From the Howard Hughes Medical Institute Laboratories, Section of Diabetes and Metabolism, Division of Endocrinology, Metabolism and Nutrition and the Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Zfp-36, the gene encoding the
putative zinc finger protein tristetraprolin (TTP), is rapidly induced
in fibroblasts by a variety of growth factors. Recent gene knockout
experiments have shown that TTP-deficient mice developed arthritis,
cachexia, and autoimmunity, all apparently mediated by an excess of
tumor necrosis factor . We recently showed that full serum
inducibility of Zfp-36 requires elements in the promoter;
in addition, removal of the single intron strikingly inhibited
serum-induced TTP expression. We show here that replacement of the
intron with unrelated sequences, or removal of 95% of the intron but
retention of the splice sites, each resulted in the maintenance of
approximately 45 and 19%, respectively, of full serum-induced
expression. In addition, deletion of intron sequences base pairs
601-655 decreased the serum-induced expression of TTP by 65%.
Sequence base pairs 618-626 bound specifically to the transcription
factor Sp1; mutation of this binding motif decreased TTP expression by
70%, suggesting that Sp1 binding to this motif contributes to serum
induction of Zfp-36. We conclude that full serum-induced
expression of Zfp-36 depends on the activation of
conventional promoter elements as well as elements in the single intron, and that the presence per se of the intron in its
natural location also contributes significantly to the regulated
expression of this gene.
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INTRODUCTION |
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Tristetraprolin (TTP)1
is a widely expressed protein containing two putative zinc fingers of
the unusual CCCH class (1-5). It is the prototype of an enlarging
group of proteins containing similar zinc fingers (6-9). Although
predominantly nuclear in quiescent fibroblasts, TTP is rapidly
translocated from the nucleus to the cytoplasm by serum and other
mitogens (10), an event that occurs concomitantly with stimulated
serine phosphorylation (11). Although TTP has no proven function,
disruption of its gene, Zfp-36, in mice leads to a complex
syndrome that includes erosive arthritis, conjunctivitis, myeloid
hyperplasia, cachexia, and autoimmunity (12). All aspects of the
syndrome were prevented by pretreating the animals with monoclonal
antibodies to tumor necrosis factor (TNF
) (12). These data
suggest that a potential function of TTP is to regulate the production
of TNF
by certain cell types. They also indicate the possibility
that defects in Zfp-36 might be involved in the pathogenesis
of certain human conditions in which TNF
excess plays a role, such
as rheumatoid arthritis and systemic lupus erythematosus.
The transcription of Zfp-36 is rapidly and dramatically
stimulated by a variety of growth factors and mitogens, but not by agents acting solely through increases in cAMP levels (1, 3, 4).
Several transcription factor-binding sites have been identified in the
Zfp-36 promoter that are each partially responsible for activating transcription in response to serum or insulin (13). In
addition, we showed that the single intron of Zfp-36 also
participates in the regulation of its transcription, since deletion of
the intron results in an 85% decrease in serum-stimulated expression (13). In the present paper, we have characterized the positive contribution of the intron in more detail. We found that the simple presence of the intron in its natural location is important for normal
mitogen-stimulated expression; in addition, we have identified an
NFB-like binding site and two Sp1 sites within the intron sequence.
The Sp1 sites are responsible for a large component of the
intron-dependent, serum-induced expression.
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EXPERIMENTAL PROCEDURES |
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Plasmid Constructions
Parent Plasmids--
The TTP mRNA expression constructs
TTP137bp and intronless TTP137bp-Int have been
described (13). These contain 77 bp of the mouse Zfp-36
promoter linked to the mRNA coding region, including the 3-most
polyadenylation signal, in the presence or absence, respectively, of
the single 677-bp intron in its natural position. TTP137bp
exhibits essentially the same extent of serum-induced expression as
constructs containing up to 1.7 kilobases of promoter (13). For
purposes of clarity, we will refer to all constructs as derivatives of
the TTP gene, rather than of Zfp-36.
