Characteristics of the Intron Involvement in the Mitogen-induced Expression of Zfp-36*

Wi S. Lai, Michael J. ThompsonDagger , and Perry J. Blackshear§

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

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha . 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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha  (TNFalpha ) (12). These data suggest that a potential function of TTP is to regulate the production of TNFalpha 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 TNFalpha 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 NFkappa B-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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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 NFkappa B-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 NFkappa 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 alpha -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 alpha -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 NFkappa B-like binding site (21); two NFkappa B binding oligonucleotides (tcgacAGGGCTGGGGATTCCCCATCTCCACAG) from the murine major histocompatibility complex class I gene H-2Kb enhancer (22), and (tcgacTCAACAGAGGGGACTTTCCGAGG) from immunoglobulin (Ig) kappa  enhancer (23), were made as described above.

The 5'-protruded ends of the DNA fragments and oligonucleotides were filled in with [alpha -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-NFkappa B rabbit IgG p65 (sc-109) and p50 (sc-114) were purchased from Santa Cruz Biotechnology. Recombinant mouse TNFalpha was from H&R Systems (Minneapolis, MN).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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')right-arrow) 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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Fig. 1.   Intron position-dependent TTP expression. A, structures of the mouse TTP transcription constructs used for transfection assays. The 79-bp promoter sequences are represented by a thick solid line. Exon I and exon II sequences are shown as hatched and open bars, respectively. The position of the mouse TTP intron is indicated by the thin line, with the arrowhead indicating the orientation. The wild-type plasmid TTP137bp contains 79 bp of 5' sequence upstream of the transcription start site. TTP137bp-Int is the same as TTP137bp except that the intron is absent; the sequences of the two exons are correctly spliced together (13). In plasmids 5'Int(5')right-arrow and 5'Int(3'), the intron is inserted 5' of the 79 bp of promoter in a 5'-3' or a 3'-5' orientation, respectively. In plasmids +46Int(5')right-arrow and +46Int(3'), the intron is inserted in the mRNA 5'-UTR after bp +46 (8 bp 5' of the translation start site) in the orientations indicated. In plasmids BglInt(5')right-arrow and BglInt(3'), the intron is inserted in the indicated orientation into the BglII restriction site in exon II, 32 bp 3' of the translation stop codon. In plasmids 3'Int(5')right-arrow and 3'Int(3'), the intron is inserted 3' to exon II in the orientations indicated. The tabulated values for expression were obtained by comparing quantified PhosphorImager values from Northern blots of TTP mRNA bands transcribed from the experimental plasmids with the wild-type plasmid in each transfection experiment. The quantified PhosphorImager values were corrected for transfection efficiency by analysis of HGH produced by a co-transfected plasmid, pXGH5. The indicated values are the means ± S.D. obtained from a number (n) of individual transfection experiments. See the text for further details. B, an example of the Northern blot analysis of TTP mRNAs expressed from the plasmids diagrammed in A. CEF were transfected with the plasmids; 24 h after transfection, the cells were serum deprived for 24 h and then treated with control conditions (C) or with 10% FBS (S) for 60 min. Each gel lane contained 20 µg of total cellular RNA, and the blot was hybridized with the 32P-labeled mouse TTP cDNA probe. Equivalent RNA loading in this and subsequent figures was confirmed by acridine orange staining (data not shown). The positions of the 18 S and 28 S ribosomal RNAs are indicated. The arrow indicates the position of spliced TTP mRNA.

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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Fig. 2.   Effect of truncated or substituted introns on TTP expression. A, the wild-type TTP137bp construct and the -Int construct are the same as shown in Fig. 1. In plasmid d(11-661), intron sequences bp 11-661 of TTP137bp have been deleted, and the two ends of the remaining sequences, which contain all the consensus donor and acceptor sequences for intron splicing (15), were ligated together, resulting in an "intron" of 26 bp. In plasmid F52Int, TTP intron sequences bp 11-661 were deleted and replaced with a 505-bp BamHI fragment (represented by a dashed line) from the mouse MRP intron (62). The quantified PhosphorImager values from Northern blots of TTP mRNA bands transcribed from these plasmids were compared with results from wild-type plasmid TTP137bp in each transfection experiment, and further corrected for transfection efficiency as described in the legend to Fig. 1. B, an example of Northern blot analysis of TTP mRNAs expressed from the plasmids diagrammed in A. CEF were transfected with the wild-type plasmid TTP137bp, or with intronless construct TTP137bp-Int, or with constructs containing an altered intron as indicated in A. Cell treatments, abbreviations, and other details are as described in the legend to Fig. 1.

