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J. Biol. Chem., Vol. 275, Issue 23, 17381-17390, June 9, 2000
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From the Institute of Histology and Embryology, University of Padova, 35100 Padova, Italy
Received for publication, January 3, 2000, and in revised form, March 21, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The region extending from Genes of the extracellular matrix are very often among targets of
terminal differentiation programs. In most cases, expression of the
genes is the result of transcriptional regulation attained by
tissue-specific enhancers. Well characterized examples are genes such
as osteocalcin, collagen I, osteopontin, and bone sialoprotein in
osteoblasts, and collagen II and XI in chondroblasts. The exclusive transcription of osteocalcin and the high level expression of Our group has undertaken the study of regulation of collagen VI in the
mouse and has identified several sequences of the 5'-flanking region of
the Col6a1 gene active in transcriptional control (7-10). In particular, analyses in transgenic mice have located three regions
responsible for tissue-specific transcription at high frequency (8).
The 0.6 kb1 just upstream of
the transcription start site drives expression in the superficial and
muscular aponeurotic system and in tendons. A second fragment, from
Plasmid Constructs--
For an easier understanding of the
results reported in this work, some of the constructs described
previously were renamed. These include p5.4
To synthesize reporter plasmids with deletions of the
For linker-scanning analysis of the E-J segment, the
p1.4(LSX)CAT plasmids (where X is a number
comprised from 1 to 16) were synthesized. These plasmids carry
15-16-bp replacements, including a NotI restriction site,
within the E-J segment and were derived by PCR-based site-directed
mutagenesis using the E-J fragment cloned into pGEM7Z (Promega) as the
template. The forward and reverse primers were 35-36 bp in length and
comprised the replacing sequence at the 5'-end, followed by 20 bp of
E-J fragment corresponding to the sequence flanking the mutagenized
bases. PCR reactions were carried out on 100-pg pGEM7Z(E-J) as
template, using the Expand High Fidelity PCR System (Roche Molecular
Biochemicals) according to the protocol supplied by the manufacturer
and the following cycling program: 5 min at 94 °C; 35 cycles,
30 s at 94 °C, 1 min annealing at 60-70 °C depending on the
oligonucleotides couple used, 10 min and 30 s elongation at
68-70 °C; 7 min at 72 °C. The PCR-amplified products were run on
a 0.8% agarose gel, and after excision of the band, the DNA was
extracted and purified using QIAEXII Gel Extraction Kit (Qiagen). DNA
was then digested with NotI to generate compatible ends,
purified by agarose gel electrophoresis, and 50 ng were ligated
overnight at 16 °C in the presence of 3 units of T4 DNA ligase.
After transformation, clones with the mutation were identified by
NotI digestion and sequenced to confirm the introduction of
the linker sequence and to verify that errors were not introduced by
the PCR into the sequences of the E-J fragment. The mutagenized
inserts were excised by digestion with SphI and
HindIII and ligated into p1.4CAT digested with the same enzymes.
Trimers of oligonucleotides spanning portions of the E-J fragment were
obtained using double-stranded oligonucleotides containing the
sequences defined in Fig. 2 and 4 bp of complementary protruding ends
appropriately chosen in the various oligonucleotides to avoid the
formation of potential binding sites for transcription factors, as
monitored by using resources of the Transfac transcription factor data
base (18). 60 picomoles of individual oligonucleotides in 30 µl were
phosphorylated with polynucleotide kinase (10 units) in 50 mM Tris-HCl, pH 8.2, 10 mM MgCl2,
0.1 mM EDTA, 5 mM dithioerythritol, 0.1 mM spermidine, 3 mM ATP, for 40 min at
37 °C, and the enzyme was inactivated for 10 min at 70 °C. 1 µl
of 10× ligase buffer (660 mM Tris-HCl, pH 7.5, 50 mM MgCl2, 10 mM dithioerythritol, 10 mM ATP) and 1 µl (2 units) of ligase were added, and
the samples were incubated at 30 °C for 10 min. After treatment with
phenol/chloroform and ethanol precipitation, the DNA was resuspended in
25 µl of distilled water, 8 µl of 5× T4 polymerase buffer (50 mM Tris-HCl, pH 8.5, 15 mM
NH4SO4, 7 mM MgCl2, 0.1 mM EDTA, 10 mM 2-mercaptoethanol, 200 µg/ml
bovine serum albumin, and 0.2 mM each dNTP) and 1 µl (1 units) of T4 polymerase were added and the sample incubated for 10 min at 37 °C. After addition of 1 µl (2 units) of Klenow polymerase, incubation was extended for 10 min at 37 °C for 5 min at
room temperature and 5 min on ice, and the enzymes were then
inactivated at 70 °C for 10 min. The sample was separated by
electrophoresis in a 2% agarose gel and molecular species in the range
from 50 to 300 bp purified using QIAEX II Gel Extraction Kit (Qiagen).
The DNAs were cloned into the SmaI site of Bluescript KS+ using standard procedures (19). To identify clones with
the correct size (3× oligonucleotide), the inserts were released by cutting with BamHI and HindIII restriction
enzymes and separated in 12% polyacrylamide gels (20 cm long) in TBE
buffer (19). The gels were fixed in 10% ethanol, 0.5% acetic acid for
10 min with shaking, incubated with 300 ml of 0.17% (w/v)
AgNO3 for 20 min, and rinsed in 300 ml of distilled water.
