In Vitro Cell.Dev.Biol.—Animal (2014) 50:358–366
DOI 10.1007/s11626-013-9692-3

Nuclear factor Y (NF-Y) regulates the proximal promoter
activity of the mouse collagen α1(XI) gene (Col11a1 )in chondrocytes
Mariko Hida & Ryoji Hamanaka & Osamu Okamoto &Kouhei Yamashita & Takako Sasaki &Hidekatsu Yoshioka & Noritaka Matsuo

Received: 20 August 2013 / Accepted: 11 September 2013 / Published online: 3 October 2013 / Editor: T. Okamoto# The Society for In Vitro Biology 2013

Abstract Type XI collagen, a heterotrimer composed ofα1(XI), α2(XI), and α3(XI), plays a critical role in cartilage
formation and in skeletal morphogenesis. However, the transcriptional regulation of α1(XI) collagen gene (Col11a1) inchondrocyte is poorly characterized. In this study, we investigated the proximal promoter of mouse Col11a1 gene inchondrocytes. Major transcription start site was located at−299 bp upstream of the translation start site, and the proximal
promoter lacks a TATA sequence but has a high guanine–cytosine (GC) content. Cell transfection experiments demonstrated that the segment from −116 to −256 is necessary for
activation of the proximal Col11a1 promoter, and an electrophoretic mobility shift assay showed that a nuclear protein isbound to the segment from −116 to −176 in this promoter.
Additional comparative and in silico analyses demonstrated thatan ATTGG sequence, which is critical for binding to nuclear
factor Y (NF-Y), is within the highly conserved region from−135 to −145. Interference assays using wild-type and mutant
oligonucleotide or with specific antibody revealed that NF-Y
protein is bound to this region. Cell transfection experimentswith reporter constructs in the absence of NF-Y binding sequence exhibited the suppression of the promoter activity. Furthermore, chromatin immunoprecipitation assay demonstrated
M. Hida : K. Yamashita : T. Sasaki : H. Yoshioka : N. Matsuo (*)
Department of Matrix Medicine, Faculty of Medicine, Oita
University, 1-1 Idaigaoka, Hasama-machi, Yufu, Oita 879-5593,
Japane-mail: matsuon@oita-u.ac.jp
R. Hamanaka
Department of Cell Biology, Faculty of Medicine, Oita University,
1-1 Idaigaoka, Hasama-machi, Yufu, Oita 879-5593, JapanO. Okamoto
Department of Dermatology, Faculty of Medicine, Oita University,
1-1 Idaigaoka, Hasama-machi, Yufu, Oita 879-5593, Japanthat NF-Y protein is directly bound to this region in vivo, andoverexpression of dominant-negative NF-Y A mutant also
inhibited the proximal promoter activity. Taken together, theseresults indicate that the transcription factor NF-Y regulates theproximal promoter activity of mouse Col11a1 gene inchondrocytes.
Keywords Type XI collagen . Chondrocyte . NF-Y .
Transcription . Promoter

Introduction
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. At the present, there
are more than 46 distinct α-chains of collagen genes, which
form at least 28 types of collagen proteins. They can bedivided into several subgroups based on their structural andfunctional properties (Ricard-Blum 2012). Among them, type
I, II, III, V, XI, XXIV, and XXVII collagens are classified into
the group of fibril-forming collagens, based on their structuraland functional features. They are divided into three subgroups, major (I, II, and III), minor (V and XI), and new(XXIV and XXVII) fibrillar collagens on the basis of theircontents in tissues and phylogenetic analyses.
Vertebrate fibrillar collagens are either restricted to cartilage (II, XI, and XXVII) or widely distributed in non-cartilage
tissues (I, III, V, and XXIV), and the quantitatively minor
collagens regulate the diameter of the major collagen fibrils
by participating in fibril assembly (Exposito et al. 2010; Fanget al. 2012). Cartilage is a highly specialized tissue and serves
as a template for the growth and development of bones. It

