From the Institute of Histology and Embryology, University of Padova, 35100 Padova, Italy
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Analysis of the chromatin of different cell types
has identified four DNase I-hypersensitive sites in the 5'-flanking
region of the Collagens are the most abundant extracellular matrix proteins of
vertebrates (1). 19 types have been characterized so far, differing in
structural features and tissue distribution. In addition to maintaining
the structural integrity of organs, collagens endow tissues with
peculiar mechanical and biological properties depending on the pattern
and the levels of expression. For this reason the regulation of
expression is a key issue in collagen biology. For most collagen genes,
transcription is the major regulatory step, and analyses of
cis- and trans-acting elements have been obtained mainly for We have recently undertaken a study of the regulation of transcription
of the Isolation of Nuclei and Analysis of DNase I-hypersensitive Sites
in the Chromatin--
NIH3T3 and C2C12 cell lines were propagated in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum. EL4 lymphocytes were grown in RPMI 1640, 10% fetal calf serum, 4 mM glutamine, and 20 µM
For each digestion, 5 A260 nuclei were adjusted
to a volume of 80 µl with buffer 4, and 10 µl of DNase I assay
buffer (400 mM Tris-HCl, pH 7.5, 60 mM
MgCl2) and 10 µl of DNase I (Sigma) diluted to a
concentration of 0-6 units/µl were added. The samples were incubated
at 37 °C and the reaction interrupted with 200 µl of stop buffer
(50 mM Tris-HCl, pH 8.0, 100 mM EDTA, 100 mM NaCl, 1% SDS). The nuclei were then treated with 3 µl
of RNase (50 µg/ml) at 37 °C for 1 h followed by the addition
of 40 µl of proteinase K (20 mg/ml) at 37 °C for one night under
mild agitation. The DNA was extracted by phenol/chloroform and
precipitated by adding 1 volume of isopropyl alcohol and 0.1 volume of
5 M NaClO4. After centrifugation, the DNA was
suspended in 10 mM Tris-HCl, pH 8.0, and 1 mM
EDTA. 5 µg of DNA was digested with the selected restriction
endonuclease and run in a 0.8% agarose gel. The DNA fragments were
transferred into nylon filters (GeneScreen Plus, NEN Life Science
Products) and hybridized with an appropriate 32P-labeled
probe (18).
DNA Constructs--
The cloning of fragments Generation and Analysis of Transgenic Mice--
lacZconstructs
were microinjected into fertilized B6D2F1 × B6D2F1 mouse oocytes
and the developing embryos analyzed at E14.0-E15.5. Transgenic embryos
were identified by dot-blot assay of DNA purified from the yolk sac,
and the transgene copy number analysis and histological examination for
Promoter Assays--
NIH3T3 and C2C12 cells were grown as
described above; 3 × 105 cells were plated into 10-cm
Petri dishes and transfected the following day with the CAT plasmids
using the calcium phosphate method (21). All subsequent manipulations
and assays were performed as detailed previously (16).
DNase I Footprinting--
The fragment Electrophoretic Mobility Shift Assay--
The synthesized
double-stranded oligonucleotides used included AP1-Col6a1, which
encompasses nucleotides Identification of a DNase I-hypersensitive Site Proximal to the
Basal Promoter of the Col6a1 Gene--
Given the frequent association
of DNase I-hypersensitive sites with regions that control
transcriptional regulation (9, 10), the hypersensitive sites located
within 7.5 kb of the 5'-flanking sequence of the Col6a1 gene
were identified. Because the presence of hypersensitive sites is
usually related to the state of transcriptional activity of a gene in a
given cell type, mapping was carried out in three cell lines that
express different levels of Characterization of the Region Corresponding to HS1--
The
hypersensitivity of chromatin to nucleases is caused by structural
features of chromatin brought about by assembly of nuclear factors at
defined sequence elements (9, 10). In a previous paper we showed that
several nuclear factors bind to nucleotides Role of the AP1 Binding Site and of the Core Promoter in
Tissue-specific Transcription in Vivo--
In previous papers we have
reported on transient transfections carried out with various CAT-Col6a1
promoter constructs (13, 16). A comparison of CAT expression from
plasmids p215CAT and p82CAT, which contain and lack the AP1 binding
site, respectively, suggested an activating role of the site. However,
the same plasmids, or similar constructs carrying the E. coli
lacZ instead of the CAT gene, were not expressed in mouse
transgenic lines, so that the function of the AP1 site in
vivo could not be determined (12, 13). To overcome this difficulty
the constructs of Fig. 4A were designed, with the rationale that the presence of the enhancer containing region Context and Cell Type Dependence of Function of the Core Promoter
and of the AP1 Binding Site--
The data reported in the preceding
paragraph suggest that the AP1 binding site is absolutely required for
transcription in some tissues in vivo and that expression in
different tissues changes when the core promoter is replaced by a
heterologous one. However, a quantitative evaluation of the stimulatory
activity of each element was not possible. In addition, the results did not allow analysis of the function of the sequences in the absence of
the The results described in this paper contribute substantial
information on the function of the proximal promoter region of the
Col6a1 gene. A DNase I-hypersensitive site (identified as HS1) was localized in the chromatin at about DNase I footprinting and band-shift assays have located a recognition
site for transcription factor AP1 at Analysis of transgenic mice carrying promoter-lacZ
constructs has shown that the frequency of expressing lines and the
average level of expression in the lines are variously affected by the AP1 binding site in different tissues. Both parameters are particularly dependent on the presence of the AP1 site in subepidermal mesenchyme, at the insertion of the superficial muscular and aponeurotic system, and in tendons. The frequency parameter can be attributed to the capacity of a cis-acting region to make chromatin accessible
to the transcriptional machinery, indicating that AP1 has an important structural role in these tissues. This function of the AP1 site is
clearly evident also in vibrissae, where the frequency, but not the
intensity, was greatly stimulated. The level of expression of a
transgene probably depends on the activating potential of the
cis-acting elements, i.e. the ability of the
factors binding to DNA modules to recruit the transcription
preinitiation complex (8). Our data lead us to conclude that AP1 is a
