(Received for publication, May 12, 1995; and in revised form, August 23, 1995)
From the
The delayed early serum response gene T1 encodes glycoproteins of the immunoglobulin superfamily with significant sequence similarity to the type 1 interleukin-1 receptor. The T1 gene is transcribed in fibroblasts into an abundant 2.7-kilobase (kb) and a rare 5-kb mRNA in response to proliferation-inducing stimuli. It gives predominantly rise to the longer transcript in the bone marrow of adult mice and in cultured mast cells. Alternative 3` processing is responsible for the two mRNA forms. The short transcript encodes a secreted protein with marked similarity to the extracellular domain of the interleukin-1 receptor, whereas the long mRNA is translated into a protein with an additional putative transmembrane and an intracellular domain.
Here we demonstrate that T1 transcription in mast cells and fibroblasts initiates at two different start sites which are 10.5 kb apart. The alternative first exons are both spliced to exon 2 which contains the translation start site. Northern blot analysis and primer extension experiments revealed that promoter usage is strictly cell type-specific. T1 transcription in mast cells is initiated exclusively at the distal promoter, whereas in fibroblasts both the short and the long T1 mRNA start at the proximal promoter. Two GATA-1 elements were identified in the 5`-flanking region of the mast cell-specific distal exon 1.
The T1 gene, also designated ST2 or DER4, was independently
isolated as a Ha-ras oncogene-responsive gene (1) and
as a gene inducible by growth factors in murine
fibroblasts(2, 3) . Growth factor-mediated T1 gene
induction requires ongoing protein synthesis, thus defining T1 as a
delayed early serum responsive gene(3, 4) . Upon
stimulation of fibroblasts with proliferation-inducing agents such as
serum, platelet-derived growth factor, fibroblast growth factor,
12-O-tetradecanoylphorbol-13-acetate, lysophosphatidic acid,
or upon oncogene expression T1 is transcribed into an abundant 2.7-kb ()and a rare 5-kb mRNA (4) . (
)The
shorter transcript encodes a primary translation product of 337 amino
acids with an NH
-terminal leader peptide
sequence(6) . The mature small T1 protein is a secreted,
heavily N-glycosylated protein (7, 8) of the
immunoglobulin superfamily with marked sequence similarity to the
carcinoembryonic antigen (6) and the extracellular portion of
the interleukin 1 (IL-1) receptors(2) . The rare 5-kb T1 mRNA
arises by alternative 3` processing of the primary transcript and
encodes a 567-amino acid protein. The corresponding cDNA (designated
ST2L) was cloned from serum stimulated BALB/c-3T3 cells(9) .
The sequences of the two T1 proteins are identical at the amino
terminus and diverge in nine amino acids in front of the COOH-terminal
end of the small protein. The extension of the large T1 protein
consists of a putative transmembrane and an intracellular domain with
significant homology to the type 1 IL-1 receptor(9) .
The in vivo expression patterns of the two T1 transcripts differ
drastically. The short mRNA has been found in experimental
Ha-ras-induced murine mammary tumors, in the developing
mammary gland 3-4 weeks after birth (10) as well as in
embryonic skin, retina, and bone(11) . Expression of the long
transcript is restricted to distinct cells of the hematopoietic organs
(embryonic liver, spleen, bone marrow) (11) and to the lung (12) throughout ontogenesis. Despite their similarity to the
IL-1 receptor, the T1 proteins neither bind IL-1 nor
IL-1
(11) , suggesting that the large protein is a novel
orphan receptor predominantly expressed in a subset of hematopoietic
cells.
Growth factor-induced T1 gene expression in fibroblasts is mediated by AP-1(4, 13) , a homodimeric or heterodimeric protein complex formed by transcription factors of the Jun and Fos families(14) . A 12-O-tetradecanoylphorbol-13-acetate-responsive element located within the T1 enhancer at position -3.6 kb is essential for gene induction(13) . Furthermore, expression of the immediate early transcription factors c-Fos and FosB resulted in the accumulation of T1 mRNA(4) . Likewise, the rat T1 homologue fit-1 was identified as a c-Fos-responsive gene in fibroblasts(15) .
Sequence analysis of cDNA clones obtained from T1 mRNA in hematopoietic cells and fibroblasts revealed different transcription initiation sites in these two cell types. Here we demonstrate that the T1 gene is transcribed from two promoters which are 10.5 kb apart. Promoter usage is strictly tissue-specific, the distal and proximal promoters being used exclusively in hematopoietic cells and fibroblasts, respectively.
