The Y-box Binding Protein YB-1 Suppresses Collagen 
1(I) Gene
Transcription via an Evolutionarily Conserved Regulatory Element in
the Proximal Promoter*
Jill T.
Norman
,
Gisela E.
Lindahl,
Kaveh
Shakib,
Abdelaziz
En-Nia§,
Emek
Yilmaz§, and
Peter R.
Mertens§¶
From the Department of Medicine, Royal Free and
University College Medical School, Sir Jules Thorn Institute for
Clinical Sciences, The Middlesex Hospital, Mortimer Street, London
W1T 3AA, United Kingdom and § Department of Nephrology and
Immunology, University of Aachen, Pauwelsstrasse 30, 52057 Aachen,
Germany
Received for publication, April 9, 2001, and in revised form, May 25, 2001
 |
ABSTRACT |
Appropriate expression of collagen type I, a
major component of connective tissue matrices, is dependent on tight
transcriptional control and a number of trans-activating
and repressing factors have been characterized. Here we identify the
Y-box binding protein-1 (YB-1) as a novel repressor of the collagen
type 
1(I) (COL1A1) gene. Collagen type I mRNA
and protein levels decreased upon overexpression of YB-1 by
transfection in NRK fibroblasts. The human, rat, and mouse
COL1A1 promoter 
220/+115 contains three putative Y-boxes, one of these sites, designated collagen Y-box element (CYE), includes a
Y-box plus an adjacent 3' inverted repeat. DNase-I footprinting and Southwestern blotting with fibroblast nuclear extract demonstrated binding of several nuclear proteins across the CYE, one of which was
identified as YB-1. Recombinant YB-1 bound the CYE sequence in gel
shift assays with a preference for single-stranded templates. The
entire sequence (88/48) was required for high affinity binding. Complex formation of endogenous YB-1 with the CYE was established by
supershift studies. COL1A1 promoter-reporter constructs
were suppressed up to 80% by cotransfection with YB-1 in a variety of
cell types. In addition, CYE conferred YB-1 responsiveness on two
heterologous promoters further demonstrating the importance of this
repressor region. Mung bean nuclease sensitivity analysis suggested that repression is most likely exerted through changes in DNA conformation.
| |
INTRODUCTION |
Collagens are major components of the extracellular matrix, where
they not only serve as structural proteins but, via interactions with
specific integrins, can modulate cell proliferation, differentiation, and function (1). Physiological processes such as wound healing are
dependent on tight spatial and temporal expression of collagen genes by
mesenchymal cells, and a broad spectrum of fibrotic conditions is associated with collagen accumulation (2). Interstitial collagen
type I is a heterotrimeric protein composed of two 1 and one 2
chains encoded by separate genes, collagen 1(I)
(COL1A1)1 and
2(I) (COL1A2), that are tightly and coordinately
regulated in a tissue- and cell type-specific manner (2, 3).
Regulation of collagen gene expression occurs primarily at the level of
transcription (2-4). A number of regulatory elements required for
constitutive and inducible expression have been identified in the
promoter and first intron of both the COL1A1 and
COL1A2 genes (2, 5, 6) with gene transcription controlled by the complex interplay of positive and negative regulatory factors. Much
of the information on cis-acting elements and their
respective binding activities is derived from work on the mouse
COL1A1 gene and, in particular, analysis of sequences
between 220 and +115. The proximal promoter region of the
COL1A1 gene is highly conserved between species (7). Known
regulatory elements are summarized in Fig. 1A and include a
TATA-box, two regions containing inverted CCAAT sequences, one of which
binds CBF (8), as well as binding sites for Sp1 and NF-1/CTF (9),
c-Krox (10), and BFCOL1 (11). The CBF binding site between 100 and
96 has been shown to be required for basal activity, and an
inhibitory factor, IF2, binds to sequences flanking this site (1). Sp1
and NF-1 have been shown to bind in a mutually exclusive manner to the
two sites located between 105 to 78 and 129 to 110. c-Krox
binds preferentially to the purine-rich region (190 to 170),
whereas BFCOL1 shows stronger affinity for the pyrimidine-rich
sequences between 160 and 130 (11).
Inverted CCAAT-boxes can form the core of binding sites for Y-box
binding proteins. The presence of two such regions in the COL1A1 promoter together with the previously reported
finding that YB-1 (also known as dbpB), a prominent member of this
family of evolutionarily conserved DNA/RNA binding factors, can
up-regulate expression of the matrix metalloproteinase-2
(MMP-2) gene in activated mesangial cells (12, 13), raised
the question whether YB-1 might also play a role in regulating COL1A1
expression. YB-1 has been implicated in the regulation of a variety of
genes (14) and may, depending on the cellular context, act as either a
transcriptional activator or repressor even of the same gene
(15). Closer inspection of the COL1A1 gene sequences between
220 and +115 revealed a third putative Y-box protein binding site
with striking similarity to the YB-1 binding element-1 (RE-1) in the
rat MMP-2 promoter (12, 13) (Fig. 1B). Notably,
the homology extended to a 3' inverted repeat also present in the RE-1
(Fig. 1C).
Based on these homologies, the present study focused on the
functionality of this site, which we have designated the collagen Y-box
element (CYE). The data show that YB-1 is expressed in the nuclei of
collagen-producing fibroblasts and that both recombinant and endogenous
YB-1 bind specifically to the sense and antisense strands of the CYE.
Furthermore, YB-1 suppresses the activity of the COL1A1
promoter through this element. A potential insight into the functional
activity of this protein is provided by the observation that YB-1
induced strand separation in the inverted repeat region of CYE, which
may prevent or disrupt binding of positive regulatory factors and
thereby repress transcriptional activity. Overexpression of YB-1
suppresses endogenous collagen gene expression and protein production.
These data identify YB-1 as a novel transcriptional repressor of
COL1A1 gene expression.
| |
EXPERIMENTAL PROCEDURES |
Cells and Culture Conditions
The rat renal fibroblast cell line NRK-49F (European Collection
of Animal Cell Culture, Porton Down, UK) was maintained in Dulbecco's modified Eagle's medium/Ham's F12 medium supplemented with 10% fetal calf serum, penicillin, streptomycin, and
amphotericin-B (Life Technologies, Inc.). Confluent cells were made
quiescent by incubation in 0.5% fetal calf serum for 48 h. A
variety of other cell types were used, which, unless otherwise
indicated, were maintained as described above: Chinese hamster ovary
cells, rat mesangial cells (15), mouse 3T3 fibroblasts, and fetal
Harlan Sprague-Dawley rat cardiac fibroblasts (passages 6-9 (16)).
Plasmids
pSG5-YB-1--
A YB-1 expression vector (pSG5-YB-1) containing
the complete human YB-1 open reading frame cloned into the expression
vector pSG5 (Stratagene) was kindly provided by J.P.-Y. Ting
(University of North Carolina).
Mouse Collagen 1(I) Promoter Constructs--
pGL1-2.3 was
constructed by subcloning a fragment containing sequences between
2310 and +115 of the mouse COL1A1 gene originating from
the genomic clone pG70 (a gift from J. Rossert, INSERM, Hopital Tenon,
Paris, France (1)) into the HindIII site of pGL3Basic (Promega). The original 3'-end XbaI site was converted to a
HindIII site by ligation to a linker, p-CAAGCTTG,
and digested with HindIII prior to ligation into pGL3Basic.
