cis Elements That Control the Expression of Chick Aggrecan*

Edward W. Pirok IIIDagger §, Judith HenryDagger , and Nancy B. SchwartzDagger ||

From the Departments of Dagger  Pediatrics, § Pathology, and  Biochemistry and Molecular Biology, the University of Chicago, Chicago, Illinois 60637

Received for publication, November 1, 2000, and in revised form, February 2, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Aggrecan is a large chondroitin sulfate proteoglycan whose expression is both cell-specific and developmentally regulated. Cloning and sequencing of the 1.8-kilobase genomic 5'-flanking sequence of the chick aggrecan gene revealed the presence of potential tissue-specific control elements including a consensus sequence found in the cartilage-associated silencers, CSIIS1 and CSIIS2, that were first characterized in the type II collagen promoter sequences, as well as numerous other cis elements. Transient transfections of chick sternal chondrocytes and fibroblasts with reporter plasmids bearing progressively deleted portions of the chick aggrecan promoter and enhancer region demonstrated cell type-specific promoter activity and identified a 420-base pair region in the genomic 5-flanking region responsible for negative regulation of the aggrecan gene. In this report, three complementary methods, DNase I footprinting assays, transient transfections, and electrophoretic mobility shift assays (EMSA), provided an integral approach to better understand the regulation of the aggrecan gene. DNase I footprinting revealed that six regions of this genomic sequence bind to nuclear proteins in a tissue-specific manner. Transient transfection of reporter constructs bearing ablations of these protected sequences showed that four of the six protected sequences, which contain the sequence TCCTCC or TCCCCT, had repressor activities in transfected chick chondrocytes. Cross-competition EMSA using nuclear protein extracted from chondrocytes or fibroblasts explored the contributions of the different sequence elements in formation of DNA-protein complexes specific to cell type. This is the first parallel examination of the EMSA patterns for six functionally defined cis elements with highly similar sequences, using protein from primary cultured cells.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Aggrecan is a large chondroitin sulfate proteoglycan found predominantly in cartilage that is essential for maintaining the integrity of this tissue. Because of its ability to bind to hyaluronate and link protein, aggrecan forms large space-filling aggregates in the extracellular matrix produced by chondrocytes, providing the resilient and compressible properties of cartilage (1, 2). Aggrecan consists of a 225-250-kDa core protein with three globular domains and two linear domains to which about 100 chondroitin sulfate chains (CS)1 and 25-30 keratan sulfate chains covalently bind (3). The genomic structure of the chick aggrecan gene consists of 18 exons; multiple exons encode the three globular domains, G1, G2, and G3, the latter being composed of epidermal growth factor, lectin, and complement regulatory protein-like domains (4, 5).

A mutation in the CS exon of the chick gene that produces a premature stop codon results in a shortened and nonfunctional protein that leads to the lethal chondrodystrophy nanomelia, the phenotype of which is most notably characterized by shortened limbs (4, 6). A mutation in the mouse aggrecan gene (cmd, cartilage matrix-deficient) produces shortened limbs in utero, cleft palate, and death shortly after birth (6-8). Furthermore, aggrecan is important for maintenance of the cartilage phenotype later in life. Aggrecan degradation, concomitant with matrix destruction, is the hallmark of osteoarthritis and rheumatoid arthritis, in which aggrecan catabolism is elevated compared with normal articular cartilage (9). Therefore, a precise understanding of the regulation of aggrecan expression is critical to investigating the mechanisms of normal development and of diseases that involve abnormalities of the extracellular matrix.

The dynamics of transcriptional control of gene expression are complex and intriguing, such that it is often the context of transcription factors and DNA elements that determines the functionality of a regulatory region. It has been proposed that some transcription factors may have multiple domains, some specific for repression and others for activation (10). Protein-protein interactions can influence the basal DNA-protein interactions observed in a regulatory region. Thus formation of protein complexes and how they interact with DNA may influence the transcriptional machinery that enables specific expression of genes at a precise time and in response to appropriate stimuli (11). An emerging paradigm is that there are a variety of mechanisms by which individual cis elements interact with trans factors. The initial interaction with a cis element may be a site of nucleation to which a series of other proteins are recruited, and it may then be this complex, rather than a single factor, that interacts with the RNA polymerase machinery. In fact, the DNA may act as a tether to enable localized and distal interactions between transcription factors.

Recently, we reported (12) the cloning and sequencing of a 1.8-kilobase genomic fragment containing the 5'-flanking sequence of the chick aggrecan gene. By using transient transfections of chick sternal chondrocytes and fibroblasts with reporter plasmids bearing progressively deleted portions of the chick aggrecan promoter and enhancer region, we demonstrated tissue-specific promoter activity and identified a 420-base pair region in the genomic 5'-flanking sequence responsible for negative regulation of the aggrecan gene. Analysis of this nucleotide sequence revealed the presence of potential tissue-specific control elements including a consensus sequence found in the cartilage-associated silencers, CSIIS1 and CSIIS2, that were first characterized in the type II collagen promoter sequences, as well as numerous other potential cis elements (13).

To continue our analysis of the chick aggrecan promoter and enhancer region, we have conducted DNase I footprinting assays, transient transfections, and electrophoretic mobility shift assays (EMSA) with the 420-base pair region previously found to confer repressor activity and with native and mutated sub-sequences drawn from that region. These three complementary methodologies provide an integrated approach to understand better the regulation of the aggrecan gene, allowing us to determine the nature and extent of some actual protein-DNA interactions and clarify which cis elements, of the many predicted, were responsible for our previous findings. By using sequences characterized in these studies, we later assayed for the binding of previously defined transcription factors and characterized novel tissue-specific binding sites that eventually led to the identification of a novel factor that binds to the aggrecan promoter and enhancer region.2

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Oligonucleotides were made with an Applied Biosystems 3808 DNA Synthesizer. Reagents for biochemical and molecular cloning experiments were of the highest quality available from commercial vendors. Restriction endonucleases were from New England Biolabs unless otherwise stated. T4 DNA ligase, T4 kinase, DNase I, and Klenow polymerase were from Promega. Taq polymerase was from PerkinElmer Life Sciences.