strong activator of transcription in cells of subepidermal mesenchyme,
at insertions of the superficial muscular and aponeurotic system, and
tendons. On the contrary, the AP1 site does influence only marginally
both the frequency and the intensity of expression of transgenes in cells of the peripheral nervous system. To explain the independence of
frequency from the AP1 site it may be hypothesized that, in the
peripheral nervous system, either the function of the site is replaced
by another site not active, and hence not detected, in the cell
cultures we have used, or opening up of chromatin is almost completely
dependent on the upstream enhancer region. An intermediate situation is
apparent in the remaining tissues, articular cartilage and
intervertebral discs, where the AP1 site increases to some extent the
frequency and intensity of expression. The in vivo data also
suggest a role for the core promoter in tissue-specific transcriptional
regulation of the Col6a1 gene, in a way similar to that of
the AP1 binding site. In fact, expression of transgenes in tendons and
at the insertions of superficial muscular and aponeurotic system was
more evident with the Col6a1 promoter than with the
In a previous report we located the region inducing transcription in
tendons and at the insertions of the superficial muscular and
aponeurotic system within 0.6 kb upstream from the RNA start site (12).
The new results point at the AP1 binding site as an important element
contributing to activation of transcription in these tissues. In the
same paper, the modules responsible for transcription in the
subepidermal mesenchyme were assigned to the The complexity of the mechanisms of tissue-specific regulation of the
Col6a1 gene observed in vivo was defined further
in transfections in vitro. The quantitative analysis of the
results leads to a conclusion similar to that of the in vivo
data: the levels and the features of transcriptional activation in
different cell types depend on the specific interactions among the core promoter, the proximal activating region, and the enhancer region. Four
distinct types of interaction could be identified by the data reported
in Table II, as outlined in Fig. 5. In
C2C12 cells the AP1 site did not interact positively with the
1(VI) collagen gene, mapping at
4.6,
4.4,
2.5,
and
0.1 kilobase (kb) from the RNA start site. The site at
2.5 kb was independent from, whereas the other three sites could be related to,
1(VI) mRNA expression. The site at
0.1 kb was present in cells expressing (NIH3T3 and C2C12) but absent in cells not expressing (EL4) the mRNA; the remaining two sites were apparently related with high levels of mRNA. DNase I footprinting and gel-shift assays with NIH3T3 and C2C12 nuclear extracts have located a binding site for
transcription factor AP1 (activator protein 1) between nucleotides
104 and
73. When nuclear extracts from EL4 lymphocytes were used,
the AP1 site-containing sequence was bound by proteins not related to
AP1. The existence of the hypersensitive site at
0.1 kb may be
related to the binding of AP1 and of additional factors to the core
promoter (Piccolo, S., Bonaldo, P., Vitale, P., Volpin, D., and
Bressan, G. M. (1995) J. Biol. Chem. 270, 19583-19590). The function of the AP1 binding site and of the core
promoter in the transcriptional regulation of the Col6a1 gene was investigated by expressing several promoter-reporter gene
constructs in transgenic mice and in cell cultures. The results indicate that regulation of transcription of the Col6a1
gene by different cis-acting elements (core promoter, AP1
binding site and enhancers) is not completely modular, but the final
output depends on the specific interactions among the three elements in
a defined cell type.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1(I),
2(I), and
1(II) genes (2-7 and references therein). Several types of regulatory regions necessary for high level
transcription have been identified in collagen genes. As for other
genes, these include the core promoter, which comprises sequence motifs
usually within
40 and +40 nucleotides from the RNA start site and may
or may not include a TATA box motif; the proximal upstream activating
region, which extends from about
50 to
200 base pairs from the RNA
start site and contains recognition sites for a subgroup of
sequence-specific DNA-binding transcription factors; and enhancers,
cis-acting DNA sequences that increase transcription in a
manner that is independent of their orientation and distance relative
to the RNA start site (8). Other important transcription control
regions, such as the locus control region, have not been identified yet
in collagen genes. The locus control region was recognized initially in
the
-globin gene cluster (9) and has now been characterized in
several other genes (8, 10). The locus control region is necessary to
convert an inactive locus to a state competent for transcription, a
condition detected by an increase in sensitivity of chromatin to
digestion by DNase I. Subsequent transcription ensues by additional
specific regulatory sequences, which, when active, usually introduce
additional DNase I-hypersensitive sites. For example, five
hypersensitive sites have been detected in the
-globin locus control
region (10), and additional hypersensitive sites are located close to
the core promoter of transcribed genes (9). Although, as stated above, no locus control regions have been defined yet, a correspondence between hypersensitive sites and actual transcription has been found
also for collagen genes, in particular
2(I) and
1(I) (5, 11). As
for the manner in which the different cis-acting regulatory elements contribute to the transcriptional regulation of a collagen gene, the available data suggest that they act in a modular way (4, 5,
12, 13). As proposed recently by Arnone and Davidson (14), this means
that each region contributes "a particular regulatory function that
is a subfraction of the overall combined regulatory function executed
by the complete system" independently from the other regions. A
corollary of this view is that tissue specificity of transcription is
contributed by enhancers and is independent of the core promoter, whose
function is the assembly of the basal transcription apparatus; hence,
in experiments with transgenic animals, promoter-reporter gene
constructs are expected to give rise to the same temporal and spatial
pattern of expression whether using the homologous or a heterologous
promoter. The few experiments addressing this issue for collagen genes
confirm the above prediction (5).