To create the pGL1-220 construct containing sequences between 220
and +115, a BglII fragment was excised from pGL1-2.3,
and the plasmid was religated. Sequencing across the cloning sites
revealed a discrepancy with the published sequence with an extra G
between positions 11 and 12; however, this is not within or close
to any putative transcription factor binding site. To avoid confusion
with the numbering of transcription factor binding sites described by
other investigators, we have used the published base pair numbering to
identify regions of interest. The transcriptional start site is from
GenBankTM accession number X54876. The sequence up
to 1627 is available from GenBankTM, and the location of
the 5'-end of pGL1-2.3 is from an unpublished sequence.2
Rat Collagen 1(I) Promoter Constructs--
pColCAT3.6/1.6
(also known as B16) was a gift from A. Lichtler and D. Rowe (University
of Connecticut Health Center). The plasmid contains rat
COL1A1 gene sequences between 3518 and +1594. It is
identical to B15 (17) except for an EcoRV to ClaI
conversion in the polylinker. To create the promoter deletion
constructs pColCAT 1.3/+1.6 and 0.4/+1.6, B16 was digested with
NheI (3382) together with Tth111I (1282) or
MunI (390), respectively, and religated. The intron-less
pColCAT 2.3/0 is identical to B47 (17) except that it lacks the 5'
HindIII fragment between 3544 and 2369 present in B16.
pGL2P-CYE and pGL3-CMV850-CYE--
A double-stranded
oligonucleotide homologous to the 95 to 45 sequence of the
mouse COL1A1 promoter, encompassing the entire CYE, was
subcloned into the KpnI and BglII sites of
pGL2Promoter (Promega) and designated pGL2P-CYE. The oligonucleotide
was also cloned between the KpnI and SmaI sites
in the pGL3B portion of pGL3-CMV850 (a gift from O. Schwickerath,
University College London, UK) after blunting of the
BglII site (designated pGL3-CMV850-CYE). This vector
exhibits high basal activity in NRK
fibroblasts.3
Electrophoretic Mobility Shift Assay (EMSA)
Nuclear extracts were prepared as described in (18). Protein
concentration was determined by the BCA assay (Pierce).
Oligonucleotides were end-labeled with T4 polynucleotide kinase
(T4-PNK; Promega) and [
-32P]ATP (3000 Ci/mmol,
Amersham Pharmacia Biotech) and gel-purified. EMSA were performed
exactly as described (13) using recombinant YB-1 (rYB-1) protein (5 ng/binding reaction) or nuclear extracts (5 µg/binding reaction).
Recombinant YB-1 was prepared from a pRSET vector (Invitrogen)
containing an insert coding for a hexahistidine T7 epitope-YB-1 fusion
protein (a gift from Dr. Chien, University of California, San Diego) as
described by Mertens et al. (12). For competition
experiments, unlabeled oligonucleotides or nonspecific DNA (500×
excess) were added to the binding reaction 15 min prior to the addition
of labeled oligonucleotides followed by a 30 min incubation period and
subsequent separation on polyacrylamide gels. Titration experiments
demonstrated that at lower competitor concentrations (100- and 250-fold
molar excess) specific competition was incomplete under the chosen
conditions. Relative binding affinities were determined by quantitation
of shifted bands using a PhosphorImager system (Bio-Rad). For
supershift assays, affinity-purified rabbit anti-YB-1 antibody raised
against a C-terminal epitope (13) was added to the nuclear extracts 30 min prior to the addition of labeled oligonucleotides, and the binding
reaction was incubated overnight at 4 °C. In controls non-immune IgG
was added.
DNase I Footprinting
A 32P-labeled probe corresponding to 220/+115 of
mouse COL1A1 was generated by polymerase chain
reaction amplification. Briefly, 4 pmol of a forward primer
(5'-CCGGGCTCGAGATCTGG-3'), corresponding to vector sequence plus the 5'
8 bases of the promoter sequence in pGL1-220, was end-labeled using
T4-PNK and [-32P]ATP. pGL1-220 was used as
template for a standard polymerase chain reaction using the
32P-labeled forward primer, the reverse GLprimer2
(Promega), 1 unit of Red Hot DNA polymerase (Abgene), and 1 mM MgCl2. The DNA probe was gel-purified
(QIAquick kit, Qiagen). A 10-bp ladder (Life Technologies, Inc.),
end-labeled with [-32P]ATP by T4-PNK, was used as a
molecular weight standard. Binding reactions were performed in a final
volume of 50 µl. 32P-labeled DNA probe (~0.1 ng of
labeled DNA) was incubated for 30 min at ambient temperature in 20 µl
of binding buffer (10% glycerol, 50 mM KCl, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 1 µg of poly(dI-dC) (Sigma)). Nuclear
extract, prepared as above, was added, and the reaction was incubated
at room temperature for 25 min. The binding reaction was transferred to
a tube containing 2 µl of RQ1 DNase I (0.5-1 unit/reaction, Promega)
and 1 µl of 250 mM MgCl2, 250 mM
CaCl2 (final concentrations 5 mM). After 3 min
at room temperature, the reaction was terminated with 100 µl of stop
solution (200 mM NaCl, 30 mM EDTA, 1% SDS),
phenol-chloroform extracted, ethanol-precipitated, and resuspended in
95% formamide buffer (Amersham Pharmacia Biotech). Samples were
electrophoresed on 6% acrylamide, 7 M urea gels in 1×
Tris borate-EDTA buffer, dried, and autoradiographed.
Mung Bean Nuclease Sensitivity Analysis
A mung bean nuclease (MBN) sensitivity analysis was performed as
described previously (12). The strictly double-stranded, asymmetrically
end-labeled probe was prepared by digesting pT4-Luc-CYE Site I with
BglII or KpnI, and the resultant overhanging
5'-ends were dephosphorylated with calf intestinal alkaline phosphatase and end-labeled with [-32P]ATP using T4-PNK. The DNA
fragment was released by BglII/KpnI digestion and
gel-purified. About 105 cpm of probe was included in the
binding reaction with either rYB-1 (10 ng) or mesangial cell nuclear
proteins (10 µg) at saturating concentrations of MBN enzyme (50 units, Promega) as determined by titration.
Southwestern Blot Analysis
Southwestern blot analysis was performed as described (15) using
NRK nuclear proteins (50 µg) and 32P-end-labeled CYE
oligonucleotide probes (106 cpm/ml).
Western Blot Analysis
Western blotting with affinity-purified rabbit anti-YB-1
antibody (1:1000) was performed as described by Mertens et
al. (15) using NRK nuclear proteins (30 µg). For Western blot
analysis of Southwestern blots, radioactivity was removed by repeated
washing in 10 mM Tris·HCl (pH 7.5), 50 mM
NaCl, 1 mM EDTA, 1 mM dithiothreitol prior to
blocking and incubation with anti-YB-1 antibody. For Western blot
analysis of collagen type I protein, lysates were prepared from NRK
cells 72 h after transfection with pSG5-YB-1 or pSG5. Proteins (30 µg/sample) were separated on a 10% SDS-polyacrylamide gel,
electroblotted, and probed with an anti-collagen type I antibody (Southern Biotechnology Associates Ltd.), diluted 1:1000 in
phosphate-buffered saline containing 0.2% Tween 20, followed by
ECL detection.