TTP Intron Location Constructs--
Two unique restriction sites
in the genomic sequences flanking the TTP intron were created in
plasmid TTP137bp by the polymerase chain reaction
primer-overlapping mutagenesis technique (14). A SalI site
was made 8 bp 5 to the splice site at the 5
end of the intron, and a
SnaBI sequence was created 5 bp 3
to the splice site at the
3
end of the intron so that, when this DNA fragment was released by
SalI and SnaBI digestion, the consensus donor and
acceptor sites (15) were included. The mouse TTP intron from this
construct was isolated, the ends filled with dNTPs, and then inserted
at various sites in the parent plasmid TTP137bp-Int.
Mutant Constructs--
Specific deletions in the intron sequence
of the TTP137bp construct were generated by using the
Site-directed in vitro Mutagenesis System (Amersham Corp.,
Arlington, IL). Substitution mutations in the intron NFB-like site
and intron Sp1 site (see below) were made by the polymerase chain
reaction primer-overlapping mutagenesis technique and the use of a
proofreading DNA polymerase pfu (Stratagene, La Jolla, CA). Mutation
primer for the NF
B-like binding site was: NFAT
(131GGTCCAGATACCGTTGATCAACTTGGACGAAAAG164).
Mutation primers for the two Sp1 mutants were: SPEV
(607GCTTTACAACAGTCGCGAGGCGACGTCACC637)
and
CHGHSp1(607GCTTTACAACAAGATATCAGCGACGTCACC637),
in which the numbers flanking the sequences are the bp numbers in
the intron (see Fig. 3), and underlined sequences were the mutated
sites. All mutant constructs were sequenced (U.S. Biochemical Corp.,
Cleveland, OH) to confirm that the appropriate deletions and mutations
had been made.
Cell Culture and Transfections
Primary chick embryo fibroblasts (CEF) were isolated and transient transfections were performed with plasmid DNA in calcium-phosphate precipitates, exactly as described previously (13). Briefly, 1 day before the transfection each 10-cm tissue culture dish was plated with 3 × 106 CEF. To each plate was added a transfection mixture containing 15 µg of test plasmid DNA and 5 µg of pXGH5 (Nichols Institute Diagnostics, San Juan Capistrano, CA). Four to six h after DNA addition and incubation at 37 °C, the cells were treated for 4 min with 4 ml of 10% glycerol in HEPES-buffered saline (pH 7.1) and washed twice with phosphate-buffered saline to remove the remaining precipitate. After a further 24-h incubation in complete culture medium, the cells were incubated for 24 h in medium containing 0.5% fetal bovine serum (FBS) to make them quiescent, and were then washed and used for the preparation of total cellular RNA. Plasmid pXGH5 was co-transfected as an internal control for transfection efficiency. Human growth hormone (HGH) released into the culture medium was measured by immunoassay (Nichols Institute Diagnostics).
RNA Preparation and Northern Blot Analysis
Total cellular RNA was prepared as described (16), with
modifications as described (17). Northern blots were prepared as
described before (1). Blots were hybridized with random primed
-32P-labeled (Stratagene) mouse TTP cDNA (1). TTP
mRNA accumulation was quantified using a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA) and the values were normalized to the amount
of HGH secreted into the medium. This was done by comparing the HGH
formed in each dish transfected with a test plasmid to cells
transfected with the same amount of vector plasmid BS+.
Nuclear Extracts and Electrophoretic Mobility Shift Assays
Nuclear extracts from CEF and NIH-3T3 cells were prepared as
described previously (18). Briefly, 10 µl of binding buffer (10 mM Tris (pH 7.5), 1 mM EDTA, 10% (v/v)
glycerol, 1 mM dithiothreitol) containing 1 µg of
poly(dI-dC) (Pharmacia Biotech Inc., Piscataway, NJ), and 20 × 103 cpm of -32P-labeled probe, were added to
5 µg of nuclear protein in 10 µl of nuclear extract buffer (20 mM Tris (pH 7.9), 20% (v/v) glycerol, 50 mM
KCl, 50 mM sodium fluoride, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride), and then subjected to binding reactions and electrophoresis
as described (18). The immunosupershift assays were performed in the
same way except that the antibodies were incubated with the nuclear
extracts at room temperature for 30 min before the radioprobe mixture
was added.