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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Fig. 3.   Sequences of TTP introns. A, nucleotide sequence of the mouse TTP intron. The first nucleotide of the intron is bp 1. Consensus recognition sequences for transcription factors are indicated by brackets. The sequence shown corresponds to bp 5108-5784 of GenBank accession number L42317. B, alignments of NFkappa B-like and Sp1-binding sites in TTP introns from three species (B, bovine; H, human; M, mouse). Binding sites are underlined. Nucleotide numbers are relative to their positions in the introns. Of the NFkappa B-like binding sites, Bttp nucleotide numbers shown correspond to bp 654-679 of GenBank accession number L42319; Http nucleotide numbers shown correspond to bp 1847-1886 of GenBank accession number M19844; Mttp nucleotide numbers shown correspond to bp 5240-5278 of GenBank accession number L42317. Of the Sp1-binding sites, Bttp nucleotide numbers shown correspond to bp 1040-1073; Http nucleotide numbers shown correspond to bp 2228-2259; and Mttp nucleotide numbers shown correspond to bp 5720-5753 in their respective GenBank sequences.


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Fig. 4.   Deletion analysis of the mouse TTP intron. A, the top of the figure depicts wild-type plasmid TTP137bp, with the intron indicated as a thin line, and the promoter and exon sequences indicated as in Fig. 1A. The two exons are correctly spliced together in the intronless plasmid (-Int). The intron sequences that were removed in each deletion mutant are shown by the space flanked by a pair of numbers that indicate the missing sequences; these numbers correspond to the sequence shown in Fig. 3. The quantified PhosphorImager values from Northern blots of TTP mRNA bands transcribed from the intron deletion plasmids were compared with the wild-type plasmid TTP137bp in each transfection experiment, and were further corrected for transfection efficiency as described in the legend to Fig. 1. B, an example of Northern blot analysis of TTP mRNAs with the plasmids diagrammed in A. CEF were transfected with the wild-type plasmid TTP137bp, or with the intronless construct TTP137bp-Int, or with constructs in which various parts of the intron had been deleted as indicated in A. Cell treatments, abbreviations, and other details are as described in the legend to Fig. 1.

Nuclear Factor Binding to Intron Sequences

NFkappa B-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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Fig. 5.  

Gel mobility shift assay for nuclear proteins binding to TTP intron sequence bp 115-221. All lanes contained 20 × 103 cpm of 32P-labeled DNA probe, in the presence of poly(dI-dC) at a final concentration of 50 µg/ml. The unlabeled competitors (indicated at the top of the figure) were present at 1 µg/reaction where indicated. Assay conditions are described in the text. A, nuclear extracts (NE, 5 µg of protein) from CEF treated with control conditions (C) or for 10 min with 10% FBS (S) were allowed to bind to a probe consisting of bp 115-221 from the mouse intron. Arrows labeled CI, II, and III refer to specific protein-DNA complexes described in the text. B, nuclear extracts (NE, 5 µg of protein) from NIH-3T3 cells treated with control conditions (C) or for 20 min with 10% FBS (S), for 20 min with 100 nM PMA (P), or for 20 min with 10 ng/ml TNFalpha (T), were allowed to bind to the probes consisting of bp 116-164 or bp 165-220 (both were subfragments of probe bp 115-221). C, nuclear extracts (NE, 5 µg of protein) from NIH-3T3 cells treated as described in B were allowed to bind to wild-type probes (bp 116-164, WT137), mutants (G137, AT137), or an authentic NFkappa B probe from Igkappa . D, nuclear extracts (NE, 5 µg of protein) from NIH-3T3 cells treated for 20 min with 10 ng/ml TNFalpha (T) were incubated with 0.4 µg of anti-NFkappa B antibody (Anti-p65) for 30 min at room temperature before probes were added. Antibody supershifted bands (SS) are indicated.

When this 116-bp intron fragment was further digested with AluI to produce two fragments, bp 116-164 and bp 165-220, there was no observable binding of bp 116-164 to proteins in CEF nuclear extracts prepared from control or serum-treated cells (data not shown). Binding of bp 165-220 revealed three complexes that showed no difference in binding pattern with nuclear extracts from control or serum-treated CEF (not shown). Since TNFalpha also activates TTP transcription in mammalian fibroblasts and macrophages,2 we used the same probes to test nuclear extracts from control, serum-, PMA-, or TNFalpha -treated NIH-3T3 cells. As shown in Fig. 5B, nuclear protein binding to probe bp 165-220 was not altered by any of these treatments, and the binding patterns resembled those obtained when CEF nuclear extracts were used (not shown). However, when probe bp 116-164 was used, a DNA-protein complex was formed with nuclear extracts of TNFalpha -treated NIH-3T3 cells that was not observed with nuclear extracts from control, serum-, or PMA-treated cells (Fig. 5B). A double-stranded oligonucleotide (WT137) containing bp 137-157 was able to compete for this TNFalpha -induced binding with probe bp 116-164, indicating that the protein-binding site is located within this stretch of sequence. While a single base mutation (G137) within this oligonucleotide at bp 145 (C right-arrow G) did not change the competiveness of the oligonucleotide, mutant (AT137), with mutations at bp 145 (G right-arrow T), 155 (C right-arrow T), 150 (C right-arrow A), and 151 (C right-arrow A), significantly weakened the ability of the oligonucleotide to compete with the binding of probe bp 116-164 (Fig. 5B).

