©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcriptional Activation of the 1(VI) Collagen Gene during Myoblast Differentiation Is Mediated by Multiple GA Boxes (*)

(Received for publication, May 11, 1995)

Stefano Piccolo Paolo Bonaldo Paola Vitale Dino Volpin (§) Giorgio M. Bressan

From the Institute of Histology and Embryology, University of Padova, 35100 Padova, Italy

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

During differentiation of C2C12 myoblasts in vitro, expression of alpha1(VI) collagen mRNA was transiently stimulated severalfold. Promoter assays on cells transfected with chloramphenicol acetyltransferase (CAT) chimeric constructs have identified a region of the alpha1(VI) collagen promoter that increases CAT activity about 8-fold during differentiation. The region, which overlaps with transcription initiation sites, was shown to contain three protected segments (A, B, and C) in DNase I footprinting assays. The contact points between nuclear factors and the protected segments were determined by methylation interference assay and included the sequence GGGAGGG (GA box) in all segments. Experiments in which CAT constructs were cotransfected with double-stranded oligonucleotides containing the GA box suggested that this motif was necessary for induction. Transfections with deletion constructs of the natural promoter and with minipromoters made of three copies of A, B, or C showed that the elements have inducing activity and that elements C and, to a lower extent, B are stimulatory for basal transcription, whereas the contribution of A in this process is limited. Electrophoretic mobility shift assays with nuclear extracts from C2C12 cells indicated that the three GA box-containing elements bound several transcription factors, including Sp1. Comparison of the properties of the bands shifted under different experimental conditions (presence of 10 mM EDTA, heating of the nuclear extracts, addition of different concentrations of competitor oligonucleotides) established that A, B, and C probes form nine, eight and five main retarded complexes, respectively, and indicated that nuclear factors binding to C and B are subsets of proteins binding to A. UV cross-linking assays identified several peptides (seven with probe A, six with B, and five with C) in the range of 150-32 kDa. Comparison of the gel retardation pattern obtained with nuclear extracts from proliferating and differentiating cells revealed a particular increased intensity of two retarded bands. The data establish that multiple GA boxes mediate induction of the alpha1(VI) collagen promoter during myoblast differentiation and suggest the attractive hypothesis that the effect may be related to variations of expression of transcription factors binding to these motifs.


INTRODUCTION

Collagens constitute a complex family of extracellular proteins that are major determinants of the mechanical properties of tissues (van der Rest and Garrone, 1991). The expression of each collagen type is specifically controlled in different tissues and is differentially activated by various stimuli. Although several conditions influencing expression of different collagen types have been described, the molecular mechanisms involved have only rarely been determined, the most notable example concerning the stimulation of collagen I chains by transforming growth factor-beta (Rossi et al., 1988; Ritzenthaler et al., 1993; Inagaki et al., 1994).

Type VI collagen is composed of three genetically distinct polypeptide chains, alpha1, alpha2, and alpha3, all of which contain several domains related to the von Willebrand type A repeats (for a recent review on this collagen type, see Colombatti et al.(1993)). The protein has adhesive properties, and several observations suggest that it plays an essential role in regulating the structural organization of the extracellular matrix through specific interactions with a number of other components. Collagen VI is particularly abundant in the pericellular space where it forms microfibrillar aggregates. Expression of the protein during development is both stage- and tissue-specific. (^1)Several studies have featured a distinct program of type VI collagen regulation. As in other collagens, the synthesis of all three chains in fibroblasts is inhibited by viral transformation, but treatment of the cells with phorbol esters does not change mRNA levels for collagen VI, although it causes a 3-5-fold reduction for collagen I and III (Schreier et al., 1988). Hyperglycemia, on the contrary, has been found to increase expression of the three collagen VI chains (Muona et al., 1993). The effect of transforming growth factor-beta and -interferon is restricted to the alpha3 chain, the former stimulating and the latter inhibiting its expression (Heckmann et al., 1989; Heckmann et al., 1992). A unique feature of the regulation of collagen VI expression is the considerable induction of protein and mRNA levels observed during differentiation of mesodermal cells like adipocytes (Dani et al., 1989), chondrocytes (Quarto et al., 1993), and myoblasts (Ibrahimi et al., 1993) and by confluence in fibroblasts (Hatamochi et al., 1989).

