Modular Arrangement of Cartilage- and Neural Tissue-specificcis-Elements in the Mouse α2(XI) Collagen Promoter*

Type XI collagen, a heterotrimer specific to cartilage matrix, plays an important role in cartilage morphogenesis. We analyzed various α2(XI) collagen promoter-lacZreporter gene constructs in transgenic mice to understand tissue-specific transcriptional regulation. The −530 promoter sequence was sufficient to direct reporter gene expression specifically to cartilage. Further deletion to −500 abolished reporter gene expression in cartilage but activated the expression specific to neural tissues such as brain and neural tube. An additional 47-base pair deletion resulted in random tissue expression patterns. A 24-base pair sequence from −530 to −507 of the α2(XI) promoter was able to switch the activity of the heterologous neurofilament light gene promoter from neural tissues to cartilage. These results suggest that the α2(XI) collagen gene is regulated by at least three modular elements: a basal promoter sequence distal to −453, a neural tissue-specific element (−454 to −500), and a cartilage-specific element (−501 to −530), which inhibits expression in neural tissues and induces expression in cartilage.

Type XI collagen, a heterotrimer specific to cartilage matrix, plays an important role in cartilage morphogenesis. We analyzed various ␣2(XI) collagen promoter-lacZ reporter gene constructs in transgenic mice to understand tissue-specific transcriptional regulation. The ؊530 promoter sequence was sufficient to direct reporter gene expression specifically to cartilage. Further deletion to ؊500 abolished reporter gene expression in cartilage but activated the expression specific to neural tissues such as brain and neural tube. An additional 47-base pair deletion resulted in random tissue expression patterns. A 24-base pair sequence from ؊530 to ؊507 of the ␣2(XI) promoter was able to switch the activity of the heterologous neurofilament light gene promoter from neural tissues to cartilage. These results suggest that the ␣2(XI) collagen gene is regulated by at least three modular elements: a basal promoter sequence distal to ؊453, a neural tissue-specific element (؊454 to ؊500), and a cartilage-specific element (؊501 to ؊530), which inhibits expression in neural tissues and induces expression in cartilage.
Cartilage is a highly specialized tissue that serves as the template for the development of skeleton and lines the joint surface. Cartilage consists of an abundant extracellular matrix maintained by chondrocytes. The collagen network provides a scaffolding for proteoglycans in the extracellular matrix and confers tensile strength important for resisting compression and shearing loads in cartilage. Cartilage collagen fibrils are heterotypic fibrils composed of types II, IX, and XI collagens.
Type XI collagen likely regulates the diameter of cartilage collagen fibrils (1,7). This putative function is supported by a null mutation in the ␣1(XI) gene of cho/cho (chondrodysplasia) mice, in which the cartilage lacks the ␣1(XI) collagen chain and perhaps collagen XI trimer molecules resulting in abnormally thick collagen fibrils (8). Furthermore, association of ␣2(XI) gene mutations with certain forms of human chondrodysplasia, Stickler syndrome, and otospondylo-megaepiphyseal dysplasia indicates that type XI collagen is intimately involved in skeletal morphogenesis (9). These observations suggest that the fidelity of spatiotemporal expression of type XI collagen is crucial for the development and maintenance of the normal structure of cartilage.
The expression patterns of the three subunits of type XI collagen molecules differ from each other. In addition to its presence in cartilage, mRNA for the ␣1(XI) chain is also found in a variety of non-cartilaginous tissues, including the bone, vitreous, heart, and cerebral neuro-epithelium (10). Similarly, mRNA for the ␣3(XI) chain is detected in various tissues besides cartilage (11). On the other hand, expression of the ␣2(XI) gene appears to be more restricted. Although low levels of alternatively spliced variants of ␣2(XI) transcripts are detected in non-cartilaginous tissues, the major transcript is predominantly found in cartilage (12,13). In addition, the level of ␣2(XI) mRNA is lower than that of ␣1(XI) mRNA and much less than that of the ␣1(II) collagen gene (14). Together, these results suggest that expression of the ␣2(XI) gene may be rate-limiting for the trimer formation of type XI collagen molecules that is critical for the subsequent lateral growth of cartilage collagen fibrils.
Recently, we cloned and sequenced the 5Ј-flanking region of the mouse ␣2(XI) collagen gene (Col11a2) 1 and found that the retinoid X receptor ␤ gene (Rxrb) is located 742 bp upstream of the transcriptional start site of the Col11a2 gene (15). We also showed that the Ϫ742-bp promoter sequence of the Col11a2 gene contains information for expression specific to the primordial cartilage of long bones and ribs (15). In the present study, we further delineated cis-regulatory elements of the Col11a2 promoter necessary for cartilage-specific expression using reporter gene constructs in transgenic mice. We found that the Ϫ530-bp sequence was sufficient for the cartilage-specific expression of the Col11a2 gene. Deletion of a sequence between Ϫ530 and Ϫ500 abolished reporter gene expression in cartilage. Interestingly, deletion of this sequence induced neural tissue-specific expression. We also showed that a 24-bp sequence in this region could activate reporter gene expression in cartilage when it was linked to the heterologous human neu-* This work was supported in part by grants from Labor Welfare Corporation of Japan and the program for Promotion of Fundamental Studies in Health Science of the Organization for Drug ADR Relief, R & D Promotion, and Product Review of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