The following DNA fragments and double-stranded synthetic
oligonucleotides containing potential sequences for DNA binding factors
were used in this study. A 116-bp fragment containing the mouse TTP
intron region bp 116-220 (see Fig. 3) was excised by digestion with
XhoI (at a site created by mutagenesis at bp 114-119) and
NcoI; the fragment thus obtained was further digested with
AluI to yield two short sequences (bp 116-164 and bp
165-220). A 132-bp fragment containing the intron region bp 326-430
was excised by digestion with HindIII (at a site created by
mutagenesis at bp 325-330) and PvuI. A 110-bp
DdeI fragment containing bp 563-673, and a 66-bp
DdeI-AhaII fragment containing bp 563-629, were
also isolated from the intron (Fig. 3). For oligonucleotide Sp1, two
complementary synthetic oligonucleotides (Life Technologies, Inc.,
Gaithersburg, MD) were annealed to form a doubled-stranded oligonucleotide corresponding to mouse intron sequence bp 617-629 (tcgacAGGGGCGGGGCGA), where underlined bases indicate the
core sequences of the consensus Sp1-binding site (19). Double-stranded oligonucleotide HGHSp1 (tcgacTGTGTGGGAGGAGCTTCTAG), which
corresponds to the Sp1-binding site at 139 to
121 in the HGH
promoter (20), was made the same way. A 5-base single-stranded tail
(SalI site) was added to both ends of the two
oligonucleotides for subcloning and fill-in labeling. Double-stranded
oligonucleotides WT137 containing intron sequence bp 137-157
(tcgacGATACCGGCGATCCCCTTGGAG), where underlined bases
indicate the NF
B-like binding site (21); two NF
B binding
oligonucleotides (tcgacAGGGCTGGGGATTCCCCATCTCCACAG) from
the murine major histocompatibility complex class I gene H-2Kb enhancer (22), and
(tcgacTCAACAGAGGGGACTTTCCGAGG) from immunoglobulin (Ig)
enhancer (23), were made as described above.
The 5-protruded ends of the DNA fragments and oligonucleotides were
filled in with [
-32P]dCTP (NEN Life Science Products,
Boston, MA) and unlabeled dATP, dGTP, and dTTP (Life Technologies,
Inc.). Unlabeled dCTP was subsequently added to the reaction. The
labeled DNA was separated from unincorporated radioactivity by
acrylamide gel purification.
The following double-stranded oligonucleotides were also prepared and used in the gel mobility shift assays: mutant Sp1 oligonucleotides (mutated nucleotides underlined) SPEV (tcgacAAGATATCAGCGAG), and CHGHSp1 (tcgacAGTCGCGAGGCGAG); mutant intron bp 137-157 oligonucleotides G137 (tcgacGATACCGGGGATCCCCTTGGAG) and AT137 (tcgacGATACCGAAGATCTTCTTGGAG); and a nonspecific competitor TIE (tcgacGAAGTGCTTTACAG). A double-stranded oligonucleotide AP2 (GATCGAACTGACCGCCCGCGGCCCAT, core sequence underlined) was from Santa Cruz Biotechnology (Santa Cruz, CA).
The Sp1 rabbit polyclonal antiserum (24) was a kind gift from Dr.
Jonathan M. Horowitz (Duke University Medical Center, Durham, NC). The
anti-NFB rabbit IgG p65 (sc-109) and p50 (sc-114) were purchased
from Santa Cruz Biotechnology. Recombinant mouse TNF
was from H&R
Systems (Minneapolis, MN).