TRANSCRIPTIONAL REGULATION OF MOUSE COL11A1 GENE IN CHONDROCYTE

contains an extensive extracellular matrix, which includes
type II, IX, and XI collagen, and provides mechanical strength
to resist compression in the joint (Mendler et al. 1989).
Type XI collagen is a fibrillar collagen mainly found in
cartilage and consists of three polypeptide chains: α1(XI),
α2(XI), and α3(XI). The α1(XI) and α2(XI) chains are genetically distinct products, whereas the a3(XI) chain is an
overgylcosylated variant of the α1(II) chain, which is encodedby type II collagen gene (Ricard-Blum 2012). Type XI collagen co-polymerizes with type II collagen within collagen
fibrils and is important for the regulation of the fibril diameterand function in connective tissue (Mendler et al. 1989). Therefore, the absence and/or mutation of α1(XI) chains results inabnormally thickened cartilage collagen fibrils. For example,
chondrodysplasia mice (cho mice), which do not synthesize
α1(XI) chains, show irregular collagen fibrils in their cartilage, causing a chondrodystrophic phenotype (Li et al. 1995).
In the case of human diseases, mutation in the COL11A1 genehas been identified in the Stickler and Marshall syndromes,
which are clinically characterized by altered facial appearance,eye abnormalities, joint alterations, and hearing loss
(Annunen et al. 1999; Myllyharju and Kivirikko 2001). Furthermore, fibrochondrogenesis, which is short-limbed skeletal
dysplasia, is also caused by mutations in the COL11A1 gene
(Tompson et al. 2010). These observations suggest that the
fidelity of Col11a1 expression is intimately involved inmaintaining normal structure and function of cartilage.
Chondrogenesis is an important biological event in skeletal
development and tissue formation. It starts from multipotent
mesenchymal cells, and then these cells proliferate, followedby differentiating into chondrocytes. During these processes,
the cells secrete chondrogenic matrices such as type II, IX, XI,
and XXVII collagens around themselves. Therefore, to understand chondrocyte differentiation and cartilage formation, it isnecessary to study the regulation of extracellular matrix(ECM) protein in cartilage (de Crombrugghe et al. 2000).
Previous investigations also indicate that the components of
ECM play important roles in maintaining the phenotype ofchondrocytes and their correct organization (Mundlos andOlsen 1997a, b). Among several ECMs in cartilage, type II
collagen, which is expressed in all chondroprogenitor cells
and at high level in chondrocytes, is well characterized as amodel gene of chondrogenic marker. Analyses of type II
collagen gene revealed multiple regulatory elements targetedby the transcriptional activator and/or repressor such as Sox9
(Bell et al. 1997; Zhou et al. 1998), Sp1/3 (Ghayor et al. 2001;
Renard et al. 2012), Egr-1 (Tan et al. 2003), c-Krox (Ghayor
et al. 2000), and Ap2 (Niebler and Bosserhoff 2013) in boththe proximal promoter and the first intron. In the type XI
collagen gene, it has been reported that two chondrocytespecific enhancer elements were identified in the proximal
promoter and the first intron of the Col11a2 gene and wereregulated by Sox9 (Bridgewater et al. 1998; Liu et al. 2000).

359

However, the transcriptional regulation of Col11a1 gene inchondrocytes is poorly examined.
In this study, we have first characterized the proximal promoter region of mouse Col11a1 gene in chondrocytes. Transient transfection assays revealed that the segment from −116 to−256 is necessary for the activation of basal transcription.
Electrophoretic mobility shift assays (EMSAs) and chromatin
immunoprecipitation (Chip) assay demonstrated that the transcription factor, nuclear factor Y (NF-Y), binds to the ATTGG
sequence within the proximal promoter region in vitro andin vivo. Furthermore, cell transfection experiments in combination with DNA-binding assay demonstrated that NF-Y regulates the proximal promoter activity of Col11a1 inchondrocytes.

