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© The Rockefeller University Press,
0021-9525/1998//1291 $5.00
The Journal of Cell Biology, Volume 141, Number 6,
, 1998 1291-1300
Articles |
Different cis-Regulatory DNA Elements Mediate Developmental Stage- and Tissue-specific Expression of the Human COL2A1 Gene in Transgenic Mice
Embryology Unit, Children's Medical Research Institute, New South Wales 2145, Australia
Expression of the type II collagen gene (human COL2A1, mouse Col2a1) heralds the differentiation of chondrocytes. It is also expressed in progenitor cells of some nonchondrogenic tissues during embryogenesis. DNA sequences in the 5' flanking region and intron 1 are known to control tissue-specific expression in vitro, but the regulation of COL2A1 expression in vivo is not clearly understood. We have tested the regulatory activity of DNA sequences from COL2A1 on the expression of a lacZ reporter gene in transgenic mice. We have found that type II collagen characteristic expression of the transgene requires the enhancer activity of a 309-bp fragment (+2,388 to +2,696) in intron 1 in conjunction with 6.1-kb 5' sequences. Different regulatory elements were found in the 1.6-kb region (+701 to +2,387) of intron 1 which only needs 90-bp 5' sequences for tissue-specific expression in different components of the developing cartilaginous skeleton. Distinct positive and negative regulatory elements act together to control tissue-specific transgene expression in the developing midbrain neuroepithelium. Positive elements affecting expression in the midbrain were found in the region from –90 to –1,500 and from +701 to +2,387, whereas negatively acting elements were detected in the regions from –1,500 to –6,100 and +2,388 to +2,855.
Abbreviations used in this paper: dpc, day(s) post coitum; nt, nucleotide(s).
DURING embryogenesis, the selective and combinatorial expression of genes signifies the developmental program and phenotype of differentiated cells. The activation and repression of gene expression is usually controlled at the level of transcription by multiple positive and negative regulatory elements (Darnell, 1982; Gutman et al., 1994).
Expression of the type II collagen gene is a hallmark of chondrogenesis in vertebrates. In chickens, rodents, and humans, the type II collagen gene is transcribed at high levels by cells of the chondrogenic lineage (Kosher et al., 1986; Cheah et al., 1991; Ng et al., 1993; Lui et al., 1995). In Xenopus, avian, and mouse embryos (Von der Mark et al., 1976; Kravis and Upholt, 1985; Kosher et al., 1986; Thorogood et al., 1986; Swalla et al., 1988; Kosher and Solursh, 1989; Cheah et al., 1991; Su et al., 1991; Wood et al., 1991; Ng et al., 1993), expression of the type II collagen gene is not restricted to chondrogenic cells. In humans, COL2A1 mRNAs are also widely expressed in tissues that are not involved in cartilage formation, such as the notochord, neural retina, heart, fetal brain, and the epithelium of the otic vesicle (Sandell, 1994; Lui et al., 1995; see Table I).
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The importance of sequence elements for tissue-specific expression of genes is often inferred from cotransfection assays in cultured cells. Although such studies are valuable, differing results may be obtained depending on whether transfection is stable or transient. It is also not possible to identify elements important for stage specificity of expression by this approach. Experiments using transgenic animals to recapitulate the normal pattern of gene expression have shown that in certain cases, such as elastase and 
1(I) collagen genes that DNA sequences identified as essential for gene regulation in transfected cells may not have any functional role in vivo (Swift et al., 1989; Sokolov et al., 1995).
The regulatory activity of some type II collagen gene sequences has been examined in vivo by transgenesis. The first intron of the mouse type II collagen gene (Col2a1) is essential for cartilage-specific expression of an Escherichia coli β-galactosidase (lacZ) reporter gene in transgenic mice (Metsäranta et al., 1995). Sequences within a 182-bp fragment of the mouse Col2a1 first intron were also shown to direct chondrogenic expression of a lacZ reporter gene when present as tandem copies (Mukhopadhyay et al., 1995; Zhou et al., 1995). However, some sequences shown to be important for enhancer activity of the first intron in cell transfection assays were found to be redundant for chondrocyte-specific expression (Mukhopadhyay et al., 1995). These studies on the regulation of the Col2a1 gene have concentrated mainly on defining minimal sequences important for chondrocyte-specific enhancer activity and on expression in the chondrogenic lineage from 10.5 days post coitum (dpc)1 in the mouse. The elements necessary for expression of Col2a1 before 10.5 dpc and in nonchondrogenic tissues in the mouse have not been determined. Furthermore, the transgenic studies performed so far have not revealed any activity of the silencing (repressor) elements that have been identified by cell transfection studies (Savagner et al., 1990; Seghatoleslami et al., 1994).