The promoter region of the three genes coding for type VI collagen chains has been recently cloned from several species (Koller et al., 1991; Koller and Trueb, 1992; Bonaldo et al., 1993; Saitta and Chu, 1994). The characterization of the chicken alpha1(VI) collagen promoter has revealed the presence of two Sp1 and one AP1 binding sites close to the transcription start sites, which are necessary for full promoter activity (Willimann and Trueb, 1994). Additional stimulatory elements are certainly contained in more 5`-end sequences (Koller and Trueb, 1992; Bonaldo et al., 1993); these elements, however, have not been characterized yet. The availability of the promoter region of different alpha-chains allows the investigation of the molecular mechanisms of transcriptional regulation possibly involved in the different conditions affecting type VI collagen expression. In this work, we have studied the transcriptional regulation of the alpha1(VI) collagen promoter during differentiation of a myoblasts cell line in vitro and have begun to define the molecular details of its activation.


MATERIALS AND METHODS

Cell Cultures and Transfections

C2C12 myoblasts (Yaffe and Saxel, 1977) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (proliferation medium) in an atmosphere of 5% CO(2). When required, cells were induced to differentiate by changing the medium to Dulbecco's modified Eagle's medium containing 2% horse serum and 0.5 µg/ml bovine insulin (differentiation medium). For transfection, cells were plated at a density of 100-200 10^3/Petri dish (10-cm diameter). The following day, DNA-Ca phosphate precipitates (Wigler et al., 1978) were added directly to the culture medium, and cells incubated for 16 h. The medium was then removed, the layer was treated for 2 min with 15% glycerol in 25 mM Hepes, 140 mM NaCl, 0.75 mM Na(2)HPO(4), pH 7.05, and, after washing with phosphate-buffered saline, fresh proliferation medium was added and incubation continued for 24 h. At this stage, one-half of the plates were scraped and processed (proliferating cells), whereas in the other half the medium was changed to differentiation medium, incubation was prolonged for two additional days, and cells were harvested (differentiating cells). Extracts were prepared by resuspending the collected cells in 0.1 ml of 0.1 M potassium phosphate buffer, pH 7.8, 2 mM dithiothreitol, and repeated freeze-thawing. CAT (^2)and luciferase assays were performed as described (Seed and Sheen, 1988; Brasier et al., 1989). The amount of DNA used for transfection of each Petri dish was 10 µg of CAT-promoter plasmid and 1 µg of Rous sarcoma virus luciferase (De Wet et al., 1987) as internal standard for differences in transfection efficiencies.

RNA Analysis

Total RNA was purified from 10-cm Petri dishes with RNAfast reagent (Molecular Systems) following the procedure recommended by the manufacturer and analyzed by Northern hybridization using standard protocols (Sambrook et al., 1989). Probes used were clone SMP1, encoding the amino-terminal end of the alpha1(VI) mRNA (Bonaldo et al., 1993), a rat cDNA clone for cardiac troponin T kindly provided by Dr. S. Schiaffino (University of Padova, Italy), and a cDNA clone for human glyceraldehyde-3-phosphate dehydrogenase (Tso et al., 1985).

Plasmid Constructions

Plasmid p1341CAT was constructed by ligating a 1382-base pair fragment spanning positions -1341 to +41 of the murine alpha1(VI) promoter (Bonaldo et al., 1993) into the promoterless vector pBL6CAT (Boshart et al., 1992). The two plasmids p215CAT and p82CAT were constructed by polymerase chain reaction amplification from plasmid p1341CAT and cloning into pBL6CAT. p215Delta(-72, -1)CAT was obtained by ligation of two polymerase chain reaction fragments into HindIII- and PstI-cut pBL6CAT. One fragment, which extended from -215 to -73, contained a HindIII site at the 5`-end, whereas the other fragment (+1 to +41) contained a PstI site at the 3`-end. Deletions of p82CAT from the 5`- and 3`-ends were developed by Bal31 digestion following established protocols (Sambrook et al., 1989). All plasmids were purified by CsCl gradient centrifugation and sequenced to verify their correct orientation and sequence identity. Artificial promoter constructs containing repetitions of elements A, B, and C identified in Fig. 5were derived by ligating synthetic double-stranded oligonucleotides, including the indicated sequences and appropriate sticky ends, and cloning the ligation products upstream of the promoter segment of p8CAT. Clones carrying three copies of the elements were selected and confirmed by sequencing.


Figure 5: Summary of the structural analyses of the alpha1(VI) collagen promoter. Regions protected in the DNase I footprinting experiments reported in Fig. 3are indicated by doublelinesabove and below the sequence. Singlelines mark the DNase I footprinting of an AP1 binding site characterized in unpublished experiments. Dots identify contact sites with transcription factors mapped by methylation interference assays. The most upstream transcription initiation site is labeled by an arrow, and transcribed sequences are lowercaseletters. Squarebrackets delimit the sequence of double-stranded oligonucleotides A, B, and C used in gel shift experiments reported in the following figures.




