From the Institut für Molekularbiologie und
Tumorforschung (IMT), Marburg 35033, Germany and
§ Departamento de Química Biológica, Facultad
de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
and Instituto de Biología y Medicina Experimental (IByME),
Consejo Nacional de Investigaciones Científicas y
Técnicas, Buenos Aires 1428, Argentina
Received for publication, September 21, 2000, and in revised form, March 21, 2001
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ABSTRACT |
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Differential splicing from the bcl-X
gene generates several isoforms with opposite effects on the apoptotic
response. To explore the mechanism controlling the balance between the
various isoforms, we have characterized the 5' region of the mouse
bcl-X gene. We identified three new promoters in addition
to the two previously described (Grillot, D. A., M., G.-G.,
Ekhterae, D., Duan, L., Inohara, N., Ohta, S., Seldin, M. F., and
Núñez, G. (1997) J. Immunol. 158, 4750-4757). These five promoters (P1-P5) would give rise to at least
five mRNAs with different 5'-untranslated region, all sharing the
same translation initiation site. Except for the product of the most
proximal promoter (P1), the other mRNAs are generated by
alternative splicing of noncoding exons to a common acceptor site
located in the first translated exon. Reverse transcriptase-polymerase chain reaction, primer extension, and RNase protection assays demonstrate a tissue-specific pattern of promoter usage. P1 and P2 are
active in all tissues analyzed, whereas the other three promoter show
tissue-specific activities. P3 is active in spleen, liver, and kidney,
P4 is active in uterus and spleen, and P5 is active in spleen, liver,
brain, and thymus. We present evidence suggesting that promoter
selection influences the outcome of the splice process. Transcripts
from P1 generate mainly the mRNA for the long isoform
Bcl-XL, whereas transcripts from P2 generate mRNAs for
the isoforms Bcl-XL, Bcl-XS, and
Bcl-X The term apoptosis refers to a physiologic and genetically
controlled program for cells to commit suicide. Control of cell survival is of central importance for the formation of various organs
during embryogenesis as well as in adult tissues with high cell
turnover such as reproductive glands and the lymphoid system. Moreover,
disabling programmed cell death may be a critical step in
tumorogenesis. Cells undergo apoptosis not only in response to external
signals but also by a cell autonomous genetic program controlled at
several checkpoints. One crucial control relies on the ability of the
cell to sense changes in the ratio between members of the Bcl-2 family.
The bcl-2 oncogene was first identified in human B-cell
lymphomas in the context of a chromosomal translocation, which placed
it within the immunoglobulin locus (2). bcl-2 promotes cell
survival by inhibiting apoptosis induced under a wide variety of
circumstances, suggesting that it is a ubiquitous inhibitor of cell
death triggered by multiple pathways (3). bcl-2 is the
founding member of a growing multigene family with many representatives
in mammals, including bcl-X, bax, bak,
and mcl-1, which can either promote or prevent apoptosis
(4-7). Bcl-2-related proteins have a putative transmembrane domain at
their carboxyl terminus and are found associated with mitochondrial,
endoplasmic reticulum, and nuclear membranes (8). The various members
of the Bcl-2 family can form homo- and heterodimers, which exert opposite effects on apoptosis. For instance, whereby Bax homodimers are
formed upon death stimuli and promote apoptosis, Bax/Bcl-2 heterodimers
or Bcl-2 homodimers prevent apoptosis (9).
It has been suggested that members of the bcl-2 gene family
control mitochondrial membrane permeability during apoptosis by regulating the electrical and osmotic homeostasis of mitochondria (10,
11). Those that prevent apoptosis inhibit release of cytochrome
c, whereas those that promote programmed cell death induce
this release. According to the mechanism of action suggested by Shimizu
et al. (10), proapoptotic bcl-2 family members
accelerate the opening of the mitochondrial porin channel (also named
voltage-dependent anion channel), whereas the
anti-apoptotic members close this channel by binding directly to it
(10).
Bcl-X is highly related to Bcl-2 and exists in several isoforms
generated by alternative splicing (4) (Fig.
