(Received for publication, July 18, 1995; and in revised form, November 2, 1995)
From the
The type II collagen gene (Col2a1) is expressed
primarily in chondrocytes. Transcription of Col2a1 is mediated
by cell-specific regulatory elements located within the promoter and
first intron. Here, we map a minimal enhancer and identify elements
that determine cartilage-specific Col2a1 expression by
analyzing the activity of a series of chimeric genes consisting of rat Col2a1 first intron deletion mutants ligated to the
chloramphenicol acetyltransferase reporter gene. We show that a
100-base pair (bp) segment within the first intron is the minimum size
necessary for high level, cell type-specific expression of Col2a1. Sequence analysis of this 100-bp Col2a1 enhancer revealed several sequence motifs similar to motifs
present within the regulatory region of the link protein gene, another
cartilage gene. These motifs include an AT-rich element, a C1 motif and
a C3 motif. Deletion of any of these elements reduced Col2a1 enhancer activity in chick embryo chondrocytes. We also tested
enhancer-mediated activity in CFK2 cells which differentiate to a
chondrogenic phenotype and begin to express type II collagen mRNA after
extended culture. In stably transfected CFK2 cells, constructs
containing the 100-bp enhancer were activated during the transition
from prechondrogenic to chondrogenic cell populations and deletions
within the enhancer strongly down-regulated activity.
Chondrocyte-specific DNA-protein complexes were identified using
nuclear extracts prepared from chick embryo chondrocytes and P-labeled oligonucleotides from these regions of the first
intron. These results suggest that interaction of chondrocyte specific
nuclear factors with multiple core elements from a small region within
the first intron are important for cell-type specific Col2a1 enhancer activity.
Type II collagen is the primary collagen in cartilage. Although type II collagen is present in the notochord, nucleus pulposa of intervertebral disks and vitreous of the eye, type II collagen is most abundant in hyaline cartilage(1, 2, 3, 4) . In hyaline cartilage, type II collagen, aggrecan, and link protein form a unique matrix that functions to absorb shock and to resist compression and shearing. Disruption of collagen expression in cartilage due to inflammatory or genetic influences may lead to degenerative joint diseases and a variety of chondrodysplasias(5, 6) . For example, defects in the type II collagen gene are associated with spondyloepiphyseal dysplasia, achondrogenesis type II, and the Stickler syndrome(7, 8, 9, 10) . Defects in type II collagen are also associated with Kniest dysplasia(11) . Together, these observations suggest that the fidelity of type II collagen expression is essential for maintaining the normal structure and function of cartilage.
The type II collagen
gene (Col2a1) ()encodes an
1(II) polypeptide
chain that is assembled into a homotrimeric molecule. Regulation of Col2a1 is complex and involves both transcriptional and
post-transcriptional
mechanisms(12, 13, 14, 15, 16, 17) .
Transcriptional regulation of Col2a1 is dependent on the
binding of several nuclear factors to both the 5`-flanking region and
an enhancer located within the first intron. Transcription of Col2a1 requires the presence of a minimal promoter that
includes at least one functional Sp1 binding site(17) .
Previously, the majority of enhancer activity was localized to a 1.5-kb
region in the central region of the first intron(15) . In
addition, the binding of chondrocyte nuclear factor(s) to a
helix-loop-helix consensus site has been demonstrated in this
region(18) .
In this study, we used two different but complementary transfection strategies to further delineate the Col2a1 enhancer and determined the minimal sequence necessary for full enhancer activity. In addition, we show that chondrocyte-specific nuclear factors bind to these functionally important regions. Several sequence motifs including an AT-rich element were identified in the enhancer. Deletion of these motifs reduced enhancer activity in both transient and stably transfected chondrocytes. Interestingly, three of these motifs are also present in the regulatory region of the cartilage link protein gene, suggesting that cartilage genes may share common elements for transcriptional regulation.