1 chain of type VI collagen, a gene that has been linked to
Bethlem myopathy in humans (15). These studies have identified several
regulatory regions within the 7.5 kb1 of 5'-flanking sequence,
including the basal promoter; module(s) activating expression at low
levels in tendons and at high levels at the insertions of the
superficial and muscular aponeurotic system within about 600 bases from
the transcription start site; enhancer modules for transcription in
articular cartilage, intervertebral discs, vibrissae, the peripheral
nervous system, and subepidermal mesenchyme, located between about
5.4 and
4.0 kb; and region(s) stimulatory for transcription in
articular cartilage, intervertebral discs, meninges, and skeletal
muscle between
7.5 and
6.2 kb (12, 13, 16). In this paper we have
identified several DNase I-hypersensitive sites in the 5'-flanking
region of the gene. One of these sites, located at about
0.1 kb from
the transcription initiation site, is detectable only in cells
expressing collagen VI mRNA and contains a recognition motif for
the transcription factor AP1. Analysis of the function of the AP1 site
in vitro and in vivo in the context of the
homologous and of a heterologous promoter indicates that both the AP1
site and the core promoter play an important role in the regulation of
tissue-specific transcription of the Col6a1 gene.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-mercaptoethanol. Nuclei were prepared from six large plates
(22.5 × 22.5 cm) of confluent NIH3T3 or C2C12 cells and from 600 ml of EL4 lymphocytes (44 × 105 cells/ml) as
described (17) with minor modifications. Adherent cells were washed
extensively with phosphate-buffered saline, scraped in the same buffer
using a Cell-Lifter (Costar), and harvested by centrifugation for 10 min at 400 × g. EL4 cells were collected by
centrifugation, resuspended twice in phosphate-buffered saline, and
centrifuged. The packed cell volume was measured and cells resuspended
in 10 × packed cell volumes of buffer 1 (15 mM
Tris-HCl, pH 7.5, 15 mM NaCl, 60 mM KCl, 1 mM EDTA, 0.5 mM EGTA, 1.9 M
sucrose, 0.1% Triton X-100, 0.5 mM spermidine, 0.15 mM spermine, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each
pepstatin, leupeptin, and aprotinin). Cells were then lysed in a Dounce
cell homogenizer with five strokes of a type B pestle, 10 packed cell volumes of buffer 2 (buffer 1 without Triton X-100) were added, and the
refractive index of the suspension was adjusted to 1.40-1.42 with
buffer 3 (buffer 1 without Triton X-100 and sucrose). The samples were
centrifuged at 12,000 × g for 10 min and resuspended in 5 packed cell volumes of buffer 4 (buffer 1 lacking EDTA, EGTA, and
Triton X-100 but containing 0.34 M sucrose). The absorbance of 5 µl of the suspended nuclei was measured at 260 nm. The nuclei were pelleted again at 12,000 × g for 10 min and
resuspended in buffer 4 at an absorbance of about 0.2 A260/ml. All manipulations were carried out at
4 °C.
82/+41 and
215/+41, which include the indicated nucleotides from the
transcription start site of the Col6a1 gene into pGEM3
vector and the fusion of the fragments into plasmid pBL6CAT to derive
p82CAT and p215CAT, was described previously (16). Both plasmids
contain the core promoter, which extends from nucleotides
75 to +25.