1). Some of these isoforms seem to have a
ubiquitous expression, whereas others are expressed in a
tissue-specific manner or in response to specific stimuli. The large
isoform, Bcl-XL, protects cells against apoptosis, whereas
a short isoform, Bcl-XS, antagonizes cell death inhibition by interacting with Bcl-XL and Bcl-2 (4). The cDNA of a
third Bcl-X isoform, Bcl-X and transcripts from P3 yield mainly mRNAs for
the isoform Bcl-X
. Our results suggest a key role of
promoter choice in determining alternative splicing and, thus, the
balance of Bcl-X isoforms.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
TM, was cloned in
mouse and shown to be generated by alternative splicing of the
carboxyl-terminal transmembrane domain of bcl-X (12). As a
consequence, the protein localizes diffusely throughout the cytosol.
Bcl-X
TM was found to prevent programmed cell
death (12). A fourth isoform Bcl-X
, promotes apoptosis
and seems to be specifically expressed in cerebellum, heart, and thymus
(13). Finally, a fifth isoform named Bcl-X
was recently
identified as an antiapoptotic protein, which is induced in lymphocytes
and correlates with T-receptor expression (14).
View larger version (12K):
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Fig. 1.
Spliced products of the mouse
bcl-X gene. The scheme at the top
shows the genomic structure of the translated region with the spliced
sites and the stop codons indicated. The various resulting Bcl-X
isoforms are indicated underneath. Further description can be found in
the text. ORF, open reading frame;
Although Bcl-2 and Bcl-XL are both apoptotic inhibitors, their functions are not redundant since they are not found in the same cell types nor at the same developmental stages (15). Moreover, contrary to the bcl-2 knockout mice, mice lacking bcl-X die around embryonic day 13 due to extensive postmitotic neuronal death (16).
The genomic structure of the human and mouse bcl-X genes
have been recently described (1). The gene has been shown to contain two distinct TATA-less promoter regions. One is located at position 149 to
142 relative to the translation initiation site and contains a consensus YYAN(T/A)YY initiator element (Inr), whereas
the other is located farther upstream (nucleotides
(nt)1 655) within a GC-rich
region and contains several consensus Sp1 binding motifs (1). However,
several lines of evidence support the existence of longer
bcl-X transcripts extending farther upstream and suggest the
presence of at least one additional promoter (8, 14).
Here we present a detailed analysis of the 5'-flanking region of the
mouse bcl-X gene, including several kilobases upstream from
the previously described promoters P1 and P2 (1). Three TATA consensus
regions were found located at positions 1886,
2721, and
3412
upstream of the translation initiation site. We named these regions as
promoter P3, P4, and P5, respectively. Each of these promoters shows
tissue-selective expression, suggesting that tissue-specific factors
may be involved in controlling bcl-X transcription. The
significance of the utilization of different promoters is not clear,
but promoter choice could play a key role in the control of
bcl-X expression in response to different stimuli by
influencing the splicing process and, thus, leading to the synthesis of
specific Bcl-X isoforms (17). RT-PCR with specific oligonucleotides for
the cDNAs belonging to each 5'-untranslated region (5'-UTR) reveals
that activation of promoter P1 generates mainly Bcl-XL,
whereas bcl-X
is generated from transcripts containing exon B or exon C and originating from promoters upstream of
P1. Our results support the notion that the generation of specific spliced products depends on the 5'-leading exon present on the mRNA
and that alternative promoter usage could be a key mechanism controlling the expression of different Bcl-X isoforms.
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MATERIALS AND METHODS |
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Plasmid Constructions and DNA Sequencing-- pNM1-9 SalI vector contains a 10-kilobase DNA fragment from the 5' region of the mouse bcl-X gene cut from the plasmid pNM1-9 (kindly provided by N. Motoyama) with SalI enzyme and subcloned into SalI site of pBluescriptKS plasmid. pNM1-9Eag I is a subclone containing a 9.6-kilobase fragment from pNM1-9 cut with EagI and religated. DNA sequencing was performed by SeqLab (Göttingen, Germany).