Materials and Methods
Cell cultures. Rat chondrosarcoma (RCS) and mouse prechondrocyte ATDC5 cells were used in this study. RCS cells
were kindly provided by Dr. Francesco Ramirez (Mount Sinai,
School of Medicine, New York, NY), and ATDC5 cells werepurchased from the RIKEN BRC through the National
BioResource of the NEXT, Tokyo, Japan. Cells were cultured
in Dulbecco's modified Eagle's medium supplemented with
10% heat-inactivated fetal bovine serum at 37°C in humidified 5% CO2 and 95% air.
RNA analysis. Total RNA was isolated from RCS and
ATDC5 cells using Isogen (Nippon Gene, Tokyo, Japan)
according to the manufacturer's instruction. Reverse transcriptions were carried out using ReverTra Ace reverse transcriptase (TOYOBO, Tokyo, Japan) with random and oligo(dT)primers. PCR was carried out using the single-stranded cDNA
molecules and gene-specific primers. Amplification conditions included a cycle at 95°C for 2 min followed by 25–35
cycles at 95°C for 30 s, 56°C for 30 s, and 72°C for 1 min anda final elongation cycle at 72°C for 5 min. The PCR products
were analyzed by electrophoresis on 1.5% agarose gel. The
primers for Col11a1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as follows: Col11a1 sense 5′GACTACTCAGATGGCATG-3′ and antisense 5′ACTTCCTGGTTTCTCCTT-3′ and GAPDH sense 5′ACCACAGTCCATGCCATCAC-3′ and antisense 5′TCCACCACCCTGTTGCTGTA-3′.
To determine the transcriptional start sites, the oligonucleotide-capping rapid amplification of cDNA ends
(RACE) was performed using the GeneRacer kit according
to the manufacturer's protocol (Invitrogen, Carlsbad, CA) withamplification primers corresponding to the sequences ofmCol11a1R1 (5′-CTCCCTCTGACCTCTCTAG-3′) and
mCol11a1R2 (5′-TGACGACTGTGAACCCAC-3′). Amplification products were subcloned into the pGEM-T Easy

360

a

HIDA ET AL.
RCS

ATDC5

Col11a1

GAPDH

b

-120
-80

---------- ---------- ----gaggag ggggggaagg
agagggaaga tgaagggtgg ctggtgggct gggcctgcac
agaagtctgg atcctccagg gagagactgg gcgggttcaa
ggctgcgggg aggcctgtga ttgggtatga ccttcagtcg
CCAAT(ATTGG)box
gcttgtcagt ttctcgggca cacaggaggc gggaggagtg
ggcggaggag ggggctgcgc gagcctcttg tccagcccac

-40

1 11
1
* **
*
ccacagcatg aagcctttag gggtgctcag tgctttcggc

-240
-200
-160

+1
+81
+161
+241
+281

8 14 11* ** **
tttttttctt gaaacccttc atctgccctc agtctgaagg
cagggtcagg cgtaggacgg gtcccggggt cgcagtctcc
tgcgggccac cccaaaggga gaggaggggg cgagagacag
gaaggaagca aaccatcaaa ttggaaaaaa aaaaattaga
gccctttgac tttttttttt tttactccct ttcccttccg
gatggctgtg tagcgaacgt aggcgacacc ttggaaggga
caaagttggt ctgcagtggc aatttcgtgg gttcacagtc
Col11a1-R2
gtgagcgtgg ggcttggagA TGGAGCCCTG GTCCAGGTGG
AAAACGAAAC GGTGGATCTG GGATCTCACC ATCTCAACCC
TCGCCTTGAC TTTTCTCTTC CAAGCTAGAG AGGTCAGAGG
Col11a1-R1
AG
AGgtaaggag

