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J. Biol. Chem., Vol. 278, Issue 35, 32763-32770, August 29, 2003
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1(XI) Collagen Gene (COL11A1)*
¶
From the
Department of Anatomy, Biology, and
Medicine, and Research Laboratory Center, Oita
Medical University, Hasama-machi, Oita 879-5593, Japan
Received for publication, May 28, 2003 , and in revised form, June 11, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Type XI collagen is a component of the collagen fibrillar network found in
cartilage (5), and consists of
three genetically distinct polypeptide chains: 
1(XI), 2(XI), and
3(XI); the last of these is thought to be overglycosylated 1(II)
chains (6). Although type XI
collagen is a relatively minor collagen and is buried 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 was demonstrated 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 cross-type 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 in both
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 functional features 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 proteins probably 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 correlated with the degree of cell differentiation in osteoblastic
and skeletal muscle cell lines, in vitro. At –395 to
–379, the core sequence of FP7 exhibits some homology with that of
FPB1 in
COL5A2, and FP7-protein complexes can be competed with an excess of
FPB oligonucleotides in an EMSA using nuclear extracts from the 1120 cell line
(17,
19). Further studies using
transfection 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 FP7-binding proteins
(20).
In this study, we have characterized the region downstream of 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 were checked 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, for substitution sites. The primers for 6 base-substitution constructs, pGL3–541E1, pGL3–541E2, pGL3–541E3, and pGL3–541E4, were as follows: E1: sense, 5'-gaattcTGGGTCTGACCCTCAGCCTG-3'; antisense, 5'-gaattcCGACTCTGGGGCGGCCCCAA-3'; E2: sense, 5'-gaattcCTCAGCCTGCTTGTCAGTTT-3'; antisense, 5'-gaattcACCCAATCACACGACTCTGG-3'; E3: sense, 5'-gaattcTGTGATTGGGTCTGACCCTC-3'; 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 pGL3-del–199/–65 were as follows: for CBF mutant construct pGL3–541CBFm: sense, GaaATacGggCTGACCCTCAGCCTGCTT; antisense, ccCgtATttCACGACTCTGGGGCGGCCC (mutated nucleotides are indicated by lowercase letters); for deletion construct pGL3-del-147/–121: sense, 5'-AGCCTGCTTGTCAGTTTCGC-3'; antisense, 5'-GGGGCGGCCCCAAGCCCGCC-3'; for deletion construct pGL3-del–199/–65: sense, 5'-GGCGGAGGAGGGGGCTGCCC-3'; antisense, 5'-GAGCAGGCCCAGCCCACCAA-3'.
Construct of the dominant-negative CBF-B/NF-YA was generated by reverse transcriptase PCR as described previously (22, 23). Two sets of primers 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 are indicated by lowercase letters.)
These PCR products were cloned into the pGEM-T Easy vector and subcloned 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 a density of 2 x 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, the transfected cells were harvested, lysed, centrifuged to pellet the debris and performed to luciferase assay as described below. For stable transfection, 1 µg 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 at a density of 1 x 103 per 100-mm dish with fresh medium containing 8 µg/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 Reporter 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 µg 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 each experiment as negative and positive controls, respectively. Relative luciferase activities (percentages) of each construct were normalized against 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 were prepared according to Dignam et al. (26) with some modifications. All buffers contained the protease inhibitors leupeptin (2 µg/ml), aprotinin (2 µg/ml), pepstatin A (2 µg/ml), phenylmethylsulfonyl fluoride (0.5 mM), and dithiothreitol (1 mM). Cells (1 x 108) were resuspended in buffer (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 °Cfor1h.The supernatants with nuclear proteins were recovered by centrifugation at 24,000 x 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 the mutant 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 make double-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 from Santa Cruz Biotechnology, Inc., used as competitors, were as follows: CBFwt, 5'-AGACCGTACGTGATTGGTTAATCTCTT-3'; CBFmt5'-AGACCGTACGAAATACGGGAATCTCTT-3' (mutated nucleotides are underlined); 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 µl 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 µg of poly(dI-dC), and
1–20 µg of nuclear extracts.