Isolation of Total RNA and RNA Analysis--
CF1 male and female
mice were sacrificed by cervical dislocation, and total RNA was
prepared from fresh tissues by the guanidinium thiocyanate-phenol-chloroform extraction method (18). RNase protection
assays were performed as described below (19). For preparing the P1
riboprobe, plasmid pNM1-9SalI was digested with MaeIII and transcribed by T3 RNA polymerase. The full-length
transcribed riboprobe was 558 bp. For preparing the P2 riboprobe,
plasmid pNM1-9EagI was digested with EcoRI and
transcribed by T7 RNA polymerase; the full-length transcribed riboprobe
was 495 bp. [-32P]CTP (Amersham Pharmacia
Biotech)-radiolabeled RNA probes were prepared using a kit according to
the instructions of the manufacturer (Promega, Madison, WI) and as
described before (20). The probes were coprecipitated with RNA samples,
dissolved in hybridization buffer, denatured at 95 °C for 10 min,
and hybridized at 52 °C for 18 h. After digestion with RNase A
and T1 followed by digestion with proteinase K, the samples
were precipitated, denatured, and subjected to electrophoresis on a 6%
denaturing acrylamide gel.
For primer extension analysis primers, E-rev
(5'-TAGATCGGAATGGACCCTGGCT-3', D-rev
(5'-AAATGAGCTATAACTCAGTTTTTCAA-3'), and C-rev
(5'-AGAAAGGGACTGGCATCGAGAC-3'), corresponding to the reverse complement
of nt 3223 to
3244 (primer E-rev), nt
2548 to
2573 (primer
D-rev), and nt
1754 to
1775 (primer C-rev), were used. RNA (30 µg) was precipitated and resuspended in 12 µl of annealing buffer
(120 mM KCl, 24 mM Tris-HCl, pH 8, 5 × 105 cpm of 32P-labeled primer), denaturated at
75 °C for 10 min, and hybridized at 39 °C for 25 min. After
hybridization, the reaction was extended with 50 units of Moloney
murine leukemia virus reverse transcriptase (Life Technologies, Inc.)
in 30 µl of reaction mixture (60 mM Tris-HCl, pH 8, 20 mM KCl, 10 mM MgCl2, 1.5 mM dithiothreitol, 0.6 mM each dNTP) at
39 °C for 1 h. The reaction was stopped by the addition of 80 µl of stop mixture (150 mM NaCl, 15 mM EDTA, 0.75% SDS) followed by phenol-chloroform extraction and ethanol precipitation. The radiolabeled DNA was electrophoresed through a 6%
polyacrylamide/urea gel in parallel with sequencing reactions from the
genomic bcl-X cloned into pBluescript or with radiolabeled pBR322 MspI size marker.
RT-PCR and Sequence of the 5'-Untranslated Region of bcl-X
cDNAs--
For reverse transcription 4 µg of total RNA was used.
The first cDNA strand was synthesized with Moloney murine leukemia
virus reverse transcriptase (Life Technologies, Inc.) and
oligo(dT)17, as reverse complementary primer. For PCR
amplification the oligonucleotide primer ATG-rev
(5'-AGGAGAGAAAGTCGACCACCAGCTCCCGGTTGCTCTGAGA-3'), corresponding
to nt +40 to +3 from the ATG, was used as the reverse complementary
primer. Primers P3L
(5'-GGACTAGTCCTAGATTGAAGGAATGTGAACCATAAACGTTCCACGCG-3'), P4L
(5'-CCAGGATCTGAGTTCCACTCTTGAACAGAATTAACGC-3', and P5L
(5'-GGACTAGTCCGTGGAGGACACACACACCATTCGGATCCCATTAGA-3') (nt 1858
to
1821, nt
2692 to
2656, and nt
3310 to
3276, respectively)
were used as forward primers. The cDNA pool (2 µl), 1.25 units of
Thermus aquaticus (Taq) polymerase (Life
Technologies, Inc.) and amplification primers (20 pmol each) in 50 µl
of PCR mixture (1× polymerase buffer, 2 mM
MgCl2, 200 mM each dNTP) were denatured 3 min
at 96 °C followed by 35 cycles of amplification by using a step
program (96 °C for 35 s, 72 °C for 1 min 20 s) and a
final extension at 72 °C, 10 min. Four percent of the initial PCR
product was reamplified with the nested oligonucleotides: P3s
(5'-CCATGTCTCGATGCCAGTCCCTTTCT-3'); P4s
(5'-TCATGTGTACGTGCCCCAAATAAA-3'), and P5s
(5'-CATTCGGATCCCATTAGAGTTGCTT-3') (nt
1779 to
1754; nt
2600 to
2577; and nt
3293 to
3269, respectively). Primer AI-rev
(5'-CCAGGATCCAAAGCCAAGATAAGGTT-3') was used as the reverse primer. PCR
was performed by 30 cycles (96 °C for 35 s, 60 °C for
20 s, and 72 °C for 1 min) and a final extension at 72 °C for 10 min.