Figure 3: DNase I footprinting analysis of the region extending from -82 to +41 nucleotides from the transcription start site. The end-labeled fragments were reacted with 80 µg of nuclear extract prepared from differentiating C2C12 cells. The areas protected from DNase I digestion are marked by brackets. Positions of protected segments were determined by comparison with a G + A sequencing reaction (G/A) and are indicated relative to the most upstream transcription start site.



DNA Binding Assays

Nuclear extracts were prepared as described (Shapiro et al., 1988). C2C12 cells were plated at a density of 11 10^3/cm^2 and either harvested the following day (proliferating cells extract) or collected after 1 day in differentiation medium (differentiating cells extract).

DNase I Footprinting

Appropriate DNA fragments were labeled at one end with [P]dNTPs and Klenow enzyme and gel purified as described (Ausubel et al., 1993). 20 10^3 cpm probe were incubated in 20 mM Hepes, pH 7.9, 50 mM KCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol with or without nuclear extract (usually 80 µg) in a total volume of 30 µl for 45 min at 4 °C. 2 µl of 125 mM MgCl(2), 25 mM CaCl(2) were then added, and the samples were maintained at room temperature for 1 min. DNase I (Sigma) was added (100-20 ng/reaction in samples containing the nuclear extract and 5-0.4 ng/reaction in samples without nuclear extract), samples were incubated for 1 min at room temperature, and the reaction was stopped by addition of 200 µl of 0.2 M NaCl, 1% SDS, 25 mM EDTA and 100 µg/ml herring sperm DNA. After phenol/chloroform extraction and ethanol precipitation, the samples were resuspended in loading buffer (80% formamide, 10 mM EDTA, 0.1% xylene cyanol, 0.1% bromphenol blue), denatured at 95 °C for 3 min, and resolved in a 8 or 10% sequencing gel. The gel was dried and exposed to x-ray film at -80 °C with intensifying screen.

Electrophoretic Mobility Shift Assay

Synthesized double-stranded oligonucleotides were radiolabeled with T4 polynucleotide kinase (Sambrook et al., 1989). 2-5 µg of nuclear extract were incubated for 10 min at room temperature in 30 µl of binding buffer (20 mM Hepes, pH 7.9, 50 mM KCl, 5 mM MgCl(2), 0.5 mM dithiothreitol, 50 µM ZnSO(4), 5% glycerol) containing 0.5 µg of poly(dI-dC). Competitor oligonucleotides and the probe (20-30,000 cpm) were added, and incubation was prolonged for an additional 20 min at room temperature. Samples were electrophoresed in 6% non-denaturing polyacrylamide gels (20:1 acrylamide to bisacrylamide) in 40 mM Tris, 190 mM glycine for 3 h at 150 V. The gels were fixed in 10% acetic acid, dried under vacuum, and exposed to x-ray films. In supershift experiments, the samples also contained 0.3-4 µg of either affinity-purified anti-Sp1 antibody (Santa Cruz Biotechnology, Inc.) or the same amount of preimmune IgG.

Methylation Interference Assay

The procedure followed was exactly that detailed by Ausubel et al.(1993).

UV Cross-linking

100,000 cpm of labeled double-stranded oligonucleotide were incubated with 10 µg of nuclear extract in 30 µl under the same conditions used for electrophoretic mobility shift assays. Samples were then exposed to UV light (250 nm) for 50 min in a Stratalinker apparatus (Stratagene), 30 µl of 2 final sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 0.1% bromphenol blue, 10% glycerol) were added, and the molecules were separated by polyacrylamide gel electrophoresis in the presence of SDS. DNA-protein complexes were revealed by exposure to x-ray film.

Southwestern Blotting

20 µg of C2C12 nuclear extract were resolved by polyacrylamide gel electrophoresis in the presence of SDS and electroblotted into nitrocellulose filters. The proteins bound to the filter were renatured, and filters were processed as described (Jackson, 1993).


RESULTS

alpha1(VI) Collagen mRNA Expression in Differentiating Myoblasts

C2C12 myoblasts can be induced to differentiate when cultured at high density in medium deprived of fetal calf serum (Yaffe and Saxel, 1977). The appearance of differentiation markers in our cultures was very rapid, as demonstrated by the increase of the mRNA for cardiac troponin T (Fig. 1), a gene that is transiently expressed also at early stages of skeletal muscle differentiation (Toyota and Shimada, 1981). Myotubes could be noted at day 2 after stimulation of differentiation and constituted the main part of the culture at day 4 (data not shown). During differentiation, the mRNA encoding the alpha1(VI) chain increased very rapidly in the first day, peaking at about 24 h, and then declined in the following days to a level that remained constant for at least 1 week (Fig. 1). The maximal increase of mRNA relative to the level detected at the time of stimulation of differentiation (time 0 in Fig. 1) was usually 5-15-fold, depending on the state of confluence of the cells. Very similar results have been described for the alpha2(VI) collagen chain during differentiation of the same cells (Ibrahimi et al., 1993).