Mutagenesis-- The Altered Sites II kit from Promega was employed for oligonucleotide-mediated mutagenesis strategies, following the manufacturer's protocol. The Ag-1(+) genomic clone was used as a template sequence for the oligonucleotide-mediated alterations of cis elements (12). Briefly, the Ag-1(+) clone was denatured and annealed with a mutant oligonucleotide, an ampicillin-repair oligonucleotide, and a tetracycline-knockout oligonucleotide. Subsequent synthesis, ligation, and selection for ampicillin resistance were used to identify mutant constructs. Positive mutants were sequenced to ensure that the correct mutation was made. The oligonucleotides used to make the mutant constructs are displayed in Table I.

Cell Cultures-- Cultures of day-14 chick sternal chondrocytes were established according to the procedures described by Cahn et al. (14) and as modified by Campbell and Schwartz (15). Cultures of fibroblasts were established from skin of day-14 chick embryos following trypsinization (15). Cells were plated at an initial density of 1.5 × 106/100-mm tissue culture dish (Falcon) in either F-12 medium (chondrocytes) or Dulbecco's modified Eagle's medium (fibroblasts) and supplemented with 10% fetal calf serum and 10 µg/ml ascorbic acid (chondrocytes). The cells were permitted to attach to the dishes, and subsequent growth (2-3 days) was maintained by a complete change of the medium every 2 days (16). On the day of transfection chondrocyte cultures were trypsinized, and single cells were suspended in F-12 medium, replated, and allowed to attach to the dishes for 3-4 h before treatment as described below.

Purification of Nuclear Proteins-- Standard protocols were used to purify nuclear proteins from either chick chondrocytes or fibroblasts. Briefly, cells from confluent monolayer or suspension cultures were scraped and collected into 50-ml conical tubes (Falcon). The cells were pelleted for 10 min in a JS-4.2 rotor at 3000 rpm, resuspended in five times the packed cell volume of phosphate-buffered saline (usually 15 ml), and then pelleted for another 5 min at 3000 rpm. The phosphate-buffered saline was poured off, and the pellets were resuspended in a hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.2 mM PMSF, 0.5 mM DTT) and allowed to swell for 10 min. The cells were lysed by Dounce homogenization, and the lysate was centrifuged at 3300 × g for 15 min. The cytoplasmic supernatant was discarded, and the nuclear pellet was suspended in a low salt buffer (20 mM HEPES, pH 7.9, 0.02 M KCl, 1.5 mM MgCl2, 25% glycerol, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT); nuclear proteins were then extracted in high salt buffer (20 mM HEPES, pH 7.9, 1.2 M KCl, 1.5 mM MgCl2, 25% glycerol, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT) for 30 min. The solution was centrifuged at 25,000 × g, and the supernatant was dialyzed at 4 °C against dialysis buffer (20 mM HEPES, pH 7.9, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT). The nuclear extracts were used immediately for DNase I footprinting experiments, gel shift assays, or further purification procedures.

Transfection-- Standard methods were followed for calcium phosphate transient transfections (17). Duplicate plates containing ~5 × 106 cells (either chondrocytes or fibroblasts) received 20 pmol of a given plasmid construct to be assayed, and incubation continued for 36 h. Five µg of a beta -galactosidase reporter plasmid were co-transfected with each experimental construct to correct for cell loss. Duplicate transfection sets were prepared three times, each time yielding similar results.

Cell Recovery and Assays-- Reagents for the luciferase and beta -galactosidase assays were purchased from Promega. Since both luciferase assays and beta -galactosidase assays were performed, Reporter Lysis Buffer from Promega was used (RBL, E3971) to prevent the inhibition of beta -galactosidase activity that occurs in buffers containing detergents such as Triton X-100. No deviations were made from the manufacturer's protocols for preparation of extracts from tissue culture cells and the enzyme activity assays. Three sample aliquots from each of the duplicate transfection plates were assayed. The enzymatic activity of luciferase was measured with a luminometer (Analytical Luminescence Laboratory, Monolight 1500). The enzymatic activity for beta -galactosidase was measured with a microplate reader (Dynatech) at 409 nm. Average luminescence values and standard deviations were determined for the set of six assays performed within each experimental group.

Preparation of DNA Probes for DNase I Footprinting-- Probes used for DNase I footprinting experiments were generated via PCR using the previously described genomic clone Ag-1(+) as template DNA, Fig. 1 (12). For probes 1 and 2, XhoI sites were introduced at the 5'-end of the amplified fragment, whereas BglII sites were introduced via the primers at the 3'-end. The defined 5'-3' orientation of the cloned PCR fragments corresponds to the native 5'-3' orientation of the Ag-1(+) clone sequence relative to the aggrecan gene. PCR fragments were purified using Qiaquick PCR Preps (Qiagen). Samples of purified and unlabeled PCR fragments were electrophoresed on agarose gels to determine sizes, and sequencing of the PCR products was done to exclude PCR artifacts in the probes. Approximately 0.3 µg of a given probe was digested overnight at 37 °C with the restriction enzyme BglII and then treated with 0.5 units of alkaline phosphatase. The digested PCR products were again purified using a Qiaquick Nucleotide Removal Kit (Qiagen). The purified double-stranded end-digested probes were end-labeled using [gamma -32P]ATP and T4 DNA kinase according to standard protocols. The previously undigested end of each probe was digested for 2 h at 37 °C using the appropriate restriction enzyme (XhoI) to ensure that only one end of the probe contained a radiolabeled phosphate. The probes were then extracted twice with phenol/chloroform, precipitated using standard protocols (12), and then stored at 4 °C in TE8 buffer (10 mM Tris, 1 mM EDTA, pH 8).