To obtain pEn82CAT and pEn215CAT, the BamHI-EcoRI
fragment extending from
5.4 to
3.9 kb (19), which acts as a strong
enhancer for expression in a specific set of tissues (12), was cloned
into p82CAT and p215CAT upstream of the promoter region. Similar fusion
constructs with the Escherichia coli lacZ gene replacing the
CAT gene were synthesized starting from the promoterless plasmid
pNSlacZ, in which the
-galactosidase sequence is fused with the
nuclear localization signal of SV40. First, the
82/+41 and
215/+41
fragments were inserted into pNSlacZ to give p82lacZ and p215lacZ, and
then the
5.4/
3.9 enhancer region was cloned upstream of the
promoter fragments to produce pEn82lacZ and pEn215lacZ. A homologous
set of CAT and lacZ constructs was also synthesized, where the human
-globin substitutes for the Col6a1 gene promoter. The
steps in the synthesis of these vectors were the release of the
fragment
215/+41 from pEn215CAT or pEn215lacZ and the cloning, in its
place, of a fragment from pBGZA, which contains sequences from
37 to
+12 of the human
-globin gene (20), thus obtaining pEn
GCAT and
pEn
GlacZ. The AP1 binding site of Col6a1 gene was added
to these plasmids by amplifying the region from
71 to
124 of
p215CAT by polymerase chain reaction and ligating it between the
enhancer region and the
-globin promoter. The resulting vectors were
identified as pEnAP1
GCAT and pEnAP1
GlacZ. Finally, the
-globin
promoter from pBGZA and the fragment containing the AP1 site fused with
the
-globin promoter from pEnAP1
GCAT were cloned into pBL6CAT to
give p
GCAT and pAP1
GCAT. All plasmids were purified by CsCl
gradient centrifugation and sequenced to verify correct cloning.
-galactosidase expression were carried out exactly as described
(12).
215/+41 was labeled at
either end with 32P-dNTPs and Klenow enzyme and purified by
agarose gel electrophoresis (22). DNase I digestion and electrophoretic
analysis of the products were carried out using established protocols
(16).
104/
73 of the
1(VI) chain gene promoter
(19); AP1-cons (5'-AAGCATGAGTCAGACAT-3'), which contains the binding
consensus sequence of AP1 (23); and AP1-mut, in which bases 10 and 11 of the previous oligonucleotide (TC) were mutated to GG. Purification
of nuclear extracts from NIH3T3 and C2C12 and EL4 cells, labeling of
oligonucleotides with [32P]ATP, and assay procedures were
as described (16). Antibodies against c-Fos, c-Jun, JunB, JunD, and
Fra-1 for supershift experiments were purchased from Santa Cruz
Biotechnology Inc., and 0.1-0.4 µl was used in each reaction.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1(VI) mRNA. These lines include
NIH3T3 fibroblasts, in which the steady-state concentration of mRNA
is the highest; C2C12 myoblasts, which contain about 10-fold less
mRNA; and the T cell line EL4, in which the mRNA is
undetectable (data not shown). Isolated nuclei were treated with DNase
I, and the purified DNA was digested with either SphI or
BamHI and analyzed by Southern blotting. Fig.
1 shows the results obtained after
digestion of DNA with SphI, but similar results were
observed after treatment with BamHI (data not shown). In addition to the 9-kb SphI-SphI fragment, the
probe hybridized with four other bands in NIH3T3 cells. One band of
about 4 kb (labeled * in Fig. 1) was similarly present in C2C12 and EL4
cells and was therefore not related to the level of expression of the
1(VI) mRNA. The corresponding hypersensitive site, which maps at
about
2.5 kb from the RNA start site, is probably caused by a region
of chromatin constitutively susceptible to DNase I digestion and not
dependent on the state of transcription of the gene, as described for
some DNase I-hypersensitive sites in the Col1a1 gene (11). A
broad band at about 6 kb was very strong in NIH3T3 fibroblasts, very
faint in C2C12 cells, and absent in EL4 lymphocytes. Rehybridization of
the filter with a probe located at the 5'-end of the
SphI-SphI fragment revealed that the
hybridization signal was composed by two bands (data not shown) and was
therefore marked HS2 and HS3 in Fig. 1. The characterization of these
two hypersensitive sites, which map at about
4.4 and
4.6 kb and
were associated with high expression of
1(VI) mRNA, will be
described in a separate report.2 Finally, the band of
about 1.6 kb was distinctive of cells expressing
1(VI) mRNA
because it was lacking in nonexpressing EL4 T cells. This band
corresponded to a hypersensitive site located at about
0.1 kb (HS1 in
Fig. 1).
View larger version (64K):
[in a new window]
Fig. 1.
Identification of chromatin DNase
I-hypersensitive sites in the 5'-flanking region of the Col6a1
gene. The region extends from about 7.5 kb upstream
(5'-SphI site) to 2 kb downstream (3'-BamHI site)
from the transcription start site (horizontal arrow).
Upper panel, summary of the location of the hypersensitive
sites found in different cell lines. The sites are indicated by
vertical arrows. p, probe used for Southern
blotting analysis shown in the lower panel. Lower
panel, Southern blotting analysis of DNA extracted from nuclei
from different cell lines, treated with various amounts of DNase I,
digested with SphI, and hybridized with probe p defined in
the upper panel. * points to a hypersensitive site
detectable in all cells analyzed, irrespective of the expression of
1(VI) mRNA. HS1, HS2, and HS3 label the position of
hypersensitive sites present only in cells expressing the mRNA. The
sites are located at
0.1,
4.4, and
4.6 kb, respectively.
M, DNA markers.