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RESULTS |
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Requirement for the Intron for the Full Serum-induced Expression of the TTP Gene
We previously reported that TTP genomic constructs containing the
single intron exhibited approximately 6-fold greater expression of TTP
mRNA in transiently transfected, serum-stimulated CEF cells than an
otherwise identical intronless construct (13). This occurred with
similar constructs prepared from the mouse, human, and bovine genes
(13). Introns in certain genes have been shown to possess enhancer
activity, which is not affected by the position or orientation of the
intron in the DNA construct (25). To test the possibility that the
intron from the TTP gene contained enhancer activity, we inserted the
mouse intron into TTP137bp-Int in both orientations and in
different positions, including immediately 5 to the minimal promoter,
8 bp before the translation start site, 32 bp after the translation
stop codon, and immediately 3
of the 3
-most TTP cDNA sequence
(Fig. 1A). Of the eight
constructs made, only the one (+46Int(5
)
) with the intron inserted
in a 5
to 3
orientation 8 bp before the translation start site showed improved (2.6-fold) serum-induced expression compared with the intronless construct (Fig. 1). This expression was still only 35% of
that seen with intact TTP137bp. The closeness of this
insertion site to the naturally occurring intron splice site (~30 bp)
may indicate the importance of this location to the regulation of serum-induced TTP gene expression. The oppositely orientated intron inserted into this site failed to improve expression, as did the insertion of the intron into the other positions in either orientation. These data indicate that the effect of the intron on the serum-induced expression of TTP does not resemble that of a simple enhancer.
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However, it was possible that the intron splicing per se was important for the full expression of TTP mRNA (26); when the intron was inserted in ectopic locations, particularly in the opposite orientation, splicing of the mRNA might not occur, despite preservation of intact donor and acceptor splice sequences. To determine whether intron splicing was important for the expression of TTP mRNA, construct TTPF52Int was created by substituting the TTP137bp intron sequence from 11 to 661 with a 505-bp BamHI fragment from the intron of the MARCKS-related protein (MRP (27); also known as F52 or MacMARCKS), an intron known to confer no serum-inducibility on the expression of MRP.2 Serum-induced expression of TTPF52Int in CEF cells transiently transfected with this construct was 45% of that seen with the native intron in TTP137bp (Fig. 2), or about 9-fold greater than that seen with the intronless construct TTP137bp-Int. A construct in which the intron sequence from 11 to 661 was deleted, leaving behind the splice donor and acceptor sequences and an "intron" of only 26 bp, also showed markedly improved serum-induced expression (3.8-fold increase) compared with TTP137bp-Int (Fig. 2). These data indicate that intron splicing per se plays an important role in the full serum-induced expression of TTP. They also indicate that the simple presence of an intron in the appropriate location, containing either 95% extraneous sequence or shortened to 5% of its original size, is sufficient to confer 45 and 19%, respectively, of the serum-induced expression of the wild-type gene.
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There are three potential Sp1 and five potential AP2-binding sites in the mouse TTP intron (Fig. 3). To investigate the possible roles of these DNA sequences in controlling the expression of TTP, seven deletion mutants were constructed by removing segments of the intron. These plasmid DNAs as well as the parent plasmid TTP137bp and the intronless construct TTP137bp-Int were transiently transfected into CEF cells, which were then stimulated with serum. TTP mRNA expression from these constructs is shown in Fig. 4. While there was no significant change in the serum-induced expression of TTP mRNA from deletion (d) constructs d(11-115), d(227-327), d(427-536), and d(535-599), expression from mutants d(116-220), d(326-430), and d(601-655) was 58, 78, and 35% of the control construct TTP137bp, respectively, indicating that these three regions in the mouse intron contained sequences that were important for the serum-induced expression of TTP.