Figure 1: Expression of alpha1(VI) collagen mRNA by differentiating myoblasts. A series of Petri dishes containing C2C12 cells were plated at a density of 150,000/dish (10 cm) in proliferation medium. Cells from groups of dishes were harvested for total RNA purification with the following schedule: 1 day after plating, which corresponds to the day before switching to differentiation medium (-1d); the day of application of differentiating conditions (time 0); at various times (hours or days) after induction of differentiation. 15 µg of RNA were run on a 1% agarose gel and analyzed by the Northern blotting procedure using cardiac troponin T, alpha1(VI), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes. The histogram is a quantitative evaluation of alpha1(VI) mRNA levels and was obtained by densitometry of specific bands and normalization to the glyceraldehyde-3-phosphate dehydrogenase signal.



Identification of the Promoter Region Activated during Differentiation

The increased production of mRNA in differentiating myoblasts could be the effect of either transcriptional or post-transcriptional regulation. To investigate the contribution of transcriptional mechanism(s), the promoter region of the mouse alpha1(VI) collagen gene was isolated (Bonaldo et al., 1993), and several CAT chimeric constructs were derived. The plasmids were transiently transfected into C2C12 cells, and their promoter activity was measured in parallel in exponentially growing cells and in cells switched to differentiating medium (see ``Materials and Methods''). Initial experiments were performed with two plasmids containing fragments extending from +41 to -1341 and from +41 to -215 base pairs from the most upstream transcription initiation site. The results indicated that CAT expression in proliferating myoblasts was about double for the longer construct and that differentiation stimulated promoter activity about 15- and 8-fold for the long and the short construct, respectively (data not shown). We therefore concentrated our study on the region -215 to +41, from which several 5`- and 3`-deletions were obtained and tested for promoter activity (Fig. 2). The data showed that the region extending from -83 to -215, although required for high levels of expression in proliferating cells, was not important for induction (compare CAT activities of p215CAT and p82CAT). On the contrary, the sequence from -1 to -82 produced low basal expression but was essential for induction (compare the results obtained with plasmid p215CAT with that of p82CAT and p215Delta(-72,-1)CAT). Stepwise removal of 5`-sequences from the region -82 to +41 gradually lowered basal CAT activity and induction properties of the constructs, indicating the presence of multiple regulatory tracts. Reduction of the 5`-end to -42 base pairs caused a drop of induction to 5-fold, and an additional cut to -8 brought to the complete loss of stimulation. The results indicated that the region -24 to -9 contributed most to induction and that the stretch -82 to -25 had low induction activity but remarkably increased basal expression. This interpretation was confirmed by assays with 3`-deletions of the promoter region, in which fragments extending from -16 to -82 and from -55 to -82 gave an identical low level of induction (Fig. 2). Induction was specific for the alpha1(VI) promoter constructs, since CAT activity expressed by the SV40 early promoter (pACAT(2)) and the tropoelastin promoter (pTE911CAT) did not change during myoblast differentiation (Fig. 2).


Figure 2: Expression of alpha1(VI) promoter CAT constructs in C2C12 myoblasts. Promoter fragments extended from the indicated base at the 5`-end to base +41 at the 3`-end from the most upstream transcription initiation site (Bonaldo et al., 1993). Deletions are indicated by Delta followed by parentheses comprising the position of the bases delimiting the deleted fragment. pBL6CAT is the vector into which the DNA fragments were cloned. Plasmid pACAT(2) contains the SV40 early promoter without the enhancer (Laimins et al., 1984). pTE911CAT is a plasmid enclosing a portion of the human tropoelastin promoter (Marigo et al., 1993). The data were derived from several experiments in which four Petri dishes were transfected with the indicated plasmids. After incubation for 1 day in proliferation medium, cells were harvested from two dishes, and CAT activity was measured (leftpanel). Differentiation medium was added to the other two dishes, and cells were grown for 30-36 h. After this time, CAT activity was tested, and induction was determined (rightpanel) as the ratio of CAT activity obtained from differentiating and proliferating cells.



Structural Analysis of the Inducible Region

DNase I footprinting with a DNA fragment extending from +41 to -82 identified two protected sequences in the upper strand: one from -75 to -59 and the second, which was very long, from bases -45 to +4. Three footprints were detected in the lower strand and comprised bases +8 to -19, -28 to -45, and -60 to -70 (Fig. 3). The interaction of the DNA fragment with transcription factors was also analyzed by methylation interference assay (Fig. 4), and the results are summarized, together with the DNase I footprinting data, in Fig. 5. The contact points (indicated by dots in Fig. 5) were concentrated in three regions and were Gs in the sequence GGGGAGGG or GGGAGGG, thus identifying the binding string as a GA box.