Figure 1. RNA expression and determination of the transcription startsite of Col11a1 in chondrogenic cells. (a) Reverse transcription PCR
analysis of Col11a1 in RCS and ATDC5 cells. The ubiquitous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was included as thenormalizing control. (b) Nucleotide sequence of the proximal promoter
region of mouse Col11a1 gene. The oligo-nucleotide-capping RACE
products are represented by asterisk, and total number of each clone isindicated by Arabic numerals. The transcriptional start site is designed as+1 and the predicted translation start codon is at position +300. The
sequence of Exon1 is shown by a capital letter, and the primers used in
the oligo-capping RACE experiments are underlined.

vector (Promega, Fitchburg, WI) and sequenced on an
ABI3130 sequencer (Applied Biosystems, Carlsbad, CA).
Construction of chimeric plasmids. All Col11a1 promoterluciferase reporter gene constructs were derived from pBACe3.6
RP24-512D13 clone. To obtain various 5′ deletion constructs,
these fragments were generated by using sets of oligonucleotide
primers, which are KpnI site-linked 5′- and XhoI site-linked 3′primers specific for the Col11a1 sequence. Amplified products
were cloned into the pGEM-T Easy vector (Promega, WI),
followed by digestion with KpnI and XhoI, and subcloned into
the KpnI/XhoI site of pGL3-basic vector (Promega, WI).
Internal deletion and nucleotide substitutions were generated by site-directed mutagenesis as described previously(Matsuo et al. 2003).

Cell transfection and luciferase assay. The cells were plated
at a density of 1.5×105 cells per 35-mm dish 18 h before
transfection. For transient transfection, RCS and ATDC5 cells
were transfected with each of plasmid DNA by using thecalcium phosphate-mediated precipitation methods with somemodification (Chen and Okayama 1987) or by using the
Lipofectamine plus reagent system according to the manufacturer's protocol (Invitrogen, CA).
Luciferase activity was assayed 24 h later by the DualLuciferaseTM Reporter Assay system (Promega, WI) using aluminometer (Lumat, 9507, Berthold Technologies, Chennai,
India). The cotransfection experiments with a dominantnegative NF-Y A subunit expression vector were performed
as previously described (Matsuo et al. 2003; Nagato et al.
2004). The pRL-TK Renilla reniformis luciferase expression
vector was used as an internal control for transfection efficiency. The relative luciferase acitivity of each construct wascompared with the shortest construct, pGL3+1+68. Results
were expressed as the mean ± SE of three independent experiments and evaluated by Student's t test.
DNA-nuclear protein binding assays. Nuclear extracts wereprepared according to Dignam et al. (1983) with somemodifications.
For the EMSA, wild-type and mutant oligonucleotide
probes were genetrated by PCR amplification using HindIIIsite-linked primers. PCR products were subcloned into thepGEM-T Easy vector (Promega, WI), cleaved with HindIII,
and radiolabeled with [α-32P]dCTP using Klenow enzyme.
DNA-nuclear protein binding reaction was carried out
according to the published protocols (Matsuo et al. 2003,
2006). For competitors and antibody interference assays,
unlabeled probes or antibodies were added to the reaction
mixture and incubated for 1 h at 4°C before the addition oflabeled probe. The anti-NF-Y A antibody (C-18) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
The DNA–protein complexes were separated in a 4.5%
nondenaturing polyacrylamide gel in 0.25% TBE (89 mM
Tris–borate, 2 mM EDTA) buffer and visualized by autoradiography using Bio Imaging Analyzer FLA-7000 (GE Healthcare,
Buckinghamshire, UK). Chip assays were performed using achromatin immunoprecipitation assay kit (Millipore, Burlington,MA) according to the published protocols. Quantitative PCR
was carried out for 35 cycles using 5 μl of reaction, and amplification products were separated in 2% agarose gel in 1× TAE(40 mM Tris–acetate, 1 mM EDTA) buffer.