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Nuclear Factor Binding to Intron Sequences
NFB-like Binding--
To further delineate the intron sequences
that affected the serum-stimulated expression of TTP, a 116-bp fragment
that includes intron region bp 116-220 was isolated and used as a
probe in electrophoretic mobility shift assays, in which the probe was
incubated with nuclear extracts prepared from CEF treated with 10% FBS
or control conditions for 10 min. Three major DNA-protein complexes
were formed (Fig. 5A). One
band, the uppermost (CI), was present in extracts from control cells
but was absent in extracts from FBS-treated cells. This region of the
intron contains one consensus AP2 site and one Sp1 site. However, when
excess double-stranded oligonucleotides containing either AP2 or Sp1
binding sequences were included in the reaction mixture, there were no
changes in the intensity of any of the shifted bands, nor did any
supershifting of the complexes occur upon addition of the anti-Sp1
antiserum (data not shown). These observations suggest that neither Sp1
nor AP2 is involved in the formation of these DNA-protein
complexes.
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Sp1 Binding-- Deletion of intron sequence bp 601-655 led to the most significant loss (65%) of serum-induced TTP expression. To determine whether this region contained sequences that bind to proteins that modulate the level of TTP transcription, a 110-bp DdeI mouse TTP intron fragment spanning bp 563 to 672, which included the whole deleted area of mutant d(601-655), was isolated and used as a probe in gel shift assays. A 66-bp DdeI-AhaII (563-629) fragment, which included much of the deleted area of mutant d(601-655), was also used.
When either probe was used in gel-shift assays, two DNA-protein complexes were formed with CEF nuclear extracts (Fig. 6A). There was no difference in the intensity of these two shifted bands when nuclear extracts were used from serum-treated or control CEF, nor from TNF
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Expression of Constructs with Mutations in the Intron Sp1-binding Site
As demonstrated above, the consensus Sp1 binding motif in the 625-679 region of the mouse TTP intron apparently binds to Sp1 contained in the CEF nuclear extract. This motif is also identical in sequence and is in a similar position within the introns from the mouse, human, and bovine genes (Fig. 3B). We next examined whether mutations in this Sp1 motif would affect the serum-stimulated expression of TTP mRNA. Fig. 7 shows that when the Sp1 sequence in this region was mutated to SPEV, the decrease in serum-induced expression (by 70% in comparison to the parent construct) was similar in extent to that seen with the d(625-679) deletion mutant (by 65%). Mutant CHGHSp1, which resembles the Sp1-binding site in the HGH promoter, did not affect the normal expression of TTP. These findings indicate that the loss of this Sp1-binding site, GGGGCGGGG, at bp 618-626, can account for the loss of TTP mRNA expression in the deletion mutant.
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We previously showed that TTP promoter element 1 (TPE1), a unique
nuclear protein binding sequence in the TTP promoter, was important for
the full serum-induced expression of TTP, along with nearby Sp1 and
AP2-like binding sequences (13). It was therefore of interest to
investigate potential interactions between TPE1 and Sp1 in the
serum-induced expression of TTP. When both binding sites in
TTP137bp were mutated, from Sp1 (at bp 618 to 626 in the
intron) to SPEV and TPE1 to TPE1 (13), the expression of TTP
mRNA from the double mutant decreased to 22.5% of control, compared with 30-31% of control for the constructs that contained either of the single mutations (Fig. 7). The apparent lack of a direct
interaction between these two DNA binding elements on the expression of
TTP mRNA was also reflected in the gel mobility shift assays, since
there was no observable binding cooperactivity in the DNA-protein
binding complexes formed. In other words, when two labeled probes were
present in the same binding mixture, there was no supershifting or
change in binding intensity of any of the formed DNA-protein complexes
(data not shown). Nevertheless, these results demonstrate that
interactions of Sp1 with its binding sites in both the promoter (13)
and in the intron are required for the full serum-stimulated expression
of TTP137bp.
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DISCUSSION |
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In a previous paper (13), we demonstrated that removal of the
single intron from the TTP gene decreased its serum-inducible expression by about 85%. The aim of the present work was to evaluate the mechanism by which the intron contributed to serum-induced expression in such a striking manner. The results demonstrate that the
intron contains an NFB-like binding element and two copies of a
promoter element, Sp1, which, when mutated or deleted, significantly
(by about 42, 70, and 22%, respectively) decreased the serum-induced
expression of TTP. In addition, the presence of an intron per
se in the appropriate location and in "spliceable" form also
contributed significantly (by about a 3.8-fold increase) to
serum-induced expression. The contribution of these factors together
appeared to account for most of the intron effect on the rapid mitogen
inducibility of the gene.