Transient Transfections
NRK fibroblasts, Chinese hamster ovary cells, and NIH3T3
fibroblasts (~70% confluent) were transfected with purified plasmid DNA (Endotoxin-Free Qiagen Maxi-Prep Kit, Qiagen) using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's
instructions. A mixture of collagen promoter-reporter constructs
(0.5-2 µg) and the YB-1 (0.1-1.0 µg) expression vector or the
pSG5 control vector was co-transfected using 6 µl of LipofectAMINE.
The total amount of transfected DNA was equalized by the addition of
pSG5 control plasmid. All transfections were carried out in triplicate. At least two different DNA preparations were tested to eliminate potential preparation artifacts. After 12 h, transfection medium was replaced with medium containing 2% fetal calf serum. Cells were
incubated for 24 to 48 h, washed with phosphate-buffered saline,
and lysed using passive cell lysis buffer (Promega) for luciferase-reporter constructs or CAT-enzyme-linked immunosorbent assay
lysis buffer (Roche Molecular Biochemicals) for CAT reporter constructs. Luciferase activity was measured using the Luciferase Assay
System (Promega) and data calculated as units of luciferase/µg of
protein (mean ± S.D.). CAT activity was measured by enzyme-linked immunosorbent assay (Roche Molecular Biochemicals) and data presented as pg of CAT/µg of protein. Protein concentration was measured using
a modified Bradford assay (Bio-Rad). Because many viral promoters are
regulated by YB-1, transfection efficiency was measured by the Hirt's
assay using [-32P]dCTP-labeled vector DNA (20) or
pEGFP-YB-1 (CLONTECH) and was determined at
50-60% (20). Rat cardiac fibroblasts (~90% confluent) and
mesangial cells were transfected according to the manufacturer's
instructions using the calcium phosphate Profection mammalian
transfection system (Promega) and Tfx-50 (Promega), respectively.
RNA Extraction and Northern Blot Analysis
Total RNA was extracted using Trizol® reagent (Life
Technologies, Inc.) according to the manufacturer's instructions.
Northern blotting using an [-32P]dCTP-labeled collagen
1(I) cDNA probe (Hf677, ATCC) was performed as described
previously (21).
HPLC Analysis of Collagen Production
Total collagen production was measured by reverse phase HPLC
as described previously (21). Procollagen production is
expressed as nM hydroxyproline/well.
| |
RESULTS |
Identification of Putative YB-1 Binding Sites in the Mouse COL1A1
Gene Promoter--
Sequence analysis of the region 220 to +115 of
the mouse COL1A1 gene (19, 22, 23) revealed three putative
YB-1 binding sites located between positions 83 and 72 (Site I),
103 and 92 (Site II), and 129 and 118 (Site III) (Fig.
1B). A comparison with the
YB-1 binding element in the rat (RE-1) and human (r1) MMP-2
genes (13, 15, 24, 25) revealed striking similarities of Site I (83
to 72) to the RE-1 with seven consecutive matching bases in the
Y-box. Site II showed stronger homology than Site I to the Y-box
consensus sequence (22) but was more divergent compared with the RE-1.
Site III showed a similar degree of homology to the Y-box consensus
sequence as Site I but greater divergence compared with the RE-1 (Fig.
1B). In addition, the 3' sequence adjacent to Site I
included an inverted repeat (IR) similar to the one present in the RE-1
(Fig. 1C). Based on these homologies the present study
focused on this region (83 to 59), which we designated the
collagen Y-box element. Comparison of CYE in mouse, rat, and
human genes showed strong sequence similarity across this element (Fig.
1D). The rat and mouse COL1A1 genes are identical across this region, although the human promoter is highly homologous with only two mismatches across the Y-box and 1 mismatch in the 3'
portion of the inverted repeat.
View larger version (28K):
[in this window]
[in a new window]
|
Fig. 1.
Identification of the Collagen Y-box element,
CYE, in the COL1A1 promoter. A, transcription factor
binding map of the 220 to +115 mouse COL1A1 gene showing
the inverted CAATT-boxes (bold), putative YB-1
binding sites, and the CYE, including the Y-box and the adjacent 3'
inverted repeat (bold italics). The CYE oligonucleotide
(Table I) is indicated by a solid line. Known
regulatory elements and transcription factor binding sites are
illustrated: TATA box, AP-1 (27), NF-1, Sp-1 (9), CBF (8), IF-2 (1),
BFCOL1 (11), and c-Krox (10). The 12-bp repeat that brackets the
proximal CCAAT motif (1) is also shown (bold dashed line).
The right-angle arrow indicates the start of transcription.
B, sequence homology between the Y-box consensus sequence,
MMP-2 RE-1, and the putative YB-1 binding regions in the proximal
promoter of the mouse COL1A1 gene (Sites I, II,
and III). C, comparison of the rat MMP-2 RE1 with
the mouse COL1A1 Site I and adjacent sequence showed homologies that
include a 3' inverted repeat (inverted repeats are marked in
bold). The region encompassing the Y-box and 3' inverted
repeat (83 to 59) was designated the collagen Y-box element
(CYE). D, sequence similarities of the CYE in the
mouse, rat, and human COL1A1 promoters.
|
|
Nuclear Protein Binding Regions within the 220 to +115 Regulatory
Sequence--
DNase I footprinting of 220 to +115 of the mouse
COL1A1 gene with nuclear extracts from NRK fibroblasts was
performed to determine the sites of protein interaction with the
promoter. Six protected regions (A-F) were identified (Fig.
2) that lie between 190 and 177
(region A), 177 and 155 (region B), 145 and 135 (region C),
130 and 105 (region D), 95 and 65 (region E), and 65 and 40
(region F). The Y-box of CYE is located within region E, and the IR
lies at the boundary between E and F and coincides with a
hypersensitive site between 70 and 60 (Fig. 2).
View larger version (17K):
[in this window]
[in a new window]
|
Fig. 2.
DNase I footprinting of the mouse COL1A1
(220 to +115) with NRK nuclear proteins. The 32P
end-labeled probe (220 to +115 of pGL1-220) was incubated
with and without NRK nuclear extract, treated with DNase I, and
electrophoresed. Lane 1, DNase I digestion of
32P-labeled probe incubated with 48 µg of nuclear
extract; lane 2, probe without nuclear extract; lane
3, 10-bp DNA ladder. Numbers on the
right show the positions relative to the start of
transcription (+1). Brackets indicate the protected regions
(regions A-F). The data show a representative gel of three
independent protein preparations.
|
|
Karsenty and de Crombrugghe (1) previously described 4 protected
regions (which they also designated A-D) in DNase I footprinting of
the same region of mouse COL1A1 with nuclear extracts
from mouse NIH3T3 fibroblasts. Region A, protected by NRK fibroblast nuclear extract, is identical to the synonymous region reported by these investigators. Our regions C, D, and E all lie within the
protected regions previously identified; however, the regions we have
designated B and F have not been described previously. For comparison,
DNase I footprinting of the 220 to +115 segment with NIH3T3 extracts
was also performed (data not shown). When 20-30 µg of NIH3T3 or NRK
nuclear proteins were used, the footprints obtained were identical to
the published ones (1). However the addition of higher protein
concentrations (48 µg) revealed the additional protected regions
described herein.