Results
RNA expression and determination of the transcriptional startsite of mouse Col11a1 gene in chondrogenic cells. Initially,
we examined the expression of Col11a1 transcript in cultured

TRANSCRIPTIONAL REGULATION OF MOUSE COL11A1 GENE IN CHONDROCYTE

chondrogenic cells using the RT-PCR analysis. In this study,
we used rat RCS and mouse ATDC5 cell lines, which represent late (mature) and early (immature) stage of chondrocyte
differentiation, respectively. As shown in Fig. 1a, the amplified PCR products were observed in both cells, and the levelof Col11a1 expression in RCS cells was higher than in
ATDC5 cells.
Next, to determine transcriptional start sites of mouse Col11a1 gene in chondrogenic cells, oligo-nucleotide-capping
RACE experiment was carried out using total RNA derived
from mouse ATDC cells. Twenty independent RACE products were cloned and sequenced in this study. As shown in
Fig. 1a, the transcriptional start sites of mouse Col11a1 gene
were broadly distributed approximately 300 bp upstream(−292 to −335) from the ATG translation initiation codon.
Among them, 8 of the 20 RACE products were generated
from the same position, which suggested that the major transcription start site is located 299 nucleotides immediately
upstream of the ATG codon. This gene has no canonical TATA
sequence and has a high GC content (Fig. 1b).

a
Luc
Luc
Luc
Luc
Luc

b

RCS
ATDC5
0

Deletion analysis of the proximal Col11a1 promoter in mousechondrocytes. To delineate the proximal regulatory regions in
the mouse Col11a1 promoter in chondrogenic cells, a series ofchimeric constructs containing 5′-end deletions linked to theluciferase gene were generated as shown in Fig. 2a, and then,luciferase assays were carried out. The promoter activity ofeach construct was compared with the shortest construct,
pGL+1+68.
As shown in Fig. 2b, the longest construct, pGL−551+68,
had strong transcriptional activity, and deletion from nucleotides −366 to −256 bp had no significant reduction in theluciferase activity. However, deletion of nucleotides between−256 and −116 bp led to a dramatic reduction (65–75%) in thetranscriptional activity compared with pGL−256+68, and further deletion of nucleotides +1 (pGL+1+68) had slightly
reduced the luciferase activity. These results indicated thatthe region between −116 and −256 bp was important for
proximal transcriptional activity of the mouse Col11a1 promoter in chondrogenic cells.
Determination of nuclear protein binding site and identificationof DNA-binding protein in the proximal Col11a1 promoter. To
identify nuclear protein binding site on the proximal promoter
region of Col11a1, we prepared two overlapping probes (G1,−117−196, and G2, −177−256) covering the proximal promoter region from −117 to −256 and carried out an EMSA. As
shown in Fig. 3, 32P-labeled G1 probe-bound nuclear protein
was extracted from the RCS cells in a dose-dependent manner;
however, G2 probe did not bind the DNA–protein complexes.
This result indicated that a 60-bp sequence from −117 to 176
in the proximal promoter region is necessary to bind nuclear
proteins.

361

2

4

6

8

10

Relative Luciferase Activity

Figure 2. Determination of the proximal promoter sequence of mouse
Col11a1 gene. (a) Schematic representation of the deletion constructs ofmouse Col11a1 promoter. (b) 5′-Deletion analysis of mouse Col11a1
promoter. A series of mouse Col11a1 promoter-luciferase constructs
were transfected into the cells with the pRL-TK vector as an internal
control for transfection efficiency. Relative luciferase activities werenormalized against the activity of the pGL+1+68 vector, and the results
are expressed as mean + SE of five independent experiments.