PCR products were purified by electrophoresis in 1.6% agarose gel, and the different bands were extracted from agarose and purified with clean up kit (DNA Clean-up system, Promega). Sequence analysis was performed by SeqLab (Göttingen, Germany).
RT-PCR and Southern Blots of the Specific bcl-X-spliced
Products--
cDNAs were prepared as described above. For PCR
amplification, the oligonucleotides were used as forward primers: P1
(5'-CCTGAAGCTCTCTCTCTCTCTTCA-3') P2L
(5'-GACTAGTCCAGGTTGTGAGGGGGCAGGTTCCTAAGCTTCGCAATTCCTCT-3') (nt
137 to
114 and nt
619 to
577, relative to the ATG translation start codon, respectively) and P3L. The oligonucleotides S/L-rev (5'-GTAGAGTGGATGGTCAGTG-3') and
-rev
(5'-TTGAACTTCCGATCCTTCTGTTTCC-3') (corresponding to the 5'-end of
each specific second translated exon) were used as reverse primers in
order to amplify bcl-XL/S and
bcl-X
isoforms, respectively. For
amplification of the bcl-X
isoform it was
necessary to perform a semi-nested PCR using, in the second round, the
oligonucleotide AII-rev (5'-ACCACCAGCTCCCGGTTGCTCTGAGACAT-3') (corresponding from nt +1 to +26 from the translation initiation site) as reverse primer. For amplification of
bcl-XS/L isoforms (corresponding to the samples
belonging to spleen, brain, and liver), it was also necessary to
perform a semi-nested PCR using, in the second round, the
oligonucleotide S/L(II)-rev (5'-CCCGTAGAGATCCACAAAAGTGTC-3') as
the reverse primer. PCR products were resolved by electrophoresis in 1.6% agarose gel and transferred to a nylon plus membrane
(Qiabrane) with NaOH (0.4 M) as the transfer
solution. Southern blots were performed using a fragment containing
exon A of mouse bcl-X as the hybridization probe. The
membranes were incubated in the prehybridization buffer (potassium
phosphate (120 mM), pH 7.2, 10% polyethylene glycol
(Mr 8000), 7% SDS, NaCl (250 mM),
50% formamide, and 100 µg/ml calf thymus DNA) at 42 °C for 2 h. Then membranes were hybridized in the same solution with the
32P-labeled probe at 42 °C overnight. A wash was carried
out 2× 5 min at room temperature in 2× SSC (1× SSC = 0.15 M NaCl and 0.015 M sodium citrate),
0.5% SDS followed by 2× 5 min at 50 °C in 2× SSC, 0.1% SDS.
Membranes were exposed between 2 and 5 days.
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RESULTS |
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Evidence for Transcription Initiation Upstream of the P2
Promoter--
An RNase protection assay with a probe encompassing the
P1 promoter (Fig. 2A) yielded
two protected fragments: a doublet of 176 nt and another of 147 nt. The
176-nt fragments originate from transcripts initiated at the P1
promoter, which were detected in all tissues tested, including uterus,
spleen, heart, liver (Fig. 2A, lanes 2-5) as
well as in lung, thymus, brain, and kidney (data not shown). The 147-nt
fragments could correspond to transcripts initiated upstream of P1 and
spliced to a consensus 3'-acceptor splice site located at position
112 from the ATG. In principle these transcripts could also originate
from initiation around this region, but there is no obvious consensus
site, and previous studies using primer extension assays did not detect
transcripts initiating around
112 (1). A comparison of the intensity
of the 147-nt and the 176-bp bands suggests that most of the
transcripts hybridizing with this probe are initiated upstream of the
P1 promoter. The origin of the minor 125-nt fragments is unclear, but
they could correspond to another previously described start site at position
96 (1) or to exon B.