For primer extension analysis, 1-2
10
cpm of end-labeled oligonucleotides TG1p
(GGGCTGGAGAGAATAAGCTCAGAGCCGTGAGGGC) or TG2
(GCTCTCTGAGGTAGGGTCCAGAAGAGAAATCAC) (purified on 7 M urea, 13%
polyacrylamide gels (30) prior to labeling) were mixed with
5-30 µg of total RNA, ethanol-precipitated, and resuspended
in 30 µl of 50% formamide, 400 mM NaCl, 10 mM PIPES (pH 6.4), and 1 mM EDTA. Annealing was performed by
incubation at 80 °C for 3 min followed by slow cooling to room
temperature. The nucleic acids were precipitated with isopropanol and
primer extension reactions were carried out in 20 µl of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl
, 5 mM dithiothreitol, 1 µg of
actinomycin D, 125 µM of each deoxynucleoside
triphosphate, 20 units of RNase Inhibitor (Boehringer Mannheim), and
300 units of RNase H
Moloney murine leukemia virus
reverse transcriptase (SuperScript(TM); Life Technologies, Inc.) at
39 °C for 1 h. For dideoxy chain termination primer extension
reactions(29) , the conditions used were identical to the above
with the following changes: the concentration of each of the three
driver dNTPs (not in competition with the ddNTP) was 10
µM, the concentration of the dNTP which was competed for
by the corresponding ddNTP was 6 µM, ddATP was 25
µM, ddCTP was 20 µM, ddGTP was 10
µM, and ddTTP was 30 µM. The reactions were
stopped by the addition of EDTA to a final concentration of 25
mM, and the RNA was degraded with 5 µg of RNase A at 37
°C for 30 min. After phenol/chloroform extraction of the mixture
and subsequent ethanol precipitation the primer extension products were
analyzed on 7 M urea, 9% polyacrylamide sequencing gels (30) .
In order to identify this start site, we first determined the genomic organization upstream of the end point of the published sequence (Fig. 1A). DNA of plasmid pETH (Fig. 1A) as well as genomic DNA of NIH 3T3 cells and of primary mast cells were subjected to Southern blot analysis to verify that the organization of the recombinant DNA corresponds to the one of the genomic DNA. The restriction fragment patterns of the different DNAs, obtained by hybridization with the radioactively labeled 0.85-kb SacI/SphI fragment (Fig. 1B), were identical, indicating that no DNA rearrangement had occurred during cloning. This result was confirmed using the 0.6-kb EcoRI fragment as a probe (data not shown).
Figure 1:
Genomic organization of the 5` part of
the T1 gene. A, different DNA fragments from a phage
containing approximately 18 kb of the T1 gene upstream of the EcoRI site in the second intron were subcloned (pETRIB, pETH,
and pETRI 17) and a restriction map established. Open and filled boxes represent nontranslated and coding regions in the
first five exons, respectively; the small ellipse marked Enh illustrates the enhancer(13) . Restriction sites,
the translation start codon, and the extent of sequences subcloned in
the plasmids pETRIB, pETH, and pETRI 17 are indicated. The bars represent the probes used for analysis of the genomic Southern
blot. d1, distal exon; p1, proximal exon 1. B, Southern blot analysis of cloned and genomic T1 sequences.
0.2 ng of plasmid DNA (pETH) as well as 5 µg of total genomic DNA
from NIH 3T3 cells and mast cells were cut with the restriction enzymes
indicated above the lines and hybridized to the 0.85-kb S/Sp probe. M, size marker. B, BamHI; E, EcoRI; H, HindIII; S, SacI; Sp, SphI.
Next we localized the mast cell-specific sequence by Southern blot hybridization utilizing as a probe the oligonucleotide TG1d which represents part of the extra sequence present at the 5` end of mast cell but not of fibroblast T1 mRNA. The hybridization pattern indicated that the novel sequence is limited to the 0.6-kb EcoRI fragment of the plasmid pETH located approximately 10.5 kb upstream of the previously described transcription start site (Fig. 2A). Sequence analysis of this restriction fragment revealed that the entire extra 5` sequence of the mast cell-specific T1 transcript is colinear with genomic DNA, thus representing an alternative first exon within this 0.6-kb EcoRI fragment (Fig. 2B). In order to accurately map the transcription initiation site, we performed primer extension analysis (Fig. 2C). A major and a minor extension product were obtained. Reactions in the presence of dideoxynucleoside triphosphates allowed the exact positioning of the transcription start site. The 5` ends determined by primer extension and by cDNA cloning coincide precisely.