The sequence elements directing human type II collagen gene expression are not well defined. We have previously shown that a 30-kbp genomic fragment containing the human COL2A1 gene can be expressed in chondrogenic tissues in the mouse (Lovell Badge et al., 1987). More recent studies on the COL2A1 gene have shown that a combination of 1.8-kbp 5' flanking DNA with a 3.0-kbp first intron or 4.5-kbp upstream sequence with 1.8-kbp of intron 1 DNA, was sufficient for chondrogenic-specific lacZ reporter gene expression in transgenic mice (Seghatoleslami et al., 1994; Cheah et al., 1995). These studies concentrated on later stages and on the chondrogenic lineage; there is little information on the cis-regulatory elements governing COL2A1 transcription in vivo or the ability of these sequences to direct expression at early stages of embryogenesis and in nonchondrogenic tissues. We have assayed such elements in the 5' flanking and intron 1 regions of COL2A1 expressed in transgenic mice.
A prerequisite for analysis of the regulation of the human COL2A1 gene in mice is that the developmental pattern of expression can be recapitulated in the transgenic animal. We have previously shown very similar patterns of expression of the endogenous human and mouse type II collagen genes in nonchondrogenic and prechondrogenic cells from stages equivalent to 9.5 dpc in mouse embryos (Ng et al., 1993; Lui et al., 1995). To provide a starting point for the identification of regulatory elements controlling appropriate COL2A1 transcription in this study, we compared expression of a genomic clone containing COL2A1 in transgenic mice with that of the endogenous mouse Col2a1 gene. In addition, we have studied the regulation of COL2A1 expression in transgenic mice during early organogenesis and fetal stages.
In the present study we identified a 309-bp fragment within the first intron of human COL2A1 which is capable of directing reporter transgene (COL2A1-lacZ) expression in prechondrogenic and chondrogenic sites as well as in a subset of nonchondrogenic tissues (such as node, neural crest cells, and notochord), characteristic of type II collagen. These sequences were sufficient for correct temporal regulation of gene expression. We show that the 5' flanking DNA and the first intron need to work synergistically to direct the expression of the transgene in tissues that normally express Col2a1. We present evidence that positive and negative tissue-specific regulatory elements in the first intron and the 5' flanking region of the COL2A1 gene are important for directing expression in the developing midbrain neuroepithelium.
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Materials and Methods |
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Reporter gene constructs were created in pPOLYIII I vector by fusing various lengths of 5'-flanking promoter sequences to a 125-bp 5' untranslated region of human COL2A1 gene and an E. coli β-galactosidase (lacZ) reporter gene cassette with an initiation translation codon and polyadenylation signal, followed by various lengths of the first intron of the human COL2A1 gene. The COL2A1 content in the different constructs were as shown in Fig. 1. For ease of reference, the intron 1 sequences analyzed in this study have been subdivided into three regions, designated X, Y and Z, the coordinates of which are +701 to +2,387, +2,388 to +2,696, and +2,697 to +2,855, respectively (see Figs. 1 and 6).
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Section and Whole Mount In Situ Hybridization
In situ hybridization to sections was carried out as described previously (Cheah et al., 1991; Nonchev et al., 1996). Two hybridization probes used were human COL2A1-specific (KH14) and mouse Col2a1-specific (KH15). KH14 and KH15 probes each contained 70 bp of relatively less conserved 3' untranslated sequence from equivalent regions of the human and mouse type II collagen genes corresponding to +31,102 to +31,171 (COL2A1) and +28,624 to +28,693 (Col2a1), i.e., KH14 is the human homologue of KH15. These probes were so designed because they contained short stretches of nonconserved bases and were species-specific when sections were treated with RNase posthybridization, and then washed at high stringency. Autoradiography and photography of sections were carried out as described previously (Cheah et al., 1991).
Whole mount in situ hybridization on 8.0–9.0 dpc CBA mouse embryos was carried out as described by Wilkinson (1992) using single-stranded, digoxgenin-UTP (Boehringer Mannheim, Mannheim, Germany)–labeled antisense riboprobes generated from pNJ61 (Ng et al., 1993), which contains exon 1–5B of Col2a1.
X-Gal Staining and Histochemistry
Mouse embryos or fetuses of various stages were harvested in PBS, pH 7.4, and then fixed for 15–60 min, according to their size, at 4°C in 4% paraformaldehyde in PBS. They were assayed for expression of lacZ by X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) staining essentially as described in Yee and Rigby (1993). After staining, fetuses were embedded in paraffin. 6-µm sections were counterstained with eosin.