Subsequent electrophoretic mobility shift assays were performed using radiolabeled oligonucleotides containing bp 137-157. Both WT137 and G137 formed DNA-protein complexes with nuclear extracts from TNFalpha -treated NIH-3T3 cells that migrated at the same position as the complex formed with the bp 116-164 probe, while no specific binding was seen with AT137 (Fig. 5C). Although the sequence within bp 116-164 that formed the specific complex with nuclear extracts from TNFalpha -treated NIH-3T3 cells contained a consensus AP2-binding site (142GGCGATCCC151), an oligonucleotide comprising an authentic AP2-binding site was not able to compete effectively for the binding with probe bp 116-164 (Fig. 5C). The appearance of this specific DNA-protein complex in this region of the intron only after stimulation of the cells with TNFalpha raised the possibility of the binding of NFkappa B, whose translocation from the cytosol to the nucleus occurs upon stimulation of cells with TNFalpha (28). An authentic NFkappa B binding oligonucleotide derived from the Igkappa light chain enhancer (23) was able to compete for the binding of bp 116-164, and the radiolabeled NFkappa B probe formed a complex with nuclear extracts from TNFalpha -treated NIH-3T3 cells that migrated to the same position as that seen with intron probe bp 116-164 (Fig. 5C). This specific complex, like that formed with the intron probe, was not effectively competed by the authentic AP2 oligonucleotide, but was eliminated when WT137 or G137 was present in the binding reactions (Fig. 5C).

These observations suggested that the DNA-protein complex formed in this region of the intron (bp 142-151) might be due to NFkappa B binding induced by TNFalpha treatment of the cells. To investigate this possibility, immunosupershift assays were performed using a polyclonal antibody to NFkappa B. In the presence of the antibody, a supershifted complex was formed with both bp 116-164 and WT137 probes, which migrated to the same position as the supershifted complex formed with the authentic NFkappa B probe (Fig. 5D). Probe G137, with a single C right-arrow G mutation that changes the sequence to one resembling the NFkappa B-binding site in the murine major histocompatibility complex class I gene (H-2Kb) (22), formed binding and supershifted complexes as strongly as those with the Igkappa NFkappa B probe (Fig. 5D).

We reported previously that the full expression of human and bovine TTP constructs also required the presence of the intron (13). Sequences resembling the NFkappa B binding motif are also found in the human and bovine TTP introns (Fig. 3B). In quiescent fibroblasts, NFkappa B is transiently activated in the G0 to G1 transition upon stimulation of fibroblasts with serum (29). We have not been able to demonstrate changes in DNA-protein interactions in this region of the intron (bp 116-220) using nuclear extracts from serum-treated cells; furthermore, our preliminary experiments have shown only a 15% reduction when the intron NFkappa B-like binding sequence in construct TTP137bp was changed from GGCGATCCCC to GTTGATCAAC (mutated bp underlined), a change that eliminated NFkappa B-like binding activity of mutant probe AT137 (Fig. 5C). Future studies will attempt to elucidate the role of this intron NFkappa B-like binding site in the TNFalpha -induced expression of TTP.

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 TNFalpha -treated or control NIH-3T3 cells (data not shown). These protein-DNA interactions were specific; they could be eliminated by competition with an unlabeled Sp1 oligonucleotide (Fig. 6A), but not by identical concentrations of the unrelated oligonucleotide TIE (not shown). Furthermore, an anti-Sp1 antiserum produced a supershift pattern with both bands (Fig. 6A).


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Fig. 6.  

Identification of the nuclear protein binding to TTP intron sequences bp 326-430 and bp 563-672 as Sp1. Nuclear extracts (NE, 5 µg of protein) from CEF treated with control conditions (C) were incubated with 32P-labeled DNA probes from the intron, as indicated at the bottom of the gel (A and C), or with a double-stranded oligonucleotide containing Sp1 sequences (B). As indicated above each lane, 1 µl of anti-Sp1 antiserum or 1 µg of unlabeled competitor oligonucleotide was included in the reaction mixture. Antibody supershifted bands (SS) are indicated. Sequences of Sp1 and HGHGSp1, as well as assay conditions, are described in the text.

