivation for 48 h, thetransfected cells were harvested, lysed, centrifuged to pellet the debris
and performed to luciferase assay as described below. For stable transfection, 1 ␮g of pGL3⫺199/⫹271 or pGL3(⫺147/⫺121)x4 luciferase
construct with 50 ng of pMAM2-BSD selection vector were transfected
into A204 cells by using the LipofectAMINE Plus reagent system (Invitrogen) and cultured for 48 h. These transfected cells were replated ata density of 1 ⫻ 103 per 100-mm dish with fresh medium containing 8
␮g/ml of blasticidin (Funakoshi, Tokyo, Japan). After an additional
cultivation for 2 weeks, the resistant colonies were isolated and further
cultured in the present of blasticidin, followed by assayed the luciferase
activity to select stable cell lines.
Luciferase activities were assayed by the Dual-LuciferaseTM Re-

porter Assay System according to the manufacturer’s protocol (Promega, Madison, WI) using a luminometer (Lumat L.D. 9507;
PerkinElmer Life Sciences). Five micrograms of firefly luciferase reporter construct and/or dominant-negative CBF-B/NF-YA expression
vector were cotransfected with 0.25 ␮g of pRL-TK Renilla reniformis
luciferase expression vector as an internal control for transfection efficiency. The pGL3-Basic and pGL3-Control vectors were used for eachexperiment as negative and positive controls, respectively. Relative
luciferase activities (percentages) of each construct were normalizedagainst the activity of pGL3-Control vector and results were expressed
as mean ⫾ S.E. of three to five independent experiments.
Electrophoretic Mobility Shift Assay (EMSA)—Nuclear extracts wereprepared according to Dignam et al. (26) with some modifications. Allbuffers contained the protease inhibitors leupeptin (2 ␮g/ml), aprotinin(2 ␮g/ml), pepstatin A (2 ␮g/ml), phenylmethylsulfonyl fluoride (0.5mM), and dithiothreitol (1 mM). Cells (1 ⫻ 108) were resuspended inbuffer (10 mM HEPES, pH 7.8, 10 mM KCl, 0.1 mM EDTA, and 0.1%
Nonidet P-40), followed by incubation on ice for 10 min and homogenized. After centrifugation at 3000 rpm for 10 min, the cell pellets were
resuspended in buffer (50 mM HEPES, pH 7.8, 420 mM KCl, 5 mM
MgCl2, 0.1 mM EDTA, and 20% glycerol) and rotated at 4 °C for 1 h. The
supernatants with nuclear proteins were recovered by centrifugation at
24,000 ⫻ g for 30 min, and protein concentration of the nuclear extracts
was determined by the Bradford’s colorimetric reagent (Bio-Rad) using
bovine serum albumin as a standard.
Wild-type and mutant probes derived from COL11A1 promoter fragment were generated by PCR using each set of HindIII site-linked
primers, and all PCR products were cloned into the pGEM-T Easy
vector (Promega). Both sense and antisense oligonucleotides of themutant fragments covering ⫺147 to ⫺121, CBFwt, CBFmt, NF-1,
C/EBP, and GATA consensus overhanging HindIII sites at the ends
were synthesized (Proligo Japan KK, Kyoto, Japan), annealed to makedouble-stranded oligonucleotides, digested with HindIII, and cloned
into the HindIII site of pBluescript SK vector. The CBFwt, CBFmt,
NF-1, C/EBP, and GATA consensus sequences, based on the data fromSanta Cruz Biotechnology, Inc., used as competitors, were as follows:
CBFwt, 5⬘-AGACCGTACGTGATTGGTTAATCTCTT-3⬘; CBFmt5⬘-AGACCGTACGAAATACGGGAATCTCTT-3⬘ (mutated nucleotides areunderlined); NF-Y, 5⬘-TTTTGGATTGAAGCCAATATGATAA-3⬘;
C/EBP, 5⬘-TGCAGATTGCGCAATCTGCA-3⬘; and GATA, 5⬘-CACTTGATAACAGAAAGTGATAACTCT-3⬘.
All plasmids were digested with HindIII and the digested fragments
were radiolabeled with [␣-32P]dCTP using Klenow fragment to fill in
the HindIII overhang sites. For EMSA, the binding reaction was performed for 30 min at 25 °C in 25 ␮l of reaction buffer (50 mM HEPES,
pH 7.8, 250 mM KCl, 25 mM MgCl2, 5 mM EDTA, and 50% glycerol)containing 20,000 –30,000 cpm of labeled probe, 3 ␮g of poly(dI-dC), and1–20 ␮g of nuclear extracts.
For competitors and antibody interference assay, unlabeled probes orantibody was added to the reaction mixture for 1 h at 4 °C before theaddition of [␣-32P]dCTP labeled probe. These antibodies against CBFA/NF-YB, CBF-B/NF-YB, CBF-C/NF-YC, C/EBP, NF-1, GATA-1, andpre-immune goat IgG were purchased from Santa Cruz Biotechnology.
The DNA-protein complexes were separated on 4.5% nondenaturing
polyacrylamide gel in 0.25⫻ Tris-borate/EDTA with 2.5% glycerol at
4 °C and visualized by autoradiography using Bio Imaging Analyzer
BAS-2000 (Fuji Film, Tokyo, Japan).
Purification of DNA Binding Protein Using DNA Affinity Latex
Beads—DNA affinity latex beads were prepared as described previously(27). To purify DNA binding proteins, the DNA-latex beads were added
to the nuclear extracts and incubated for 24 h at 4 °C. After centrifugation at 15,000 rpm for 5 min, the beads were washed three times with
0.1⫻ buffer A (50 mM Tris-HCl. pH 7.9, 20% glycerol, 0.1 M KCl, 1 mM
EDTA, 1 mM dithiothreitol, and 0.1% Nonidet P-40) containing 0.1 M
KCl. To elute the binding proteins from the DNA-latex beads, the beads
were resuspended in 0.4⫻ buffer A containing 0.4 M KCl and incubatedfor 15 min at 4 °C. After centrifugation at 15,000 rpm for 5 min, thesupernatant containing DNA binding proteins was recovered and concentrated by microcon-10 (Millipore).
Chromatin Immunoprecipitation Assay—Chromatin immunoprecipitation assays were performed using a chromatin immunoprecipitation
assay kit (Upstate Biotech, Lake Placid, NY) according to the manufacturer’s protocol. All solutions were contained with 1 mM phenylmethylsulfonyl fluoride, 1 ␮g/ml aprotinin, and 1 ␮g/ml pepstatin A. A204 cells
were inoculated at a density of 1 ⫻ 106 per 100-mm dish 16 h before
formaldehyde cross-linking and chromatin immunoprecipitation. Cells
were fixed with 1% formaldehyde for 15 min at 37 °C. After washing
twice with PBS, cell pellets were resuspended in SDS lysis buffer,Human ␣1(XI) Collagen Proximal Promoter