Recombinant YB-1 Binds to CYE Oligonucleotides--
First, a
series of DNA binding studies were performed with rYB-1 and
oligonucleotides containing the complete CYE sequence (88 to 48;
Table I), both sense (SS1) and antisense
(SS2) strands. As has been reported previously, double-stranded
probes demonstrated only weak rYB-1 binding activity (data not shown),
and therefore these studies focused on single-stranded binding assays.
rYB-1 formed several specific complexes with both SS1 and SS2
consisting of closely migrating bands (SS1, lane
1, and SS2, lane 5 in Fig. 3A). Competition experiments
were performed with truncated CYE oligonucleotides encompassing either
the Y-box (Y, Table I) or the inverted repeat (IR, Table I). Homologous
competitor DNA completely blocked rYB-1 binding to the CYE
(SS1, lane 4, and SS2, lane
8), whereas competitor oligonucleotides Y and IR only partially
inhibited binding. The Y oligonucleotide diminished the fastest
migrating bands formed with SS1 and SS2 (lanes 2 and 6), whereas the other complexes were unaffected. The IR
oligonucleotide did not compete for rYB-1 binding to SS2 (lane
7). Thus, maximal rYB-1 binding to the CYE is dependent on the
whole sequence including the Y-box and 3' adjacent IR. To substantiate
this finding, a direct comparison of rYB-1 binding to the SS1 of CYE,
Y, and IR was performed (Fig. 3B). Specific complex
formation was observed with Y (compare lane 1 with
lanes 4 and 5), whereas IR was not bound by rYB-1
(lane 2). A comparison of the relative binding affinities
revealed an ~10-fold higher affinity of rYB-1 for the complete CYE
(lane 3) compared with the Y-box alone (lane 1). These results emphasize the importance of the whole sequence context for high affinity YB-1 binding. To extend this finding and further define the sequence requirements for binding, specific mutations (G to T substitutions) were introduced into the CYE sense strand in either the Y-box (CYEmut1, Table I) or the IR (CYEmut2, Table I). As
seen in Fig. 3C, rYB-1 binding was not, as expected,
diminished with the introduced mutations in the Y-box but was even
increased. In contrast, binding of rYB-1 to the CYEmut2 oligonucleotide
was somewhat weaker than binding to the wild type oligonucleotide. These results emphasize the importance of the whole sequence context for high affinity YB-1 binding and indicate that simple base
substitutions in the Y-box sequence do not necessarily result in weaker
binding. An oligonucleotide spanning the region 112 to 83
encompassing the Site II Y-box at 100 to +96 (Fig. 1A)
failed to bind rYB-1 (lane 4 in Fig. 3C). This
finding again supports the notion that YB-1 binding not only requires a
Y-box but is dependent on more complex sequence determinants, and
although Site II displays high homology to the YB-1 binding consensus
sequence, it is not a functional binding site for the recombinant
protein.
View this table:
[in this window]
[in a new window]
|
Table I
CYE oligonucleotides
Sequences for the sense (SS1) and antisense (SS2) oligonucleotides
spanning the CYE (88 to 48) showing the Y-box (bold) and the 3'
inverted repeat (bold italics, arrows). Oligonucleotides spanning the
Y-box (Y), inverted repeat (IR), and with mutations were sense
oligonucleotides.
|
|
View larger version (37K):
[in this window]
[in a new window]
|
Fig. 3.
Recombinant YB-1 binds single-stranded CYE
oligonucleotides. A, EMSA of rYB-1 with single-stranded
CYE oligonucleotide probes shows binding to both sense (SS1,
lanes 1-4) and antisense (SS2, lanes
5-8) oligonucleotides. Binding was competed by homologous
oligonucleotide (lanes 4 and 8). Shorter
oligonucleotides Y (lane 2) and IR (lane 3) as
competitors reduced the intensity of all complexes formed with SS1.
With SS2, Y diminished the fast migrating band but had no effect on
other complexes (lane 6), whereas IR had no effect on
protein binding (lane 7). B, a comparison of
rYB-1 binding to CYE SS1, Y, and IR oligonucleotides showed specific
complexes formed with SS1 (lane 3) and Y (compare lane
1 with lanes 4 and 5) but not with IR
(lane 2). rYB-1 bound SS1 with ~10-fold higher affinity
than Y (lane 3). C, a comparison of rYB-1 binding
to CYE oligonucleotides with G to T mutations in the Y-box
(mut1, Table I) and the 3' portion of the inverted repeat (mut2, Table
I). Mutations in the Y-box increased binding of rYB-1 to CYE (compare
lanes 1 and 2), whereas mutations in the inverted
repeat decreased binding compared with wild type CYE (compare
lanes 1 and 3). An oligonucleotide (112 to
83) spanning Site II (Fig. 1B) was not bound by
recombinant protein (lane 4).
|
|
Sizing of the CYE Binding Activities--
Western blot analysis
showed that YB-1, a protein with a calculated molecular size of 35 kDa
and apparent size of 52 kDa in SDS-polyacrylamide gel (23), is
expressed in nuclear extracts from quiescent and proliferating NRK
cells (Fig. 4A). Anti-YB-1 antibody also cross-reacted with two other proteins (~200 and 35 kDa,
indicated by asterisks).
View larger version (25K):
[in this window]
[in a new window]
|
Fig. 4.
YB-1 binding activity in NRK fibroblast
nuclear extracts. A, Western blot analysis of nuclear
extracts (30 µg) from quiescent (lane 1) and proliferating
(lane 2) NRK fibroblasts with anti-YB-1 antibody showed the
presence of YB-1 protein (52 kDa; arrow). The antibody
detected two additional bands (*) with apparent molecular size of >200
and 35 kDa. Lane 3, control without primary antibody. The
position of the molecular size markers, 14-200 kDa, is shown.
B, Southwestern blotting of nuclear extracts (50 µg) from
quiescent (lanes 1 and 3) and proliferating
(lanes 2 and 4) NRK cells with SS1 and SS2 CYE
oligonucleotides showed binding of several nuclear proteins. The
presence of YB-1 (54 kDa; arrow) was confirmed by Western
blotting of the same membrane with anti-YB-1 antibody. The position of
the molecular size markers, 14-200 kDa, is indicated.
|
|
Southwestern blotting of nuclear proteins was used to determine the
sizes of the CYE-binding proteins (Fig. 4B). Single-stranded sense (SS1) and antisense (SS2) CYE oligonucleotides bound several proteins from NRK fibroblasts with estimated molecular sizes of 90-200, 54, and 35 kDa. Two of these activities were of the same size
as those detected by the antibody (see above). Some differences in the
binding were observed with SS1 compared with SS2, with additional SS2
binding activities of ~69 and 14 kDa. A comparison of extracts from
quiescent and proliferating cells showed differential regulation of
some CYE-binding proteins with the appearance of a novel high molecular
weight protein binding to SS2 in proliferating cells. Subsequent
Western blotting of the same membrane with anti-YB-1 antibody confirmed
the presence of endogenous YB-1 (52 kDa; arrow in Fig.
4B) with similar levels of the protein in quiescent
and proliferating cells. The identity of the other CYE-binding proteins remains to be established. Data base searches of this region for transcription factor binding sites showed potential binding sites for
Sp1, NF-1, LBP-1, and Ets family members.
To elucidate protein-DNA complex formation under nondenaturing
conditions, EMSAs were performed with mesangial cell (a cell type known
to constitutively express YB-1 (15)) or NRK nuclear proteins and SS1
and SS2 CYE oligonucleotide probes. As shown in Fig.