Next, we carried out the comparative sequence analysis ofmouse Col11a1 proximal promoter region with rat and humangenes and then performed in silico analysis using
TFSEARCH (www.cbrc.jp/research/db/TFSEARCH) andGenomatix (www.genomatix.de) to identify putativetranscription factor binding sites.
As shown in Fig. 4a, the nucleotide sequence between mouseand rat gene is very similar (97%, rat vs mouse), while that ofhuman gene is different (70%, human vs mouse). Most highly
conserved region is 11 bp nucleotide sequence located at −135 to−145 (underlined). Furthermore, two in silico analyses indicatedthat this conserved region has a CCAAT (ATTGG sequence) box
which is a potential binding site for the transcription factor NF-Y
(as shown in Fig.1b). We therefore focused on the characterization of NF-Y in the proximal Col11a1 promoter.
To examine whether NF-Y binds to this region, we
performed an interference assay using wild-type or mutant
oligonucleotide. An excess of the wild-type (NF-Y wt) probe
inhibited the binding of the 32-P-labeled G1 probe (Fig. 4b,lane 3), whereas the mutant (NF-Y mut) probe failed tointerfere with DNA–protein complex formation (Fig. 4b, lane4). To further characterize the binding protein, another362

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NF-Y A mutant subunit also suppressed the proximal promoter activity in both cells (Fig. 5b). Taken together, our data
indicate that the transcription factor NF-Y regulates the proximal promoter activity of mouse Col11a1 gene inchondrocytes.

Discussion

Figure 3. Identification of nuclear protein binding region in mouse
Col11a1 proximal promoter. EMSA analysis using overlapping probe
in the Col11a1 proximal promoter. 32P-labeled G1 (−117 to −196) and G2(−177 to −256) were incubated with nuclear extract from RCS cells andseparated on a 4.5% nondenaturing polyacrylamide gel.

interference assay using specific antibody against NF-Y wascarried out. As shown in Fig.4b (lane 6), the DNA–protein
complex was supershifted by anti-NF-Y antibody, whereas
control IgG failed to supershift the DNA–protein complex(Fig. 4b, lane 5).
Finally, we performed Chip assays to clarify whether NF-Y
binds to the mouse Col11a1 proximal promoter in vivo. As
shown in Fig. 4c, DNA fragments immunoprecipitated with
NF-Y antibody could be amplified by PCR with a set of P1/P2
primers as well as input, whereas those immunoprecipitatedwith normal IgG could not. Consistent with the EMSA data,
Chip assay also provided evidence that the transcriptional
factor NF-Y directly binds to the ATTGG sequence in theproximal promoter of mouse Col11a1 gene.
Functional analysis of NF-Y in the proximal Col11a1 promoter. To
confirm whether NF-Y regulates the proximal promoter activity of mouse Col11a1 gene in chondrocytes, we carried out
cell transfection experiments using internal deletion and/or
mutation luciferase constructs. As shown in Fig. 5a, bothinternal deletion and mutation constructs had significantly
reduced the luciferase activity (approximately 40–60%). Consistent with these data, overexpression of dominant-negative