FIG. 1. Deletion analysis of the human COL11A1 promoter inthe luciferase assay. A, schematic representation of the deletion
constructs of the human COL11A1 promoter. B, 5⬘-deletion analysis of
the human COL11A1 promoter. A series of human COL11A1 promoterluciferase constructs were transfected into the cells with the pRL-TK
vector as an internal control for transfection efficiency. Relative luciferase activities (percentages) were normalized against the activity ofthe pGL3-control vector, and results are expressed as mean ⫾ S.E. of
three independent experiments.
incubated for 10 min at 4 °C, and sonicated four times for 10 s using
ultrasonic homogenizer VP-5S (TAITEC, Tokyo, Japan). After centrifugation, the supernatant was diluted in chromatin immunoprecipitation dilution buffer and then incubated overnight at 4 °C with antiCBF-A/NF-YB, CBF-B/NF-YA, and CBF-C/NF-YC antiserum (Santa
Cruz Biotechnology). Immune complexes were recovered by the addition of 60 ␮l of salmon sperm DNA/protein A-agarose slurry, followed byincubation for 2 h at 4 °C. After washing the beads with both low- andhigh-salt buffer, then LiCl buffer, and finally Tris/EDTA buffer, theimmune complexes were eluted by incubation for 15 min at 25 °C with200 ␮l of elution buffer (1% SDS, 100 mM NaHCO3, and 1 mM dithiothreitol). To reverse the cross-linking of DNA, the elutes were added to
8 ␮l of 5 M NaCl and incubated for 4 h at 65 °C, followed by treatment
with proteinase K for 1 h at 45 °C. DNA was recovered by phenolchloroform extraction and ethanol precipitation, and then the pellets
were resuspended in 50 ␮l of Tris/EDTA buffer. Quantitative PCR was
carried out for 35 cycles using 5 ␮l of sample DNA solution, and PCR
products were separated on 2% agarose gels in 1⫻ Tris-acetate/EDTA.
RESULTS