100 ml of developer (3% NaOH, 0.1% formaldehyde) were added, slowly
swirled to disperse the cloudy precipitate that had formed, discarded,
and 300 ml of the same solution added. The gel was incubated at room
temperature with mild agitation until dark bands were apparent (usually
within 15 min) and rinsed and stored in 5% acetic acid. The expected structure of the constructs was confirmed by sequencing. The
oligonucleotide trimers were excised by BamHI and
HindIII and cloned into the SphI site of pBLCAT6
(20) after blunting ends of both insert and vectors with T4 polymerase
and Klenow enzymes to give constructs p1.4(Ix3)CAT to
p1.4(VIIIx3)CAT.
Cells--
Cells were used for RNA and chromatin structure
analysis, transient transfections, and production of nuclear extracts.
Established cell lines included NIH3T3 fibroblasts, C2C12 myoblasts,
EL4 lymphocytes (10), MC615 (21), and RCS (22) chondroblasts, SCT-1
(23) and RN22 (24) Schwann cells. The cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, with
the exception of EL4 cells, which were maintained in RPMI 1640, 10%
fetal calf serum, 4 mM glutamine, and 20 mM
2-mercaptoethanol. Primary cultures of sternal chondroblasts and tendon
fibroblasts from 16-day-old chick embryos were prepared and grown as
described (25, 26).
Transfections and Promoter Assays--
3 × 105
cells were plated into 10-cm Petri dishes and transfected the following
day with the CAT constructs and the control plasmid pRSV-luciferase (10 and 2 µg, respectively) using the calcium phosphate method (27). All
subsequent manipulations and assays were performed as described
previously (7).
EL4 lymphocytes were transfected by electroporation. 16 × 106 cells in 0.8 ml of phosphate-buffered saline containing
2 mM 2-mercaptoethanol were transferred into a 0.4 cm-path
cuvette; 16 µg of CAT construct and 2 µg of control plasmid
pRSV-luciferase were added, and the sample was pulsed in a Gene-Pulser
apparatus (Bio-Rad) set at 320 V, 500 microfarads. The cells were then
kept for a few minutes on ice, diluted in RPMI medium (see above), and
maintained in culture for 2 days. The cells were then harvested by
centrifugation and processed for CAT and luciferase assays (7).
Generation and Analysis of Transgenic Mice--
LacZ constructs
were microinjected into fertilized B6D2F1 × B6D2F1 mouse oocytes,
and the developing embryos were analyzed at embryonic day 14.4-16.5.
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 Other Assays--
Northern blotting, DNase I footprinting,
identification of chromatin DNase I-hypersensitive sites, and
electrophoretic mobility shift assays were performed exactly as
described (7, 10).
Analysis of Activating Properties of In order to define sequences of the 
5.4 to 3.9 kilobase
pairs from the transcription start site of the Col6a1 gene
has been previously shown to contain sequences activating
tissue-specific transcription in articular cartilage, intervertebral
disks, subepidermal, and vibrissae mesenchyme and peripheral nervous
system (Braghetta, P., Fabbro, C., Piccolo, S., Marvulli, D., Bonaldo,
P., Volpin, D., and Bressan, G. M. (1996) J. Cell
Biol. 135, 1163-1177). Analysis of expression of deletions of
this region in transgenic mice has identified the 383-base pair
fragment E-L as the most active sequence of the region.
Linker-scanning mutagenesis analysis of segment E-J, which spans the
5' 245 base pairs of E-L and is sufficient for high frequency
expression in articular cartilage, showed that all the mutations
reduced transcription considerably, suggesting that the integrity of
the entire cluster of elements is necessary for enhancer activity.
Electrophoretic mobility shift assays with nuclear extracts derived
from various sources showed that fragment E-J binds numerous
transcription factors (at least 22). These factors are present in most
cells, expressing and nonexpressing 
1(VI) collagen mRNA, but in
different relative proportions, and none of them appears to be cell
type-specific. Several lines of evidence indicate that sequence
elements of the enhancer may have different functional roles in various
cells. The data configure the 5.4/3.9 region of the
Col6a1 gene as a new type of tissue-specific enhancer,
characterized by a variety of tissues supporting its activation and by
the dependence of its function only on ubiquitous transcription
factors. This type of enhancer is postulated to be particularly
important for genes such as those of the extracellular matrix, which
are often expressed with broad tissue specificity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1(I)
collagen, osteopontin, and bone sialoprotein are controlled by
sequences binding Osf2/Cbfa1, a transcription factor
necessary for the differentiation of osteoblasts (1), whereas
transcription of 1(II) and 2(XI) genes requires sequences
recognized by Sox9 and other members of the high mobility group class
of transcription factors, which are involved in cartilage
differentiation (2-6). Thus, the identification and analysis of
enhancers responsible for tissue-specific expression of extracellular
matrix components are important not only to understand the regulation
of their genes but also to clarify the genetic control of
differentiation programs.
7.5 to 6.2 kb, induces transcription in joints, intervertebral
disks, vibrissae, skeletal muscle, and meninges. Finally, the sequence
between about 5.4 to 3.9 kb from the transcription start site
contains information for expression in articular cartilage,
intervertebral disks, nerves, vibrissae, and subepidermal mesenchyme.