Figure 4: Determination of contact sites of nuclear factors within the -82 to +41 region by methylation interference assay. The non-coding strand of the fragment was end-labeled with P and 100,000 cpm reacted with dimethyl sulfate, incubated with 20 µg of nuclear extract from differentiating C2C12 cells, and resolved by polyacrylamide gel electrophoresis. The major retarded band (R) and the free probe (F) were purified, fragmented by piperidine treatment and analyzed in an 8% sequencing gel (rightpanel). G/A, Maxam and Gilbert G + A sequencing reaction.



Functional Analysis of GA Box-containing Elements

To establish the functional importance of this repeated motif on induction of promoter activity, myoblasts were cotransfected with construct p215CAT and a molar excess of double-stranded oligonucleotides derived from the sequence of the GA box-containing elements marked by brackets in Fig. 5. Induction of CAT expression during myoblast differentiation was completely inhibited by the three oligonucleotides A, B, and C, which contain the wild type sequence, whereas it was not affected by C*, in which the GA box of C was mutated (Fig. 6). On the contrary, none of these oligonucleotides had any major effect on the CAT activity of proliferating cells. These data suggest that the GA box motif is necessary for induction of the alpha1(VI) collagen promoter during myogenesis. To evaluate the inductive potency of the individual A, B, and C elements (Fig. 5), artificial promoters were created in which trimers of each element were fused upstream of the region -8 to +41, which contains the transcription initiation sites (Bonaldo et al., 1993). Induction was observed for all three elements, with A having the highest and C the lowest activity (Fig. 7). Three copies of A in the antisense orientation were also stimulatory (plasmid pAsCAT in Fig. 7). On the contrary, mutation of the GA box abolished induction (plasmid pB*sCAT in Fig. 7). The experiments described in Fig. 7gave also important information on the function of the three promoter elements in basal transcription. In proliferating myoblasts, three copies of C produced a very high level of CAT expression, which was 30-40 times that of the A trimer and 5-10 times that of the B trimer and of the natural promoter.


Figure 6: The GA box motif is necessary for induction of the alpha1(VI) collagen promoter during myogenesis. C2C12 myoblasts were transfected with the construct p215CAT (defined in Fig. 2) in the absence or presence of the double-stranded oligonucleotides A, B, C (defined in Fig. 5), and C*, in which the GA box of C was mutated (GGGGAGGG to GGACATGG). The molar ratio of the oligonucleotides to the p215CAT construct was 800-fold. Dishes of proliferating and differentiating cells were processed for CAT activity, and induction was determined as described in the legend of Fig. 2. CAT activity is expressed as cpm butyryl-[^14C]chloramphenicol formed/h/10^6 light units.




Figure 7: Functional properties of artificial promoters carrying multiple copies of the individual GA box-containing elements. C2C12 myoblasts were transfected with the indicated constructs, and CAT activity from proliferating and differentiating cells and induction was determined as described in the legend of Fig. 2. Constructs (pAsCAT, pBsCAT, pCsCAT, and pB*sCAT) contain three copies of element A, B, C, and mutated B, respectively, in the sense orientation fused with the region +8 to -41 of the natural alpha1(VI) collagen promoter. In B*, the GA box (GGGGAGGG) was mutated to GGACATGG. pAsCAT differs from pAsCAT for the antisense orientation of the A trimer. p215CAT is defined in Fig. 2.