Figure 2: Precise mapping of the distal exon 1 and determination of the transcription initiation site. A, 10 ng of DNA from the plasmids pETRIB and pETH (Fig. 1A) were digested with the restriction endonucleases indicated above the lines and subjected to Southern blot hybridization with the oligonucleotide TG1d. This probe is complementary to the 3` part of the distal exon 1 (Fig. 4A). The lengths of the hybridizing restriction fragments are indicated. B, BamHI; E, EcoRI; H, HindIII; S, SacI. B, the nucleotide sequence of the 0.6-kb EcoRI fragment at position -10.5 kb. The highlighted 57 nucleotides represent the extra sequence identified at the 5` end of cDNAs derived from mast cells but not fibroblasts. The two putative TATA boxes for the major and minor transcription start sites are underlined, and putative binding sites for the GATA transcription factors and SP1 are in brackets and double underlined, respectively. C, mapping of the 5` end by primer extension. The primer TG2 (Fig. 4A) was extended on 25 µg of total mast cell RNA. The reverse transcription reactions were performed with or without dideoxynucleoside triphosphates. The ddNTPs included in the extension reactions are indicated at the top of the figure. The deduced sequence (complementary to the one depicted in Fig. 2B) is indicated. N designates unidentified nucleotides.
Figure 4: Usage of the proximal and distal promoter is cell type-specific. A, schematic representation of the T1 promoter region. Both the proximal and distal exon 1 are spliced to exon 2. The positions of the oligonucleotides used for Southern blot analysis (TG1d) and for primer extension experiments (TG2, TG1p) as well as the size of the exons (minor transcription start site in parenthesis) are indicated (top). The expected extension products are illustrated (bottom). B, products of primer extension reactions with oligonucleotide TG2 (Fig. 4A) and 18 µg of total RNA isolated from anisomycin-treated NFR-2 cells (lane 1), serum-stimulated NIH 3T3 cells (lane 2), and 15V-T2 mast cells (lane 3) or a mixture of 18 µg of total RNA each from serum-stimulated NIH 3T3 fibroblasts and 15V-T2 mast cells (lane 4). A sequencing reaction of an unrelated DNA fragment was run in parallel on the polyacrylamide gel as a size marker (left panel). The lengths of the extension products are indicated (left margin). C, Northern blot analysis of the same RNAs as used for the primer extension reactions shown in Fig. 4B. Lanes 1-4 correspond to the same lanes in Fig. 4B. The amount of loaded RNA from fibroblasts (lanes 1, 2, and 4) and mast cells (lanes 3 and 4) was 5 µg. The bottom panel depicts part of the ethidium bromide-stained gel around the 18 S rRNA region. D, primer extension reactions with the oligonucleotide TG1p. 10 µg of total RNA extracted from serum-stimulated NIH 3T3 cells (lane 1), serum-starved Swiss 3T3 cells (lane 2), anisomycin-treated NFR-2 cells (lane 3), and primary mast cells (lane 4) were used as templates. The position for the 75-bp extension product, which is characteristic for transcription start at the proximal promoter, is indicated.
Northern blot analyses of RNA extracted from primary and established mast cells, fibroblasts, and adherent primary bone marrow cells were performed to address this question (Fig. 3). The probes used for hybridization recognized either the T1 open reading frame, the exon 2, or the proximal or distal exon 1. As expected, serum-stimulated fibroblasts mainly expressed the short 2.7-kb mRNA, whereas the 5-kb transcript accumulated predominantly in mast cells and, to a much lower extent, in primary adherent bone marrow cells.
Figure 3: Northern blot analysis of T1 transcripts using probes which discriminate between the proximal and distal promoters and others which do not. Total RNA from different established mast cell lines (first four lanes, see ``Experimental Procedures''), serum-stimulated NIH 3T3 cells, anisomycin-treated NFR-2 cells, primary mast cells, and adherent primary bone marrow cells were analyzed on Northern blots. Four identical blots were prepared with 5 µg of RNA/lane. The filters were independently hybridized to probes corresponding to the T1 whole open reading frame of the 2.7-kb mRNA (T1-ORF), the exon 2, and either one of the alternative exon 1. The positions for the short and long T1 mRNA are indicated. Bottom panel, ethidium bromide-stained 18 S rRNA from one of the gels. Right panel, 2.5 µg of poly(A) mRNA from serum-stimulated NIH 3T3 fibroblasts and anisomycin-treated NFR-2 cells were hybridized to the probe which is specific for the proximal exon 1.