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Results |
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Identification of Minimal 5' Flanking Sequence for COL2A1 Expression
The 6.1-kbp 5' flanking sequence was systematically examined in conjunction with 2.1 kbp of the intron 1 sequence (regions X, Y, and Z) to determine the minimal upstream DNA required to regulate appropriate lacZ expression. The 5' flanking sequence was reduced to 1.5 kbp (–1 to –1,500) (pKL10, and pKL10 which differs by the orientation of the intron 1 sequence, refer to Fig. 1) and also to 90 bp (–1 to –90, pKL12, refer to Fig. 1). None of these constructs showed any significantly different expression patterns from one another (Fig. 3, l–n; Table I) or that of pKL9 (Fig. 3, c–e). Constructs pKL9 and pKL80.3 were expressed in chondrogenic tissues (Fig. 4, a–d and e–j; Table I) and were appropriately downregulated in the hypertrophic chondrocytes like the endogenous Col2a1 (Fig. 4, i and j; Ng et al., 1993; Lui et al., 1995). pKL10, pKL10R, and pKL12 transgenes were expressed in the same prechondrogenic and chondrogenic tissue types as pKL9 and pKL80.3 (Table I). Reduction of the 5' flanking sequence of pKL9 to 1500 (–1 to –1,500, pKL10 and pKL10R) or 90 bp (–1 to –90, pKL12) did not seem to affect the expression of the transgene (Fig. 4, k–m at 13.5 dpc). Transgene expression was found in the notochord when the transgene was regulated by 6.1-kbp, 1.5-kbp, or 90-bp upstream sequence in combination with 2.1-kbp first intron (Fig. 3, c and e, pKL9; Fig. 3 l, pKL10; Fig. 3 m, pKL10R; Fig. 3 n, pKL12). Similar notochord expression was also achieved with region Y and 6.1-kbp 5' flanking DNA (Fig. 3, f–k, pKL80.3). Therefore, the 5' flanking sequence –1 to –90 with 2.1-kbp intron 1 sequence is sufficient for COL2A1 expression, and 6.1 kbp is required if only region Y of intron 1 is present.
Separate Regulatory Elements Direct Expression in Different Components of the Cartilaginous Skeleton
The pKL10 construct was modified by deleting region Y+Z from the intron 1 fragment (refer to Fig. 1, pKL10X). This transgene was expressed in sites that normally would express Col2a1 (Fig. 5, a–e; e.g., craniofacial mesenchyme, paraxial mesoderm, and prevertebrae), except the notochord, the second branchial arch (Fig. 5, a and b, 9.5 dpc), and the digits of the limbs (Fig. 5, c and f, 14.5 dpc). Transgene expression was generally patchy (Fig. 5 a) and reduced in limbs, digits, the upper trunk, vertebral cartilage, and rib cage (Fig. 5 c). A similar expression pattern was observed (Fig. 5, g–l) for construct pKL12X containing the minimal 90-bp (–1 to –90) 5' flanking sequence and region X (refer to Fig. 1). Together, these results suggest that at least two separable modules of DNA are present in intron 1. Region X may contain regulatory elements for the first branchial arch, facial skeleton, and lower trunk vertebrae. Indeed, deletion of region X from pKL10R (Fig. 1, pKL10RX) led to loss of expression in most tissues except the limbs and digits (Fig. 5, m–q; Table I). Region Y+Z may contain regulatory elements for regulating COL2A1 expression in the second branchial arch, notochord, upper trunk (axial), and limb (appendicular) cartilage.
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Although pKL12 was not expressed in the midbrain of the transgenic embryo, the removal of region Y+Z from pKL12 (i.e., pKL12X) has resulted in the reexpression of the lacZ reporter in the midbrain (Fig. 5, g and h; Table I d). Exchanging region X for Y+Z (pKL10RX) resulted in loss of midbrain expression (Table I). These findings therefore suggest that negative regulatory elements controlling midbrain expression may be localized in region Y+Z. These elements may act in an orientation-independent manner, since midbrain expression was similar for pKL10 and pKL10R (refer to Fig. 3, l and m). Deletion of the same region (Y+Z) from pKL10 (i.e., pKL10X), which was expressed in the midbrain, has no effect on the expression of the reporter (compare Fig. 3 l with Fig. 5, a and b). Therefore, it suggests that the positive elements found between –90 and –1,500 could overcome the negative effect of region Y+Z.