75 to +8 from the RNA
start site (16), a region that partially overlaps with the
Col6a1 core promoter (see "Discussion"). To locate other
possible transcription factors binding sites close to the region where
HS1 maps, DNase I footprinting assays were carried out with a probe
spanning nucleotides
215 to +41 and nuclear extracts from NIH3T3
cells. One protected sequence was identified extending from
104 to
73 (Fig. 2, upper panel).
The sequence contained the core motif of the binding site for
transcription factor AP1 (TGAG/CTC/AA) (Fig. 2, lower panel)
(23). Actual binding of AP1 to the protected sequence was tested by
gel-shift assay, in which a probe including the AP1 site of the
Col6a1 gene gave rise to one retarded band in the presence
of proteins isolated from NIH3T3 nuclei (Fig.
3A). The formation of the band
was inhibited by the cold oligonucleotide (lanes labeled
AP1-Col6a1 in Fig. 3A) and by an oligonucleotide
with the consensus sequence of the AP1 binding site (22) (AP1-cons in
Fig. 3A), but not by an oligonucleotide with a mutated
version of the consensus motif (AP1-mut in Fig. 3A).
Supershift assays with antibodies against the molecular components of
AP1 factor c-Fos, Fra-1, c-Jun, JunB, and JunD revealed that the
complex contained JunD (Fig. 3B). A retarded band with
similar characteristics was detected with nuclear extracts purified
from C2C12 cells (data not shown). On the contrary, the retarded bands produced by EL4 nuclear extracts with the AP1-Col6a1 probe had completely different properties: they were not competed by the AP1-cons
oligonucleotide (Fig. 3C), and none of the antibodies mentioned above induced supershifting (data not shown). Parallel gel-shift experiments using the AP1-cons oligonucleotide as probe were
also performed. These experiments established that the band retarded by
incubation with NIH3T3 or C2C12 nuclear extracts was competed by both
AP1-cons and AP1-Col6a1 oligonucleotides and that the band was
supershifted only by antibodies against junD (data not shown).
Incubation of the AP1-cons probe with EL4 nuclear proteins produced one
major band that was competed by cold oligonucleotide AP1-cons and,
unexpectedly also by AP1-Col6a1 (Fig. 3D). The band was
supershifted by antibodies to fra-1 and junD (data not shown). These
results suggests that the AP1 recognition site of the Col6a1 gene has the potential to bind AP1 complexes of EL4 cells, although, as
shown in Fig. 3C, direct binding could not be detected.
View larger version (68K):
[in a new window]
Fig. 2.
DNase I footprinting analysis of the region
extending from 215 to +41 base pairs from the transcription start
site. Upper panel, separation of DNA fragments in
denaturing 8% polyacrylamide gel. 100-20 ng and 5-0.4 ng of DNase I
were added to samples with and without nuclear extract, respectively.
50 µg of nuclear extract purified from NIH3T3 cells was used in the
indicated reactions. The protected sequences are indicated by
slashed boxes. Lower panel, sequence of the
Col6a1 gene promoter spanning the protected region. The
lines over and under the nucleotide sequence represent the
protection of the coding and noncoding strand, respectively. The
sequence of the putative AP1 binding site is boxed.
View larger version (63K):
[in a new window]
Fig. 3.
Analysis of nuclear factors binding
using electrophoretic mobility shift assays. Double-stranded
oligonucleotide AP1-Col6a1, which spans the protected sequence
identified by DNase I footprinting in Fig. 2, bases 104 to
73, was
used as probe in panels A-C. Double-stranded
oligonucleotide AP1-cons, which contains the binding consensus sequence
of AP1 (23), was used as probe in panel D. 2-4 µg of
nuclear extracts from NIH3T3 cells (panels A and
B) or from EL4 lymphocytes (panels C and
D) was employed in each reaction. Competition assays
(panels A, C, and D) were carried out
with cold oligonucleotide AP1-Col6a1 and with oligonucleotides AP1-cons
and AP1-mut, which contain inactivating mutations of the consensus
binding sequence for AP1. For supershift experiments (panel
B), either preimmune Ig (p-Ig) or the indicated
antibodies against the molecular components of AP1 were added to the
reaction mixture.
5.4 to
3.9 (12) would overcome silencing of the
basal promoter, with or without the AP1 site, in vivo. Moreover, to test whether or not the function of the AP1 site and of
the enhancer region was dependent on the type of basal promoter, the
constructs depicted in Fig. 4B were synthesized, in which
the
-globin promoter, which contains a TATA box, replaced the core
promoter of Col6a1, which lacks a TATA box. The four constructs were microinjected into fertilized oocytes, and
-galactosidase expression was examined in the founder transgenic
embryos. The presence of the AP1 binding site increased the percentage
of expressing mouse lines, and the effect was particularly relevant
(3-fold) with the constructs containing the
-globin basal promoter
(Fig. 4). Although the pattern of expression of the transgenes
resembled that described previously (12), the histological analysis
revealed interesting functional features of the AP1 binding site (Table I). The parameters considered to estimate
the effect of the AP1 site were the percentage of lines expressing in
one particular tissue over the total of expressing lines (frequency)
and the average level of expression attained in expressing lines
(intensity), evaluated by the relative amount of
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside-positive nuclei in a defined tissue on an arbitrary scale as defined previously (12). The presence of the AP1 site was particularly important for
expression at high frequency and high intensity in subepidermal mesenchyme, insertion of superficial and muscular aponeurotic system,
and tendons with both the
-globin and the
1(VI) promoter. A
stimulating effect of the AP1 site on frequency and intensity was also
noted in articular cartilage and intervertebral discs with both
promoters. The site was also required for high frequency expression in
vibrissae. In the peripheral nervous system expression from the
Col6a1 gene promoter was increased slightly by the AP1; on
the contrary, expression was apparently not different with or without
the AP1 site for constructs carrying the
-globin promoter. The data
of Table I also show that expression in different tissues was dependent
on the core promoter. Thus, compared with the
1(VI) promoter, the
-globin promoter was less efficient in tendons, insertions of
superficial and muscular aponeuroses, articular cartilage, and
intervertebral discs; however, it induced a higher frequency of
expression in the peripheral nervous system.