EXPERIMENTAL PROCEDURES
Construction of Col11a2-lacZ Transgenes-The transgene constructs, 742lacZ and 453lacZ, which bear 742-and 453-bp Col11a2 promoter sequences, respectively, linked to the ␤-galactosidase reporter gene (lacZ) were described previously (15). Other transgene constructs containing various lengths of the Col11a2 promoter region were prepared by cloning into the expression vector pNASS␤ (CLONTECH), which contains a SV40 RNA splice site, the ␤-galactosidase gene, and a SV40 polyadenylation signal. All constructs were confirmed by DNA sequencing.
For the reporter gene construct containing the promoter of the human neurofilament light gene (16), a DNA segment between Ϫ292 and ϩ15 of NF-L was amplified by PCR with human genomic DNA as a template using primers Ne-1 containing an EcoRI site and a XhoI site and Ne-2 containing a SalI site. After digestion with EcoRI and SalI, the PCR fragment was cloned into the EcoRI/XhoI sites of pNASS␤ to create DNA construct NF-lacZ. For generation of the fusion promoter construct, complementary oligonucleotides 24-1 and 24-2 containing three tandem copies of a sequence (Ϫ530 and Ϫ507) of Col11a2 were synthesized and annealed to generate a double-stranded DNA fragment. The annealed fragment was cloned into the EcoRI/XhoI sites upstream of the neurofilament promoter (NF-lacZ) to create the construct 24ϫ3-NF-lacZ. The primers used were as follows: Ne-1, CCGG-AATTCCCGCTCGAGAAGGATCCAAGTGTCACGGGGTCTGGG; Ne-2, TGGTAGTCGACATGGATGGCTGTGTGCGGCTCGGCGCCG; 24-1, AATTCCAGGGAGGAGGGAGAGCGGCTGCTCAGGGAGGAGG-GAGAGCGGCTGCTCAGGGAGGAGGGAGAGCGGCTGCT; and 24-2, TCGAAGCAGCCGCTCTCCCTCCTCCCTGAGCAGCCGCTCTCCCTC-CTCCCTGAGCAGCCGCTCTCCCTCCTCCCTGG.
Generation of Transgenic Mice-A DNA fragment free from the plasmid sequence was prepared from each construct by digesting with EcoRI and PstI and microinjected into the pronuclei of fertilized eggs from F1 hybrid mice (C57BL/6 ϫ C3H) as described (17). Generation 0 (G 0 ) embryos were sacrificed at 13.5 d.p.c. (days postconception) and processed for expression analysis of the reporter gene. Transgenic embryos were identified by PCR analysis of genomic DNA extracted from the placenta as described previously (15).
Staining for ␤-Galactosidase Activity-␤-Galactosidase activity was analyzed by staining with X-gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside; Sigma) according to the previously described technique (18). Briefly, the embryos were fixed in 4% paraformaldehyde for 20 min and stained with X-gal for 2-7 h at 30°C. The reaction was stopped by washing in phosphate-buffered saline and postfixing in 4% paraformaldehyde. For histological examination, stained embryos were dehydrated, embedded in paraffin, and sectioned on a microtome. Then the sections were counterstained with eosin.