The T1 gene has
previously been identified as a delayed early serum response gene based
on the observation that ongoing protein synthesis is a prerequisite for
its stimulation. However, in certain cell lines such as NIH 3T3
fibroblasts, T1 induction is possible in the presence of the protein
synthesis inhibitors cycloheximide and anisomycin (32) . We even observed that anisomycin on its own led to a very
substantial accumulation of T1 mRNA (data not shown). We found
particularly high levels of the long 5-kb T1 transcript in the
Ha-ras (EJ)-transformed NIH 3T3 cell line NFR-2(33) .
Therefore, analysis of T1 transcription initiation in this cell line
allowed us to distinguish between transcript-specific versus tissue-specific promoter usage. The Northern blot analyses shown
in Fig. 3demonstrate that all T1 transcripts contain sequences
downstream of the exon 1/exon 2 boundary (top two panels),
whereas hybridization to the distal and proximal exon 1-specific probes
occurred only with RNA derived from mast cells and fibroblasts,
respectively, independent of the transcript size. Hybridization with
the probe which recognizes the proximal exon 1 is rather inefficient
probably due to the small size of this exon (Fig. 3, third
panel). We therefore analyzed poly(A)-enriched RNA to detect the
5-kb T1 transcript in fibroblasts (Fig. 3, right
panel).
Primer extension experiments were performed to confirm these results at the level of transcription initiation. The primers used in these experiments as well as the expected elongation products are depicted in Fig. 4A. The oligonucleotide TG2, which is complementary to the 5` region of exon 2, was extended on RNA derived from NFR-2 cells (Fig. 4B, lane 1), NIH 3T3 cells (Fig. 4B, lane 2), and mast cells (Fig. 4B, lane 3). The extension product indicative for transcriptional initiation at the proximal promoter was obtained with RNA from the two fibroblast cell lines. In contrast, primer extension on mast cell-derived RNA gave rise to the two extension products characteristic for initiation at the distal promoter (Fig. 4B). Thus, transcription initiation seems to be strictly cell type-specific. This implies that synthesis of the 5-kb T1 mRNA starts at the distal (Fig. 4B, lane 3) as well as the proximal (Fig. 4B, lane 1) promoter. However, one could argue that the long T1 transcript in fibroblasts is initiated at the distal promoter but that the corresponding extension product could not be detected due to its low abundance. To disprove this argument, we performed a primer extension experiment with a mixture of RNA derived from serum-stimulated NIH 3T3 fibroblasts and from mast cells. Northern blot analysis demonstrated that the ratio of the 2.7-kb to the 5-kb T1 transcript in this mixture is approximately the same as in anisomycin-treated NFR-2 cells (Fig. 4C, compare lanes 1 and 4). We obtained extension products indicative of transcription initiation at the proximal and distal promoter (Fig. 4B, lane 4). Even a mixture with a 4-fold reduced amount of mast cell RNA gave rise to easily detectable primer extension products derived from the distal exon 1 (data not shown). Hence, if the long T1 mRNA in NFR-2 cells were initiated at the distal promoter, we should have detected it by primer extension (Fig. 4B, lane 1). Therefore, we conclude that synthesis of the large majority of both T1 transcripts in NFR-2 fibroblasts is started at the proximal exon 1 and that transcript size is independent of the promoter used.
Furthermore, primer extension experiments with the oligonucleotide TG1p, which anneals to the proximal exon 1, yielded a product with RNA from fibroblasts but not from mast cells (Fig. 4D). This confirms that T1 gene transcription in mast cells is exclusively initiated at the distal promoter.
In this report we have demonstrated that the synthesis of T1
mRNA starts at different promoters in fibroblasts and in mast cells.