The conclusions concerning the presence per se of the intron
are based largely on experiments in which constructs containing a
foreign intronic sequence (TTPF52Int), a minimal intron of
only 26 bp, or the TTP intron inserted into the 5-UTR of the TTP
mRNA were able to confer greater serum inducibility than that of
the intronless construct TTP137bp-Int. It has been
demonstrated previously that adding an intron to the 5
-UTR can
significantly increase expression of cDNAs in transient
transfection studies, in which the mechanism of stimulation was not
intron-specific but dependent on splicing per se (31). Other
studies have shown that splicing can increase the levels of both
nuclear and cytoplasmic poly(A) RNA (32), implying that the requirement
for the intron is at the post-transcription level. Huang and Gormann
(26) have shown that there was increased expression of transiently
transfected reporter cytoplasmic RNA and protein in the presence of a
synthetic intron inserted into the 5
-UTR. Their study suggested that
splicing is coupled to a polyadenylation/transport pathway. The
increased accumulation of mRNA in the presence of a heterologous
intron has also been attributed to the intron's positive influence on the rate of transcription, even though there was no evidence that the
intron employed contained any general enhancer-like elements (33).
There was no improvement in serum-induced TTP expression when the
intron in either orientation was placed into the vector immediately 5
of the minimal promoter sequence, or inserted 3
to the stop codon, or
downstream of the polyadenylation site in the cDNA. Similar
negative results2 were observed in experiments in which
transcription was driven by a silent heterologous promoter, with the
TTP intron in different positions and orientations in plasmid Glo48TTP
(13). These observations indicate that the mechanism by which the TTP
intron facilitates serum-induced expression is different from that of
classical transcriptional enhancers, which exert their effects
regardless of their location or orientation relative to the transcribed
gene (34, 35).
Many examples of modulation of gene activity by sequences within introns have been described. In some cases, the modulation of gene activity was under the control of GC-rich sequences in the introns (36-41). The GC-box sequence that binds transcription factor Sp1 is one of the most ubiquitous regulatory DNA elements in eukaryotic genes (19). Sp1 is a nuclear protein that binds to GC-rich sequences by means of three zinc fingers, and activates transcription via glutamine-rich domains (42). A GT-box motif present in several viral and eukaryotic promoters has also been shown to bind factors related to Sp1 (41, 43-45).
There are three consensus Sp1-binding sites within the mouse TTP
intron. When the sequence containing the first Sp1 site in the intron
was eliminated (116-220), there was a modest (42%) inhibition of
serum-induced TTP expression. However, a bona fide Sp1 site
oligonucleotide was not able to compete with any of the protein-DNA
complexes formed in gel mobility shift assays when this fragment of
intron DNA was used as a probe, nor did an anti-Sp1 antiserum
supershift any of the complexes. These observations indicate that Sp1
is not likely to be involved in the modulation of TTP expression by
this region of the intron. However, one of the complexes formed with
the bp 116-220 probe changed in intensity when nuclear extracts
prepared from serum-treated CEF were used. The possibility that other
DNA binding factors, such as NFB, could bind to this intron fragment
and modulate TTP expression was therefore investigated.