Deletion Analysis of the Human COL11A1 Promoter—To delineate the proximal regulatory regions in the human
COL11A1 promoter, a series of chimeric constructs containing
5⬘-end deletions linked to the luciferase gene were generated,
and then luciferase assays were carried out (Fig. 1A). The
activity of each construct was compared with the longest construct, pGL3⫺291/⫹271. As shown in Fig. 1B, the promoter
activity of pGL3⫺199/⫹271 was not significantly reduced compared with that of pGL3⫺291/⫹271. However, deletion to ⫺88
produced a significant reduction in the transcriptional activity;
furthermore, deletion to ⫹1 produced activity similar to that ofthe pGL3-Basic negative control vector. This result indicatesthat the segment spanning the ⫺199 to ⫹1 region is importantfor basal transcriptional activity of the human COL11A1
promoter.

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Definition of the Nuclear Proteins Binding Site in the Proximal COL11A1 Promoter—On the basis of the aforementioned
data, we examined the binding proteins that interact in thisregion. We prepared three overlapping oligonucleotides, GS1–
GS3, covering the ⫺199 to ⫹1 region (Fig. 2A), and carried out
EMSAs. As shown in Fig. 2B, 32P-labeled GS2 probe, covering⫺147 to ⫺65, and GS3 probe, covering ⫺199 to ⫺121, boundnuclear proteins extracted from A204 cells in a dose-dependent
manner. Both probes were found to form DNA-protein complexes with the same mobility in an EMSA. To determine
whether GS2 and GS3 oligonucleotides bind the same proteins,these probes were used with each other in a competition assay.
The 32P-labeled GS2-protein complexes could be competedaway not only by the corresponding unlabeled probe (Fig. 2C,
lane 4), but also by the unlabeled GS3 oligonucleotide (Fig. 2C,
lane 5). Likewise, the 32P-labeled GS3-specific band also disappeared when either unlabeled GS3 or GS2 oligonucleotides
were added as competitors (Fig. 2C, lanes 9 and 10); however,an excess of the unlabeled GS1 (⫺88 to ⫹1) oligonucleotides did
not compete for the binding of 32P-labeled GS2 and GS3 probes(Fig. 2C, lanes 3 and 8). To further dissect this protein-binding
region, another three oligonucleotides covering the GS2 andGS3 regions were generated (Fig. 2A). Fig. 2D shows that32
P-labeled GS4 probe, covering ⫺147 to ⫺121, formed DNAprotein complexes in a dose-dependent manner. However, neither 32P-labeled GS5 nor GS6 probes, which do not possess the
GS4 region, could bind protein. These results indicate that a
27-bp sequence from ⫺147 to ⫺121 in the proximal COL11A1
promoter region is necessary to bind nuclear proteins. We
subsequently carried out EMSAs using substitution mutation
probes within the 27-bp sequence to narrow down the criticalregion for binding (Fig. 3A). The 32P-labeled GSE1 and GSE4
probes, which contain 6 single bp substitutions, could not form
DNA-protein complexes, whereas the 32P-labeled GSE2, GSE3,
and GSwt probes bound protein in a dose-dependent manner.
In addition, Fig. 3C shows that an excess of the unlabeled
GSE2 and GSE3 probes inhibited the binding activity in amanner similar to that of GSwt, whereas both GSE1 and GSE4
could not compete with the 32P-labeled GSwt-specific band. To
confirm a core-binding site, we further performed competition
experiments using oligonucleotides with 2 single bp mutations.
As shown in Fig. 3D, an excess of unlabeled GSM1, GSM2,
GSM3, and GSM4 failed to inhibit the binding, whereas GSM5
and GSM6 blocked the binding of nuclear proteins (Fig. 3C).
These results suggest that the GTGATTGG sequence within
the 27-bp region was the critical binding site within the proximal COL11A1 promoter.
Identification of DNA-Binding Proteins in the Proximal
COL11A1 Promoter—To identify the nuclear binding proteinsin the proximal COL11A1 promoter, we searched the computer
data base and found four candidate transcription factors:
CCAAT binding factor CBF/NF-Y, CCAAT transcription factor
NF-1/CTF, CCAAT/enhancer binding protein C/EBP, and
GATA (Fig. 4A). An excess of the consensus oligonucleotides of
CBF/NF-Y inhibited the binding of the 32P-labeled GSwt probe(Fig. 4B, lane 2), whereas NF-1, C/EBF, GATA consensus, and
CBFmt oligonucleotides could not abolish the binding activity
observed (Fig. 4B, lanes 3– 6). To further characterize the binding protein, we performed an interference assay using specificantibodies against CBF/NF-Y, NF-Y, C/EBP, and GATA1. As
shown in Fig. 4C, the DNA-protein complex was only supershifted by anti-CBF antibodies, namely anti-CBF-A/NF-YB(Fig. 4C, lane 3), CBF-B/NF-YA (Fig. 4C, lane 4), and CBF-C/
NF-YC (Fig. 4C, lane 5). Specific antibodies against the otherproteins (Fig. 4C, lanes 6 – 8) and control IgG (Fig. 4C, lane 9)
failed to inhibit the supershift (Fig. 4B).