Figure 1: Map of the Col2a1-CAT plasmid constructs and CAT activity in transfected CEC. A, schematic representation of the rat Col2a1 first intron. The EcoRI site within the BamHI fragment is arbitrarily designated +1. B, several 5` and 3` first intron deletion mutants were transfected into CEC. CAT activity was analyzed 48 h after transfection. CAT activity was expressed as a percentage of the activity obtained with pDAS1BB5. Transfections were performed in duplicate in at least three independent experiments. *Values are expressed relative to the level of CAT activity of pDAS1BB5 in CEC. #Values are expressed relative to the level of CAT activity of pcDNA3/CAT in COS-7 cells.
Figure 3: Mapping of the 100-bp enhancer. Schematic representation of DNA constructs used to map regulatory regions of the Col2a1 first intron. See ``Materials and Methods'' for details. CAT activity was analyzed 48 h after transfection. *CAT activity was expressed as a percentage of the activity obtained with pDAS1BB5. Transfections were performed in duplicate in at least three independent experiments.
For electrophoretic
mobility shift assays, 3 µg of nuclear extract was incubated for 30
min at room temperature in mobility shift buffer (12 mM Hepes
(pH 7.9), 50 mM KCl, 4 mM MgCl, 1 mM EDTA, 0.1 mM ZnSO
, 1 mM dithiothreitol, 5% glycerol and 2 µg
poly(dI-dC)
poly(dI-dC)) with 30,000 cpm of
P
end-labeled double-stranded DNA probe in a 30-µl volume.
DNA-protein complexes were resolved on a 5% nondenaturing
polyacrylamide gel containing 4.5 mM Tris-HCl (pH 7.5), 4.5
mM boric acid, and 1 mM EDTA. The gels were dried and
exposed to x-ray film (Amersham Corp.).
Figure 2:
Nucleotide sequence of the
enhancer-containing segment. Nucleotide sequence of the 822-bp EcoRI-BamHI fragment of the rat Col2a1 first
intron. The EcoRI site is arbitrarily designated +1. The
100-bp minimal Col2a1 enhancer is in bold. Core
motifs are underlined. Inverted repeats are dashed.
This sequence has been submitted to GenBank (accession no.
L48618).
To evaluate the significance of these sequence motifs, internal deletions and base substitution mutations within the 100 bp enhancer were created and examined for their enhancer activity (Fig. 3). Deletions of the AT-rich element (OS-3) and C1 motif (OS-12) reduced CAT activity to 64 and 53% of the 100-bp enhancer, respectively. OS-9, a deletion of 10 bp downstream of the C1 motif, did not significantly alter CAT activity. Elimination of one C2 motif (OS-83) decreased CAT activity to 10%, while a deletion of C3 (OS-80) decreased CAT activity to 8%. A 2-bp substitution (OS-20, TT to GG) in the overlapping region of the C2 and C3 motifs reduced CAT activity to 12%, whereas a substitution of CC with AA (OS-25) in the C2 motif decreased CAT activity to only 85%. These results suggest that all the identified sequence motifs are critical for full enhancer activity.
CAT activity expressed by pIE55D and OS-30 transfected cells increased 6-fold during the course of differentiation of CFK to dCFK2 cells (Fig. 4). The increase in CAT activity from either pIE55D or OS-30 transfected CFK2 cells paralleled the increase in endogenous Col2a1 mRNA levels during extended cell culture (Fig. 5). After 9 days in culture, CFK2 cells did not express detectable Col2a1 mRNA. By day 14, Col2a1 mRNA was expressed and expression continued to increase for the duration of the culture period. When the AT-rich element or the C3 motif of the Col2a1 enhancer was deleted to generate the OS-35 and OS-80 constructs respectively, expression of CAT activity increased slightly over time (Fig. 4). However, even in the differentiated state, CAT activity in OS-35 and OS-80 transfected cells was less than 20 counts/min/h/µg protein and remained 4-6-fold lower than the activity from OS-30-transfected cells. Thus, these results suggest that Col2a1 enhancer elements located between 311-331 bp (AT-rich element) and 390-398 bp (C3 motif) are responsive to factors expressed as the cells progress through the developmental transition from a prechondrocytic (CFK2) to a chondrocytic phenotype (dCFK2) in vitro.