Characterization of the Nuclear Factors Binding to GA Box

Nuclear factors binding to GA boxes were examined by mobility shift assays. Incubation of double-stranded oligonucleotides A, B, or C (defined in Fig. 5) with nuclear extracts from C2C12 cells produced several retarded bands (Fig. 8, lanes2, 7, and 12). When the GA box of B or C was mutated (GGGGAGGG to GGACATGG), the bands disappeared or were dramatically decreased (data not shown), indicating that all the factors binding to B and C required at least part of the GA box sequence. No mutation of the A oligonucleotide was attempted, since it is entirely formed by repeated GGGAGGG motifs, and changes of the GA box would have altered the entire sequence. To better identify each single band and define the possible correspondence of the bands obtained with the three different probes, the conditions of the assays were varied: divalent cations were chelated by EDTA (Fig. 8, lanes3, 8, and 13) or an antibody against Sp1 was added (Fig. 8, lanes4, 9, and 14) or the nuclear extract was heated for 5 min at 95 °C just before the assay (Fig. 8, lanes5, 10, and 15) or cold competitor oligonucleotides were included in the mixture (Fig. 9). Representative results are reported in Fig. 8and Fig. 9, and the data from several experiments are summarized in Table 1. The use of the different conditions allowed the clear identification of nine, eight, and five retarded bands with oligonucleotide A, B, and C, respectively. The analysis suggested that complexes observed with B and C were due to subsets of proteins binding to A, and, likewise, C bound a group of factors recognizing B. We have labeled the bands in Fig. 8and 9 and in Table 1on the basis of the probe used (A, B, or C) and with progressive numbers that are equal for bands that we propose are retarded by the same protein(s) on the basis of their properties reported in Table 1. With all probes, band 1 (Fig. 8, lanes2, 7, and 12) was composed of two complexes (bands 1a and 1b), one of which was supershifted by an antibody against Sp1 (Fig. 8, lanes3, 8, and 13). The presence of two bands is perceptible with probe C (Fig. 8, lane12) and is also deduced from the fact that increasing the concentration of anti-Sp1 antibody used in Fig. 8did not alter the amount of the supershifted complex (data not shown). Band A4 (Fig. 8, lane2) also included two overlapping associations, bands A4a and A4b, of which only the former corresponded to a complex with similar properties in assays with probes B and C (bands B4 and C4, respectively). This conclusion was derived from several observations. After heating the nuclear extract, complex A4 did not disappear completely (Fig. 8, lane5), identifying a heat-resistant band unique for probe A (A4b in Table 1). This band was EDTA sensitive, since addition of this reagent after heating completely suppressed complex A4 (data not shown). On the contrary, treatment with EDTA without heating did not abolish band A4 (Fig. 8, lane3), thus distinguishing an EDTA-resistant, heat-sensitive association (band A4a in Table 1). Finally, cold oligonucleotide A inhibited all the complexes generated with probe B (Fig. 9, lanes15-17), whereas a band with the mobility of the A4 complex persisted when probe A was incubated with an excess of cold B (Fig. 9, lanes5-7), suggesting that A binds all the factors recognized by B and, in addition, a protein(s) that contributes to the formation of band A4. The disappearance of bands in the presence of EDTA was reversible since the complexes reformed by readdition of zinc ions (data not shown).


Figure 8: Electrophoretic mobility shift assays analyzing the binding properties of GA boxes containing oligonucleotides A, B, and C (see Fig. 5). 20,000 cpm of each probe were incubated with 4 µg of nuclear extract from differentiating cells under the indicated conditions, which included the presence of 10 mM EDTA or an antibody against transcription factor Sp1 (alphaSp1) or heating of the nuclear extract for 5 min at 95 °C just before the assay. The bands that could be identified on the basis of the data reported in this figure and in Fig. 9are indicated. The criteria followed to identify the bands are summarized in Table 1.




Figure 9: Competition gel mobility shift assays identifying common associations of sequences A, B, and C (see Fig. 5) with nuclear factors from C2C12 myoblasts. 20,000 cpm of each probe were incubated with 4 µg of nuclear extract from differentiating cells. S is an oligonucleotide containing the recognition sequence for the transcription factor Sp1 (Briggs et al., 1986) and C* and B* represent oligonucleotides C and B with a mutated GA box (GGGGAGGG substituted with GGACATGG). The criteria followed to identify the bands are summarized in Table 1.





Competition between some of the nuclear factors for binding to the GA box motifs is evident in Fig. 8and Fig. 9. Thus, the intensity of band A7 increased after inactivation of heat-sensitive factors (Fig. 8, compare lanes2 and 5) or in the presence of anti-Sp1 antibodies (Fig. 8, lane4). The same band was more prominent when access of proteins to probe A was inhibited by cold oligonucleotides (the most clear examples are lanes9, 23, and 26 of Fig. 9). Similarly, band A4b became very strong when the formation of all the other complexes was blocked by cold oligonucleotide B (Fig. 9, lane7). An interesting competition concerned associations A1-A6; when formation of complexes A1 and A2 was abolished by oligonucleotide S, which contains the recognition sequence of Sp1, bands A5 and A6 (and also A7) appeared very prominent and bands A3 and A4 remarkably fainter (Fig. 9, compare lane21 with lanes26 and 27). A phenomenon similar to that described for A was also detected with probe B; the presence of S lowered intensity of B3 and B4 and enhanced B5 and B6 (data not shown). A 1000-fold molar excess of cold C* reproduced partially the effect of S (Fig. 9, lane23). This result was not surprising, in view of the fact that labeled C* gave rise to a single, very faint band corresponding to C1 and should therefore slightly inhibit complex A1 at high concentrations. It must also be noted that additional bands became apparent at low concentrations of inhibitor oligonucleotides (Fig. 9, lanes2, 5, 6, 12). This may indicate the existence of potential recognition sites for other transcription factors in the oligonucleotides used.