To understand the transcriptional regulation of mouse a1(XI)
collagen gene (Col11a1) in chondrocytes, we first identified
the transcriptional start site by oligo-capping RACE experiments. As shown in Fig. 1b, this promoter has no canonical
TATA motif and a high (GC) content, resulting in multiple
transcriptional start sites of mouse Col11a1 gene. These structural properties in the proximal promoter are almost identicalto those of the other minor fibrillar collagen genes, except for
Col5a2 (Fang et al. 2012). Consistent with these properties,
molecular phylogenetic analyses revealed that vertebrate fibrillar collagens are divied in the three independent clades: thetype A clade consists of α1(I), α2(I), α1(II), and α2(V), thetype B clade contains minor collagens, α1(V), α3(V), α1(XI),and α2(XI), with the exception of α2(V), and the type C clade
consists of newly identified collagens, α1(XXIV) andα1(XXVII) (Wada et al. 2006). These observations suggestthat the proximal promoter and the exon structures of minor
collagen genes are evolutionally conserved.
Cell transfection experiments in combination with DNAbinding assays demonstrate that mouse Col11a1 gene has an
ATTGG sequence (a CCAAT box) in the proximal promoter
region, and NF-Y regulates the proximal promoter activity of
Col11a1 gene in chondrocytes. NF-Y is a heterometric transcription factor composed of three subunits, NF-YA, NF-Y B,
and NF-Y C, and is specifically binds to the CCAAT (orATTGG) motif. This transcription factor is able to regulate awide range of genes, both ubiquitous and/or tissue-specificgenes (Mantovani 1999). In fibrillar collagen genes, the previous reports demonstrated that NF-Y regulates the proximal
promoter activity of α1(I), α2(I), α1(V), and α3(V) genes infibroblasts and/or osteoblasts, which are widely distributed in
non-cartilage tissues (Collins et al. 1997; Lindahl et al. 2002;
Sakata-Takatani et al. 2004; Wu et al. 2010). Furthermore, we
previously reported that NF-Y regulates the proximal promoter activity in the human COL11a1 gene in non-cartilage cells(Matsuo et al. 2003). On the other hand, α1(II) and α2(XI)
genes, which are restrictedly expressed in cartilage, have no
CCAAT motif in these promoter, and therefore, NF-Y is notnecessary for their transcriptional regulation in chondrocytes.
However, this study showed that NF-Y also regulates theproximal promoter activity of mouse Col11a1 gene inchondrocytes. These results indicate that NF-Y regulates theproximal Col11a1 promoter activity in both cartilage and non-

TRANSCRIPTIONAL REGULATION OF MOUSE COL11A1 GENE IN CHONDROCYTE
Figure 4. Identification of DNAbinding protein in mouse
Col11a1 proximal promoter. (a)Comparative analysis of mouse
Col11a1 proximal promoter
sequence (−176 to −117) with rat
and human gene. Bold letter
represents conservation in all
species, and most highly
conserved sequence is underlined(11 bp, −145 to −135). Thisregion has a CCAAT (ATTGG)
box as shown in Fig.1b. (b)Interference assays using
consensus oligonucleotide andspecific antibody. 32-P labeled G1
probe was incubated with nuclear
extract from RCS cells in thepresence of competitors, NF-Y wt(lane 3) or NF-Y mut (lane 4),
and in the presence of antibody,anti-NF-Y (lane 6) or control IgG(lane 5). Arrow indicates thesupershifted band. (c) Chip
analysis of the relevant Col11a1
proximal promoter sequence
using NF-Y antibody. Positive
control is input and negative
control is normal IgG,
respectively. All
immunoprecipitated DNA
fragments were analyed by PCR
with the indicated primers. The
P1/P2 primers are located in therelevant proximal promoter ofmouse Col11a1 gene, and P3/P4
primers are located approximately
2.0 kb upstream region as
negative control, respectively.

363

a
-176
-117
Mouse gactgggcgggttcaaggctgcggggaggcctgtgattgggtatgaccttcagtcggctt
gRat-----------c---------------------------------g-------------Human -cgg-------c-tgg---c--cca---t-g-----------c-----c----cct----

b
-145

-131
tgtgATTGGgtatga----AAATACGGG--

NF-Y wt
NF-Y mut

Antibody

-

-

-

-

Competitor

-

-

wt

mut

-

-

N.E.