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Human ␣1(XI) Collagen Proximal Promoter
CBF/NF-Y Binds to the COL11A1 Proximal Promoter both inVitro and in Vivo—To examine whether CBF/NF-Y binds directly to the COL11A1 proximal promoter, we performed two invitro and in vivo binding experiments. For the in vitro binding
analysis, four tandem copies of the ⫺147 to ⫺121 GS4 oligonucleotide were generated and immobilized on latex beads. These
were then used for the purification of binding proteins from
A204 cell nuclear extracts, and the purified protein was subsequently used in EMSA analyses. As shown in Fig. 5, the 32Plabeled GS4 probe bound to the affinity-purified proteins (lane4) at levels similar to those of the crude extracts (lane 1), andthe bands were supershifted by anti-CBF-A/NF-YB (lane 6),
CBF-B/NF-YA (lane 7), and CBF-C/NF-YC (lane 8) antibodies.
Furthermore, the affinity-purified proteins strongly bound to
the CBF/NF-Y consensus oligonucleotides (lane 9) and wereclearly supershifted by anti-CBF/NF-Y antibodies (lanes 10 –12). Next, we performed a chromatin immunoprecipitation
analysis to examine whether CBF/NF-Y binds to the COL11A1
proximal promoter in vivo. Protein-DNA complexes were immunoprecipitated with antibodies, the cross-links reversed,
and the recovered DNA fragments were monitored by PCR
using primers for the ⫺291 to ⫹1 region of the COL11A1
promoter. DNA fragments immunoprecipitated with polyclonal
antibodies against CBF/NF-Y could be amplified by PCR using
the indicated primers (Fig. 6, lanes 5 and 6) as well as the
positive control (lane 2), whereas those immunoprecipitatedwith normal goat IgG (lane 4) or without antibody (lane 3) could
not. These results indicate that CBF/NF-Y binds directly to the
ATTGG region in the COL11A1 proximal promoter.
Functional Analysis of CBF/NF-Y in the Proximal COL11A1
Promoter—To confirm our findings, we carried out a functional
assay of CBF/NF-Y in the proximal COL11A1 promoter. We
examined by luciferase assay whether CBF/NF-Y could function in our system as an effective transcription factor. The
pGL3⫺199/⫺65 exhibited transcriptional activity at ⬃10% of
the relative luciferase activity, compared with that of pGL3
⫹1/⫹271 (⬃2– 4%), and the activity of two copies of the ⫺199/⫺65 fragment doubled the activity (⬃20%) compared with thatof one copy. Furthermore, pGL3(⫺147/⫺121)x4 exhibited
higher promoter activity (⬃30%) than pGL3⫺199/⫺65 andpGL3(⫺199/⫺65)x2 constructs. A series of substituted and deleted luciferase mutation constructs were transfected into A204
cells and analyzed for luciferase activities. The activities of theconstructs containing a mutated or deleted CBF/NF-Y binding
site, pGL3⫺541E1, ⫺541E4, CBFm, del⫺147/⫺121, anddel⫺199/⫺65, were suppressed, whereas in pGL3⫺541E2 and-E3 constructs, the activities did not change compared withthat of pGL3⫺541/⫹271. Finally, we tested the dominant-negative effect using a CBF-B/NF-YA subunit, which was overexpressed in both transient and stable transfected cells. For thetransient cell experiment, the dominant-negative CBF-B/
NF-YA expression vector pCXN-CBF/NF-Ymut or empty vector
pCXN was cotransfected into A204 cells with pGL3(⫺147/⫺121)x4 or pGL3⫺199/⫹271 luciferase constructs. For the stable cell experiment, pGL3(⫺147/⫺121)x4 or pGL3⫺199/⫹271
luciferase construct was also cotransfected into A204 cell witha pMAM2-BSD selection vector. After selection with blasticidin, the resistant clones were isolated and used in luciferase
assays as stable cell lines. Overexpression of dominant-nega-