View larger version (22K):
[in a new window]
Fig. 4.
Constructs used to generate
transgenic mouse lines to analyze the function of the core promoter and
of the AP1 binding site of Col6a1 gene in
vivo. All of the constructs include the enhancer region of
the Col6a1 gene (En) identified previously (12),
which extends from about 5.4 to
3.9 kb from the RNA start site.
Constructs in panel A contain sequences of the
Col6a1 promoter indicated by the numbers;
therefore both constructs include the core promoter (nucleotides
75
to +25), whereas the AP1 binding site (nucleotides
104 to
73) is
present only in En215lacZ. Constructs in panel B contain the
human
-globin core promoter (
G) (nucleotides
37 to
+12); EnAP1
GlacZ contains, in addition, nucleotides
124 to
73 (AP1 box) of the Col6a1 promoter, which span the AP1
binding site. The fractions indicate the number of
expressing over the total of transgenic mouse lines produced. The
percentage is given in parentheses.
Histological analysis of expression of different promoter-lacZ
constructs in vivo
-D-galactopyranoside.
Dot-blot assays of the DNA purified from the yolk sacs were performed
to identify transgenic embryos and to determine the transgene copy
number (12). The intensity of
-galactosidase staining was evaluated
by microscopic examination of serial sections on an arbitrary scale as
described (12). Only tissues for which the presence of activating
sequences in the 5'-flanking region of the Col6a1 gene was
previously clearly established (12) have been considered.
5.4/
3.9 enhancer region, which was necessary for expression in vivo. These issues were addressed by transient promoter
assays in cultured cell lines. The constructs used were similar to
those described in Fig. 4 but carried the CAT instead of the
lacZ gene. Four additional constructs lacked the upstream
enhancer region
5.4/
3.9 and contained only the
-globin or the
1(VI) basal promoter with or without the AP1 site (the constructs
are defined under "Experimental Procedures"). The cell cultures
chosen were NIH3T3, in which DNase I HS2 and HS3 were very strong (Fig.
1), and C2C12, in which HS2 and HS3 were barely detectable (Fig. 1). The results are shown in Table II. In
constructs with the
-globin promoter the AP1 site did not increase
transcription in the absence of the enhancer region (compare p
GCAT
with pAP1
GCAT) in both NIH3T3 and C2C12 cells. When the enhancer
region was added, transcription was only slightly (2-3-fold) increased
in C2C12 myoblasts with or without the AP1 site (compare p
GCAT with
pEn
GCAT and pEnAP1
GCAT), suggesting that the only activating
interaction was between enhancer and promoter. On the contrary, in
NIH3T3 fibroblasts the enhancer region stimulated transcription about
20-fold in the absence (compare p
GCAT with pEn
GCAT) and 80-fold
in the presence (compare p
GCAT with pEnAP1
GCAT) of the AP1 site.
In this case the mutual interactions among the AP1 binding site, the
enhancer region, and the
-globin promoter can be defined as
synergistic, because transcription elements synergize when their
combination produces a transcriptional rate that is greater than the
sum of the effects produced by individual elements. In our experiments
the amount of transcription reached in the presence of the three
elements was 3.5-fold greater (fold synergism) than the sum of the
effects produced when the
-globin promoter was combined separately
with either the AP1 site or the enhancer region. The results were
completely different with constructs containing the basal promoter of
the Col6a1 gene. Transcription from enhancerless constructs
was stimulated about 5-6-fold in both NIH3T3 and C2C12 cells by the
AP1 site (compare p82CAT with p215CAT). The enhancer region increased
transcription 7-fold in C2C12 myoblasts (compare p82CAT and pEn82CAT),
and the presence of both elements, AP1 site and enhancer region,
resulted in an additive stimulation of about 12-fold (compare p82CAT
with pEn215CAT). On the other hand, expression of pEn82CAT and
pEn215CAT was similar in NIH3T3 fibroblasts, suggesting that the
stimulating function of the AP1 binding site was abolished in the
presence of the
5.4/
3.9 enhancer region in these cells.