5A, several distinct complexes
formed with SS1 and NRK nuclear proteins (lane 3), with
complexes 2 and 3 exhibiting the same mobilities as those formed with
mesangial cell nuclear extract (lane 2). Incomplete
competition was observed with specific competitor, i.e.
complex 3 was lost, whereas complex 2 was diminished (lane 4). Nonspecific competitor (500×) did not affect complex
formation (lane 5). When the IR and Y oligonucleotides were
used as competitors, IR did not compete for binding, whereas Y
partially competed for complex formation (lanes 6 and 7). In
parallel experiments using SS2 as probe (Fig. 5B), three
distinct complexes of similar mobilities could be detected with
mesangial cell (lane 2) and NRK (lane 3) nuclear
proteins. Homologous competitor DNA diminished all bands with NRK
proteins (lane 4), whereas heterologous DNA had no effect (lane 5). With IR or Y oligonucleotides as competitors
(500×), complexes 2 and 3 were diminished but not completely inhibited (lanes 6 and 7). Taken together, these results
demonstrate the formation of several closely migrating complexes with
both strands of the CYE. Major complex formation is dependent on the
whole sequence, as the Y and IR sequences only partially diminish
complex formation.
View larger version (66K):
[in this window]
[in a new window]
|
Fig. 5.
Endogenous YB-1 participates in specific
complex formation with CYE. A, EMSA of mesangial cell
(MC NE, lane 2) or NRK (lane 3)
nuclear extracts with CYE SS1 oligonucleotide showed the formation of
several complexes (complexes 1-4) that were partially competed by
specific DNA (500× SS1, lane 4) and Y oligonucleotide
(lane 7) but not with nonspecific or IR oligonucleotides
(lanes 5 and 6). B, EMSA of mesangial
cells (lane 2) or NRK (lane 3) nuclear extracts
with CYE SS2 oligonucleotide showed the formation of three complexes
(complexes 1-3) that were competed by specific competitor (lane
4) but not by nonspecific DNA (lane 5). The IR and Y
oligonucleotides diminished the intensity of complex 2 and 3 (lanes 6 and 7). C, supershift
analyses were performed using an anti-YB-1 antibody. Visualization of
supershifts required a prolonged exposure time of 36 h
(SS1, lanes 4-6; SS2, lanes
10-12). Complexes formed are indicated by numbers to
the left of each panel (SS1, lanes 1 and
4, complexes 1-6; SS2, lanes 7 and
11, complexes 7-11). Inclusion of the antibody led to
additional low mobility complexes (*, lanes 5 and
11), whereas several complexes (complexes 4, 5, and
9-11) were diminished. With SS2, inclusion of the antibody led
to the formation of a high mobility complex designated as 12. Unrelated
rabbit IgG was added to control reactions (lanes 3, 6, 9,
and 12)
|
|
To test for YB-1 participation in this regard, supershift studies were
performed using a specific anti-YB-1 antibody (Fig. 5C). The
anti-YB-1 antibody used in this study is directed against epitopes in
the C terminus of the protein (13), which also contributes to
DNA binding (26). As a result this antibody mainly disrupts DNA
binding, leading to diminished bands (bands designated
4 and 5 in lane 2 with SS1 and
9, 10, and 11 in lane 8 with SS2),
whereas supershifts are rather weak and are visualized only with
prolonged autoradiography (indicated by asterisks in
lanes 5 and 11). The supershift with SS1 in
lane 5 nearly coincided with preformed complexes
(2>) and exhibited a similar mobility as that observed with SS2
in lane 11. In addition, a high mobility band (designated 12>) appeared with SS2, most likely representing a protein that interacts with YB-1 and by itself binds the probe. Unrelated rabbit IgG
had no effect on complex formation. These findings indicate that
endogenous YB-1 binds to both CYE strands forming several distinct complexes.
YB-1 Promotes Single-stranded DNA Conformation in the
CYE--
Previous studies have suggested that YB-1 can promote strand
separation in a sequence-specific fashion (22). MBN sensitivity analysis, which specifically detects single-stranded DNA regions, was
used to test for strand separation within the CYE (Fig.
6). A strictly double-stranded probe
containing the entire CYE was generated and labeled asymmetrically
at the 5'-end. The absence of banding upon addition of MBN to the
probe alone (lane 2) confirmed the double-stranded nature of
the probe. In the presence of rYB-1, MBN produced a distinct banding
pattern that was confined to the 3' IR (lane 3) with no
banding in the Y-box motif. Nuclear extracts from quiescent and
serum-stimulated mesangial cells induced a similar banding pattern to
that observed with rYB-1, which was enhanced with prolonged serum
exposure (lane 4-6). Strong bands at the 3'-end indicated
the presence of exonuclease activity. To confirm the specificity of the
DNA structural changes and exclude phasing of the enzyme to the ends of
the probe, experiments were repeated with probes labeled at the 5'-end
of the opposite strand. A similar pattern was obtained with these
probes, i.e. single-stranded regions appeared in the
IR region (lane 7), confirming that YB-1 induces DNA strand
separation in the inverted repeat region of CYE.
View larger version (38K):
[in this window]
[in a new window]
|
Fig. 6.
YB-1 induces single-stranded regions in
CYE. MBN sensitivity analysis was performed to identify regions of
single-strandedness. Incubation of the double-stranded
-32P end-labeled CYE oligonucleotide probe alone
(lane 1) or with MBN (lane 2) had no effect.
rYB-1 (10 ng) induced banding in the 3' inverted repeat sequence
(arrows) but not in the Y-box (lane 3). A similar
pattern was detected with nuclear proteins from serum-synchronized
mesangial cells (MC-NE, lanes 4-6). Banding in the IR
region was also observed when mesangial cell nuclear extract
was added to probes labeled at the 5'-end of the opposite strand
(lane 7).
|
|
YB-1 Suppresses Collagen 1(I) Promoter Activity--
In NRK
cells co-transfected with mouse COL1A1 promoter-reporter
constructs and a eukaryotic YB-1 expression vector, YB-1 suppressed the
activity (2-3-fold) of both pGL1-2.3 (containing 2.3 kb of 5'
sequence) and pGL1-220 (containing 220 bp of 5' sequence) (Fig.
7A). Suppression of
pGL1-220 by YB-1 was concentration-dependent (Fig.
7B). Although co-transfection of the empty vector, pSG5, caused some decrease in mouse COL1A1 promoter activity, the
YB-1-dependent effect was more marked and
concentration-dependent. YB-1 overexpression suppressed
pGL1-220 activity in a variety of cell types including rat
mesangial cells (13.1% of control cells co-transfected with the empty
pSG5 vector and the collagen promoter construct), primary cultures of
rat cardiac fibroblasts (27.6% of control), mouse 3T3 fibroblasts
(21% of control), and Chinese hamster ovary cells (49% of control)
(Fig. 7C).
View larger version (14K):
[in this window]
[in a new window]
|
Fig. 7.
Overexpression of YB-1 suppresses the
COL1A1 promoter via CYE. A, NRK
fibroblasts were co-transfected with mouse COL1A1
promoter-reporter constructs (pGL1-2.3 and
pGL1-220, 2 µg/well) and either pSG5 or pSG5-YB-1 (0.5 µg/well). Luciferase activity was measured 24 h after
transfection. B, NRK cells were transfected with
pGL1-220 (2 µg/well) and increasing concentrations of pSG5-YB-1.