Production of Transgenic Mice Bearing
Col11a2-lacZ Constructs-A series of ␤-galactosidase reporter gene constructs containing various deletions between Ϫ742 and Ϫ453 of the promoter region were prepared to analyze tissue-specific activity of the ␣2(XI) gene promoter (Fig. 1). The DNA constructs were microinjected into fertilized ova, and generation 0 (G 0 ) embryos were sacrificed at 13.5 d.p.c. and stained with X-gal to analyze the expression patterns of the reporter gene. The numbers of transgenic mice and mice expressing ␤-galactosidase are summarized in Table I.

Expression of the lacZ Reporter Gene Driven by Col11a2 Promoter Constructs in Transgenic
Mice-The 742lacZ construct containing the Ϫ742-bp promoter showed expression in the cartilage of transgenic mice, which is inconsistent with our previous finding ( Fig. 2A) (15). Further 5Ј-deletions of the Col11a2 promoter revealed that the constructs, 680lacZ and 530lacZ, containing the Ϫ680and Ϫ530-bp promoter, respectively, were also capable of directing reporter gene expression specifically to the primordial cartilage in limbs and in ribs (Fig.  2, B and C). Histological analysis confirmed that X-gal staining of these mice was restricted to chondrocytes (Fig. 2D). All reporter gene expressing transgenic mice with 742lacZ, 680lacZ, and 530lacZ showed similar expression specifically in cartilage (Table I).
A 5Ј 12-bp deletion of the Ϫ530-bp promoter (518lacZ) resulted in no reporter gene expression in cartilage, but expression was activated in neural tissues such as brain and neural tube (Fig. 2, E and F). Strong staining was observed in the roof of the neopallial cortex, in the primitive ectomeninx, and in a medial portion of the neural tube. An internal 16-bp deletion between Ϫ516 and Ϫ501 (530⌬lacZ) in the 530lacZ construct also abolished expression in cartilage but induced neural tissue-specific expression (Fig. 2, G and H). The entire roof of the neopallial cortex, roof of the midbrain, and neural tube showed strong staining. A 5Ј 30-bp deletion from 530lacZ (500lacZ) also resulted in the expression of ␤-galactosidase in neural tissue, but not in cartilage (Fig. 2, I and J). Intense X-gal staining was detected in the roof of the neopallial cortex. The overall ␤-galactosidase expression patterns of transgenic embryos for these three constructs, 518lacZ, 530⌬lacZ, and 500lacZ, were limited to neural tissues, although there were some variations in the relative staining intensity. Histological analysis confirmed specific expression of the reporter gene in neural tissue in transgenic mice bearing these constructs (Fig. 2K). The 453lacZ construct, which had an additional 57-bp deletion from 500lacZ at 5Ј-end, showed ␤-galactosidase expression in various tissues. No consistent tissue-specific reporter gene expression patterns were found in the five transgenic mice with this construct, agreeing with previous reports (Fig. 2L; Ref. 15). These results suggest that the whole 30-bp sequence (Ϫ530 to Ϫ500) of the ␣2(XI) gene is required for cartilage-specific expression.