Role of core promoters, AP1 site, and 5.4/
3.9 enhancer region on
transcription in different cell types
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
0.1 kb from the transcription initiation site. HS1 was detectable only in cell lines
that express
1(VI) collagen mRNA, suggesting that a
rearrangement of the chromatin structure in the proximal 5'-region is a
necessary condition for transcriptional activation of the gene. The
analysis of DNase I-hypersensitive sites also suggested that distinct
levels of expression in different cell types were achieved by
additional rearrangements of the chromatin at other sites. Thus, high
amounts of mRNA were detected in NIH3T3 fibroblasts, where three
hypersensitive sites were easily detectable at
4.6,
4.4, and
0.1
kb, whereas 10-fold lower levels of
1(VI) mRNA were found in
C2C12 myoblasts, where the site at
0.1 kb was strongly and the other
two sites very weakly sensitive to DNase I. Regions containing a
defined set of DNase I-hypersensitive sites in chromatin are usually
required for position-independent transcription of transgenes in
vivo (9, 10). When tested alone, sequences corresponding to HS1
were completely inadequate to overcome the constraints of chromatin structure. On the other hand, they improved the function of other sites, as indicated by the relative increase of mouse transgenic lines
expressing the lacZ reporter gene (from 54 to 87% for
constructs of Fig. 4A and from 23 to 75% for those of Fig. 4B) when
the AP1 binding site was present. As indicated by the high percentage of expressing lines in Fig. 4, hypersensitive sites HS2 and HS3 are
very efficient in making chromatin transcriptionally competent at the
site of insertion of transgenes. However, the data also point out that
the hypersensitive sites detected were not sufficient for complete
independence of transgene expression from the insertion site.
Therefore, additional regulatory sequences and DNase I-hypersensitive sites should be identified to understand fully the transcriptional regulation of the Col6a1 gene.
104 to
73 base pairs, close to
where HS1 maps, suggesting that this site and probably the GA
box-containing sequences identified previously between
75 and +8 (16)
play an important role in determining DNase I hypersensitivity of
chromatin. An AP1 binding site proximal to the basal promoter is a
conserved feature of the Col6a1 gene since the site has been
found also in chicken and in human (24, 25). In addition, a similar
element was recognized in the chicken
2(VI) collagen gene (26),
suggesting that an AP1 binding site may be a key element in the
regulation of collagen VI genes. In NIH3T3 and C2C12 cells, which
express the
1(VI) mRNA, the site was actually bound by an AP1
factor complex containing JunD. In contrast, in nuclear extracts from
EL4 cells, which do not express the
1(VI) mRNA, the same
sequence was recognized by factor(s) not related to AP1, although the
cells contain various molecular forms of the AP1 transcription factor.
An obvious speculation stimulated by these results is that the presence
or absence of DNase I HS1 may be determined by the difference of
nuclear factor binding at sequences including the AP1 site. One
possibility is that the AP1 factors of EL4 lymphocytes bind with low
affinity to the Col6a1 gene promoter, whereas the molecular
form(s) present in NIH3T3 and C2C12 cells have high affinity for the
site. Differences in the molecular composition of AP1 binding to
distinct promoters have already been observed in various cell types
(27). Alternatively, EL4 cells might contain peculiar transcription
factors that are absent in the other cells and compete with AP1 protein
for binding to the site. Future studies will elucidate this issue.
-globin promoter. Conversely, the frequency of expression in the
peripheral nervous system was higher with the
-globin promoter. The
core promoter of the Col6a1 gene was partially characterized
in previous work and was shown to exhibit several unusual features
among the TATA-less promoters (16, 19). The RNA start sites are spread
on a sequence of more than 70 base pairs, and the most upstream site
has been denoted as +1. The major transcription initiation site is at
base +21 and a second strong site at base +9. These sites resemble, but
do not match exactly the consensus sequence proposed for the initiator element (+21 site: 8Py C A+1 G C 3Py; +9 site: 9Py
G+1 G C T 8Py; consensus sequence for initiator: Py Py
A+1 N T/A Py Py; where Py indicates a pyrimidine) (28).
Because it has been noted that a large number of pyrimidines
surrounding the start site can impart low levels of initiator activity
in the absence of either the A at +1 or the T at +3 (29), it is very
likely that the sequences around +21 and +9 constitute weak initiators.
These initiator elements, however, do not drive transcription unless
they are linked to an upstream sequence, containing repeated GA boxes,
which extends from
75 to +8 (16). This region has intrinsic promoter
activity, as suggested by the observation that the fragment
82 to +41
is equally active in both
orientations,3 a property not
shared by initiators (28). Considering both the putative initiator
sites and the GA box-rich region, the core promoter of the
Col6a1 gene extends from
75 to +25, a sequence that
closely corresponds to that used to synthesize our Col6a1 core promoter constructs (
82/+41).
5.4/
3.9 enhancer
region. The present data show that expression in this tissue is
strongly dependent on the homologous promoter and on the presence of
the AP1 binding site. Thus, it may be speculated that transcription in
the subepidermal mesenchyme requires a synergistic action of the three
regulatory elements: the core promoter, the AP1 site, and the enhancer
region. The overall message coming from the in vivo
experiments is, therefore, that transcription in different tissues
depends on a peculiar interplay among the three regulatory elements.