For competitors and antibody interference assay, unlabeled probes or
antibody was added to the reaction mixture for 1 h at 4 °C before the
addition of [-32P]dCTP labeled probe. These antibodies
against CBF-A/NF-YB, CBF-B/NF-YB, CBF-C/NF-YC, C/EBP, NF-1, GATA-1, and
pre-immune goat IgG were purchased from Santa Cruz Biotechnology. The
DNA-protein complexes were separated on 4.5% nondenaturing polyacrylamide gel
in 0.25x 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 0.1x 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 0.4x buffer A containing 0.4 M KCl and incubated for 15 min at 4 °C. After centrifugation at 15,000 rpm for 5 min, the supernatant 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 µg/ml aprotinin, and 1 µg/ml pepstatin A. A204 cells were inoculated at a density of 1 x 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, 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 anti-CBF-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 µl of salmon sperm DNA/protein A-agarose slurry, followed by incubation for 2 h at 4 °C. After washing the beads with both low- and high-salt buffer, then LiCl buffer, and finally Tris/EDTA buffer, the immune complexes were eluted by incubation for 15 min at 25 °C with 200 µl 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 µl 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 µl of Tris/EDTA buffer. Quantitative PCR was carried out for 35 cycles using 5 µl of sample DNA solution, and PCR products were separated on 2% agarose gels in 1x Tris-acetate/EDTA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 this region. 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, bound nuclear 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 competed away 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 and GS3 regions were generated (Fig. 2A). Fig. 2D shows that 32P-labeled GS4 probe, covering –147 to –121, formed DNA-protein 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 critical region 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 a manner 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.
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Identification of DNA-Binding Proteins in the Proximal COL11A1 Promoter—To identify the nuclear binding proteins in 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 specific antibodies 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 other proteins (Fig. 4C, lanes 6–8) and control IgG (Fig. 4C, lane 9) failed to inhibit the supershift (Fig. 4B).
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CBF/NF-Y Binds to the COL11A1 Proximal Promoter both in Vitro and in Vivo—To examine whether CBF/NF-Y binds directly to the COL11A1 proximal promoter, we performed two in vitro 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 32P-labeled GS4 probe bound to the affinity-purified proteins (lane 4) at levels similar to those of the crude extracts (lane 1), and the 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 were clearly 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 immunoprecipitated with 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.
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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 that of one copy. Furthermore, pGL3(–147/–121)x4 exhibited higher promoter activity (+30%) than pGL3–199/–65 and pGL3(–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 the constructs containing a mutated or deleted CBF/NF-Y binding site, pGL3–541E1, –541E4, CBFm, del–147/–121, and del–199/–65, were suppressed, whereas in pGL3–541E2 and -E3 constructs, the activities did not change compared with that 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 the transient 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 with a 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-negative 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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 a downstream
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 CCAAT-binding
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 specific
antibodies (Fig. 4), and
demonstrated direct binding to this region 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 high transcriptional 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 the proximal 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 basal
transcriptional 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 composed of a large number of heterotrimeric
[1(I)]22(I) or a small number of homotrimeric
[1(I)]3 molecules. It is also the most abundant 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 other putative 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 the presence 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 I collagen, as described previously, and it has been reported that the
5'-flanking proximal region of the 1(II) gene has a weak promoter
activity, and the transcription of COL2A1 is regulated 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, and further analysis of
1(III) proximal promoter indicates that two distinct positive factors,
other than CBF-NF-Y, regulate the transcription of type III collagen
(33). These observations in
major fibrillar groups suggest that the mechanisms for basal transcriptional
regulation of types II and III collagen may be 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 the proximal 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 neural
tissue-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 the same as
each other, as described previously
(17,
18,
39,
40).
Type XI collagens also form a heterotypic molecule that is composed of
[1(XI)]22(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 the basal 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 basal
transcriptional 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, found in 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
TATA-containing and TATA-less promoters
(46) and regulates on a
case-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 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 from a 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 be some 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 is control 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-specific
collagen, 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
and Cbfa1. 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
tissue-specific transcription factors deserves further study to fully
understand the mechanism of transcriptional regulation in the 1(XI)
collagen gene.
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FOOTNOTES |
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¶ To whom correspondence should be addressed. Tel.: 81-97-586-5670; Fax: 81-97-549-6302; E-mail: hidey{at}oita-med.ac.jp.
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.
2 K. Sakata-Takatani, N. Matsuo, T. Tsuda, and H. Yoshioka, unpublished
data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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