DNase I Footprinting-- For each footprinting experiment ~0.015 µg of end-labeled probe was added to 25 µl of binding buffer A (10 mM Tris, pH 8, 5 mM MgCl2, 1 mM CaCl2, 2 mM DTT, 50 µg/ml bovine serum albumin, 2 µg/ml calf thymus DNA, 100 mM KCl) with the addition of 25 µg of nuclear protein from either chick chondrocytes or fibroblasts in 25 µl of dialysis buffer. Control samples that had no protein added included only 25 µl of dialysis buffer. Binding mixtures were kept at room temperature for 90 min. Prior to digestion with DNase I, 50 µl of 5 mM CaCl2, 10 mM MgCl2 was added, and the mixture was incubated for 60 s. Control lanes that contained no nuclear protein were digested with 0.06 units of DNase I, and lanes with nuclear protein were digested with 0.12 units of DNase I for 90 s at room temperature. The reaction was stopped with 90 µl of 200 mM NaCl, 30 mM EDTA, 1% SDS, and 250 µg/ml yeast RNA. Reactions were extracted with equal volumes of phenol/chloroform and precipitated with 2 volumes of 100% EtOH. Precipitated DNA was washed once with 70% EtOH, and the pellets were resuspended in gel loading buffer. The products were electrophoresed on 6% polyacrylamide sequencing gels. Dideoxynucleotide termination sequencing reactions were done simultaneously for each DNA fragment and run next to the control footprinting reactions to match the observed footprints with the published sequence.

EMSA-- 100 pmol of each single-stranded oligonucleotide complementary pair was incubated in annealing buffer (100 mM Tris, pH 7.5, 500 mM NaCl, and 250 mM MgCl2) at 90 °C for 5 min and then allowed to cool slowly for ~1 h. Standard methods were employed to end label the resulting double-stranded oligonucleotides. Briefly, 10 pmol of probe was incubated for 30 min at 37 °C in kinase buffer (Promega) containing 20 units of T4 kinase, and [gamma -32P]ATP (PerkinElmer Life Sciences). To stop the labeling reaction, 2 µl of 0.5 M EDTA was added to the reaction, and the labeled probes were purified with a Nucleotide Removal Kit (Qiagen) to remove the excess unincorporated [gamma -32P]ATP. For each binding reaction, 20 fmol of the probe were incubated with varying amounts of nuclear protein (between 0.1 and 10 µg) for 30 min at 4 °C in a 15-µl reaction mixture containing 1.5 µl of binding buffer (50 mM Tris, pH 7.5, 5 mM EDTA, 5 mM DTT, 375 mM KCl), 2 µg of dI-dC (Amersham Pharmacia Biotech), 3.75 µl of 20% Ficoll, and water. For competition experiments, excess unlabeled double-stranded probes were added to the binding reaction (amounts ranging from 0.2 to 1.0 pmol, or as defined in the figure legend). The DNA-protein complexes were electrophoresed on 5% polyacrylamide gels in TBE buffer.

Sequence Search for Transcription Factor Binding Sites-- The Wisconsin Package (version 9, Genetics Computer Group, Madison, WI) program FINDPATTERNS was used to scan the protected footprint sequences for cis elements listed in the Transcription Factor Database.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DNase I Footprinting with Probes Spanning the Aggrecan Repressor Region-- Fig. 1 shows a representation of a portion of the 1.8-kb aggrecan gene genomic 5'-flanking sequence. The 420-bp region from -1200 to -780 relative to the most 5' transcription start site was previously shown to play a negative regulatory role (12). This sequence was searched against the Transcription Factor Database using the program FINDPATTERNS, and some of the resulting predicted cis elements embedded in that sequence are represented in the diagram. DNase I footprinting experiments were conducted using two PCR-generated, single-end 32P-labeled probes (Fig. 1), which span 430 bp of sequence and overlap with each other by 18 bp. Each of these probes was incubated with nuclear proteins extracted from primary cultures of either day-14 chick sternal chondrocytes or day-14 chick fibroblasts. Fig. 2, A and B, shows the results of these experiments using probe 1 and probe 2, respectively.


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Fig. 1.   Schematic representation of the 1.8-kb chick aggrecan promoter and enhancer region and DNase I footprinting strategy. The 1.8-kb genomic fragment Ag-1 is shown in relation to a 420-bp promoter sequence involved in the negative regulation of the chick aggrecan gene. Within the box are predicted cis motifs in order as defined using the program FINDPATTERNS or from published papers. The bars below the box represent two of the DNase I footprinting probes used with indication of which end of the probe contained the radiolabeled phosphate. The names and positions of the resulting protected sequences (Fig. 2) are indicated below each probe. Note the diagram is not drawn to scale. An additional protected sequence, L (-730 to -703), was identified using a probe taken from sequence adjacent to probe 2.


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Fig. 2.   Footprinting analysis, sequences, and embedded cis elements. Probes 1 and 2 were used for footprinting analysis in A and B, respectively. Lanes 1 and 3 contained DNA not incubated with nuclear protein. Lanes 2 and 4 contained DNA protected by nuclear protein extracted from either day-14 chick chondrocytes or fibroblasts (lanes 1 and 2 and lanes 3 and 4, respectively). DNase I digestion was performed on all as described under "Experimental Procedures," and the products were electrophoresed on 6% polyacrylamide sequencing gels. C lists the nucleotide sequences protected by day-14 chick sternal chondrocyte or fibroblast extracts. The 1st column shows the protected sequence; the 2nd column lists the designated footprint name; and the 3rd column lists the consensus sequences for potential transcription factor binding sites that were identified using the program FINDPATTERNS or from published reports. The L protected sequence resulting from footprinting with a third probe (not shown) is included.