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RNase protection assays with a probe encompassing the P2 promoter
revealed three main bands (Fig. 2B). Two of them, the 104-nt and 124-nt bands, represent protected products starting at the P2
promoter located 656 nt upstream of the translation start site (Fig.
2B, top). The 124-nt fragment extends to the end
of the probe. The 104-nt fragment corresponds to a transcript extending to a 5'-donor splice site located at nt 553 from the ATG start codon.
The identity of this fragment was confirmed by sequencing the cDNA,
which we named bcl-X B1 and which encompasses the sequences of exon B spliced to the 3'-splice acceptor site of exon A (see the
scheme in Fig. 5 and the nucleotide sequence in Fig. 6A). Transcripts from P2 were detected in spleen and heart (Fig.
2B: lanes 3 and 4) and in very low
amounts in uterus and liver (lanes 2 and 5). The
P2 promoter was also active in other tissues, such as thymus and kidney
(data not shown). The protected 400-nt band could correspond to
transcripts of exon B initiated upstream of P2 and extending to the 5 '-splice donor site at
553. This signal was stronger than the
transcripts initiated at P2 in uterus, heart, and liver but weaker in
spleen, suggesting differential promoter usage in different tissues.
The existence of these kind of transcripts was supported by RT-PCR
cloning and sequencing of a fragment extending from
1310 to
460
(data not shown). The larger fragments at the top of the gel correspond
to the residual intact probe. Other minor bands were not reproducibly observed.
Three Novel Promoter Regions--
To explore the presence of
additional promoter regions upstream of P2, we sequenced 3.42 kilobases
upstream of the ATG start codon in mouse bcl-X gene (Fig.
3). Recently, a nucleotide sequence of
the 5'-region (6447 nt) of the mouse bcl-X gene was
deposited at the GenBankTM (accession number
AF088904) that is virtually identical to the one that we determined.
Inspection of the nucleotide sequence revealed a putative 3'-acceptor
splice site at position 1468 from the ATG start codon, suggesting a
maximal length for exon B of 1271 bp (from nt
1468 to
197; see the
nucleotide sequence in Fig. 3 and the scheme in Fig. 5). In addition,
several putative TATA boxes were observed between the nt
3420 and
1870, of which only three, located at
1886 (P3),
2721 (P4), and
3412 (P5), showed activity in transcript analysis. Primer extension
assays were performed to map the initiation sites of transcripts
corresponding to these three TATA boxes. Three reverse complementary
oligonucleotides with sequences located downstream P3 (primer C-rev),
P4 (primer D-rev), and P5 (primer E-rev) were used as primers. Reverse
transcriptase extended fragments using primer C-rev (Fig.
4 A) placed a main initiation
site at position
1856 (30 bp downstream of the P3 TATA box) and two
other minor start sites 4 and 6 nucleotides farther upstream. The P3
transcripts were found in spleen (lane 3), liver (lane
5), and kidney (lane 6) but not in uterus (lane 2) or thymus (lane 4). Reverse transcriptase-extended
fragments using primer D-rev (Fig. 4B) placed another
initiation site at position
2696 (25 bp downstream of the P4 TATA
box). The existence of this transcript was confirmed by RT-PCR and
sequencing of a 146-bp fragment generated by using the P4L and D-rev
oligonucleotides as forward and reverse primers, respectively (data not
shown). The P4 transcripts were detected only in uterus (lane
2) and spleen (lane 3), whereas no detectable
transcripts were observed in liver (lane 4) and very little,
if any, in kidney (lane 5). Finally, reverse-extended
fragments using primer E-rev (Fig. 4C) located a major
initiation site at position
3387 (24 nt downstream of the P5 TATA
box). The existence of this transcript was confirmed by RT-PCR,
cloning, and sequencing of a 90-bp fragment generated by using the P5L
and E-rev oligonucleotides as forward and reverse primers, respectively
(data not shown). Again, the P5 transcripts were observed only in some
tissues, such as spleen (lane 3), liver (lane 5),
and brain (lane 6), but not in others, such as uterus (lane 2). Very small amounts of P5 transcripts were detected
in thymus (lane 4).