The molecular weight of proteins binding to the GA box-containing elements were investigated by UV cross-linking and southwestern blotting assays. With the first technique, probe A produced seven bands of about 150, 105, 76, 54, 50, 46, and 31 kDa (Fig. 10, lane1). All the complexes were specifically abolished by an excess of the same oligonucleotide (Fig. 10, lane2). Probes B and C gave rise to six (105, 76, 54, 50, 46, and 31 kDa) and five (105, 76, 54, 46, and 31 kDa) specific bands, respectively (Fig. 10, lanes3-6). Southwestern blotting assays with probe A revealed five proteins of 105, 65, 52, 43, and 34 kDa (Fig. 10, lane7). Competition experiments confirmed that binding of the probe to these proteins was specific (Fig. 10, lane8), whereas Western blotting on the same filters allowed the identification of the 105-kDa species as Sp1 (Fig. 10, lanes9 and 10).


Figure 10: Biochemical characterization of proteins binding to GA box-containing elements. Lanes1-6, UV cross-linking assays in solution. Radiolabeled double-stranded oligonucleotide A, B, and C (Fig. 5) (100,000 cpm) were incubated with 10 µg of nuclear extract from differentiating myoblasts and without or with the indicated inhibitor oligonucleotide (400-fold molar excess). After treatment with UV light, the samples were separated in a 10% SDS-polyacrylamide gel, and proteins bound to the probe were identified by autoradiography. Lanes7-10, Southwestern (S.-W.) and Western (W.) blotting assays. Nuclear proteins (20 µg) from differentiating cells were separated in a 12% SDS-polyacrylamide gel and transferred to nitrocellulose membrane. The filters were then hybridized with radiolabeled double-stranded oligonucleotide A in the absence (lane7) or in the presence (lane8) of an excess (400 ) of unlabeled oligonucleotide. The strips of lane7 and 8 were probed with either polyclonal antibodies against Sp1 (lane9) or preimmune IgG (lane10). Arrows on the left of the panels indicate the migration of radiolabeled complexes. Numbers on the right mark the mobility of proteins of known molecular mass (given in kDa).



Given the activation of the alpha1(VI) collagen gene during differentiation, it was of interest to test if there was any obvious difference in the binding activity of nuclear extracts to the GA box-containing elements in proliferating and differentiating myoblasts. Electrophoretic mobility shift assays using probes A, B, and C revealed enhanced intensity of a few bands in differentiating cells. The most significant variation concerned complexes 1 and 4: A1 and A4 (Fig. 11, lanes1 and 2), B1 and B4 (Fig. 11, lanes5 and 6), and C1 and C4 (data not shown). Addition of anti-Sp1 antibodies established that the increase of bands 1 was mainly due to the b component (Fig. 11, lanes3, 4, 7, and 8 and data not shown). Densitometric quantitation indicated a relative increment of 3-4-fold for bands A1b, B4, and C4 and of about 2-3-fold for complexes A4, B1b, and C1b.


Figure 11: Electrophoretic mobility shift assays comparing GA box binding activity in proliferating and differentiating myoblasts. 20,000 cpm of radiolabeled oligonucleotides A and B (defined in Fig. 5) were reacted with 6 µg of nuclear extract prepared from proliferating (P) or differentiating (D) C2C12 cells under the conditions indicated and resolved in a 6% polyacrylamide gel. Symbols on the left identify the different bands as referred in Table 1. The gels were overrun to allow better separation of the bands.




DISCUSSION

This study has identified a region of the promoter of the alpha1 chain of type VI collagen that plays a major role in activation of the gene during myoblast differentiation. The region extends from +8 to -75 base pairs from the transcription initiation site and is homopyrimidine/homopurine rich. Functional and structural characterization has restricted the activity to three elements (identified as A, B, and C) whose common feature is the presence of the sequence GGGAGGG (GA box). A key experiment in defining the function of this sequence is that reported in Fig. 6, which implies that GA boxes are essential for induction of promoter activity during differentiation; mutation of the GA box in cotransfected oligonucleotides encompassing the sequence protected in DNase I footprinting assays abolishes inhibition of CAT activity expressed by promoter constructs. The same mutation also prevents binding of the nuclear factors to the oligonucleotides. Promoter assays with 5`- and 3`-deletions of the region suggest that the three elements are not functionally equivalent; A has the strongest inducing activity, whereas C is important for high basal expression. Additional information on the functional properties of the three GA box-containing elements have been obtained from analysis of CAT expression from constructs carrying artificial promoters formed by repeated A, B, and C sequences. These experiments confirm the high inducing capacity of A and show that B and C are also inducers; in addition, they prove that C is the most effective in enhancing basal transcription. In fact, CAT activity expressed by the minipromoter with three copies of C is 30-40- and 5-10-fold higher than that produced by similar constructs containing an equal number of copies of A and B, respectively. It is important to note that a single A copy is very active in inducing transcription (Fig. 2, plasmid p24CAT) and that additional copies of either A (Fig. 7) or B plus C (Fig. 2) further enhance induction only to a limited extent. Owing to these data, it can be proposed that, in the context of the natural promoter, element A is mainly responsible for induction, whereas B and C are important for basal expression. In the light of this hypothesis, it is surprising to find that B and C oligonucleotides do not decrease CAT expression in proliferating myoblasts when cotransfected with p215CAT (Fig. 6). This apparent contradiction can be reconciled by assuming that basal expression and induction depend on distinct regulatory events and that the latter process is more easily altered by inhibiting binding of nuclear factors to the GA box-containing elements.