Probe 1 yielded three protected segments with chondrocyte extracts, designated footprints G-I, which spanned the sequences -1054 to -1020, -1015 to -996, and -960 to - 937, respectively (Fig. 2A, lanes 1 and 2). Footprint G was observed with fibroblast extracts (Fig. 2A, lanes 3 and 4), but the resultant protection was less than in parallel experiments utilizing chondrocyte protein (Fig. 2A, lanes 1 and 2). Experiments using fibroblast extracts yielded no observable footprint in the chondrocyte-protein-protected sequence H under four different buffer conditions, as well as at other DNase I concentrations (data not shown). Finally, fibroblast extracts produced footprint I, with protection comparable to that obtained with chondrocyte extract.

Two major segments of probe 2 were protected by fibroblast extracts, spanning the regions -878 to -831 and -808 to -791 (Fig. 2B, lanes 3 and 4); these protected sequences were designated as footprints J and K, respectively. One protected segment, footprint K, had greater DNase I protection in its 5' portion using fibroblast versus chondrocyte extracts under otherwise identical conditions (Fig. 2B, lanes 1 and 2). The upper region of footprint J varied slightly; the protected region designated as J observed for fibroblast extracts was replaced by two smaller non-overlapping footprints that spanned the same overall region when chondrocyte nuclear protein was used. Similarly, another footprint, L (Fig. 2C), was obtained using a third probe from sequence just 3' of the probe 2, from -730 to -703 (data not shown). This segment was protected by both chondrocyte and fibroblast nuclear protein.

Comparison of the protected sequences with the Transcription Factor Database search results determined that a number of previously defined cis element consensus sequences were contained within the observed footprint regions (Fig. 2C). Footprint G included MalT_CS, H2A, P5, Aldosterone_CAP_box, D4(rev), and D1 sequences. Footprint H had an SP1_CS4 sequence in the reverse orientation. Footprint I contained a Zeste sequence, also in the reverse orientation. The large footprint J is composed of multiple sites, most importantly a CIIS2- (CACCTCC) containing sequence in addition to a MalT_malPp (TCCTCC) and a CK-8-mer sequence. Finally, footprint K contained a second MalT-malPp sequence.

Sequence comparison among the protected sequences revealed several common motifs largely composed of various combinations of the bases thymine and cytosine. Both footprints J and K contain the sequence CTCCTCC (which includes the above-mentioned MalT-malPp site), and footprint J had two repeats of the sequence TCCCC, which occurs once in the footprint L sequence. Furthermore, J contained a CTTCAC sequence, whereas L contained the very similar sequence, CTTCAG, and both contained a CACCTCC sequence.

Functional Analysis via Mutagenesis of the Aggrecan Promoter Repressor Region-- The extent to which these protected sequences contribute to the previously reported repressor activity of this promoter segment was addressed by introducing mutations into the native 1.8-kb Ag-1(+) sequence, which preserved the nucleotide spacing but altered the bases putatively involved in nuclear protein binding. Alternating series of either As and Ts (poly(dA)·poly(dT)) or Cs and Gs (poly(dC)·poly(dG)) were substituted for the normal footprint sequences (Table I). Sternal chondrocytes and fibroblasts were transfected with these mutant sequences in reporter plasmid constructs; initial experiments suggested that the simple introduction of either a series of As and Ts or a series of Cs and Gs did not alter activity in itself nor did it matter which substitution sequence was used (data not shown). These experiments included use of either poly(A-T) or poly(G-C) at a given protected sequence mutation site with highly similar results. Use of these sequence blocs to mutate other locations in Ag-1(+) produced either the opposite or no effect on reporter expression. Finally, in the results described below, it was found that a given mutational sequence produced different effects depending on which protected sequence was mutated. In sum, effects of mutations correlated with their location and not with the type of sequence bloc used.

                              
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Table I
Oligonucleotides used to form mutant constructs for transfection experiments

Since the single, large protected sequence observed for footprint J with fibroblast extract appeared as two discrete regions when protected by chick chondrocyte protein, two mutations were made in the J sequence, mJ.2 that altered the nucleotides -878 to -86, and mJ.1 that altered the sequence spanning -860 to -830. The mutant construct mJ.2 ablated the CIIS2-containing sequence and the MalT_malPp consensus sequence, whereas the mutant construct mJ.1 removed only the CK-8-mer site. There is a high degree of similarity, and a 3-base overlap, between the CIIS2 and MalT_malPp sequences in footprint J. To evaluate these elements separately, we mutated the sequence that corresponds to footprint K, which also contained a consensus sequence for a MalT_malPp site but not the very similar CACCTCC sequence. A second observed CIIS2 sequence, located in footprint L, was mutated to determine to what extent that sequence alone was affecting activity, thus mK ablated a MalT_malPp site, mL removed a CIIS2 sequence, and mJ.2 mutated the composite sequence CACCTCCTCC.

Transient transfections of chondrocytes or fibroblasts with the construct Ag-1(+) (the forward orientation of the 1.8-kb insert in the promoter/enhancer-free pGL-2-Basic reporter vector) were compared with Ag-1(+) constructs bearing the various footprint mutations. Mutations of the sequence described as footprint G had little effect on reporter expression in transfected chondrocytes when compared with the normal Ag-1(+) construct (Fig. 3A); however, construct mG produced a 30% increase in luciferase activity compared with that for Ag-1(+) in transfected chick fibroblasts (Fig. 3B). Similarly, mutation of protected sequence H, which had an Sp1_CS4 site in the reverse orientation, had little effect in transfected chondrocytes, whereas reporter activity in transfected fibroblasts dropped nearly 25% when construct mH was compared with the normal Ag-1(+) construct. Disruption of the protected sequence I, which contains a Zeste-like sequence, caused a significant increase in reporter activity in both chondrocytes and fibroblasts, to nearly 400 and 175%, respectively. Alteration of the large protected sequence J caused substantial increases in luciferase activity in both transfected chondrocytes and fibroblasts. The construct mJ.2, which mutated the overlapping CIIS2 and MalT-maLPp sites, exhibited the highest increases in relative reporter activity, i.e. transfected chondrocytes showed an increase in luciferase activity of nearly 600%, and luciferase activity in transfected fibroblasts peaked at 230%. Removal of the CK-8-mer site, in construct mJ.1, increased relative reporter activity by nearly 400% in transfected chondrocytes but only 35% in transfected fibroblasts. Interestingly, both constructs mK and mL, which ablated separate MalT-malPp and CIIS2 sites, respectively, produced large reporter activity increases in both cell types, but neither of these equaled the activity increases for the mutant construct mJ.2 which eliminated overlapping sequences of each cis element. Construct mL exhibited greater activity than mK in transfected fibroblasts; this reporter activity was similar to that observed for the mutant construct mJ.2 in fibroblasts. Whereas mL exhibited less activity than mK in transfected chondrocytes, in both cell types alteration of the sequence CACCTCC (mL) caused a significant increase in luciferase activity, suggesting the presence of repressor, silencer, or modulation complexes acting at that site alone.