The data are from a representative experiment of six repeats with a
mean of 3 wells/sample. C, pGL1-220 and either pSG5 or
pSG5-YB-1 were transfected into different cell types: 3T3 fibroblasts
(3T3), rat cardiac fibroblasts (RCF), rat
mesangial cells (MC), Chinese hamster ovary cells
(CHO). Luciferase activity was measured after 24 h. The
activity in cells transfected with pSG5-YB-1 was calculated relative to
cells transfected with pSG5. Because absolute luciferase activity
varied in different cell types, the data are presented as % activity
relative to control values set as 100%. Data are from a representative
experiment with a mean of 3 wells/sample. D, NRK fibroblasts
were transfected with rat COL1A1 promoter constructs
extending up to 3.6 kb 5' of the start of transcription and either pSG5
or pSG5-YB-1. CAT activity was measured 24 h after transfection.
Data were confirmed in five independent experiments with triplicate
determinations.
|
|
The proximal COL1A1 promoter sequence is highly conserved
between species (27). To determine whether YB-1 specifically affects the COL1A1 gene via proximal promoter sequences in other
species, three rat COL1A1-CAT reporter constructs extending
over the region 3.6 to +1.6 kb (3.6 to +1.6; 1.3 to +1.6; 0.4
to +1.6) were tested in co-transfection assays with the YB-1 expression
vector. There were some differences in the basal activity between these constructs; however, the activity of all three constructs were suppressed by YB-1 in a dose-dependent manner (Fig.
7D). Although the first intron has been shown to be
important in regulating COL1A1 gene expression (5), a
construct lacking intronic sequences (pCOLCAT2.3/0) was also suppressed
by YB-1 (data not shown). Together these results show that YB-1
trans-represses COL1A1 gene transcription via
proximal sequences. Furthermore subcloning of the CYE oligonucleotide upstream of either the SV40 promoter or the CMV
promoter conferred YB-1 repression on both these heterologous
promoters.3
Over-expression of YB-1 Suppresses Endogenous Collagen Gene
Expression and Collagen Production--
Northern blot analysis of NRK
cells transfected with the YB-1 expression vector showed that
overexpression of YB-1 suppressed endogenous COL1A1 mRNA levels in
a concentration- and time-dependent manner (Fig.
8A). The decrease in collagen
gene expression was reflected in decreased collagen production as
measured by HPLC (Fig. 8B). Western blotting with an
anti-type I collagen antibody also showed a trend toward decreased
protein levels with a decrease of 17.8 ± 1.25% (mean ± S.D. of two experiments with triplicate wells in each experiment) in
pSG5-YB-1 transfected cells compared with controls.
View larger version (27K):
[in this window]
[in a new window]
|
Fig. 8.
YB-1 overexpression suppresses endogenous
COL1A1 expression and collagen protein production in NRK cells.
A, Northern blot analysis of COL1A1 mRNA expression in
NRK cells transfected with either pSG5 or pSG5-YB-1 (0.5-2 µg
DNA/well) for 24 and 48 h. Duplicate samples are shown for the
48-h sample. Ethidium bromide staining of the 28 S rRNA was used to
normalize for variations in loading. The numbers indicate
the relative densitometric intensities of the signal in pSG5-YB-1
transfected cells compared with the signal obtained in cells
transfected with pSG5, assigned an arbitrary value of 1. Data are from
a representative experiment of four repeats. B, HPLC
analysis of total collagen production in NRK cells measured 48 h
after transfection with either pSG5 or pSG5-YB-1. Data are the mean of
duplicate experiments.
|
|
| |
DISCUSSION |
An elucidation of the molecular mechanisms governing collagen
production is essential to understanding not only normal tissue morphogenesis and homeostasis but also disease processes associated with under- or over-expression of this protein. The present study focused on the regulation of the collagen 1(I) chain in renal fibroblasts (NRK-49F) as a model collagen-producing connective tissue
fibroblast. Previous studies on the COL1A1 gene sequence between 220 and +115 have demonstrated its importance in basal and
inducible gene regulation, and a number of cis-regulatory elements and their cognate binding proteins have been identified (Fig.
1A). The presence of two inverted CCAAT-boxes in this
region, which can form the core for the binding sites of Y-box binding factor-1 (YB-1), together with the previously reported finding that
YB-1 can up-regulate MMP-2 gene expression (13), raised the
question of whether YB-1 might also play a role in regulating COL1A1 expression.
Previously, binding of NF-1 and CBF/NF-Y to the inverted
CCAAT-boxes at 100 to 96 (Site II; Fig. 1B) and 126 to
122 (Site III) in the COL1A1 promoter has been
demonstrated (8, 9) but, apart from one report (6), the potential role
of Y-box binding factors in regulating collagen gene expression has not been well studied. This prompted a closer inspection of the 220 to
+115 region for potential YB-1 binding sites, and revealed a third
putative site (83 to 72) with striking similarity to the
YB-1-binding element in the MMP-2 gene (13). The present study focused on the functionality of this region (83 to 59), which
we designated the collagen Y-box element.
DNase I footprinting of the 220 to +115 sequence of COL1A1
with NRK nuclear proteins generated a closely arranged pattern of
footprints extending from 190 to 40 similar to that described by
other investigators using nuclear proteins from mouse NIH3T3 fibroblasts (1). Titration of NRK nuclear proteins to saturate the
binding sites revealed two additional, novel footprints between 177
and 155 and between 65 and 40. A strong hypersensitive site at
the boundary of footprints E and F coincided with CYE and implied the
presence of potentially important regulatory elements in this region.
Notably, the Y-box of CYE is located in region E and the more 3'
portion of the inverted repeat in region F. In further support of the
functional importance of this site, the CYE region shows strong
inter-species homology (7) suggesting evolutionary conservation of this element.
YB-1 is a prominent member of the highly conserved Y-box binding factor
family with an ever-increasing number of target genes (19). Previously,
trans-activation of the rat COL1A1 gene
expression by another member of the Y-box transcription factor family,
chkYB-1b (6), has been demonstrated. However this protein binds a more 5' region between 200 and 133, and the relatedness of chkYB-1b to
YB-1 is still unclear. Our data suggest different modes of action of
the two proteins in the context of the COL1A1 promoter.
To elucidate the functionality of CYE, we first examined binding
of rYB-1 to this region. In EMSA, rYB-1 bound to CYE oligonucleotides with a preference for single-stranded sequences of both coding and
non-coding strands. Sequence specific binding of YB-1 and other Y-box
proteins to single-stranded DNA has been demonstrated previously (14,
19, 28-31). Although YB-1 was originally cloned as a CCAAT binding
factor (32), there does not seem to be an absolute requirement for this
motif, and many YB-1 binding sites contain either an imperfect
CCAAT-box or lack one altogether, suggesting an important role for
flanking sequences in DNA recognition by this protein. Interestingly,
an oligonucleotide (112 to 83) encompassing a bona fide
inverted CCAAT-box (100 to 96) in the COL1A1 promoter
(Site II) failed to bind rYB-1 despite the fact that sequence
comparison showed a near perfect match to the Y-box consensus. The
importance of flanking sequences in the CYE is reinforced by the
diminished, or absent, binding of truncated oligonucleotides Y and IR.
