(Received for publication, May 11, 1995)
From the
During differentiation of C2C12 myoblasts in vitro,
expression of 1(VI) collagen mRNA was transiently stimulated
severalfold. Promoter assays on cells transfected with chloramphenicol
acetyltransferase (CAT) chimeric constructs have identified a region of
the
1(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
1(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.
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- (Rossi et al., 1988;
Ritzenthaler et al., 1993; Inagaki et al., 1994).
Type VI collagen is composed of three genetically distinct
polypeptide chains, 1,
2, and
3, 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. (
)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-
and
-interferon is restricted to the
3 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 1(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
-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
1(VI)
collagen promoter during differentiation of a myoblasts cell line in vitro and have begun to define the molecular details of its
activation.
Figure 5:
Summary of the structural analyses of the
1(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.
Figure 1:
Expression of 1(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,
1(VI), and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes. The
histogram is a quantitative evaluation of
1(VI) mRNA levels and
was obtained by densitometry of specific bands and normalization to the
glyceraldehyde-3-phosphate dehydrogenase
signal.
Figure 2:
Expression of 1(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
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
pA
CAT
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.
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.
Figure 6:
The GA box motif is necessary for
induction of the 1(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-[
C]chloramphenicol formed/h/10
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 1(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.
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 (Sp1) 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 1(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.
This study has identified a region of the promoter of the
1 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 1(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
1(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 1(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.