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Fig. 3.   Effects of footprint sequence mutations on Ag-1(+) promoter activity. This figure shows the relative luciferase activities resulting from transfections with the Ag-1(+) construct and mutated forms of Ag-1(+) produced with the oligonucleotides described under "Experimental Procedures." Ag-1(+) is the 1.8-kilobase promoter/enhancer region from the aggrecan gene placed in the reporter vector pGL2-Basic. Duplicate plates were transfected, and each plate was assayed for luciferase activity three times. An average value and standard deviation were determined for all six assays at each data point. Results were normalized by co-transfection of 5 µg of a beta -galactosidase reporter gene (Promega). Both day-14 chick sternal chondrocytes (A) and day-14 chick fibroblasts (B) were transfected. At the time of transfection cell density per dish was ~5 million. The transfection was allowed to proceed for 36 h.

Examination of DNA-Protein Interactions by EMSA-- To explore further the nature and extent of the DNA-protein interactions at the observed protected sequences in chick chondrocytes, double-stranded probes corresponding to these sequences were made for use in EMSA experiments. Table II shows the sequences, names, and sizes of the eight oligonucleotide pairs used in combination with their respective complements to form double-stranded 32P-labeled probes that correspond to the repressor region footprint sequences. Fig. 4A shows the results of incubating five of those probes with nuclear protein extracts derived from day-14 sternal chondrocytes seeking to determine if there are any similarities in the DNA-protein complexes formed. The 48-base pair probe J, which contains CIIS2, MalT_malPp, CTTCAC, and two TTCCCC sequences, yielded three specific DNA-protein complexes, labeled B-D, although production of complex B appeared variable for the full-length probe J in repeated experiments. The 30-base pair probe J.1 produced the same DNA-protein band patterns as the full-length probe J, although lacking the CIIS2 and MalT_malPp sequences. It did, however, contain the sequences CTTCAC and TTCCCC, in addition to a CK-8 site. Probe J.2 contains three of the shared sequences, CIIS2, TTCCCC, and a CTCCTCC, yet it only forms two complexes, A and C, when incubated with chick sternal chondrocyte extract. EMSA with probe K, which contains a CTCCTCC sequence, produced only the prominent band A, which co-migrates with the slower migrating complex observed with probe J.2. The only similarity between probes K and probe J.2 is the presence of a CTCCTCC sequence, implying band A can be attributed to nuclear proteins binding to that sequence. Probe L produces two bands, B and C. Sequence comparison between L and J.1, which produces those same EMSA bands, reveals the presence of a CTTCAG and a TCCCC sequence in both. Band C is also present in reactions incubated with probe J.2, which contains a TCCCC but not a CTTCAG sequence, suggesting that the TCCCC motif produces the DNA-protein complex C. Surprisingly, probes J.2 and L, which both contain the CIIS2 site, do not generate a unique band that can be definitively associated with this sequence alone. Indeed they both produce band C but that is also present for probe J.1, which does not contain the CIIS2 site. In sum, both probe K and J.2 contain the sequence CTCCTCC and produce band A. Probes J, J.1, J.2, and L produce band C, and all contain the sequence TCCCC. Probes J, J.1, and L share the sequence CTTCA(G/C), and each produces the band B. The D complex was only seen for probes J and J.1 suggesting that it is unique to this larger sequence that is not included in the other probes.

                              
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Table II
Nucleotide sequences used to form double-stranded EMSA probes


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Fig. 4.   EMSA using probes J, J.1, J.2, K, L, G, H, and I. A shows the results for probes J, J.1, J.2, K, and L (Table II) incubated with nuclear extracts from day-14 chick chondrocytes. Bands that appear to be affected by protein concentration and addition of excess of cold probe in other experiments are labeled A---D, and the free probe position is marked. B shows the bands resulting when probes G, H, and I (Table II) were incubated with nuclear extracts from day-14 chick chondrocytes. The major DNA-protein complexes are labeled 1-3.

Probes that corresponded to the protected sequences G-I were also synthesized; Fig. 4B represents the EMSA patterns that are observed when these probes bind with day-14 chick sternal chondrocyte nuclear proteins. Incubation with probe G produced three specific complexes (labeled 1-3) that do not align with any other observed bands, suggesting that these DNA-protein complexes are unique. Probe H produced one band that migrated between the complexes 1 and 2 observed for probe G. Probe I produced one specific band that similarly migrated between the complexes 1 and 2 observed with probe G. None of the 5 complexes from probes G-I aligned with bands B-D observed for probe J, nor did they co-migrate with band A produced by probes J.2 and K (data not shown). Sites H and I contain only one predicted cis element each, a Sp1(rev) site and a Zeste (rev) site, respectively. Thus, it is not surprising that each produces only one specific complex. Footprint G contained 6 potential cis elements yet generated only 3 specific complexes.