The complexity of the sequence requirement for binding is further
emphasized by mutational analysis. Previous studies have suggested that
guanine bases are important for YB-1 binding (22); however, G to
T substitutions in either the Y-box or the inverted repeat of CYE did
not abolish rYB-1 binding to CYE. These data suggest that YB-1 may
contact the promoter over a number of base pairs, making mutational
analysis using limited base substitutions difficult to interpret.
Endogenous YB-1 also bound CYE oligonucleotide probes, forming several
distinct complexes on both sense and antisense strands. The nature of
the multiple complexes is not clear but may represent the formation of
multimers, as has previously been shown to occur with rYB-1 (15, 22).
Moreover, on Western blots of nuclear proteins the anti-YB-1 antibody
cross-reacted with three bands, one with the expected molecular size
for YB-1 (52 kDa) as well as a smaller protein, potentially a
proteolytic fragment of YB-1 (33, 34), and a third, larger protein that
has not been characterized. Alternatively, the multiple bands may
be produced by the interaction of as yet undefined proteins with the
oligonucleotide or complex formation between YB-1 and other factors.
Southwestern blotting suggested that there are a number of
single-stranded DNA-binding proteins of different molecular
sizes that can interact with this region. Data base searches of the CYE
region for transcription factor binding sites (35) showed potential
binding sites for Sp1, NF-1, LBP-1, and Ets family members. Analysis of
YB-1 interactions with other transcription factors was not performed in
the present study. However, in other systems, YB-1 has been shown to
act indirectly by affecting the activity of other regulatory proteins
including Sp1 (36-39). Sp1 is known to activate COL1A1
transcription (3), and an Sp1 binding site lies in close proximity to
CYE raising the possibility of reciprocal regulation by Sp1 and
YB-1.
In transient co-transfections, YB-1 dose-dependently
suppressed COL1A1 promoter activity via sequences within the
220 to +115 region. A similar suppression of activity by YB-1 was
observed with constructs extending 2.3 kb 5' of the transcription
start-site, suggesting that the dominant repressive site lies in the
proximal region and arguing against upstream sites capable of
over-riding this repression. Repression of mouse COL1A1
promoter-reporter constructs by YB-1 in a number of different cell
types implies a conserved regulatory mechanism. Furthermore, YB-1 also
repressed activity of the rat proximal promoter. Unlike the mouse
COL1A1 promoter, the rat promoter appears to be further repressed by YB-1 when the sequence is extended up to 3.6 kb, suggesting
additional responsive upstream sites in the rat gene. In support of
this idea is a recent report by Stoddart et al. (40) in
which they describe (as unpublished observations) the cloning and
characterization of an inhibitory protein identified as YB-1, which
binds to the TGF-
activation element at 1624 bp in the rat
COL1A1 promoter (40). However, sequences in this region show
little homology in the rat and mouse genes. The CYE also conferred YB-1
responsiveness on two different viral promoter sequences, SV40 and CMV
(data not shown), indicating that the region can mediate YB-1
repression independent of the promoter context.
Numerous single-stranded DNA-binding proteins have been identified
(41-45), raising the question of how these proteins approach their
binding motifs within the DNA. YB-1 has both double- and single-stranded DNA binding properties and, most importantly, can
induce DNA strand separation (12, 22, 46). This observation led to a
model in which the repressive YB-1 effect is explained by this protein
opening up the DNA and thereby preventing binding of other
transcription factors to the double-stranded sequence (22). In the
COL1A1 promoter, MBN sensitivity analysis showed that YB-1
specifically induced strand separation in the inverted repeat sequence
of CYE, suggesting that this model of transcriptional repression by
YB-1 may also apply to the COL1A1 gene.
In conclusion, our data identify YB-1 as a novel repressor of
COL1A1 gene transcription acting via an evolutionarily
conserved element, the CYE, in the proximal promoter. In normal intact
tissues, collagen type I is generally expressed at low levels, and it
is likely that repressor elements, particularly in the minimal
promoter, play an important role in maintaining tight transcriptional
control. Further, we speculate that lifting of YB-1 repression may be
important in the activation of gene transcription. Future studies aimed at characterizing the mechanism by which YB-1 regulates collagen gene
transcription should provide additional insights into the regulation of
this major constituent of the extracellular matrix and provide new
strategies for modulating its expression.
| |
ACKNOWLEDGEMENTS |
We are grateful to those individuals
cited in the text who kindly provided reagents. We are indebted to J. Palmen (Cardiovascular Genetics, Department of Medicine, Royal Free and
University College Medical School) for help with DNA sequencing and to
L. Reynolds (Richard Dimbleby Imperial Cancer Research Fund,
Department of Cancer Research, St. Thomas's Hospital) for help with
HPLC analysis.
| |
FOOTNOTES |
*
This work was supported by Deutsche Forschungsgemeinschaft
SFB542, Project C4 (to P. R. M.), Interdisziplinäres Zentrum
für Klinische Forschung "BIOMAT"; Bundesministerium für
Bildung und Forschung Project 01 KS9503/9 (to P. R. M.);
National Institutes of Health Grant 5 RO1 K54602-02 (to J. T. N.);
the Wellcome Trust, UK, Grant 044502/Z/95/Z/040 (to G. E. L.); and a
research fellowship from the Royal College of Surgeons, London (to
K. S.).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: Dept. of
Nephrology and Immunology, Medical Clinic II, RWTH Aachen,
Pauwelsstrasse 30, 52057 Aachen, Germany. Tel.: 49-241-8089532; Fax:
49-241-8888446; E-mail: pmertens@post.klinikum.rwth-aachen.de.
Published, JBC Papers in Press, June 6, 2001, DOI 10.1074/jbc.M103145200
2
J. Rossert, personal communication.
3
J. T. Norman, unpublished observations.
| |
ABBREVIATIONS |
The abbreviations used are:
COL1A1, collagen
1(I);
YB-1, Y-box binding protein-1;
rYB-1, recombinant YB-1
protein;
CYE, collagen Y-box element;
IR, inverted repeat;
CMV, cytomegalovirus;
MMP-2, matrix metalloproteinase-2;
RE-1, response
element-1 (YB-1 binding element in the rat MMP-2 promoter);
CBF, CAAT
binding factor;
NF-1, nuclear factor-1;
BFCOL1, binding factor of a
type-I collagen promoter protein;
NRK, NRK-49F rat kidney fibroblasts;
MBN, mung bean nuclease;
T4-PNK, T4 polynucleotide kinase;
CAT, chloramphenicol acetyltransferase;
HPLC, high pressure liquid
chromatography;
EMSA, electrophoretic mobility shift assay;
kb, kilobase(s);
bp, base pair(s).
| |
REFERENCES |
| 1.
|
Karsenty, G.,
and de Crombrugghe, B.
(1990)
J. Biol. Chem.
265,
9934-9942
|
| 2.
|
Trojanowska, M.,
LeRoy, E. C.,
Eckes, B.,
and Krieg, T.
(1998)
J. Mol. Med.
76,
266-274
|
| 3.
|
Karsenty, G.,
and Park, R. W.
(1995)
Int. Rev. Immunol.
12,
177-185
|
| 4.
|
Kinniburgh, A. J.,
Firulli, A. B.,
and Kolluri, R.
(1994)
Gene
149,
93-100
|
| 5.
|
Bornstein, P.
(1996)
Matrix Biol.
15,
3-10
|
| 6.
|
Dhalla, A. K.,
Ririe, S. S.,
Swamynathan, S. K.,
Weber, K. T.,
and Guntaka, R. V.