Expression of the lacZ Reporter Gene by the Fusion Promoter of the Col11a2 and NF-L Genes-Because a sequence between
Ϫ530 and Ϫ500 of the Col11a2 promoter region appeared to be capable of converting reporter gene expression from neural tissue-specific to cartilage-specific expression, we investigated this activity with an heterologous promoter from the NF-L gene, which can direct expression in neural tissues. The 24ϫ3-NF-lacZ construct was prepared by placing three copies of a 24-bp sequence spanning Ϫ530 to Ϫ507 of the ␣2(XI) gene upstream of the NF-L promoter (Ϫ292 to ϩ15). The NF-L promoter alone (NF-lacZ) specifically expressed ␤-galactosidase in the nervous system (Fig. 3, A and B), in agreement with a previous report (16). Inclusion of the 24-bp segment to the NF-L promoter (24ϫ3-NF-lacZ) showed activation of the reporter gene expression in the primordial cartilage of ribs, scapula, and neural arch (Fig. 3, C and D). Although reporter gene expression was observed in some other tissues such as the primordium of follicle of vibrissa at nose and some surface ectoderm in the limbs, the 24-bp sequence down-regulated neural tissue-specific expression directed by the NF-L promoter and activated the expression predominantly in cartilage.

DISCUSSION
In this study, we have defined the minimal sequences responsible for cartilage-specific expression of the Col11a2 gene using a series of deletions in transgenic mice. We found that the Ϫ530-bp promoter sequence was sufficient to direct reporter gene expression specifically to cartilage. Further deletions showed that the Ϫ500-bp promoter sequence exclusively directed expression to the neural tissue, whereas the Ϫ453-bp promoter sequence expressed the reporter gene in various tissues. These results suggest that the Col11a2 promoter consists of three modular elements: the basal promoter sequence located downstream from Ϫ453 conferring ability to activate transcription without any tissue specificity, the neural tissuespecific cis-element between Ϫ454 and Ϫ500, and the cartilagespecific cis-element between Ϫ501 and Ϫ530 of the ␣2(XI) promoter.
The neural tissue-specific expression patterns of transgenic mice bearing 518lacZ and 530⌬lacZ suggest that the 30-bp sequence (Ϫ530 to Ϫ500) of the Col11a2 gene is necessary for cartilage-specific expression. A GGAGGAG sequence (Ϫ527 to Ϫ521) in this region may be important, because a homologous sequence was found in the complementary sequence of a silencer of the ␣1(II) collagen gene (19). At the 3Ј-end of the 30-bp region of the ␣2(XI) promoter, there is a potential Sox9 binding motif for SRY and SOX proteins (TTCAAAG, Ϫ505 to Ϫ499). Sox9 was shown to bind to the enhancer of the ␣1(II) collagen gene and to activate its expression (20,21). Thus, this site may be important for activation of the ␣2(XI) gene.
It was surprising to find that a sequence within the ␣2(XI) promoter directed the expression of the reporter gene in neural tissues, because Col11a2 expression is restricted to cartilage and is hardly found in neural tissues (13). Inclusion of the  30-bp sequence to the Ϫ500-bp promoter changed the expression pattern from neural tissues to cartilage. These results may suggest that the 30-bp cartilage-specific cis-element has dual activities, one for inactivation of transcription in neural tissues and the other for activation of transcription in cartilage. These activities were partly supported by the finding that the reporter gene expression was active in cartilage but inactive in neural tissues in transgenic mice bearing the fusion promoter construct, 24ϫ3-NF-lacZ, containing the 24-bp sequence and the NF-L promoter. Because both cartilage and neural tissues are highly differentiated tissues, it is possible that genes expressed in these tissues may share common regulatory motifs and binding factors, which are responsible for tissue-specific gene expression. Nuclear factors bound to these sites remain to be elucidated.
In addition to the 30-bp sequence in the promoter, the first intron sequence of the Col11a2 gene is also important for cartilage-specific expression. The first intron sequence induces cartilage-specific expression when combined with the Ϫ453-bp basal promoter of the gene. It also enhances the expression in cartilage when combined with the Ϫ742 promoter sequence (15). It is of interest to note that homologous sequence motifs to the 30-bp sequence are found within the first intron sequence. The regulation through multiple modulators for the ␣2(XI) collagen gene is clearly different from that for ␣1(II) collagen gene, which is regulated by a relatively compact enhancer region in the first intron. This diversity of the regulatory mechanisms between the two cartilage genes may have been evolved because different levels of ␣2(XI) collagen may be necessary for optimal function in different cartilage tissues.