Because all of the EMSA experiments described above were done with one concentration of nuclear protein, and protein-DNA interactions can be affected by relative concentrations of both the DNA and protein, varying amounts of nuclear protein were added to each of the probes to determine if any other complex formation could occur. Indeed, nuclear extracts may contain a limiting amount of cofactors needed to allow proper protein-protein interactions to form complexes that then jointly bind to the DNA elements. The footprint sequences, H, I, J, J.1, J.2, and K, did not exhibit significant differences in band formation upon addition of excess nuclear protein, except for the expected increase of band intensities (data not shown). However, addition of varying amounts of nuclear protein to probes G and L had significant effects on complex formation by these two probes.

For probe G, 3 bands were observed that increased in intensity in a dose-dependent manner and that could be competed out with excess unlabeled double-stranded probe G (Fig. 5A). Bands 1 and 3 appear with chondrocyte extract at protein concentrations of 1 µg per reaction. Band 2 did not appear distinctly until higher concentrations of nuclear protein were added. Band 3 was of lower intensity (or more diffuse) than band 1 at low concentrations, but the converse was observed at higher concentrations of proteins (compare lanes 1 and 3). Band 3 was the most difficult to compete out with excess unlabeled probe (persisting with a 100-fold excess), whereas bands 1 and 2 competed out at a 25-fold excess. A faster migrating band was present in all of the samples and was not dependent on protein concentration or amount of unlabeled probe, suggesting that it represents nonspecific binding. A protein dose-dependent change in complex formation was also seen for probe L (Fig. 5B). With increasing concentrations of chick chondrocyte nuclear protein, a complex appeared that migrated slower than band B (labeled with an asterisk). Concomitant with the appearance of this upper band, there is a visible decrease in the amounts of complexes B and C. The new band was distinct from band A, seen in the probe K lane.


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Fig. 5.   Dose dependence of probe G and L EMSA bands. A shows EMSA results for probe G (Table II) incubated with various amounts of chick chondrocyte nuclear protein. Reactions for lanes 1-3 contained 1, 5, and 10 µg of nuclear protein, whereas the lane 4 and 5 reactions contained 5 µg of nuclear protein with the addition of increasing concentrations of excess unlabeled probe G. Bands that appear to be affected by protein concentration are labeled, and the free probe location is indicated. B shows the EMSA results for probe L (Table II) incubated with various amounts of chick chondrocyte nuclear extract. Lane K is a reference lane with probe K to denote the position of complex A. The lanes L5, L10, and L20 reactions contained 5, 10, and 20 µg of protein, respectively. Bands that appear to be affected by protein concentration are labeled.

Different cis Elements with Similar Sequences May Interact with the Same Trans Factor(s) to Cause Repression-- Additional competition-EMSA studies were carried out with chondrocyte extracts and radiolabeled probes J.1, J.2, K, and L (20 fmol), using as unlabeled competitor the same probes at either 10- or 50-fold (200 fmol or 1 pmol) molar excess. Radiolabeled probe J.1 (Fig. 6A) incubated with 5 µg of chondrocyte nuclear protein yielded three complexes. As expected, the formation of complexes B-D was competed against by unlabeled probe J.1 at 50-fold molar excess. Complex B could not be competed for by unlabeled probes J.2 and K at 10-fold molar excess. Surprisingly, with 50-fold molar excess of unlabeled probes J.2 or K, which contain the MalT-malPp site, complex B increased in intensity concomitant with a reduction of complex C. Probe L was the only oligonucleotide sequence that competed for complex B and C formation at both 10- and 50-fold. No competition was observed with a 50-fold molar excess of the TA oligonucleotide.


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Fig. 6.   Competition EMSA analyses with probes J.1, J.2, K, and L. A-D display the results of incubating 20 fmol of 32P-labeled probes J.1-L (Table II) with 5 µg of day-14 chick chondrocyte nuclear extract, either alone or with excess unlabeled competitor probes. For each panel, the control (C) lane reaction contained only the labeled probe for that panel. Pairs of lanes designated with the same probe name were from reactions that contained 200 fmol (left) or 1 pmol (right) of that probe as unlabeled competitor. Lanes marked TA contained reactions with 1 pmol of unlabeled TA probe (Table II) as nonspecific DNA competitor.

The competition EMSA results suggest that probe L has a greater affinity for complexes B and C than any of the other probes, as it competed for these complexes more strongly than the native J.1 sequence. Probe J.2 competed only for complex C; the sole similarity it has with probe J.1 is the presence of a TCCCC sequence. This finding supports the previous EMSA outcome that the TCCCC sequence is required to form the DNA-protein interactions resulting in band C. Since band B increased in intensity with the addition of probes J.2 and K, whereas band C weakened in intensity, it may be that, when in excess, these probes disrupted the dynamics of the normal J.1 DNA-protein interactions. Both probes J.2 and K contain the sequence TCCTCC, which bears similarity to TCCCC. Perhaps TCCTCC has a stronger affinity for some component of complex C than does the TCCCC sequence, hence the loss of band C with the addition of probes J.2 and K.

Probe J.2 (Fig. 6B) yielded two bands, complexes A and C. As expected, complex C formation was competed for by the addition of 10- and 50-fold excess probe J.1, but complex A was more difficult to compete out at either concentration than complex C. Addition of J.2 at 10-fold molar excess diminished band C, whereas 50-fold excess seemed to somewhat diminish the intensity of A and further reduce band C. Probe K at 10-fold molar excess slightly competed for band C, and a higher molar excess (50-fold) nearly ablated it. Complex A, however, was only slightly reduced by the addition of 50-fold unlabeled probe K. Thus in two separate instances, for labeled probes J.1 and J.2, addition of K competitor reduces the intensity of complex C permitting formation of the slower migrating complexes A (probe J.2, Fig. 6B) or B (probe J.1, Fig. 6A). These results further support the notion that the DNA sequence TCCTCC competes for a complex C component.