The strong activating capacity of the 5.4- to 3.9-kb sequence has
been confirmed by experiments with transgenic mice, where promoter-CAT
fusions including this region are expressed in several tissues over 100 times more efficiently than constructs lacking it (9). One interesting
feature of the 5.4/3.9 region is that information controlling
transcription in cells with different embryological origin and
function, including articular chondrocytes, Schwann
cells,2 and fibroblasts, is
enclosed in a relatively small DNA fragment, 1.5 kb, a size compatible
with that of a single enhancer (11). The question therefore arises
whether the 5.4/3.9 region contains only one enhancer dictating
different tissue specificities or multiple enhancers, each one
responsible for transcriptional activation in only one tissue. Such
enhancers are usually formed by multiple transcription factor binding
elements, including positively acting factors that are spatially
localized in the organism in addition to ubiquitous ones (12). Data
accumulated so far on genes of the extracellular matrix conform to this
condition. Studies on cis-acting sequences of collagen I
genes have identified distinct enhancers for activation in cells of
calcified tissues, skin, and fascial and interstitial fibroblasts. When
characterized, as in bone, the active sequences have been found to bind
tissue-specific nuclear factors (1, 13-16). Similarly, discrete
elements recognizing tissue-specific transcription factors are
necessary for high level transcription of 1(II) and 2(XI) genes
in chondrocytes (2-6). In this study we have analyzed functional and
structural features of the 5.4/3.9 region. Unexpectedly, the region
neither contains a unique enhancer sequence for all five tissues nor
several distinct enhancers for the different tissues; rather, sequences
controlling transcription in different tissues overlap extensively but
do not coincide. In addition, no cell type-specific nuclear factors could be identified. These criteria identify a novel class of enhancers
responsible for tissue-specific transcription.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(4.0-1.4)lacZ and
p5.4(4.0-1.4)CAT (8, 9), renamed p1.4(A-P)lacZ and
p1.4(A-P)CAT.
5.4/3.9
enhancer region, the DNA extending from the BamHI site at about 5.4 to the EcoRI site at about 3.9, identified
also as segment A-P (Figs. 1 and 2), was excised from a
SphI subclone of the genomic clone MG3 (17) and cloned into
the SmaI site of pGEM3 after blunting with Klenow enzyme.
5'- and 3'-end deletions of A-P, including segments A-G, A-K, B-P,
E-P, I-P, and L-P (Figs. 1 and 2), were generated by exonuclease III
("Erase a base" kit, Promega) using the procedures recommended by
the manufacturer. Segments F-H, H-M, and N-O were derived from A-P
by cutting with restriction enzymes indicated in Fig. 2. Segments E-L,
E-I, I-L, and E-J were generated from A-P by PCR with appropriate
primer oligonucleotides. The segments were cloned in p1.4lacZ (8) upstream of the lacZ gene, producing the constructs
described in Fig. 1. Some of the segments were also cloned in p1.4CAT
(17) to give the corresponding CAT constructs.
-galactosidase expression were carried
out exactly as described (8).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5.4/3.9 Enhancer
Region in Vivo
5.4/3.9 region responsible
for transcriptional activation in different tissues, transgenic mice
were produced with the constructs outlined in Fig.
1a. In these constructs the
lacZ gene containing a nuclear localization signal is fused
to the proximal 1.4-kb 5'-flanking sequence and to different deletions
of the 5.4/3.9 region of the Col6a1 gene. The sequence
of the 5.4/3.9 region and the position of the start and end points
of the deletions are reported in Fig. 2.
The region extends from the BamHI site at 5.4 kb (site A)
to the EcoRI site at 3.9 (site P). Embryos were dissected
usually at 15.5 days, whole mount-stained with X-gal, and the
distribution of transgene-positive cells determined by histological
examination of serial sections. As expected by the presence of the
proximal 1.4-kb fragment, all expressing embryos exhibited staining in
superficial and muscular aponeurotic system and tendons (8) (data not
shown), a condition that allowed easy identification of the number of
expressing embryos. The results of the analysis of distribution of
staining is summarized in Fig. 1a for only those tissues
that were previously shown to require the 5.4/3.9 region for high
level and high frequency expression of the transgene, i.e.
articular cartilage, peripheral nervous system, intervertebral disks,
vibrissae, and subepidermal mesenchyme (8). In addition to constructs
with deletions of the 5.4/3.9 region, the published data obtained
with constructs containing the entire region (construct (A-P)lacZ) or
lacking it completely (constructs with 1.4-3.9 kb of flanking
sequences) are also reported for comparison in Fig. 1a.
Expression patterns from these two types of constructs constitute
positive and negative controls, respectively, for the deletion
constructs. The results are analyzed here separately for each tissue or
group of tissues.
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Fig. 1.