An oligonucleotide probe comprising an authentic Sp1-binding site also produced a similar doublet of shifted bands when used with CEF extracts; these were also supershifted by the anti-Sp1 antiserum (Fig. 6B). Binding was effectively competed by authentic non-radioactive Sp1 oligonucleotide, but not by SPEV, an oligonucleotide in which the core Sp1-binding site has been replaced by unrelated sequences (Fig. 6B). Oligonucleotide CHGHSp1, which contains 3-bp changes in the prototype Sp1 binding sequence, but whose sequence highly resembles the Sp1-binding site in the human growth hormone promoter, also successfully competed for the protein binding.

When the oligonucleotide containing the Sp1 binding sequence from the HGH promoter (20) was used as a probe with CEF extracts, it formed protein binding complexes in the same pattern as the Sp1 oligonucleotide corresponding to bp 618-626 in the TTP intron (Fig. 6B). Both sets of DNA-protein complexes were also supershifted by anti-Sp1 antiserum. As with the Sp1 probe, double-stranded non-radioactive oligonucleotides Sp1 and HGHSp1 effectively competed for protein binding to both probes, but mutant oligonucleotide SPEV could not. These data indicate that the two specific binding complexes formed with CEF nuclear extracts and the bp 601-655 intron fragment probe are likely to represent the binding of transcription factor Sp1. This protein often appears on SDS-polyacrylamide gels as two distinct species of Mr 95,000 and 105,000, apparently resulting from differential phosphorylation (30).

Deletion of intron sequence bp 326-430 resulted in a small but significant decrease (22%) in serum-induced TTP expression. There are one potential Sp1 and two potential AP2 sites in this region (Fig. 3). When a 132-bp fragment that includes intron bp 326-430 was used in gel mobility assays, two DNA-protein complexes were formed with CEF nuclear extracts (Fig. 6C). These two complexes exhibit the characteristics of Sp1 complexes, i.e. they were supershifted by the anti-Sp1 antiserum and were competed by oligonucleotide Sp1 and HGHSp1 but not by SPEV. These two binding complexes formed with the radiolabeled probe were also effectively competed by a non-radioactive AP2 oligonucleotide, indicating that both Sp1 and AP2 could bind to this intronic fragment and potentially regulate serum-induced expression of TTP.

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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Fig. 7.   Effect of mutating intron Sp1 sites on the expression of TTP. A, the parent plasmid TTP137bp is shown, along with constructs in which TPE1 sequences in the promoter region, or the Sp1 site located within bp 601-655 of the intron, or both, were deleted (d) or mutated (Delta ). Base pairs changed from the parent plasmid are underlined. The mean quantified PhosphorImager values from Northern blots of TTP mRNA bands transcribed from the deletion or mutation plasmids were compared with the wild-type plasmid TTP137bp in each transfection experiment, and were corrected for transfection efficiency as described in the legend to Fig. 1. B, an example of Northern blot analysis of TTP mRNAs transfected from the plasmids diagrammed in panel A. CEF were transfected with the wild-type plasmid TTP137bp, with intronless construct TTP137bp-Int, or with constructs containing the altered sequences as indicated in A. Cell treatments, abbreviations, and other details are as described in the legend to Fig. 1.

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 Delta 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.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 NFkappa B-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 NFkappa B, could bind to this intron fragment and modulate TTP expression was therefore investigated.

NFkappa B was originally discovered in a complex binding to the enhancer sequence in the immunoglobulin kappa  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 TNFalpha in mammalian fibroblasts and macrophages.2 The accumulation of TTP mRNA is rapid and transient upon TNFalpha treatment of the cells, with magnitudes of increase similar to those observed with serum. Since TNFalpha 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 TNFalpha 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 TNFalpha -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 NFkappa B binding motif found in H-2Kb (22). The binding complex formed with bp 137-157 using nuclear extracts prepared from TNFalpha -treated NIH-3T3 cells could be supershifted with antibody to p65, and was eliminated by competition with an Igkappa NFkappa B binding oligomer that has a preferential binding affinity for p65 (21). These data indicate that p65 is one of the components of the NFkappa 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 NFkappa 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).

NFkappa B is sequestered in the cytoplasm by binding to Ikappa B. Activation of cells by various agents, including phorbol esters, leads to dissociation of the NFkappa B·Ikappa B complex and migration of NFkappa B to the nucleus (49-51). We were not able to demonstrate the formation of an NFkappa B DNA-protein complex with nuclear extracts from PMA-treated NIH-3T3 cells using probes from either the Igkappa 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 TNFalpha ; and TNFalpha has been shown to activate NFkappa 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 Delta 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 TNFalpha -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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger 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; TNFalpha , tumor necrosis factor alpha ; bp, base pair(s); UTR, untranslated region.

2 W. S. Lai and P. J. Blackshear, unpublished data.

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Discussion
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