NFB was originally discovered in a complex binding to the enhancer
sequence in the immunoglobulin
light chain intron (23), and is now
considered to be a ubiquitous transcription factor that plays an
essential role in the activation of many cellular genes (for review,
see Refs. 46-48). Activation of TTP transcription is stimulated by
TNF
in mammalian fibroblasts and macrophages.2 The
accumulation of TTP mRNA is rapid and transient upon TNF
treatment of the cells, with magnitudes of increase similar to those
observed with serum. Since TNF
treatment brought about such a
typical and prominent induction of TTP in NIH-3T3 cells, and failed to
stimulate CEF (probably due to the lack of receptors for mammalian
TNF
in these cells), we evaluated the possible involvement of intron
bp 116-220 in the transcriptional control of TTP expression using
nuclear extracts from control and TNF
-treated NIH-3T3 cells. The
electrophoretic mobility shift assays using radiolabeled DNA fragments
revealed a regulatory element within intron bp 137-157, GGCGATCCCC,
closely resembling the NF
B binding motif found in
H-2Kb (22). The binding complex formed with bp
137-157 using nuclear extracts prepared from TNF
-treated NIH-3T3
cells could be supershifted with antibody to p65, and was eliminated by
competition with an Ig
NF
B binding oligomer that has a
preferential binding affinity for p65 (21). These data indicate that
p65 is one of the components of the NF
B complex binding to this
region of the TTP intron. It has been shown that sequences similar to
this region of the TTP intron, e.g. the NF
B site on
H-2Kb, preferentially bound p50 (21); however,
we were unable to show supershifting in the presence of an anti-p50
antiserum when intron probe WT137 was used (data not shown).
NFB is sequestered in the cytoplasm by binding to I
B. Activation
of cells by various agents, including phorbol esters, leads to
dissociation of the NF
B·I
B complex and migration of NF
B to
the nucleus (49-51). We were not able to demonstrate the formation of
an NF
B DNA-protein complex with nuclear extracts from PMA-treated NIH-3T3 cells using probes from either the Ig
light chain enhancer or bp 137-157. We are not certain of the reason for this failure under
our experimental conditions. However, it has been reported that
PMA-inducible protein kinase C isoforms were not common mediators of
both phorbol esters and TNF
; and TNF
has been shown to activate NF
B in protein kinase C-depleted cells or in the presence of protein
kinase C inhibitors (52, 53).
The elimination of sequences containing the second Sp1 binding motif and the fourth and fifth consensus AP2-binding sites (326-430) caused a slight decrease (22%) in serum-induced TTP expression. In electrophoretic mobility shift assays with a DNA probe containing this region of the intron, there was evidence of Sp1 binding to this fragment. Although it is unknown at present whether the binding of AP2 to this region of the intron is involved in the activation of the TTP gene, it is possible that the Sp1 binding motif present in this intron segment is responsible for this modest but reproducible change in serum-stimulated expression.
Intron sequences containing the third Sp1 motif (601-655), deletion of
which led to a more dramatic decrease (of about 65%) in the
serum-induced expression of TTP, are highly conserved among the mouse,
human, and bovine introns (Fig. 3B). We used electrophoretic mobility shift assays with DNA fragments derived from this region of
the intron and several competitor sequences to analyze proteins that
bound to this site in CEF nuclear extracts. We also tested the
biological significance of this site by mutating the putative protein-binding site and examining the level of serum-induced expression in transient transfection assays. Only one specific protein-binding site was identified in the bp 601-655 region of the
intron by gel shift assays. On the basis of competition studies with
oligonucleotides specific for Sp1, as well as the ability of anti-Sp1
antiserum to supershift the binding complexes, we concluded that the
protein binding to this site was Sp1. Mutational analysis showed that
when protein binding to this region was prevented, serum-induced
expression of the mutant plasmid EVSP(TTP137bp) decreased by 70% compared with the parent plasmid
TTP137bp, indicating that this specific Sp1-binding site in
the intron plays a major role in controlling serum-induced expression
of TTP.