Figure 4: CAT activity in stably transfected CFK2 cells. CAT activity was measured in differentiating CFK2 cells carrying Col2a1-CAT constructs pIE55D, Os-30, OS-35, and OS-80. Stably transfected CFK2 cells were plated in six-well dishes and were cultured for up to 28 days. During this extended culture period, CFK2 cells undergo a developmental maturation to a chondrocytic phenotype in vitro. Each value is the mean ± the standard error of the mean (error bars) for six samples.
Figure 5:
Levels of endogenous Col2a1 mRNA in cultured CFK2 cells. Total RNA extracted from cultured
CFK2 cells was subjected to Northern blot analysis. 10 µg of total
RNA were separated on a 1% agarose-formaldehyde gel and transferred to
a nylon membrane using capillary action. Filters were hybridized with a P-labeled probe corresponding to the carboxyl-terminal
procollagen cDNA. The lower panel shows ethidium bromide
staining of 28 S and 18 S ribosomal RNAs.
Figure 6:
EMSA shows cell type-specific complex
formation between the Col2a1 enhancer fragments and nuclear
factors. A, the P-labeled probe A(311-340)
was incubated with nuclear extracts (3 µg) obtained from chick
embryo chondrocytes (CEC, lanes 2, 3, and 4), human tongue fibroblasts (HTF, lane 5),
osteoblastic MC3T3-E1 cells (OB, lane 6), and human
epithelial tumor cells (HeLa, lane 7). Competition
studies were performed with 100-fold molar excess of either the A (lane 3) or B probe (lane 4). The filled
arrowheads mark the cell type-specific DNA-protein complexes whose
formation can be abrogated by the addition of unlabeled probe A, but
are unaffected by nonspecific competition with the B probe. No specific
DNA-protein complexes are formed with nuclear extracts from
nonchondrogenic cells. Open arrowheads represents DNA-protein
complexes that are not cell type-specific. B, EMSA of a
P- labeled probe A and nuclear extracts from CEC. Lane
1, free probe; lane 2, probe A; lane 3, mutant
probe A containing nucleotide substitutions within the AT-rich
element. C, DNA-protein complex formation and competition with
B fragment (375-410 bp) and nuclear extracts from CEC (lanes
1, 2, and 3) and HeLa cells (lanes 4, 5, and 6).
In the present study, we have defined a 100-bp segment within the first intron of the Col2a1 gene as the minimum size for cell type-specific enhancer activity. Further, several core enhancer elements were identified within this 100-bp segment. We have used two different, but complementary, transfection strategies to map the minimal chondrocyte-specific enhancer of the Col2a1 gene. Transient transfection of CEC with constructs containing various deletion mutants of the enhancer and 310 bp of the Col2a1 promoter revealed that first intron sequences from 311 to 410 were required for enhancer activity. To confirm the deletion mutation analysis in transiently transfected CEC, we tested a limited number of Col2a1-CAT constructs in stably transfected CFK2 cells. Extended culture of CFK2 cells results in an increase in phenotypic markers of a differentiated chondrocyte including the appearance of glycosaminoglycans in discrete nodules and increases in mRNA levels for type II collagen, link protein, and aggrecan(24) . Therefore, CFK2 cells provide a differentiating cell culture model in which Col2a1-CAT expression can be studied. The pattern of CAT expression in stably transfected CFK2 cells complements the results in transient transfection experiments in CEC and confirms the requirement for two regions within the first intron for cell differentiation-dependent expression of Col2a1. Moreover, these studies also displayed the differential use of Col2a1 enhancer elements during the course of development to a chondrocytic phenotype. During the initial cell culture period (7 days), all four Col2a1-CAT constructs expressed low CAT activity in CFK2 cells. The low level of CAT activity after 7 days in culture was consistent with the low to undetectable levels of endogenous Col2a1 mRNA expressed after a similar time in culture. However, as CFK2 cells differentiated into dCFK2 cells, primary enhancer activity was observed in the pIE55D and OS-30 constructs. CAT activity also increased in the OS-35 and OS-80 transfected cells during extended cell culture, but the activity from these deletion mutants was low and remained between 4- and 6-fold less than that expressed by OS-30-transfected cells. The observation that CAT activity from the OS-30 construct was similar to the activity expressed from the much larger pIE55D construct, supports the notion that the DNA elements within the 100-bp enhancer contain all of the elements essential for enhancer mediated-expression of Col2a1.