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Multiple mRNAs Derived from the bcl-X Gene--
The structure
of the bcl-X 5'-flanking region is shown schematically in
Fig. 5. If all five promoters were used
and the transcripts spliced to the coding region, at least five
mRNAs differing in the 5'-noncoding leader exons (exons A-E) would
be expected. Until now, mRNAs containing exons D and E had not been
detected by cloning. However, we have detected and sequenced four
mRNAs, one derived from the promoter P1, two containing exon B, and
one containing exon C. The 5' structures of these mRNAs are shown
in Fig. 6, along with the nucleotide
sequence around the start site. As previously described (1), we
detected mRNA A, derived from the P1 promoter in all tissues
analyzed.
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There are at least three different mRNAs containing exon B
sequences of various lengths, which originate from the use of three alternative 5'-donor splice sites. All these three mRNAs share the
same 3'-acceptor splice at position 112 in exon A. Using cDNA
cloning with RT-PCR, we detected only two of these mRNAs, one of
which uses nt
553 as the 5' splice site (mRNA B1) and the other
of which splices at nt
397 (mRNA B2). mRNA B1 was obtained from total RNA of mouse uterus and spleen by RT-PCR with the
oligonucleotides P2L and ATG-rev (Fig. 5) as forward and reverse
primers, respectively. mRNA B2 was also previously described (1).
Fang et al. (12) report evidence of the existence in B and T
lymphocytes of a third mRNA containing exon B sequences with a
5'-splice site located at nt
197 (mRNA B3) (GenBankTM
accession number MMU10102). We did not detect this mRNA in any of
the tissues tested.
mRNA transcripts containing exon C were detected by nested RT-PCR of total RNA from mouse spleen using primers P3L and ATG-rev for the first PCR and P3S and AI-rev in the second round (Fig. 6, mRNA C). The nucleotide sequence of all the transcripts we have detected did not show any open reading frames located upstream of the known coding region (Fig. 6) (4).
We could not detect any mRNA transcripts containing exon D or exon E when we performed RT-PCR with primers P4L/P5L and ATG-rev followed by nested PCR with primers P4S/P5S and AI-rev from total RNA of tissues in which P4 and P5 were active as judged by primer extension assays (uterus and spleen for P4 and spleen, liver and brain for P5). These results suggest that either the proportion of these mRNAs is very low in the total RNA or that the PCR products were too long to be efficiently amplified in our assays. Moreover, RT-PCR products were generated with the primers P5L/E-rev and P4L/D-rev, and their sequences corresponded to exons E and D, respectively (data not shown).
Use of Different bcl-X Promoters Generates Specific Spliced
Products and Bcl-X mRNA Isoforms--
The results described above
showed tissue-dependent activities of the multiple promoter
regions present in the bcl-X gene (Figs. 2 and 4). P1, P2,
and P4 are active in uterus and heart; only P1 and P2 are active in
thymus; P1, P2, P3, and P5 are active in liver; and all promoters are
active in spleen. The question arises as to the physiological relevance
of alternative and tissue-specific promoter usage. Our results suggest
that the selection of promoter usage would be involved in the
regulation of the levels of the various bcl-X mRNAs. The
differences in the 5'-noncoding exons of the transcripts may stabilize
different secondary structures, which could play a role in generating
specific spliced products. To test this possibility, we performed
RT-PCR using forward primer oligonucleotides corresponding to each of
the noncoding regions (primers P1, P2L, and P3L for exons A, B, and C,
respectively) and a common reverse primer that hybridized specifically
with the second translated exon (primer S/L-rev) (Fig.
7). We choose RNA from four tissues in
which the different transcripts are expressed (spleen, liver, brain,
and thymus). Activation of P1 generated mainly
bcl-XL and very little, if any,
bcl-XS (Fig. 7A, lanes 2,
3, 4, and 8). Transcripts containing
exon B generated both bcl-XL and
bcl-XS mRNAs, but with a higher proportion of
the former in all tissues tested (Fig. 7B, lanes
2, 3, 4, and 6). Transcripts
containing exon C were only detected in thymus and corresponded to both
isoforms but with a higher proportion of bcl-XS
relative to bcl-XL (Fig. 7C, lane
8). This was unexpected, as P3 is active in spleen, liver, and
brain (Fig. 4A). Thus, a bcl-X isoform different
from bcl-XL/Sis likely generated
from these transcripts. RT-PCR using oligonucleotide P3L as forward
primer and a reverse primer whose sequence hybridized specifically with
bcl-X (primer
-rev) revealed the presence
of bcl-X
mRNAs containing exon C in all
the tissues analyzed (Fig.