Tissue-specific Expression Requires Both COL2A1 5' and Intron 1 Sequences
To investigate whether region Y needs to interact specifically with the COL2A1 promoter, we replaced the 6.1-kbp 5' flanking sequence of pKL80.3 with heterologous promoter fragments from the human β-globin and mouse type X collagen (Col10a1) genes (refer to Fig. 1, pKL21 and pWP16 constructs). The human β-globin promoter fragment has been shown to cooperate with tissue-specific elements from heterologous genes, such as myogenin (Yee and Rigby, 1993) and Hoxa2 (Nonchev et al., 1996), to drive tissue-specific lacZ expression in 9.5-dpc transgenic mice embryos. The Col10a1 promoter fragment can direct lacZ expression in developing bone of 14.5-dpc transgenic mouse fetuses (Poon, W.S., and K.S.E. Cheah, unpublished data).
The β-globin promoter (pKL21) was unable to direct transgene expression in the Col2a1 pattern despite the presence of region Y of intron 1 (seven expressing embryos showing low and nonspecific expression, data not shown). Furthermore, type X collagen promoter was also unable to substitute for the COL2A1 5' flanking sequence and no expression was seen in seven transgenic embryos. The specific requirement for intron 1 sequence is further demonstrated by the inappropriate expression of pKL28 construct, in which the intron 1 fragment was replaced by the SV-40 enhancer (refer to Fig. 1). Only one of six transgenic embryos expressed lacZ and was not in a pattern characteristic of the Col2a1 (pKL9/pKL80.3) pattern (data not shown).
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Discussion |
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In our experiments, neither the first intron nor the COL2A1 5' flanking DNA could be substituted by enhancer or promoters of unrelated genes. These results are consistent with the report that the promoter and first intron of the rat type II collagen gene act synergistically (Savagner et al., 1995), but contrast with another report showing that mouse Col2a1 intron 1 sequences linked to the human β-globin or adenovirus major late promoters could direct transcription in chondrocytes (Mukhopadhyay et al., 1995; Zhou et al., 1995). These contradictory findings could be related to differences on the strength of the promoter–enhancer interaction and the proximity of the promoter to sequences in the first intron, which may affect the efficiency of transcription. In the constructs tested in the present study, the first intron sequences are separated from the promoter sequence by at least 3 kbp of lacZ DNA. This may hamper the formation of the correct promoter-enhancer loop, and thus diminishes the activity of the heterologous promoters. Furthermore, in other studies, multiple intron 1 sequences are placed immediately upstream of heterologous promoters. This arrangement may facilitate the stabilization of promoter–intron interactions allowing the expression of the transgene (Mukhopadhyay et al., 1995; Zhou et al., 1995).
The requirement for proper physical configuration and/ or differing combination of integrity of regulatory elements for transgene expression has also been shown in the present study. Appropriate expression of the lacZ transgene is achieved by a combination of either the 6.1-kbp 5' flanking fragment and region Y, or a 90-bp 5' flanking sequence and the 2.1-kbp intron 1 sequence containing X+Y+Z. Therefore, regions X and Z may contain some elements that interact with the short (90-bp) 5' flanking sequence. The deletion of sequences –1,500 to –6,100 resulted in the loss of lacZ expression. Both combinations of regulatory elements were equally effective for correct tissue-specific expression, despite sequence differences between modules. The results are consistent with the notion that sufficient lengths of specific combinations of the 5' flanking sequence and intron 1 of COL2A1 are essential to achieve the appropriate conformation for establishing the necessary heteromeric complex to activate transcription.
COL2A1 Activity in the Midbrain Is Regulated by Positive and Negative Regulatory Elements
Transgene expression in the midbrain is found to be modulated by a combination of positive and negative elements. DNA sequences in the upstream region –1,500 to –6,100 and region Y+Z of intron 1 seem to exert a suppressive activity, whereas the sequences in the –1 to –1,500 region and region X are required for the expression of the COL2A1 transgene (Fig. 6). The data also suggest that expression of COL2A1 in different skeletal elements may be mediated by different combinations of regulatory elements. For example, 1.5-kbp with region Z+Y gave limb expression only (Fig. 5 m) but 1.5-kbp region X gave expression in the axial skeleton but not in the limbs (Fig. 5 c). Recently, cis-acting elements that control 2(XI) collagen gene expression in different cartilaginous components of the skeleton (Tsumaki and Kimura, 1995) and separable DNA modules for tissue and site-specific expression of the 1(I) (Sokolov et al., 1995; Rossert et al., 1995, 1996) and 1(VI) collagen genes (Braghetta et al., 1996) have been described. The differential impact of selective enhancer and repressor elements of the COL2A1 gene on lacZ reporter activity in the brain suggests lineage-specific or region-specific deployment of regulatory elements may be a common mechanism of gene regulation.