Functional analysis of 5.4/3.9 sequences
in vivo. a, transgenic mice were
generated with promoter constructs carrying different deletions of the
5.4/3.9 region and expression in embryos determined by X-gal
staining and histological examination of serial sections. All
constructs contain the proximal 1.4-kb sequence of the
Col6a1 gene promoter, which drives high level transcription
in superficial and muscular aponeurotic system and tendons and allows
easy detection of expressing lines. Results are reported only for those
tissues in which expression was previously shown to depend on the
5.4/3.9 region (8). Sites comprising various deletions are
indicated by capital letters (A-P) and are
defined in Fig. 2. The complete 5.4/3.9 region is coincident with
fragment A-P. Data previously obtained for p1.4(A-P)lacZ and
lacZ constructs lacking the A-P region are also included
for comparison (8). AC, articular cartilage; PNS,
peripheral nervous system; ID, intervertebral disks;
Vibr., vibrissae mesenchyme; SM, subepidermal
mesenchyme. b, summary of distribution of sequences of the
5.4/3.9 region exhibiting autonomous inducing activity in the
various tissues. The height of boxes is proportional to the frequency
of expression of transgenes with the indicated deletion. Careful
deletion analysis was performed only for segment E-L by producing
transgenic embryos with subfragments E-I, E-J, I-L, F-H, and H-M
in addition to E-L itself, and the figure indicates the smallest
fragment inducing expression at highest frequency. HS2 and
HS3 mark the position of DNase I chromatin hypersensitive
sites 2 and 3 (see Fig. 6).
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Fig. 2.
Sequence of the 5.4/3.9 region. The
region extends from the BamHI site at 5.4 (site
A) to the EcoRI site at 3.9 (site P). The
letters indicate the position of sites defining deletions
used in constructs for production of transgenic mice (see Fig. 1).
Sequences marked by horizontal lines were chosen to derive
oligonucleotides employed in electrophoretic mobility shift assays and
are marked by roman numbers I to XII.
Articular Cartilage--
Sequences driving expression in articular
cartilage were detectable only in fragment E-P. A portion of this
fragment, labeled E-L, exhibited the highest frequency (7/10). The
5'-half of the E-L (fragment E-I) was much less active (2/11).
Likewise, the 3'-half of E-L (segments I-L and H-M, which is only a
few base pairs longer) was expressed at lower frequency (3/10 and
0/5 respectively, overall 3/15). Among subfragments derived from E-L
(E-I, I-L, F-H, H-M, and E-J), only E-J was expressed with a
frequency comparable to E-L itself (5/6). One conclusion coming from
these results is that the main sequence necessary for transcription in
articular cartilage is included in segment E-J and that integrity of
this fragment is required for high levels of expression. The data
indicate that distinct elements are located within E-I and I-L and
that neither element reaches high frequency expression in the absence of the other, suggesting synergism. Very weak but detectable inductive activity was also found in segment L-P (1/8) and could be narrowed down to N-O. However, a fragment containing both the E-L and the L-P
segments (construct E-P) was not more active than E-L alone (3/5 and
7/10, respectively). As the expression frequency of the entire
5.4/3.9 region in articular cartilage (construct A-P) was 100%,
this suggests that full induction requires not only elements located in
E-L but also elements comprised within L-P and A-E, which, when
tested per se, are weakly or not inductive.
Peripheral Nervous System-- Expression in this tissue at high frequency required the presence of the entire E-L region, as found for constructs (B-P)lacZ (5/5), (E-P)lacZ (4/5), and (E-L)lacZ (5/10). Constructs not containing any portion of the E-L region did not express in this tissue. Finally, constructs enclosing only portions of the E-L region ((A-G)lacZ, (A-K)lacZ, (I-P)lacZ, (E-I)lacZ, (I-L)lacZ, (E-J)lacZ, (F-H)lacZ, and (H-M)lacZ) expressed with very low frequency (only 3 over a total of 60 lines produced). Thus, the main regulatory region for PNS is E-L. However, as B-P is the only deletion with 100% expression frequency, full activation probably requires additional sequences comprised in either L-P and/or B-E.
Intervertebral Disks-- The E-L region was strongly inducing, whereas its subfragment E-J was less efficient, indicating that, at variance with articular cartilage, elements in the J-L sequence are necessary for high frequency activation in this tissue. Unlike articular cartilage, A-E had autonomous activity, although at low frequency (1/8), in intervertebral disks. Fragment N-O was also weakly inductive in this tissue.
Vibrissae and Subepidermal Mesenchyme--
The distribution of
activating regions for these two tissues was similar, although
frequencies of expressing over total transgenic lines were not exactly
coincident. The E-L region was strongly activating; however, the
shorter E-J segment was equally effective, suggesting that sequences
of the J-L region are not crucial for high frequency expression in
these tissues. As for intervertebral disks, segment A-E induced
-galactosidase expression at low frequency (1/8 in the two tissues).
A weak inducing region with slightly different activity in the two
locations was contained in fragment N-O (3/15 and 1/15, respectively).
The main conclusion that can be drawn from these experiments is
that high level expression requires the simultaneous presence of
elements contained within segment E-I and I-L and that these distinct
elements act in a synergistic manner. This is true for all the five
tissues examined, although at different extents. The effect is strong
for the peripheral nervous system in which splitting of the E-L
fragment abolishes transcription completely, whereas in the other
tissues the individual halves of E-L maintain a low degree of
activity. A second conclusion is that, in addition to E-L, sequences
with weak or no inducing activity when tested in isolation are
necessary to reach the maximal enhancer activity measured for the
entire 5.4/3.9 region. It is apparent that strongly and weakly
activating sequences controlling transcription in the five tissues
overlap, but do not coincide (Fig. 1b), suggesting that
transcriptional activation in the various tissues examined requires the
cooperation of distinct but partially common sets of nuclear factor
binding elements within the 5.4/3.9 enhancer region.