3.9/
5.4 kb enhancer region (Fig. 5, A and B).
When the
-globin promoter was used, the only interaction was between
the promoter and the enhancer (Fig. 5A). On the other hand,
the homologous promoter was stimulated by both the AP1 site and the
enhancer, and the final induction of transcription achieved with the
three modules together was the sum of those obtained from the separate
combinations of the promoter with the other modules (Fig.
5B). A completely different situation was apparent in NIH3T3
cells. The use of the
-globin promoter resulted in a synergistic
activation of about 3.5-fold when all of the modules were present. The
synergism can be explained by assuming that the protein complex
assembled at each module interacted positively at the same time with
those brought together by the other modules as indicated in Fig.
5C. By replacing the TATA-containing
-globin promoter
with the TATA-less promoter of the Col6a1 gene, synergism
did not take place, and a fourth type of interaction of modules was
observed, tentatively identified as competitive (Fig. 5D).
Namely, although the AP1 site stimulated considerable transcription
from the promoter, expression in the presence of the enhancer was
similar with or without the AP1 site. This condition can be accounted
for by hypothesizing that an activating interaction takes place between
the homologous promoter and the AP1 site if the enhancer is inactive
(or absent) and that this interaction is disrupted when the enhancer is
turned on and binds to the core promoter.
View larger version (46K):
[in a new window]
Fig. 5.
Representation of different types of core promoter,
enhancer region, and AP1 binding site interactions identified in
transfection experiments with different cells. The three regulatory
elements are bound by specific protein complexes: the core promoter,
either from -globin (
G) or from the Col6a1
gene (Col6a1), is depicted in association with the basal transcription
apparatus (BTA); the AP1 binding site is occupied by a
molecular form of the AP1 transcription factor containing JunD; the
enhancer region from
5.4 to
3.9 of the Col6a1 gene
(En) is hypothesized to bind a cell type-specific
enhanceosome (33). In panel D the two mutually exclusive
interactions of the BTA are represented: when the enhancer region is
inactive or absent, the AP1 factor binds to the BTA (dashed
line); this interaction is disrupted when an active enhancer
region binds to the BTA (solid line). For definition of
various types of interactions, see "Discussion" and Table II.
The model of Fig. 5 differs considerably from the view of DNA
regulatory elements acting in a modular way to control transcription deduced from studies on expression of transgenes in vivo by
several authors including ourselves (2-5, 12, 14). The modularity of
the function of cis-acting elements in these reports only
applies to the fact that, for genes expressed in several tissues, such as most collagens, different enhancer regions activate transcription in
specific subsets of tissues. A closer look at the function of the
regions involved, however, shows the existence of more complex
interactions among regulatory elements, which may explain peculiar
features of a gene's regulation. One example is enhancer-promoter selectivity, in which the activation of only one of multiple promoters by a nearby enhancer depends on cognate interactions between the two
elements (8, 30). As for our experiments, these results provide
evidence that different core promoters possess distinct regulatory
activities. The model of Fig. 5 is also consistent with the present
knowledge on the molecular mechanisms of transcription activation, in
which the final result is the consequence of specific interactions of
protein complexes bound to different cis-acting regulatory
elements. In a simplified view, the core promoter associates with the
general transcription factors (31), whereas activators are bound at
proximal activating sequences or at enhancers. Enhancers are usually
made of specific clusters of binding sites for nuclear factors, which
impose a precise alignment of the proteins on the DNA, resulting in the
formation of a stable, highly stereospecific three-dimensional
nucleoprotein complex called enhanceosome (32, 33). The interaction
between the general transcription factors and the enhanceosome (or
single activators at isolated binding sites) then determines the
recruitment of the RNA polymerase II holoenzyme and the formation of a
stable preinitiation complex (33, 34). It is clearly apparent from this
model that any change in the composition of the three types of protein
complexes (general transcription factors, enhanceosome, and activators
bound at the proximal activating region) could influence RNA polymerase II recruitment. The molecular analysis of the interactions among the
cis-acting regulatory regions of the Col6a1 gene
in different cell types will require the delineation of binding of
general transcription factors to the core promoter and the
characterization of the protein complex assembled at enhancer regions.
These studies are presently in progress.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Peter W. J. Rigby for the gift
of the human -globin promoter, Dr. Miriam Zanetti for help in
Southern blotting analysis, Dr. Paolo Bonaldo for critical reading of
the manuscript, and Mauro Ghidotti for the maintenance of mouse colonies.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a grant from the Progetto Finalizzato Biotecnologie of the Italian CNR and by Grants E22 and E704 from Telethon-Italy.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 correspondence should be addressed: Institute of Histology
and Embryology, Via G. Colombo 3, 35100 Padova, Italy. Tel.:
39-049-827-6086; Fax: 39-049-827-6079; E-mail: bressan{at}civ.bio.unipd.it.
The abbreviations used are: kb, kilobase(s); AP1, activator protein 1; CAT, chloramphenicol acetyltransferase; HS, hypersensitivity site.
2 D. Girotto, P. Braghetta, C. Fabbro, P.Vitale, D. Volpin, and G. M. Bressan, in preparation.
3 S. Piccolo, unpublished observations.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|