Excess unlabeled probe L competed with J.2 for formation of both DNA-protein complexes A and C, which suggests that complex A contains a protein that can bind to the TCCCC sequence or another consensus sequence embedded in probe L. However, probe L does not form complex A by itself so probe L may not be acting directly on complex A, rather it may compete away a protein common to both complexes A and C making the formation of complex A impossible. Addition of the TA probe did not change the intensity of the complexes when compared with control lane C, Fig. 6B.

Probe K incubated with 10 µg of nuclear protein (Fig. 6C) yielded one major complex, A. Unlabeled J.1, J.2, or L probes at 50-fold molar excess competed for the formation of this complex. Again, probe L competed for this complex more strongly than the other probes, exhibiting competition at 10-fold molar excess. In fact, probe L competed for band A at both excess levels more strongly than did the unlabeled probe K. The TA probe did not compete with probe K formation of band A.

Radiolabeled probe L (Fig. 6D) produced two complexes, bands B and C. Even at high molar excess, probes J.1, J.2, and K could not compete for either complex formed by probe L. The bands were competed for by unlabeled native sequence L at 10- and 50-fold excess. Interestingly, the upper band B was competed before the lower band C by a 10-fold excess of unlabeled competitor and continued to be reduced with a 50-fold excess. Again, the TA probe had no competitive effect; thus it is unlikely that any of the observed competition resulted simply from nonspecific interactions with the excess competitor probes.

Nuclear protein from primary cultures of day-14 chick fibroblasts, a cell type of mesenchymal origin that does not express aggrecan, produced some differences in DNA-protein interactions when compared with chick chondrocyte nuclear extracts but, perhaps more significantly, some similarities (Fig. 7). Band C, which is associated with the sequence (T)TCCCC(T), was also present for chick fibroblast extracts only with probe L; the appearance of complex C was not observed with fibroblast extract and probes J.1, J.2, or J, even though incubation of chick chondrocyte nuclear protein results in the formation of complex C for all of these probes. Thus the sequence (T)TCCCC(T) in probes J.1, J.2, and J is not sufficient to form complex C with chick fibroblast extract, but it is capable of forming a similar band in the context of probe L. Another similarity is that the band labeled D appears to be identical for probes J and J.1 with both fibroblast and chondrocyte extracts. Interestingly, the faster migrating complexes observed with probes J, J.1, and L are seemingly identical in both chondrocytes and fibroblasts, suggesting that these bands may represent a primary DNA-protein interaction, perhaps with the same transcription factor(s). The higher mobility bands seen for each of the probes exhibit differences that are unique to each cell type, suggesting that other proteins, specific to each type, are also binding to these cis elements or to the aforementioned primary complexes, causing slower migrating DNA-protein(s) complexes. The fundamental basis of aggrecan promoter DNA-protein interactions could be identical in these two mesenchyme-derived cell types, but the multiple protein-protein interactions appear unique to each.


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Fig. 7.   EMSA analyses with chick chondrocyte and fibroblast extracts. A and B show the bands resulting when probes G-L were incubated with nuclear extracts from day-14 chick chondrocyte and fibroblast cultures. The lowercase letters c and f, appended to the names of the probes and the major complex bands, refer to the source of nuclear protein (chondrocytes or fibroblasts, respectively) in the binding reactions.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DNA-Nuclear Protein Interactions in the Aggrecan Promoter-- The approaches used to survey trans factor-cis element interactions in the -1200 to -780 region of the chick aggrecan promoter yielded much mutually supportive information. Five of the six DNase I footprint sequences observed (excepting H) were protected to varying degrees by both chondrocyte and fibroblast nuclear proteins. Four of those five sequences (I-L) have negative roles in the transcriptional regulation of aggrecan, as evidenced by the increases in reporter activity observed for mutated Ag-1(+) constructs that preserved the native spacing while ablating individual footprint regions. All of the protected sequences were found to contain known cis elements, and the J-L regions were found to share, pairwise, a number of identical or similar sequence elements. Comparison of EMSA results for the various footprint sequence probes produced correlations of certain sequence elements to specific DNA-protein complex bands. Additionally, comparison of EMSA band patterns obtained with chondrocyte and fibroblast proteins revealed differences for all footprint probes, implying that cell type-specific transcription factor complexes were binding. For all four of the repression-associated footprint sequences, the chondrocyte/fibroblast EMSA difference is a reduction in mobility for the chondrocyte extract band compared with the slowest band observed with fibroblast extract (Fig. 7). Since chondrocytes express aggrecan (at various levels) throughout life, whereas fibroblasts do not express aggrecan at any time (1), a plausible interpretation of the EMSA data is that up-regulation of aggrecan expression in chondrocytes is accomplished in part by substitution or addition of specific proteins in/to the complexes that act at the repressor region cis elements in fibroblasts. Other bands that occur for both cell types may represent core complexes with which the cell type-specific factors interact.

Alternatively, perhaps the complexes seen with chick fibroblast extracts are more tightly bound, thus preventing aggrecan expression. These complexes could individually inhibit transcription, or the four binding sites could act synergistically to inhibit more strongly aggrecan expression. In chick chondrocytes this region, as shown in earlier work (12), can act as a modulator of high expression. If the cells are exposed to injury or are in the developmental context where greater amounts of aggrecan need to be expressed rapidly, removal of the trans complexes from these conserved cis sites could allow rapid up-regulation of the gene. It is tempting to speculate that such removal could be at least a two-stage process because both probes J.2 and K produce complex A, whereas probes L and J.1 produce complex B. Removal of a functional site by repression, squelching, or quenching (10) could increase aggrecan expression. Thus, it is conceivable that an individual cis element could interact with both negative and positive transcription factors.