Linker-scanning Mutagenesis Analysis of the E-J Region
To map the active sequences of the enhancer region better, a
linker-scanning mutagenesis analysis of the E-J segment was carried out. The choice of E-J was dictated by the fact that this was the
shortest segment exhibiting considerable inducing activity in
vivo in four out of five tissues investigated (Fig. 1) and in
transfected cell lines in vitro (Fig. 6). Although E-J was a very weak activator in the peripheral nervous system (only 1 expressing in 26 lines generated with sequences internal to the fragment, which include E-J, E-I, and F-H, Fig. 1), it contains a
large portion (about 2/3) of E-L, the smallest fragment highly expressed in this tissue. The results of these experiments are reported
in Fig. 3. Each one of the mutations
introduced into E-J lowered significantly (at least 50%)
transcription compared with either p1.4(E-J)CAT or p1.4(A-P)CAT,
indicating that all sequences included elements or part of elements
important for full activation. For constructs containing LS11, LS13,
LS15, and LS16, CAT expression was similar to that of the enhancerless
plasmid p1.4CAT, suggesting that these mutations abolished completely enhancer performance. Comparison of the corresponding sequences with
those of transcription factor binding site data bases (28) identified a
consensus sequence for AP1 in the fragment covered by LS11. No
significant homologies with known binding sites for transcription
factors were found in the sequence spanning LS13. As for LS15 and LS16,
no potential sites were revealed when the individual corresponding
sequences were compared; when the data base was probed with the merged
sequences a C/EBP-binding site was identified at the boundary of the
two segments. Thus, the loss of enhancer activity in both p1.4(LS15)CAT
and p1.4(LS16)CAT might be accounted for by the mutation of a
transcription factor binding site spanning part of the two
mutations.
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To investigate further the activating properties of elements in the
E-J region, CAT constructs, in which the proximal 1.4 kb were fused
with three copies of oligonucleotides I to VIII marked in Fig. 2, were
transfected into NIH3T3 fibroblasts and sternal chondroblasts, and CAT
activity was determined. Addition of three copies of oligonucleotides
to the 1.4-kb proximal sequence was usually activating, but only weakly
(
2-fold), compared with the addition of the E-J segment (
5-fold)
(data not shown). This was also the case of oligonucleotide VIII, which
included the potential AP1-binding site mutated in LS11. These results
favor the idea that the integrity of the entire E-J segment is
necessary for full induction; they also suggest that the enhancer
function is not dependent on one in particular but rather requires
cooperation of different transcription factors.
Complexity of Transcription Factors Binding to the E-J Segment
The results of the linker-scanning mutagenesis analysis suggest
that the E-J segment binds a considerable number of nuclear factors.
To test this prediction, protein-DNA interaction assays were performed.
DNase I footprinting assays of the E-J region produced weak
protections, the major of which encompassed about 90 nucleotides of the
E-I fragment (footprint 1 in Fig.
4) including completely the LS4 to LS8
segments defined in linker-scanning experiments (see Fig. 3). Other
protected regions were shorter and much weaker, and two examples are
given in Fig. 4 (footprints 2 and 3). Although
the DNase I footprinting experiments showed that a considerable portion
of the E-J region binds nuclear factors, the weak intensity of the
protections prevented further analysis of the complexity of the binding
transcription factors. This aspect was therefore investigated by
mobility shift assays using the 12 overlapping double-stranded
oligonucleotides defined in Fig. 2, which together span the entire E-J
region. A representative gel shift pattern generated with nuclear
extracts from chick embryo sternal chondroblasts is given in Fig.
5. Similar patterns were obtained with
nuclear extracts from other cell types, and the data are summarized in
Table I. Main features of the pattern of
band distribution were the absence of retarded bands specific for only
one of the cell types considered and the variation of the relative
intensity of bands with different cell extracts. Moreover, some bands
were common to different cell types but absent in one strain of the
same cell type, thus increasing the variation among cell strains. One
notable example is band Xc, which, in addition to other cell types, is
found in RCS, but not in sternal chondroblasts, and in tendon, but not
in NIH3T3 fibroblasts. To define better the complexity of nuclear
factors binding to the E-J segment, the bands were classified on the
basis of their sensitivity to EDTA and heat and of the pattern of
oligonucleotides inhibiting their formation in electrophoretic mobility
shift assay competition experiments (Table
II). The number of bands differing for at
least one of these classification criteria is 22, corresponding to an estimate of the minimum number of different types of transcription factors that can bind to the E-J fragment.
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Chromatin DNase I Footprinting Assays
Expression of promoter constructs in vivo and the
different intensity of gel shift bands in various cell types (Table I) stimulate to hypothesize that elements binding transcription factors in
the E-J segment have distinct regulatory relevance in different cells.