Sequence analysis revealed that the enhancer segment contains several motifs that are similar to sequences also found in the regulatory region of the promoter for the link protein gene. These include an AT-rich element, and C1 and C3 motifs. An identical 9-bp AT-rich element is present at -906 to -914 in the minus strand of the link protein gene(25) . A comparable AT-rich element is also found in the promoter of the growth hormone (26) and the muscle creatine kinase genes (27) (Fig. 7). Deletion of the AT-rich element from the Col2a1 enhancer significantly reduced enhancer activity. Furthermore, mobility shift assays suggest that the Col2a1 AT-rich element binds nuclear factors present in CEC. A similar reduction in the promoter activity of the link protein gene was found when the AT-rich element was deleted or mutated by base substitutions(25) . Thus, the AT-rich element is likely to be functionally important for transcriptional regulation of both the Col2a1 and link protein genes. It has been shown that Mhox, a mesodermally restricted homeodomain protein, and myocyte enhancer factor-2, binds to an AT-rich element in the muscle creatine kinase enhancer. Binding of myocyte enhancer factor-2 to the site in the muscle creatine kinase gene is required for enhancer activity in muscle cells(27) . Equivalent factors, specific for chondrocytes, may bind to the AT-rich element of the Col2a1 and link protein genes. It is interesting to note that the C1 and C3 motifs are also present in the regulatory region of the link protein gene. These elements appear to be important for transcription of the link protein gene since deletions of these elements reduce promoter activity in the link protein gene(25) . The two C2 motifs that are located in an inverted orientation in the Col2a1 enhancer are not found in the link protein promoter. The rat sequence of this 100-bp enhancer region is highly conserved with both mouse and human(28, 29) . The rat sequence shared a 98 and 92% identity with the mouse and human sequences, respectively. All motifs identified in this study are perfectly conserved, suggesting that these motifs may play significant roles in the type II collagen and link protein genes.
Figure 7: Comparison of the AT-rich elements of the Col2a1 enhancer with those in regulatory regions of other genes. Sources of sequences are: link protein(25) , growth hormone (26) , and muscle creatine kinase(27) . Shaded box denotes nucleotide identity. r, rat; m, mouse. Numbers indicate nucleotide positions.
Initiation of transcription involves complex interactions between nuclear proteins and cis-acting elements located in the promoter and enhancer regions(30) . It is becoming increasingly appreciated that these protein-DNA interactions occur at multiple sites in a given gene and may involve the interaction of accessory factors that may not directly bind DNA(31, 32, 33) . Several published studies show that the Col2a1 gene shares this complex transcriptional regulation. Transcriptional activity has been identified in sequences within the 5`-flanking sequences and the first intron of the Col2a1 gene(15, 17, 18) . Together, our findings, as well as previously published data, suggest that multiple functional elements are found within a relatively narrow region of the first intron. The function of discrete elements may depend on culture conditions or the assay system used. Alternatively, multiple DNA-binding proteins may be functioning through these elements. Our demonstration of enhancer activity in both transient and stable transfections strengthen the notion that the minimum 100-bp enhancer is functional. Since there are limitations of the interpretations of the results obtained in vitro, an in vivo assay using a transgenic mouse model will be help clarify this complex transcriptional regulation.
The use of multiple regulatory sites in transcriptional regulation is recognized for many genes. Because our current knowledge of possible cooperative or synergistic interactions in this region of the enhancer remains incomplete, the control of Col2a1 transcription in chondrocytes will likely involve other regulatory elements in addition to the regions described here. However, since we define an enhancer region that is operational in both transiently transfected chondrocytes and stably transfected CFK2 cells in which the transgenes have been incorporated into chromatin, we believe that this provides evidence for a minimal enhancer sequence required for cell type-specific expression of Col2a1. Because transfection experiments do not always define which regulatory elements will be functional in vivo(34, 35) , the regulatory sites identified in this study will be used to generate transgenic mice in order to study regulation of Col2a1 in the context of a developing animal.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L48618 [GenBank]