(1998)
Biochem. J.
336,
373-379
|
| 7.
|
Ravazzolo, R.,
Karsenty, G.,
and de Crombrugghe, B.
(1991)
J. Biol. Chem.
266,
7382-7387
|
| 8.
|
Maity, S. N.,
Golumbek, P. T.,
Karsenty, G.,
and de Crombrugghe, B.
(1988)
Science
241,
582-585
|
| 9.
|
Nehls, M. C.,
Rippe, R. A.,
Veloz, L.,
and Brenner, D. A.
(1991)
Mol. Cell. Biol.
11,
4065-4073
|
| 10.
|
Galera, P.,
Park, R. W.,
Ducy, P.,
Mattei, M. G.,
and Karsenty, G.
(1996)
J. Biol. Chem.
271,
21331-21339
|
| 11.
|
Hasegawa, T.,
Takeuchi, A.,
Miyaishi, O.,
Isobe, K.,
and de Crombrugghe, B.
(1997)
J. Biol. Chem.
272,
4915-4923
|
| 12.
|
Mertens, P. R.,
Alfonso-Jaume, M. A.,
Steinmann, K.,
and Lovett, D. H.
(1998)
J. Biol. Chem.
273,
32957-32965
|
| 13.
|
Mertens, P. R.,
Alfonso-Jaume, M. A.,
Steinmann, K.,
and Lovett, D. H.
(1999)
J. Am. Soc. Nephrol.
10,
2480-2487
|
| 14.
|
Wolffe, A. P.
(1994)
Bioessays
16,
245-251
|
| 15.
|
Mertens, P. R.,
Harendza, S.,
Pollock, A. S.,
and Lovett, D. H.
(1997)
J. Biol. Chem.
272,
22905-22912
|
| 16.
|
Butt, R. P.,
Laurent, G. J.,
and Bishop, J. E.
(1995)
Eur J. Cell Biol.
68,
330-335
|
| 17.
|
Breault, D. T.,
Lichtler, A. C.,
and Rowe, D. W.
(1997)
J. Biol. Chem.
272,
31241-31250
|
| 18.
|
Firth, J. D.,
Ebert, B. L.,
Pugh, C. W.,
and Ratcliffe, P. J.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
6496-6500
|
| 19.
|
Swamynathan, S. K.,
Nambiar, A.,
and Guntaka, R. V.
(1998)
FASEB J.
12,
515-522
|
| 20.
|
Clark, I. M.,
Rowan, A. D.,
Edwards, D. R.,
Bech-Hansen, T.,
Mann, D. A.,
Bahr, M. J.,
and Cawston, T. E.
(1997)
Biochem. J.
324,
611-617
|
| 21.
|
Orphanides, C.,
Fine, L. G.,
and Norman, J. T.
(1997)
Kidney Int.
52,
637-647
|
| 22.
|
MacDonald, G. H.,
Itoh-Lindstrom, Y.,
and Ting, J. P.
(1995)
J. Biol. Chem.
270,
3527-3533
|
| 23.
|
Grant, C. E.,
and Deeley, R. G.
(1993)
Mol. Cell. Biol.
13,
4186-4196
|
| 24.
|
Bian, J.,
and Sun, Y.
(1997)
Mol. Cell. Biol.
17,
6330-6338
|
| 25.
|
Frisch, S. M.,
and Morisaki, J. H.
(1990)
Mol. Cell. Biol.
10,
6524-6532
|
| 26.
|
Izumi, H.,
Imamura, T.,
Nagatani, G.,
Ise, T.,
Murakami, T.,
Uramoto, H.,
Torigoe, T.,
Ishiguchi, H.,
Yoshida, Y.,
Nomoto, M.,
Okamoto, T.,
Uchiumi, T.,
Kuwano, M.,
Funa, K.,
and Kohno, K.
(2001)
Nucleic Acids Res.
29,
1200-1207
|
| 27.
|
Lee, H. W.,
Klein, L. E.,
Raser, J.,
and Eghbali-Webb, M.
(1998)
J. Mol. Cell. Cardiol.
30,
2495-2506
|
| 28.
|
Wada, M. R.,
Ohtani, Y.,
Shibata, Y.,
Tanaka, K. J.,
Tanimoto, N.,
and Nishikata, T.
(1998)
Dev. Growth Differ.
40,
631-640
|
| 29.
|
Wolffe, A. P.,
Tafuri, S.,
Ranjan, M.,
and Familari, M.
(1992)
New Biol.
4,
290-298
|
| 30.
|
Swamynathan, S. K.,
Nambiar, A.,
and Guntaka, R. V.
(1997)
J. Virol.
71,
2873-2880
|
| 31.
|
Swamynathan, S. K.,
Nambiar, A.,
and Guntaka, R. V.
(2000)
Biochem. J.
348,
297-305
|
| 32.
|
Didier, D. K.,
Schiffenbauer, J.,
Woulfe, S. L.,
Zacheis, M.,
and Schwartz, B. D.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
7322-7326
|
| 33.
|
Diamond, P.,
Shannon, M. F.,
Vadas, M. A.,
and Coles, L. S.
(2001)
J. Biol. Chem.
276,
7943-7951
|
| 34.
|
Stenina, O. I.,
Poptic, E. J.,
and DiCorleto, P. E.
(2000)
J. Clin. Invest.
106,
579-587
|
| 35.
|
Prestridge, D. S.
(1991)
Comput. Appl. Biosci.
7,
203-206
|
| 36.
|
Chernukhin, I. V.,
Shamsuddin, S.,
Robinson, A. F.,
Carne, A. F.,
Paul, A.,
El-Kady, A. I.,
Lobanenkov, V. V.,
and Klenova, E. M.
(2000)
J. Biol. Chem.
275,
29915-29921
|
| 37.
|
Safak, M.,
Gallia, G. L.,
Ansari, S. A.,
and Khalili, K.
(1999)
J. Virol.
73,
10146-10157
|
| 38.
|
Safak, M.,
Gallia, G. L.,
and Khalili, K.
(1999)
Mol. Cell. Biol.
19,
2712-2723
|
| 39.
|
Chen, N. N.,
Chang, C. F.,
Gallia, G. L.,
Kerr, D. A.,
Johnson, E. M.,
Krachmarov, C. P.,
Barr, S. M.,
Frisque, R. J.,
Bollag, B.,
and Khalili, K.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
1087-1091
|
| 40.
|
Stoddart, J. H., Jr.,
Ladd, D.,
Bronson, R. T.,
Harmon, M.,
Jaworski, J.,
Pritzker, C.,
Lausen, N.,
and Smith, B. D.
(2000)
J. Cell. Biochem.
77,
135-148
|
| 41.
|
Michelotti, G. A.,
Michelotti, E. F.,
Pullner, A.,
Duncan, R. C.,
Eick, D.,
and Levens, D.
(1996)
Mol. Cell. Biol.
16,
2656-2669.
|
| 42.
|
Michelotti, E. F.,
Sanford, S.,
and Levens, D.
(1997)
Nature
388,
895-899
|
| 43.
|
Tomonaga, T.,
and Levens, D.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
5830-5835
|
| 44.
|
Tomonaga, T.,
Michelotti, G. A.,
Libutti, D.,
Uy, A.,
Sauer, B.,
and Levens, D.
(1998)
Mol. Cell
1,
759-764 |
TOP
ABSTRACT
INTRODUCTION