8B). In addition,
bcl-X
was found as a spliced product of
transcripts containing exon B (Fig. 8A) in all tissues
tested, whereas no amplified product of this isoform was detected with
transcripts derived from the P1 promoter (data not shown). Taken
together the results suggest that independently of the cell type, the
use of a specific promoter generates spliced transcripts specific for
different Bcl-X isoforms.
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DISCUSSION |
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The Bcl-2 family is known to control apoptosis induced by a wide range of agents. Some members of the family like Bcl-2 and Bcl-XL prevent apoptosis, whereas others, like Bax and Bcl-XS, induce cell death by dimerizing with Bcl-2 or Bcl-XL or by forming Bax-Bax dimers (4). In this way, the ratio between proapoptotic and antiapoptotic dimers acts as a "rheostat" which determines whether a given cell is going to die or to survive. Members of the Bcl-2 family with similar functions are not found in the same cell at the same time (15). Thus, a pair of proteins with opposite effect, either Bcl-2/Bax or Bcl-XL/Bcl-XS depending on the cell type or developmental stage, forms the rheostat. To ensure a correct control of apoptosis, the levels of proteins present in a given specific rheostat must be precisely regulated. Bcl-XS and Bcl-XL are not the unique splice products from the bcl-X gene. In mice and rat, five different isoforms have been described, with opposite effects on programmed cell death (4, 12-14). The control of apoptosis requires expression of the correct amount of each isoform, suggesting not only an accurate regulation of transcription but also of splicing of the bcl-X gene.
The experiments summarized in this paper demonstrate that the mouse
bcl-X gene exhibits a complex structure, in particular around its 5'-UTR, which contains at least four different exons located
upstream the unique open reading frame. Grillot et al. (1)
report that the mouse bcl-X gene is transcribed from two TATA-less promoters located between 149 and
142 (P1) and between
655 and
727 (P2) (1). Our present study demonstrates that there are
at least three additional promoters farther upstream: P3 located at
1886, P4 at
2721, and P5 at
3412 upstream of the translation
initiation codon. These three novel promoters contain a TATA consensus
sequence according to the weight matrix descriptions of eukaryotic RNA
polymerase II promoter elements (21). Very recently, the existence of
transcripts originating from a promoter corresponding to our P3 has
been reported for the human bcl-X gene and seems to be
conserved in the mouse (22).
Using immunohistochemical analysis, Krajewski et al. (15) report a strong Bcl-X expression in neurons from brain, in epithelial and cortical cells of the thymus, in red pulp granulocytes from spleen, in hepatocytes, in the loop of Henle of the kidney, and in endometrial cells (15). We did not analyze all of these cell types, but we found bcl-X expression in all the tissues tested. In addition we have observed that the use of the different promoters varied markedly in the tissues analyzed. We found that P1 and P2 were active in all tissues tested, whereas P3, P4, and P5 showed a tissue-dependent pattern of activity. According to RNase protection and primer extension assays, P3 was active in spleen, liver, and kidney, P4 in uterus and spleen, and P5 in spleen, thymus, liver, and brain.
Analysis of the nucleotide sequence of the 5'-flanking region of the
mouse bcl-X gene led to the identification of several consensus motifs for the binding of transcription factors. The physiological significance of these motifs remains to be determined. It
has been described that expression of the different isoforms of
bcl-X gene is controlled by several stimuli acting via
various transcription factors, including Ets-1 and 2, GATA-1, NF-B,
AP-1, and STAT (signal transducers and activators of transcription) factors (23). For instance, the Ets-2 transcription factor inhibits apoptosis induced by colony-stimulating factor 1 deprivation of macrophages through a Bcl-XL-dependent
mechanism (23). In particular, several reports show that the
bcl-XL isoform is induced upon the activation of the
Jak-Stat-signaling pathway (24-29). On the other hand, the involvement
of 5'-flanking NF-
B-like sites in regulation of bcl-X
gene expression has been recently reported (30).