FIG. 2. EMSA analysis of nuclear proteins in the proximal
COL11A1 promoter. A, schematic representation of the probes used
for EMSA. B, binding assay using overlapping probes in the COL11A1
promoter. 32P-labeled GS1 (lanes 1– 4), GS2 (lanes 5– 8), and GS3 (lanes
9 –12) were incubated with nuclear extracts from A204 cells and separated on a 4.5% nondenaturing polyacrylamide gel. C, interference

assays using an excess of competitor oligonucleotide. 32P-labeled GS2(lanes 1–5) and GS3 (lanes 6 –10) were incubated with nuclear extracts
from A204 cells in the presence of competitors, GS1 (lanes 3 and 8), GS2(lanes 4 and 9), and GS3 (lanes 5 and 10). D, binding assays using
dissected probes in the proximal COL11A1 promoter. 32P-labeled GS4(lanes 1–3), GS5 (lanes 4 – 6), and GS6 (lanes 7–9) were incubated with
nuclear extracts from A204 cells. N.E., nuclear extracts.

Human ␣1(XI) Collagen Proximal Promoter

32767

tive CBF-B/NF-YA significantly suppressed the proximal
COL11A1 promoter activities in both transient (Fig. 7C, top)and stable cells (Fig. 7C, bottom). From these results, it was
concluded that CBF/NF-Y binds directly to the proximal
COL11A1 promoter and activates the basal transcriptional
activity.
DISCUSSION

To understand the transcriptional regulation of the human
␣1(XI) collagen gene (COL11A1), previous studies have characterized the structure and function of the ⫺541 to ⫺199 segment of the promoter (17, 18). In this study, we further characterized the proximal promoter region corresponding to adownstream region, the ⫺199 to ⫹271 segment, by transfection
assay using a luciferase reporter gene and EMSA. We demonstrated that the segment spanning from ⫺199 to ⫹1 was necessary for the basal transcriptional activity of COL11A1 (Fig.
1) and also demonstrated that the ATTGG sequence, an inverted CCAAT motif, was a critical binding site for nuclear
proteins (Figs. 2 and 3). This pentanucleotide sequence is present in ⬃30% of eukaryotic promoters, and several CCAATbinding proteins have been reported, including c/EBP, CTF/
NF-I, CDP (CCAAT displacement protein), and CBF/NF-Y.
Among these proteins, we showed that CBF/NF-Y bound to the
ATTGG sequence in the proximal promoter region of COL11A1
gene by EMSA using consensus oligonucleotides and specificantibodies (Fig. 4), and demonstrated direct binding to thisregion both in vitro and in vivo (Figs. 5 and 6). Furthermore,functional analysis by transfection with luciferase constructs
showed that an increase in CBF/NF-Y binding produced hightranscriptional activity, whereas the lack of binding activity,
and the overexpression of a dominant-negative form, decreased
the activity (Fig. 7). These results indicate that CBF/NF-Y
plays an important role in the transcriptional regulation of theproximal promoter in the human COL11A1 gene.
In collagen genes, CBF/NF-Y binds to the ATTGG sequence
located between ⫺100 and ⫺96 in the ␣1(I), and between ⫺84
and ⫺80 in the ␣2(I) promoter, and stimulates their basaltranscriptional activities, but it is not responsible for the transcriptional regulation of other collagen genes (28, 29). It is
plausible that the structures and mechanisms on both proximal
promoters are very similar, because this molecule is composedof a large number of heterotrimeric [␣1(I)]2␣2(I) or a small
number of homotrimeric [␣1(I)]3 molecules. It is also the mostabundant and is widely expressed in vertebrate tissues, except
for cartilaginous tissues. In fact, both ␣1(I) and ␣2(I) proximal
promoters have a TATA box and a CCAAT box, and otherputative transcription factors, such as c-krox, BFCOL1, IF-1
and Sp1, in addition to CBF/NF-F, are regulated on both proximal promoters (30). However, other major fibrillar collagens,such as the ␣1(II) and ␣1(III) genes, have no CCAAT box in thepresence of a TATA box; thus CBF/NF-Y is not necessary for
their transcriptional regulation (31–33). The expression pattern of type II collagen is clearly distinguished from that of type