-

+

+

+

+

+

2

3

4

5

6

1

IgG NF-Yc
P3(-2516)

P4(-2317)

P1(-256)

P2(+68)

ATTGG

Input

IgG

NF-Y

P1/P2

P3/P4

cartilage tissues. As compared to other fibrillar collagengenes, the regulation of α1(XI) collagen gene might be complex than expected because the expression pattern of α1(XI)

collagen gene is more broad than others, in both cartilaginousand non-cartilaginous tissues. Additional findings of NF-Y
have been reported that two CCAAT boxes were identified in364

HIDA ET AL.

a

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+68

pGL-366+68 (Wt)tgATTGGgtatga
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Deletion (Del)

Relative luciferase activity

Mutation (Mut)--AAATACGGG--

RCS
ATDC5

1

*

*

0.5

*

*

0

Wt

Del

Mut

b
RCS
ATDC5

Relative luciferase activity

1

*

*

0.5

0

pCXN

pCXN-DN

Figure 5. Functional analysis of NY-F in the proximal Col11a1 promoter. (a) Deletion and/or mutation analysis of the mouse Col11a1 proximal
promoter. Deletion and/or mutation luciferase constructs were transfected
into the cells with the pRL-TK vector as an internal control for transfection efficiency. Relative luciferase activities were normalized against ofWt (pGL−366+68) vector, and the results are expressed as mean + SE offive independent experiments. p <0.01 compared with pGL−366+68(Wt). (b) Effect of overexpression of dominant-negative NF-Y on themouse Col11a1 promoter activity. Dominant-negative NF-Y expression
vector (pCXN-DN) or pCXN empty vector was cotransfected into thecells with pGL−366+68 luciferase construct. Relative luciferase activities
were normalized against the activity of pCXN empty vector, and theresults are expressed as mean + SE of five independent experiments. p <0.01 compared with pCXN.

Runx2 , and Dlx2 to promote cartilage development inzebrafish (Chen et al. 2009). These studies suggest that NFY may regulate, at least in part, chondrocyte differentiation,
cartilage formation, and skeletal development in addition to
the regulation of collagen gene expression.
One of other mechanisms of the transcriptional regulationis controlled by cis-acting element, such as enhancers andsilencers. In cartilage, Sox9, one of the HMG-box containing
transcription factors, is essential for chondrocyte differentiation and skeletal development (Akiyama 2008). As described
previously, cartilage collagens are composed of type II, IX,
and XI collagens. In the α1(II) gene, it has been reported that a
48-bp chondrocyte-specific enhancer element is identified,
and the expression of Col2a1 gene is regulated by Sox9
(Bell et al. 1997; Zhou et al. 1998). The α2(XI) gene has
two chondrocyte-specific enhancer elements in the proximal
promoter and the first intron and is regulated by Sox9 as the
Col2a1 gene (Bridgewater et al. 1998; Liu et al. 2000). TypeIX collagen gene also has Sox9 binding elements in theproximal promoter, resulting in the activation of the proximal
promoter by Sox9 (Zhang et al. 2003; Genzer andBridgewater 2007). On the other hand, there is no direct
evidence that Sox9 regulates the expression of α1(XI) collagen gene. However, Lincoln et al. have shown that Sox9 is
required for heart valve development as well as Col11a1 and
Col5a1, suggesting that it is possible that Sox9 may regulate
the expression of Col11a1 and Col5a1 genes (Lincoln et al.
2006, 2007). Recently, it has been reported that lymphocyte
enhancer-binding factor 1 indirectly activates the proximal
promoter Col1a1 gene in osteoblasts and suppressed terminal
osteoblast differentiation (Kahler et al. 2008). However, thechondrocyte-specific cis-acting elements have not been identified in α1(XI) gene yet. Thus, further study on the identification of tissue-specific transcription factor would clarity themechanism of transcriptional regulation in the α1(XI) collagen gene.
In conclusion, we first characterized the proximal promoter
region of mouse Col11a1 gene in chondrocytes. Our results
demonstrated that NF-Y was directly bound to this region andregulated the proximal promoter activity of mouse Col11a1
gene in chondrocytes.
Acknowledgments We thank Ms. S. Adachi for the technical support
and the staff members of Research Promotion Institute, Oita University.
This work was supported by Grants-In-Aid for Scientific Research (no.
21591952 to N.M.) from the Ministry of Education, Culture, Sports,
Science and Technology of Japan.

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