The two alternative first exons are 10.5 kb apart, and both are spliced
to exon 2 where translation starts. Alternative 3` processing gives
rise to a short 2.7-kb and a long 5-kb mRNA that encode a secreted and
a putative trans-membrane protein, respectively. In fibroblasts the
short transcript predominates, but small amounts of the long mRNA are
always observed. Under certain conditions, such as treatment with
anisomycin, substantial amounts of the long T1 mRNA are synthesized in
fibroblasts, particularly in ras-transformed NIH 3T3 cells.
This finding allowed us to demonstrate that all T1 transcripts are
initiated at the proximal promoter in fibroblasts, irrespective of
their processing mode (Fig. 4, B and C). This
is in line with the observation that the two T1 transcripts accumulate
in parallel under all inducing conditions in fibroblasts and thus are
apparently controlled by the same transcriptional regulatory elements.
The large T1 mRNA predominates in mast cells, but substantial amounts
of the 2.7-kb T1 transcript could be observed under certain conditions
such as treatment with Ca ionophores (data not
shown). The synthesis of both T1 mRNAs is initiated at the distal
promoter in mast cells.
Expression of fit-1, the rat homologous gene of T1, is initiated at two tissue-specific promoters as well, the transcription start sites being 14 kb apart(12) . The model has been put forward that transcription initiation is tightly coupled to 3` processing. Accordingly, whenever transcription starts at the distal promoter, the long mRNA is produced, whereas initiation at the proximal promoter instructs the transcription machinery to terminate in exon 8, giving rise to the short transcript. However, this model is rendered unlikely by our finding that substantial amounts of the long T1 mRNA are observed in fibroblasts under some conditions and that these transcripts are exclusively initiated at the proximal promoter. Rather, we suggest that transcription initiation and 3` processing (poly(A) selection) are two independent tissue-specific events.
Sequence analysis of the genomic DNA around the mast
cell-specific first exon revealed the presence of two GATA elements and
one putative SP1 binding site (Fig. 2B). The GATA
consensus motif (T/A)GATA(A/G) is the recognition site of the GATA
transcription factor family of which six members have so far been
described. The patterns of both sequence preferences for DNA binding
and expression in tissues as well as cell lines are overlapping, but
distinct for these zinc finger proteins. Thus, the GATA transcription
factors might exert differential gene regulation by distinct tissue
distribution, selective binding to DNA target sequences or by different
interactions with other nuclear proteins. Among the GATA factors,
expression of GATA-1(34, 35) , GATA-2 and GATA-3 (35) was detected in several mouse and rat mast cell lines but
not in mouse 3T3 fibroblasts. These three GATA proteins are reported to
regulate the promoter of the carboxypeptidase A gene in mast
cells(35) . Hence, we consider the possibility that GATA
factors direct T1 transcription in mast cells to the distal promoter.
It is also striking that two of the four important GATA elements in the
murine -globin promoter (36) are at almost identical
relative positions to those in the T1 gene (positions -210 and
-75 in the
-globin and positions -210 and -76 in
the T1 promoter). A further indication that the GATA elements might be
important for mast cell-specific T1 transcription is the finding of a
putative SP1 site adjacent to them. Cooperation of SP1 and GATA
transcription factors has been observed for several
genes(37, 38, 39) . Promoter studies have
been initiated to test whether these GATA elements and the SP1 binding
site are indeed instrumental for mast cell-specific T1 gene expression
and whether they work in concert with the T1 enhancer. This AP-1
binding regulatory element is centrally located between the two
alternative first exons at position -3.6 kb, and it is essential
for the induction of the proximal T1 promoter(4, 13) .
Moreover, Gong et al. have demonstrated that the transcription
of the
-globin promoter requires both promoter bound
GATA-1 and enhancer bound AP-1/NF-E2, whereas GATA-1 acts solely as a
mediator of the enhancer effect on transcription(5) .
In summary, the T1 gene is transcribed from two promoters which are 10.5 kb apart. Their usage is strictly cell type-specific, as initiation at the distal and the proximal promoter exclusively occurs in mast cells and fibroblasts, respectively. Further characterization of the distal promoter containing the two GATA elements and one SP1 binding site will help to identify the transcription factors that orchestrate tissue-specific 5` initiation. Differential expression of the putative T1 receptor protein and its soluble form is achieved by two independent tissue-specific events: transcription initiation at two promoters and alternative 3` processing. T1 is therefore a particularly useful tool for gene regulation studies to reveal the mechanisms responsible for the tissue-specific expression of a transmembrane versus a secreted form of a protein.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U38789[GenBank].