FIG. 3. Definition of the nuclear protein-binding region in theproximal COL11A1 promoter by EMSA. A, oligonucleotide sequences of various probes used for EMSA. GSwt is the sequence from⫺147 to ⫺121 in the proximal COL11A1 promoter. In the mutatedprobes, identical and mutated nucleotides are indicated by dots andlowercase letters, respectively. B, binding assay using substituted mutation probes. 32P-labeled GSwt (lanes 1–3), GSE1 (lanes 4 – 6), GSE2
(lanes 7–9), GSE3 (lanes 10 –12), and GSE4 (lanes 13–15) were incubated with nuclear extracts from A204 cells and separated on a 4.5%
nondenaturing polyacrylamide gel. C, interference assays using 6 single
bp substituted mutation probes as competitors. 32P-labeled GSwt was
incubated with nuclear extracts from A204 cells in the presence ofcompetitors, GSwt (lane 3), GSE1 (lane 4), GSE2 (lane 5), GSE3 (lane 6),
and GSE4 (lane 7). D, interference assays using 2 single bp substituted
mutation probes as competitors. 32P-labeled GSwt was incubated with
nuclear extract from A204 cells in the presence of competitors GSwt
(lane 3) and GSM1-6 (lane 4–9).

32768Human ␣1(XI) Collagen Proximal Promoter

FIG. 5. Affinity-purified proteins bind to CBF/NF-Y consensus
oligonucleotides and anti-CBF/NF-Y antibodies, in vitro. Fourtandem copies of the ⫺147 to ⫺121 fragment were immobilized on latex
beads, and used to purify DNA-binding proteins from A204 crude nuclear extracts. 32P-labeled GS4 (lanes 1– 8) and CBFwt consensus oligonucleotides (lanes 9 –12) were incubated with A204 crude nuclear
extract (lane 1), without nuclear extract (lane 2), flow-through fraction(lane 3), or affinity-purified fraction (lanes 4 –12) in the presence ofanti-CBF-A/NF-YB (lanes 6 and 10), anti-CBF-B/NF-YA (lanes 7 and11), and anti-CBF-C/NF-YC (lanes 8 and 12) antibodies. Arrows, arrowheads, and an asterisk indicate the DNA-protein complexes, the supershifted bands, and the nonspecific bands, respectively. A204, crude
A204 nuclear extracts; F.T., 0.1 M KCl flow-through fraction; B.P., 0.4 M
KCl-eluted binding proteins.

FIG. 4. Identification of DNA-binding proteins in the proximal
COL11A1 promoter by EMSA. A, oligonucleotide sequences of various consensus probes used for EMSA. GSwt is the sequence from ⫺147
to ⫺121 in the proximal COL11A1 promoter and CBF, NF-1, C/EBP,
and GATA consensus sequences are based on the oligonucleotide data
sheets of Santa Cruz Biotechnology Inc. For CBFmt consensus sequences, identical and mutated nucleotides are indicated by dots andunderlines, respectively. B, interference assays using various consensus
oligonucleotides as competitors. 32P-labeled GSwt was incubated with
nuclear extracts from A204 cells in the presence of competitors, CBFwt(lane 2), CBFmt (lane 3), NF-1 (lane 4), C/EBP (lane 5), and GATA (lane6). C, interference assays using specific antibodies against varioustranscription factors. 32P-labeled GSwt was incubated with nuclear
extracts from A204 cells in the presence of various antibodies, antiCBF-A/NF-YB (lane 3), anti-CBF-B/NF-YA (lane 4), anti-CBF-C/NF-YC(lane 5), anti-NF-1 (lane 6), anti-C/EBP (lane 7), anti-GATA1 (lane 8)
and control IgG (lane 9). Arrowhead and arrows indicate the DNAprotein complexes and the supershifted bands, respectively.