In an attempt to further establish the importance of Sp1 binding to
intron bp 618-626 in serum-induced expression of TTP, we created a
substitution mutant in which the Sp1 binding motif was replaced by a
binding site for the yeast trans-activator protein GAL4
(5-CGGAAGACTCTCCTCCG-3
(54)). Thus, co-transfection of a recombinant
construct expressing a fusion protein containing the GAL4-binding
domain and the functional activation domain of Sp1 might be able to
restore the expression of this mutant TTP gene to the level of that of
the wild-type construct. However, in the CEF, co-transfection of as
little as 0.5 µg of the GAL4-Sp1 recombinant construct (55)
suppressed the expression of the TTP constructs to about 20% of the
level seen in the absence of the fusion construct. This inhibitory
effect of the GAL4-Sp1 recombinant construct was seen with both
wild-type and mutant TTP constructs alike (not shown). Furthermore,
when a plasmid containing only the GAL4-binding domain (56) was
co-transfected with either the wild-type or mutant TTP constructs, the
expression of TTP was also decreased (not shown). We do not know the
reason for these apparently nonspecific inhibitory effects; perhaps in
this cell system, the GAL4 constructs replicated much more rapidly than
the TTP constructs, resulting in decreased expression of TTP. The
reduction in expression was probably not due to the GAL4-Sp1 fusion
protein or GAL4-binding domain per se, since TTP constructs that did not contain the GAL4 recognition site were also affected. Further experiments, probably in a different transfection system, will
be necessary to validate this approach to the importance of the intron
Sp1 site to TTP transcriptional regulation.
Sp1 is a ubiquitously expressed transcription factor that generally
stimulates transcription by binding to GC-rich promoter elements
located in a wide variety of cellular and viral promoters (19, 57, 58).
In a few cases, the levels or binding activity of Sp1 could be
dynamically regulated in response to stimulants such as phorbol esters
(59) and glucose (60), leading to activation of genes that respond to
these agents. However, we were unable to demonstrate a change in
complex intensity using this intron segment and nuclear extracts from
serum-treated CEF, or serum-, PMA-, or TNF-treated NIH-3T3 cells,
suggesting that Sp1 involvement may be more constitutive than acutely
regulated in this case.
Our inability to demonstrate any serum-inducible activity of the TTP
intron when it was moved to other regions of the TTP gene (with the
lone exception of placement in the 5-UTR) suggests that the TTP intron
probably does not function as a typical enhancer. Intronic sequences
that increase transcriptional activity when placed in the correct
position relative to their promoters have been observed in other genes
(32, 61). One mechanism by which these introns might influence
transcription could involve an interaction between regulatory sequences
in the intron and in the 5
-flanking promoter. Such an interaction
could be mediated by trans-acting factors that bind to both
sequences (57). In our previous study, we identified four closely
spaced serum regulatory elements, EGR-1, TPE1, AP2-like, and Sp1, in
the 5
-flanking region of the TTP gene (13). These elements, together
with the single TTP intron, are likely to function in a concerted
fashion, since the full serum-induced mRNA expression required the
presence of all these elements. However, our present data do not
provide evidence for a direct physical interaction between the promoter
elements and the intron elements, except the rather weak evidence that
the intron must be positioned near the promoter to increase
serum-stimulated expression.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Jonathan M. Horowitz for the Sp1 antiserum. We also thank Jane S. Tuttle for preparing and maintaining the CEF cells.
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FOOTNOTES |
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* This work was supported in part by Grants T32-DK-07012 (to W. S. L.) and K11-DK02227-02 (to M. J. T.) from the National Institutes of Health.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.
Present address: Division of Diabetes, Dept. of Medicine,
University of Massachusetts Medical Center, Worcester, MA 01655.
§ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: National Institute of Environmental Health Sciences, MD-A2-05, 111 Alexander Dr., Research Triangle Park, NC 27709. Tel.: 919-541-4899; Fax: 919-541-4571.
1
The abbreviations used are: TTP,
tristetraprolin; PMA, phorbol 12-myristate 13-acetate; HGH, human
growth hormone; CEF, chicken embryonic fibroblast; FBS, fetal bovine
serum; TPE1, TTP promoter element 1; TNF, tumor necrosis factor
;
bp, base pair(s); UTR, untranslated region.
2 W. S. Lai and P. J. Blackshear, unpublished data.
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REFERENCES |
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