To confirm this hypothesis chromatin DNase I footprinting and in
vitro promoter assays were performed. The first kind of assays
were carried out with the rationale that distinct assemblies of protein
complexes binding to the enhancer region would imply a different
organization of chromatin in the various tissues. Mouse cells employed
for this analysis comprised lines derived from tissues where the
5.4/3.9 enhancer region is stimulatory such as NIH3T3 fibroblasts,
MC615 chondroblasts, and the Schwann cell line SCT-1 (8, 9 and see
below); all these lines expressed the 1(VI) collagen mRNA, which
was particularly abundant in NIH3T3 and MC615 (Fig.
6a). The analysis was also
carried out in C2C12, a cell line derived from the myogenic cell
lineage, where the enhancer region is only weakly active (9). C2C12
cells produced low levels of mRNA (Fig. 6a). A final
cell line included EL4 lymphocytes that did not produce detectable
levels of mRNA (Fig. 6a). Four DNase I-hypersensitive
sites were detected in the segment extending from 7.5 to +1.5 kb from
the transcription start site (Fig. 6, b and c).
As previously observed, one, indicated by * in the figure, was an
invariant site present in all cells, whereas a second site, HS1,
mapping at about 0.1 kb, was detectable in all cells expressing 1(VI) mRNA (10). Two other sites (HS2 and HS3) mapped at about 4.6 and 4.4 kb and were located within segments E-I and I-L, respectively (Figs. 6, b and c and Fig.
1b). These sites were absent in EL4 and very weak in C2C12
cells. On the contrary, at least one of them was strong in NIH3T3,
MC615, and SCT-1 cells. However, remarkable differences depending on
the cell type could be noted; both sites were equally evident in NIH3T3
fibroblasts, whereas HS2 was strong and HS3 weak in MC615
chondroblasts. In SCT-1 HS3 was undetectable, and HS2 was interestingly
shifted to more upstream regions by increasing DNase I concentration, due to shortening of the DNA fragment detected by the probe used (Fig.
6, b and c). These results indicate differences
of chromatin structure within the enhancer region in various cell
types.
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Transient Transfections with Deletions of the 5.4/3.9
Region
This assay allows a quantitative evaluation of relative inducing
properties of regulatory regions and is expected to be informative in
our analysis aimed at establishing whether similar sequences of the
enhancer have slightly different cell-specific activity. Constructs
were synthesized similar to those of Fig. 1a, but containing the CAT instead of the lacZ gene, and were transfected in
various cells. SCT-1 cells were poorly transfected in our experiments and were excluded. Transfections were more efficient with a rat Schwann
cell line (RN22), but the entire enhancer region, plasmid p1.4(A-P)CAT, induced CAT activity only 2-fold compared with the enhancerless construct (p1.4CAT) (data not shown), a level too low for
a reliable quantitative comparison of the activating effect of deletion
constructs. C2C12 myoblasts were not used in this experiments because
we had previously demonstrated that there is no significant difference
of CAT expression between p1.4(A-P)CAT and p1.4CAT (9). On the
contrary, the whole enhancer region increased transcription
considerably in NIH3T3, MC615, and sternal chondroblasts from chick
embryos (Fig. 7). The pattern of
expression of deletion constructs transfected in these cells was
clearly cell type-dependent. In NIH3T3 and sternal
chondroblasts the inductive properties of the entire enhancer region
were as high as those of segments E-J and E-L (compare p1.4(A-P)CAT,
p1.4(E-J)CAT, and p1.4(E-L)CAT with p1.4CAT). On the contrary, in
MC615 cells, CAT activity produced from the deleted constructs
(p1.4(E-J)CAT and p1.4(E-L)CAT) was only about one-third that from
the plasmid with the whole enhancer region (p1.4(A-P)CAT). Moreover,
by comparing CAT expression of p1.4(E-L)CAT with that of p1.4(E-I)CAT
and p1.4(I-L)CAT, it can be calculated that E-I and I-L act
synergistically in NIH3T3 and sternal chondroblasts (about 2.5-fold
synergism) but additively in MC615. Consistent with data in
vivo (Fig. 1), the A-E and L-P segments were poorly inductive
when tested in isolation; in fact, expression attained with
p1.4(A-E)CAT and p1.4(L-P)CAT was not significantly different from
that reached by p1.4CAT in all the cell types. However, these segments
contributed to produce the high level induction of the complete
5.4/3.9 (A-P) region in MC615 chondroblasts (Fig. 7). This
property parallels the activating function of segments outside E-L
revealed by the in vivo data (Fig. 1). Finally,
transfections with the constructs of Fig. 7 were also performed with
EL4 lymphocytes. This experiment was of particular interest as this
cell line does not express the endogenous gene (Fig. 6), yet it
contains the same set of factors binding to the E-J segment detected
in expressing cells (Table I). The pRSV-luciferase internal control
plasmid was highly transcribed in EL4 lymphocytes. On the contrary CAT
activity from all the constructs was identical to the background
measured in non-transfected samples (data not shown). This result was
highly dissimilar from those obtained with the expressing cell lines,
where CAT activity reached by the least active plasmid, p1.4CAT, was
considerably higher (usually >100-fold) than that measured on
non-transfected samples. These data suggest that transcription of the
Col6a1 gene is completely silenced in lymphocytes. Overall,
the transfection experiments add further support to the suggestion that
the function of active sequences of the 5.4/3.9 enhancer region on
transcription of the Col6a1 gene varies in different cell
types.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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This work represents the first characterization of the 5.4/3.9
enhancer region of the Col6a1 gene. This region increases transcription in cells of five tissues (articular cartilage,
intervertebral disks, peripheral nervous system, vibrissae, and
subepidermal mesenchyme) differing for embryological origin, anatomical
location, and specific function (8). The results show very clearly that the 5.4/3.9 (A-P) fragment does not contain a separate enhancer specific for each one of the five tissues controlled. Instead, high
level expression in the five tissues requires sequences located in one
segment of 383 bp, designated E-L. In addition, our analysis suggests
that activating elements are located in the 5.4/3.9 (A-P) region
also outside the E-L fragment. These elements have very weak or no
autonomous activity, but they increase enhancer performance in
vivo when linked to E-L. These elements could not be studied in
detail due to their low intrinsic activity.