FIG. 6. CBF/NF-Y binds the ATTGG region in the COL11A1
proximal promoter, in vivo. Chromatin immunoprecipitation analysis was performed to monitor the binding of CBF/NF-Y to the COL11A1
proximal promoter, in vivo. Protein-DNA complexes were incubatedwith polyclonal antibodies against CBF-A/NF-YB, CBF-B/NF-YA, and
CBF-C/NF-YC, and isolated by immunoprecipitation (lanes 5 and 6).
Positive control was prepared before immunoprecipitation (lane 2) andnegative controls were isolated by immunoprecipitation with normal
goat IgG (lane 4) or without antibody (lane 3), respectively. All immunoprecipitated DNA fragments were analyzed by PCR with the indicated primers, and a 100-bp ladder was used as a molecular weightmarker (lane 1).

I collagen, as described previously, and it has been reported
that the 5⬘-flanking proximal region of the ␣1(II) gene has aweak promoter activity, and the transcription of COL2A1 isregulated by several enhancers or silencers (34). Although type
III collagen is coordinately expressed with type I collagen in
most non-cartilaginous tissues, the ratio of type I to type III
collagen varies in many tissues and during development, andfurther analysis of ␣1(III) proximal promoter indicates that two
distinct positive factors, other than CBF-NF-Y, regulate thetranscription of type III collagen (33). These observations inmajor fibrillar groups suggest that the mechanisms for basaltranscriptional regulation of types II and III collagen may beHuman ␣1(XI) Collagen Proximal Promoter

FIG. 7. Functional analysis of CBF/NF-Y in the proximal
COL11A1 promoter. A, one or two copies of ⫺199/⫺65 and four tandem copies of the ⫺147/⫺121 region containing the CBF/NF-Y bindingsite activate the luciferase activity. B, loss of CBF/NF-Y binding site
suppresses the activity. Underline indicates CBF/NF-Y binding site.
A204 cells were transfected with the constructs together with pRL-TK
vector as an internal control for transfection efficiency. Relative luciferase activities (%) were normalized against the activity of a pGL3control vector, and results are expressed as mean ⫾ S.E. of three
independent experiments. C, overexpression of dominant-negative
CBF/NY-Y suppresses the proximal COL11A1 promoter activity in bothtransient and stable cells. For the transient cell experiment (top), dominant-negative CBF/NF-Y expression pCXN-CBF/NFYm or empty vector pCXN was cotransfected into A204 cells with pGL3(⫺147/⫺121)x4
or pGL3(⫺199/⫹271) luciferase construct by lipofection. For the stable
cell experiment (lower), pCXN-CBF/NFYm or pCXN vector was transfected into the (⫺147/⫺121)x4 cells or the ⫺199/⫹271 cells by lipofection. Relative luciferase activities (percentages) were normalizedagainst the activity of pCXN empty vector and results are expressed asmean ⫾ S.E. of three independent experiments. p values were significantly different at ⬍ 0.05.