Some Regulatory Motifs Identified by Cell Transfection Assays Have No Function In Vivo
Region Y contains the analogous sequences to those in the rodent type II collagen genes shown to be important for enhancer activity in transfected chondrocytes (Wang et al., 1991; Krebsbach et al., 1996; Lefebvre et al., 1996) but not all the motifs identified were present in this region. An AT-rich element previously shown to be important for the enhancer activity and for transcriptional regulation of the Col2a1 gene (Krebsbach et al., 1996) was absent in region Y, suggesting that this sequence is not essential for tissue-specific expression of COL2A1 in vivo.
The 90-bp upstream region of COL2A1 is highly conserved among human, mouse, and rat and contains many potential transcription factor–binding motifs, including a potential Sp1-binding site (–75 to –80) (Vikkula et al., 1992). In the rat Col2a1 gene, a potential Sp1-binding site outside this 90-bp region (–114 to –119) interacts with the first intron enhancer via a complex consisting of Sp1 factor and a zinc finger protein, CIIZFP (Savagner et al., 1995). Our results showing appropriate transgene expression in the absence of this Sp1-binding site (construct pKL12) suggest either that the site is redundant or the other potential Sp1-binding site (–74 to –80) or yet undefined elements within the 90-bp sequence are sufficient for gene expression. The increased specific binding activity to Sp1 motifs found for nuclear extract prepared from non- Col2a1–expressing cells such as fibroblasts and dedifferentiated chondrocytes suggest that the Sp1-binding site may mediate negative regulation of the gene (Dharmavaram et al., 1997).
The rat type II collagen gene contains two sequence elements at positions –421 to –436 and –657 to –666 which behave as silencers in cell transfection assays (Savagner et al., 1990). This region is not well conserved between human and rat type II collagen gene (Vikkula et al., 1992) and the silencing activity has not been tested in vivo. Omitting the –90 to –1,500 region of COL2A1 did not change the tissue-specificity of transgene expression, suggesting that the putative silencer elements in the 5' flanking DNA do not play any role in the regulation of COL2A1 activity in transgenic mice.
Molecular Mechanisms for Regulation of COL2A1 Expression: A Model of the Organization of COL2A1 Lineage-specific Regulatory Elements
The main structural and functional features of the DNA modules regulating lineage-specific expression of human COL2A1 (Fig. 6) are: (a) early organogenesis and chondrogenic expression are regulated by the interaction of elements in the upstream (–1 to –6,100) region and the 309-bp region Y of the first intron and of elements in –1 to –90 region and region X of the first intron; (b) expression in the midbrain tissue is mediated by interactions between positive elements in the –1 to –1,500 region and the negative elements in –1,500 to –6,100 and region Y+Z of the first intron; (c) critical regulatory elements for gene expression in the notochord are found in the region –1 to –1,500 and region Y of the first intron.
Such an interplay of positive and negative regulation to achieve defined expression patterns has been found for many developmentally regulated genes (Ip and Hemavathy, 1997). Transcription factors such as Bicoid, Dorsal, snail, and Elf-1 regulate patterning in the Drosophila embryo by restricting gene expression to specific sites (Novina and Roy, 1996; Ip and Hemavathy, 1997). We and others have recently shown that SOX9 directly regulates the mouse and human type II collagen genes via SRY/SOX binding motifs found in the Y region of the first intron (Bell et al., 1997; Lefebvre et al., 1997). However, the expression of SOX9 does not always transactivate the Col2a1 gene, especially in nonchondrogenic tissues, suggesting that other factors are also required (Bell et al., 1997). Future challenges lie in identifying those additional factors which bind to and interact with the various activating/repressing DNA modules in vivo, to direct appropriate tissue-specific expression of COL2A1 during key differentiation events in organogenesis and chondrogenesis.
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Acknowledgments |
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This work was supported by grants from the Research Grants Council (Hong Kong) (HKU356/94M), the Croucher Foundation (Hong Kong) and the Arthritis and Rheumatism Council (UK) (CO523).
Submitted: 10 September 1997
Revised: 4 May 1998
Address all correspondence to Professor Kathryn S.E. Cheah, Department of Biochemistry, The University of Hong Kong, Sassoon Road, Hong Kong. Tel.: (852) 2819-9240 or 2819-9170. Fax: (852) 2855-1254. E-mail: hrmbdkc{at}hkumd1.hku.hk
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