To identify elements responsible for transcriptional induction,
linker-scanning mutagenesis experiments were performed on E-J, a
245-bp fragment representing the most active portion of E-L. This
analysis could not define a limited number of discrete sequences
responsible for induction; on the contrary, all mutations reduced
transcription significantly. The important conclusion derived from
these experiments is that multiple elements are necessary for the
function of the E-L segment and that these elements must be
simultaneously present for full activation. This conclusion is not
contradicted by the observation that a variable degree of reduction of
transcription, ranging from 50 to 100%, was detected with different
mutations (Fig. 3). In fact, activation reached by multiple copies of
sequences corresponding to the most effective mutations was very poor,
indicating the need of the contribution of other elements. Experimental
evidence suggests that cooperative interactions among elements is a
main feature of the E-L enhancer; the frequency of transgene
expression in vivo of each half of the E-L segment,
fragments E-I and I-L, is dramatically reduced compared with the
entire sequence. In addition, a 2.5-fold synergism between E-I and
I-L can be measured in transient transfections of NIH3T3 and sternal
chondroblasts. In conclusion, the E-L enhancer should be viewed as a
cluster whose full transcriptional output depends on the integrity of
the entire cluster. This feature characterizes several types of
enhancers, the most studied is -interferon (29).
As the enhancer is active in a number of cell types and is formed by
several elements, one obvious question is whether each element of the
cluster has the same functional role in different cells. The data
indicate that this is not the case; on the contrary, they support the
idea that the contribution of elements to the enhancer output varies in
different tissues. The first evidence of this comes from the in
vivo data. A clear example is the variation of the frequency of
expression attained in the five tissues by deleting 137 bp of the
3'-part of E-L. The resulting fragment, E-J, is equally highly
expressed in articular cartilage and vibrissae, whereas it is not
expressed in the peripheral nervous system, and the efficiency is
considerably lowered in intervertebral disks. A second evidence comes
from transient transfections in vitro, where a synergistic
effect between E-I and I-L could be measured in NIH3T3 cells and in
sternal but not MC615 chondroblasts. This result may seem surprising in
view of the fact that both sternal and MC615 chondroblasts produce high
levels of 1(VI) mRNA (Fig. 6).3 It could well be,
however, that regulation of mRNA expression is different in the two
types of cells. The 5.4/3.9 region is not the only enhancer active
in chondroblasts, and a distinct enhancer for these cells has been
previously located at 7.5/6.2 (8). MC615 chondroblasts are
transformed cells containing an active SV40 large T antigen. This may
influence the activity of the two enhancers compared with normal cells.
Alternatively, high levels of mRNA in MC615 may be due to
post-transcriptional regulation, resulting in stabilization of the
molecule. An indirect evidence that the contribution of elements of
E-L to the enhancer output may vary in different tissues comes from
the analysis of protein binding to the E-J segment by gel shift assays
(Tables I and II). The total number of factors binding to E-J in
different cells was estimated to 22, none of which exhibited a cell
type-specific distribution. Not all factors were present in each cell
line, and their relative proportion was varied in different cells.
Unfortunately, the weak intensity of protection in DNase I footprinting
experiments did not allow us to confirm the high number of binding
transcription factors using this assay coupled with oligonucleotide
competition and to perform an extensive analysis of structural
interactions in different cells by using the corresponding nuclear
extracts. However, an indication of the different composition of
protein complexes binding to the E-J region in various cells comes
from DNase I footprinting of chromatin, showing that the intensity and
features of the two hypersensitive sites located within E-L depend on
the cell line. The formation of hypersensitive sites by transcription
factors bound to DNA has been proposed to be due to either direct
alteration of DNA structure (30) or to the increased probability of
keeping a region nucleosome-free (31). In both hypotheses, variability
of features of the hypersensitive sites are assumed to be the
consequence of differences in the binding of transcription factors. The
properties described delineate the E-L region of Col6a1
investigated as a new variant of tissue-specific enhancer whose main
features are as follows: (i) the region controls transcription in
several cell types; (ii) it comprises a large number of elements
binding transcription factors with broad distribution; and (iii) high
level tissue-specific transcription is achieved by cooperative
contribution of sets of regulatory elements that are partially distinct
in different tissues. It should be noted that tissue specificity of
transcription distinguishes this type of enhancer from those, such as
the interferon- enhancer, that are dependent on ubiquitous factors
and require cooperative interactions among elements (29) but whose
activation is not cell type-specific (32).