32769

distinct from those of type I collagen genes in regard
to CBF/NF-Y.
In the case of type XI collagen genes, previous studies indicated that the structure of the proximal promoter regions in the␣1(XI) and ␣2(XI) genes exhibited the features of housekeeping
genes, which have several GC boxes and no TATA box (17,35–37). Our results first demonstrated that a functional
CCAAT box exists in COL11A1 gene by directly binding to theproximal promoter region, whereas the 5⬘ flanking region of
COL11A2 has no CCAAT box. In the transgenic mice experiments, the 742-bp promoter segment of Col11a2, located upstream of the retinoid X receptor gene, is able to direct transcription in most cartilaginous tissues (38). Further studies
indicated that Col11a2 is regulated by several cis-acting elements, the cartilage-specific element (⫺530 to ⫺501), a neuraltissue-specific element (⫺501 to ⫺454), and a basal promoter
element (39) and a Kruppel-associated box zinc-finger protein
repress the cartilage-specific promoter activity of Col11a2 (40).
In addition, structural analysis of the two genes indicates that
the COL11A2 promoter region shares limited homology (⬍43%
identity) with the COL11A1 gene except for two homologous
regions located within ⫺93 to ⫺49 (60%) and ⫺301 to ⫺285(65%) of the COL11A2 (40). This indicates that the functional
cis-acting elements in both proximal promoters are not thesame as each other, as described previously (17, 18, 39, 40).
Type XI collagens also form a heterotypic molecule that iscomposed of [␣1(XI)]2␣2(V) in non-cartilaginous tissues (9 –14).
Previous studies indicated that two cis-acting elements, Foot
Printing A (⫺114 to ⫺95) and FPB (⫺149 to ⫺95), were responsible for the regulation of the human COL5A2 proximal promoter (19), and the sequence of FPB in the COL5A2 gene
exhibited some homology with FP7 in COL11A1 (17). However,the latest study demonstrated that FPB-binding proteins in the
COL5A2 gene differed from FP7-binding proteins in the
COL11A1 gene, identified as PBX, PREP, and HOX proteins(20). We preliminarily examined whether FPA-binding protein
was in fact CBF/NF-Y by EMSA. However, the band of FPA
complexes was different from that of CBF/NF-Y complexes,
with a distinct mobility by EMSA (data not shown), thus indicating that CBF/NF-Y proteins may not be required for thebasal promoter of the COL5A2 gene. However, our recent study
indicated that CBF/NF-Y is also responsible for the basal promoter activity of the ␣1(V) collagen gene.2 These observations
suggest that CBF/NF-Y plays an important role in the basaltranscriptional regulation of some fibrillar collagens (␣1(XI)and ␣1(V) in addition to ␣1(I) and ␣2(I), as described previously) that are expressed in non-cartilaginous tissues.
Transcriptional regulation of various eukaryotic genes requires both sequence-specific transcription factors proximal to
the transcription start site and cis-acting elements at a distance to the genes (41). CBF/NF-Y was originally identified as
the proteins bound to the MHC class II conserved Y box in Ea
promoters (42), the yeast CYC1 UAS CCAAT box (43), and the␣2(I) collagen gene (44) in the proximal promoter region. CBF/
NF-Y is one of the most ubiquitous transcription factors, foundin a variety of eukaryotes, and it is highly conserved throughout evolution (45). In unrelated promoters, CBF/NF-Y binds to
CCAAT (40%) and ATTGG (60%) sequences in both TATAcontaining and TATA-less promoters (46) and regulates on acase-by-case rather than an all-or-none basis (43– 45, 47– 49).
We demonstrated that CBF/NF-Y bound to the ⫺147 to ⫺121
region but could not find any protein-DNA complex in the ⫺199
to ⫹271 region. These data are not consistent with the previous

2
K. Sakata-Takatani, N. Matsuo, T. Tsuda, and H. Yoshioka, unpublished data.

Human ␣1(XI) Collagen Proximal Promoter

32770

experiments using DNA footprinting techniques, in which
three areas, FP1 (⫺63 to ⫺51), FP2 (⫺111 to ⫺95), and FP3(⫺171 to ⫺159), were protected using the nuclear extracts froma primary line of smooth muscle cells, called 1120 (14). We do
not know the reason for this discrepancy so far. It may be
because of the different techniques or cells used or there may besome other reasons. However, other factors may also be involved in generating a basal promoter activity, because pGL3–
88/⫹271 has still promoter activity (Fig. 1B).
One of the other mechanisms of transcriptional regulation iscontrol by cis-acting elements, such as enhancers and silencers.
Sox9, one of the HMG-box-containing transcription factors,
binds to a 48-bp chondrocyte-specific enhancer element in the␣1(II) collagen genes (50, 51) and is required for chondrocyte
differentiation and for the expression of chondrocyte-specificcollagen, such as ␣1(II), ␣2(IX), and ␣2(XI) genes (52, 53). In
osteoblast differentiation and bone formations, Cbfa1/Runx2 is
one of the positive regulators of type I collagen expression (54,55). These findings suggest that the specific transcriptional
mechanisms of some collagen genes, at least in part, are regulated by tissue-specific transcription factors, such as SOX9 andCbfa1. Although the ␣1(XI) gene is expressed in both cartilage
and bone (10, 11, 13), the tissue-specific cis-acting elements
have yet to be identified. Thus, the identification of tissuespecific transcription factors deserves further study to fully
understand the mechanism of transcriptional regulation in the␣1(XI) collagen gene.
Acknowledgments—We thank Y.-H. Wang for technical assistance,
M. Mori for preparation of the manuscript, and the staff of the Radioisotope Center at Oita Medical University, where some of these experiments were performed.
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