Electrophoretic mobility shift assays indicate that the three GA box-containing sequences associate with a common set of five nuclear factors, including Sp1. Three additional protein complexes are bound by B and A but not by C element. Finally, one complex is recognized only by fragment A. Therefore, although the GA box is essential for binding of all the factors, sequences flanking this motif also contribute to the binding specificity of the three elements. The relative affinity of the nuclear proteins and the competition between the factors for binding to overlapping sequences could determine the overall properties of each GA box-containing element. The complexity of nuclear factors recognizing the homopyrimidine/homopurine-rich region of the alpha1(VI) collagen promoter is apparent also from the partial biochemical characterization. Seven, six, and five polypeptides in the range 150-31 kDa are found to associate with elements A, B, and C, respectively, by UV irradiation. Again, some are common to A, B, and C, one is common to A and B, and one is unique to A. Due to the complexity of the patterns, a correspondence between the gel-shifted bands and the UV-cross-linked peptides could not be deduced. The formation of five, four, and three complexes in band shift assays with probes A, B, and C, respectively, required the presence of zinc ions, suggesting that an important group of transcription factors binding to the alpha1(VI) collagen promoter is represented by proteins with zinc-finger domains. Accordingly, our data show that one of the EDTA-sensitive proteins is Sp1. As pointed out above, the functional data have established that A, B, and C have inductive activity and that C is particularly important for basal expression. A simple correlation of these functional data with the pattern of bands detected in electrophoretic mobility shift assays would predict that induction is mediated by the factors that bind all three elements (complexes 1-4) and that the function of the additional factors, which recognize A and B (bands 5-7), may rather depress basal expression.

The molecular mechanism of transcriptional activation of the alpha1(VI) collagen gene during myoblasts differentiation remains to be established and will require full characterization of the GA box-binding factors. The finding of a consistent severalfold increase of intensity of some bands, especially 1b (A1b, B1b, C1b) and 4 (A4, B4, C4) in gel retardation assays stimulates the hypothesis that induction is due to increased synthesis or binding activity of these complexes. Gel mobility shift experiments show that all the nuclear factors require the GA box for significant binding and also indicate that the nuclear factors compete for binding to the DNA. It is attractive to speculate that induction during differentiation is not only due to higher availability of key factors but is also the consequence of the fact that the increased factors displace from the promoter proteins, which are inhibitory for transcription.

Myoblast differentiation is just one of several situations characterized by increased expression of collagen VI. Others include differentiation of adipocytes (Dani et al., 1989) and chondroblasts (Quarto et al., 1993) and confluence of fibroblasts (Hatamochi et al., 1989). All these conditions are characterized by a decrease of cell proliferation, which may be a major determinant of the effect, as previously suggested (Ibrahimi et al., 1993). Thus, it is not unlikely that collagen VI induction in these other cells is also mediated by factors binding to GA boxes, whose activity may be modulated by biochemical pathways depending on the cell cycle.


FOOTNOTES

*
This work was supported by grants from Telethon and Associazione Italiana Ricerca sul Cancro, from the Progetti Finalizzati, Invecchiamento, and Applicazioni Cliniche della Ricerca Oncologica of the Italian Consiglio Nazionale delle Ricerche, and from the Bridge BIOT-CT91-0260 project. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Institute of Histology and Embryology, University of Padova, Via Trieste 75, 35100 Padova, Italy. Tel.: 39-49-828-6613; Fax: 39-49-828-6601; volpin{at}cribi1.bio.unipd.it.

(^1)
D. Marvulli, P. Braghetta, S. Piccolo, C. Fabbro, P. Bonaldo, D. Volpin, and G. M. Bressan, manuscript in preparation.

(^2)
The abbreviation used is: CAT, chloramphenicol acetyltransferase.


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