If the function of the Col6a1 enhancer is based on
ubiquitous nuclear factors, why is it not active in all cell types? The answer to this question will require future work, but a few
possibilities will be considered here. In cells that do not express
collagen VI, the Col6a1 enhancer may be inactive because the
entire gene is repressed. One example may be lymphocytes, in which
Col6a1 constructs, with or without the enhancer, are equally
not transcribed, as indicated by the finding that CAT activity was the
same as non-transfected controls. In a previous paper (10) we observed that a sequence at about 0.1 kb from the transcription start site
binds AP1 in collagen VI-expressing cells but is a specific factor not
related to AP1 in lymphocytes. This site corresponds to a
hypersensitive site of chromatin (HS1) present in all cells expressing
Col6a1 and absent in lymphocytes. We can assume that this
lymphocyte-specific factor is a repressor that turns off completely
transcription of the Col6a1 gene in these cells by altering
the structure of chromatin close to the promoter. We are presently
testing this possibility.
Different conditions may be responsible for the lack of expression of
the 5.4/3.9 enhancer in some tissues that produce collagen VI. This
is the case, for example, of connective tissues associated with
viscera, particularly the digestive tract and the lung, for which we
have previously assumed the existence of a separate enhancer(s) (8).
One hypothesis is that these tissues contain different variants or
isoforms of same transcription factors binding to the 5.4/3.9
region. Given that the interaction of elements in this enhancer region
is cooperative, these variants and isoforms may not be equally
effective in the assembly of a protein complex on the enhancer
sequences. It has become increasingly apparent in recent years that
enhancers stimulate transcription by driving the cooperative assembly
of specific three-dimensional DNA-protein complexes called
enhanceosomes, which increase recruitment of the transcription
machinery to the basal promoter (29, 33, 34). Genes containing
enhancers are transcribed at high levels only when the appropriate set
of nuclear proteins is present and a specific higher order structure
(enhanceosome) is assembled (35). The lack of activation of the
Col6a1 enhancer examined here in viscera could be an
exemplification of this principle. A second hypothesis is that some of
the transcription factors must be activated by specific signal
transduction pathways in order to bind to the enhancer and that these
pathways are at work only in particular cells. In other words, the
activation of the Col6a1 enhancer would be dependent on the
so-called "cellular context," a concept indicating the complex of
signaling molecules and signal transduction pathways present in a given
cell (36). The tissue-specific activation of the Col6a1
enhancer region investigated here could reflect the permissive or
non-permissive properties of the cellular context.
The final definition of the number and nature of transcription factors binding to the E-L enhancer region will require cloning of their cDNA. We have undertaken this study using the one-hybrid method with constructs carrying trimers of the oligonucleotides used for gel shift assays. At present we have completed the procedure only with oligonucleotide III. This oligonucleotide gives rise to three of the major retarded bands in gel shift assays (see Fig. 5), and we had estimated a binding capacity for a minimum of two proteins (Table II). Three different nuclear factors were isolated by the one-hybrid method and shown to bind specifically oligonucleotide III in gel shift assays. One is a ubiquitous factor; the other two belong to transcription factors for which several isoforms have been described in different tissues. This may well be a lucky coincidence, but it certainly fits very well with both our appraisal of the number of binding proteins and our suggestion that the factors are either present in all cells or represent tissue variants/isoforms of widely distributed transcription factors.
The distribution of cells supporting activation of the
Col6a1 enhancer is not ubiquitous but, nevertheless,
includes a wide variety of tissues. Despite the broad spectrum of
expressing cells, the molecular set up controlling transcription is
likely to be sufficiently different in each of them to allow cell
type-specific characteristics of expression. Thus, enhancers such as
that studied here are expected to confer particular flexibility of
expression to the genes they control. Enhancers characterized so far in
genes of the extracellular matrix, such as those responsible for
collagen I in calcified tissues and collagen II in cartilage, are
active only in a restricted number of related cell types, and their
mechanism occurs through binding of tissue-specific factors (1-6) and
therefore differ from that examined in this study. However, given the
widespread expression of the majority of extracellular matrix
components, we expect that enhancers like that of Col6a1
studied here will be identified in many other genes.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. J. Kimura and Dr. B. de Crombrugghe for supplying the RCS cell line and Dr. F. Mallein-Gerin and Dr. A. Messing for the gift of MC615 and SCT-1 cell lines, respectively. We also thank Dr. Miriam Zanetti for performing the dot blot assays and Mauro Ghidotti for the maintenance of mouse colonies.
| |
FOOTNOTES |
|---|
* This work was supported by Telethon Grants E22 and E704 and from the Italian CNR Target Project on Biotechnology.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@civ.bio.unipd.it.
Published, JBC Papers in Press, March 27, 2000, DOI 10.1074/jbc.M000075200
2 P. Vitale, P, Braghetta, D. Volpin, and G. M. Bressan, manuscript in preparation.
3 D. Girotto, C. Fabbro, P. Braghetta, P. Vitale, D. Volpin, and G. M. Bressan, unpublished results.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
kb, kilobase pair(s);
CAT, chloramphenicol acetyltransferase;
bp, base pair(s);
X-gal, 5-bromo-4-chloro-3-indolyl--D-galactoside;
PCR, polymerase chain reaction.
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REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
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