We have previously shown that steroid hormones dexamethasone and
progesterone induce bcl-X expression and increase the
bcl-XL/bcl-XS ratio in
endometrial cells (20). Studies from other authors confirm that steroid
hormones control bcl-X transcription in different tissues
(31-33). Transient transfection experiments with a reporter gene under
the control of the mouse bcl-X P1, P2, or P3 promoters have
demonstrated a hormone-dependent
expression,2 although no
consensus hormone regulatory element motifs are found in the
neighborhood of these promoters. This suggest that steroid receptors
might control bcl-X expression by interacting with other transcription factors, such as AP1, NF-B, and STAT5, rather than by
a specific interaction with hormone regulatory element, as has been
recently shown in other systems (34-36).
The activation of the five promoters described in this study would
generate at least five mRNAs with different 5'-UTRs. Multiple promoter usage is a mechanism that offers the possibility of responding to distinct combinations of factors present in the different tissues. Using RT-PCR, we have found only some of these transcripts. The mRNA A, which is generated by activation of P1, starts at nt 144 in exon A and is expressed in all analyzed tissues. We also identified two different types of mRNAs containing exon B sequences that differ in the selection of the 5'-donor splice site. We cloned mRNA
B1 from the spleen, whereas mRNA B2 had been described in several
reports and corresponds to transcripts generating three isoforms, long,
short, and
(GenBankTM accession numbers U51278, U51279,
and U51277, respectively), as confirmed here by RT-PCR. A third type of
transcripts containing exon B sequences (mRNA B3) can been found in
GenBankTM (accession number MMU10102). Its sequence
corresponds to bcl-X
TM, a
bcl-X isoform lacking the transmembrane domain (Fig.
1A) (12). In our study we did not detect this mRNA, but
we found an mRNA containing exon C sequences (mRNA C). mRNA
C generated only bcl-X
in spleen, brain, and
liver, whereas in thymus it generated the two ubiquitous isoforms,
bcl-XS and bcl-XL. According to
the primer extension assays, P3 is not active in thymus, suggesting
that the detected amplification products are derived from promoter
regions located farther upstream.
Given the important role of bcl-X in controlling programmed
cell death, its expression should be precisely regulated. We suggest that multiple promoter usage in the mouse bcl-X gene
provides for tissue specificity and variations in expression levels of the different spliced products. The existence of multiple transcripts differing in their 5'-UTR and likely in their secondary structure could
determine the generation of specific spliced products. In this way,
external signals, by influencing promoter selection in a specific
tissue, would determine the formation of specific Bcl-X isoforms. It
has been described that promoter sequences influence the selection of
the splice site through an interaction between the transcription
machinery and serine-arginine proteins (SR) involved in the splicing
process (17). Although far from being complete, our results describe
for the first time the complexity of the structure of the 5'-flanking
region of the bcl-X gene and provide the basis for future
studies on the physiological relevance of multiple promoter usage and
alternative splicing in controlling programmed cell death.
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ACKNOWLEDGEMENTS |
---|
We thank N. Motoyama, Howard Hughes Medical Institute, Washington University, St. Louis, MO, for the mouse bcl-X clone pNM1-9 and Joerg Klug and Alberto R. Kornblihtt for helpful discussions.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fond der Chemischen Industrie (to M. B.) as well as by Universidad de Buenos Aires para Ciencia y Tecnica (UBACYT) Grant TX 82, Fon dos para la Ciencia y Tecnica Grant PICT 970256, and Proyectos deInvestigacion Plurianuales Grant 4404 (to J. L. B.), UBACYT Grant JX 05 and Programa de Regulacion Hormonal y Metabolica-Consejo Nacional de Investigaciones Científicas y Técnicas (to A. P.), and Fundacion Antorchas-Deutsche Akademische Austauschdienst (to M. B. and J. L. B.).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.
¶ An established researcher from the Consejo Nacional de Investigaciones Científicas y Técnicas.
To whom correspondence should be addressed. Tel.:
49-6421-28-6286; Fax: 49-6421-28-5398: E-mail:
beato@imt.uni-marburg.de.
Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M008665200
2 A. Pecci and M. Beato, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: nt, nucleotide(s); 5'-UTR, 5'-untranslated region; RT-PCR, reverse transcriptase-polymerase chain reaction; RNase, ribonuclease.
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