Footprints J.1, J.2, and L exclusively contain the sequence TCCCC and are the only probes to produce band C in EMSAs using chondrocyte protein, but when fibroblast extract is used only probe L forms band C. If TCCCC is in fact the cis element directing complex C formation, as seems likely, then it is clear that the sequences adjoining a cis element can influence its behavior in different cell types. Similarly, probe L out-competed J.1 in formation of bands B and C, J.2 for bands A and C, and K for band A. Probe L shares various sequence elements with the other three, so the difference in affinity for particular complex components likely stems from the exact arrangement of elements. Also, since L can compete against band A formation even though it does not form that complex itself, it is possible that bands A and B, although never observed together, share some protein components.

Examination of the Defined Repressor cis Elements in the Context of Other Genes and Exogenous Stimuli-- Footprint J contained the sequence (CACCTCC), a motif present in the 80- and 100-base pair silencers CIIS1 and CIIS2 defined in the rat type II collagen promoter (13). Studies on the collagen alpha 1(II) promoter defined this site in the context of a 100-bp sequence abolishing transcription in fibroblasts and HeLa cells but not in chondrocytes (13). Our studies demonstrate that this sequence is acting as a repressor of the aggrecan gene in both mesenchyme-derived cell types. Most likely, the sequence (CACCTCC) that is present in footprints J and L and the rat collagen alpha 1(II) CIIS1 and CIIS2 regions is sufficient to produce silencer activity in both chondrocytes and fibroblasts, and the CACCTCC sequence is a minimal core or half-site element responsible for the binding of a general mesenchyme-specific repressor. EMSA results for both chondrocytes and fibroblasts suggest that elements flanking this sequence are important in the cell type-specific interactions observed in our study and can produce cell type-specific DNA-protein complexes.

Footprint K contained the similar MalT-malPp consensus sequence (TCCTCC), a site important in the positive regulation of the malT gene (18). The malT gene is regulated by Mlc, which has homology to the protein NagC, a gene regulator functioning as a repressor of enzymes that control the uptake and release of N-acetylglucosamine (19). Regulation of the malT gene can be modulated by the amount of internal free glucose or glucose derived from disaccharides (20-22). It is tempting to speculate that these aggrecan repressor sites are in some way linked to the availability of sugar precursors since aggrecan is extensively glycosylated. Furthermore, the TCCTCC motif has also been found in the human COL1A2 promoter (23) and the decay-accelerating factor gene (DAF) (24). When the TCCTCC site was mutated in the COL1A2 promoter, there was a significant reduction of basal promoter activity in transfection experiments using human fibroblasts (23); deletion of a region containing this motif in the DAF gene resulted in complete loss of promoter activity as determined by chloramphenicol acetyltransferase assays in transfected COS cells (24). In these promoters then, the TCCTCC motif appears to bind an activator of transcription. Interestingly, when the same group put the TCC-containing motif in the opposite direction to drive the expression of a thymidine kinase promoter system, basal promoter activity was significantly increased, suggesting that in a different context and system this motif has the capacity to act as a repressor. The authors (23) suggest that these phenomena could be due to conformational changes induced by the trans factors that bind to this site, indicating that this sequence has a dynamic role that probably involves precise positioning relative to adjacent sequences.

The human COL1A2 promoter also has a TCCCCC motif, mutation of which increased promoter activity by nearly 6-fold (23). Interestingly, the TCCCCC motif was located at -159 in the human genomic clone, only 31 base pairs from the TCCTCC motif at -128. A similar pattern is seen in the chick aggrecan promoter with the TCCTCC sequence contained in protected regions J.2 and K, with very close proximity to the TCCCC sequences found in sequences J.1 and L.

In sum, we have dissected a functionally significant 420-bp region of the aggrecan promoter that was previously shown to repress chick aggrecan gene expression (12). We have identified cis elements mediating the observed repressor activity and have characterized the propensity of these sequences to form cell type-specific DNA-protein interactions with nuclear proteins derived from day-14 chick chondrocytes or fibroblasts. DNase I footprinting protected several forward-strand sequences primarily composed of combinations of the bases thymine and cytosine; three of these were found by EMSA to participate in specific DNA-protein interactions as follows: the sequence CTCCTCC produced band A, CTTCA(G/C) produced band B, and TCCCC produced band C in chondrocytes. Band C was observed with chick fibroblast nuclear proteins only in the context of probe L, which was shown to have the strongest affinity for nuclear proteins in cross-competition EMSA experiments; clearly the location of a TCCCC sequence greatly influences its binding behavior. Three of the four demonstrated repressor areas have a CCTCC motif, alluded to in other reports in a variety of capacities as a silencer or even an enhancer, and thus important in binding to transcription factors in a number of genes. To our knowledge this is the first parallel examination of the EMSA patterns for six functionally defined cis elements with highly similar sequences, using protein from normal primary cells. By purifying and characterizing transcription factors that bind to these sequences, we are now able to investigate how these elements function to regulate aggrecan expression during development and in response to either chemical stimuli by cytokines or physical stresses on cartilage tissues, both in normal and disease states.

    ACKNOWLEDGEMENTS

We thank Dr. Miriam Domowicz for helpful discussion, Glenn Burrell for manuscript preparation, and James Mensch for advice and manuscript review.

    FOOTNOTES

* This work was supported by United State Public Health Service Grants AR-19622 and HD-09402 (to N. B. S.), Training Grant HL-07237, and a Fellowship from the Markey Program in Molecular Medicine (to E. W. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom the correspondence should be addressed: The University of Chicago, 5841 S. Maryland Ave., MC 5058, Chicago, IL 60637. Tel.: 773-702-6426; Fax: 773-702-9234.

Published, JBC Papers in Press, February 28, 2001, DOI 10.1074/jbc.M009944200

2 E. W. Pirok III and N. B. Schwartz, unpublished data.

    ABBREVIATIONS

The abbreviations used are: CS, chondroitin sulfate; EMSA, electrophoretic mobility shift assay; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; PCR, polymerase chain reaction; kb, kilobase pair; bp, base pair.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.