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J. Biol. Chem., Vol. 275, Issue 21, 15613-15620, May 26, 2000
The Promoter for Constitutive Expression of the Human ICln
Gene CLNS1A*
Elke
Scandella ,
Ulrich Olaf
Nagl§,
Bernhard
Oehl,
Fredericke
Bergmann,
Martin
Gschwentner,
Johannes
Fürst,
Andreas
Schmarda,
Markus
Ritter,
Siegfried
Waldegger§,
Florian
Lang§,
Peter
Deetjen, and
Markus
Paulmichl¶
From the Department of Physiology, University of
Innsbruck, Fritz-Pregl Strasse 3, A-6020 Innsbruck, Austria and the
§ Department of Physiology I, University of Tübingen,
Gmelinstrasse 5, D-72076 Tübingen, Germany
Received for publication, January 11, 2000
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ABSTRACT |
The ICln protein is expressed ubiquitously in
mammals. Experiments designed to knock down the ICln protein in NIH 3T3
fibroblasts as well as in epithelial cells led to the conclusion that
this protein is crucially involved in volume regulation after
cytoplasmic swelling. Reconstitution of the ICln protein in lipid
bilayers revealed the ion channel nature of ICln. Here we describe a
new human promoter sequence, composed of 89 nucleotides, which is responsible for a highly constitutive expression of the ICln protein. The promoter sequence lacks a TATA box, and the transcription can be
effected at multiple sites. In addition to the starting sites, upstream
sequence elements are mandatory for an efficient transcription of the
ICln gene (CLNS1A). These new nucleotide elements were
defined by site-directed mutagenesis.
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INTRODUCTION |
The increase of the cell volume results in the activation of
pathways that effect the reduction of osmotically active molecules and
ions in the cytoplasm, thus leading to compensatory cell shrinkage (1,
2). The loss of potassium and chloride caused by the activation of
swelling-activated channels
(SAC)1 plays a major role in
regulatory volume decrease, evidenced by the consistent activation of
SAC in mammalian cells after swelling (2). Using the expression cloning
technique, we identified a protein (ICln) that, expressed in
Xenopus laevis oocytes, leads to a current that is similar
to SAC found in several cell types in terms of its kinetics,
pharmacology, and relative selectivity for anions (4). The assumption
that ICln is functionally tightly linked to SAC is supported further by
the finding that the knock-down of the ICln protein in fibroblasts and
epithelial cells by the use of antisense oligodeoxynucleotides specific
to ICln mRNA can seriously hamper the activation of
swelling-dependent anion channels (5, 6). Using
fluorescence in situ hybridization, we identified two
different human gene loci that carry the coding region for ICln. One
locus at position 6p12, termed CLNS1B, contains an
intronless gene (7), whereas the second gene locus at position
11q13.5-14.1, termed CLNS1A, is segmented by introns. The
exon sequences of CLNS1A are identical with the cloned human
cDNA, suggesting transcription of this gene (8). Because ICln is
expressed ubiquitously and transcribed constitutively in mammalian
cells (9), and volume regulation is an inherent regulatory entity of
living cells, it seems likely that this protein is part of one of the
housekeeping regulatory machineries of the cells. However, preliminary
experiments indicate that this constitutive transcription of the
CLNS1A gene is modulated by the volume stress placed upon
the cells and, in addition, during their progression in the cell cycle.
A prerequisite of the attempt to investigate these regulatory
mechanisms for CLNS1A transcription is the identification of
the promoter for the constitutive transcription of this gene.
Furthermore, the constitutive expression of the ICln protein is
remarkably high. Therefore, the identification of the minimal promoter
region needed for its transcription should lead to the identification
of a highly efficient human promoter that could also be used for an
effective expression of other proteins in human cells. Such a promoter
would be very instrumental in avoiding the use of viral promoters for the expression of proteins in human cells. To characterize the minimal
promoter responsible for the constitutive expression of the ICln gene,
we subcloned and analyzed the 5'-flanking region of the human
CLNS1A gene.
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EXPERIMENTAL PROCEDURES |
Cloning of the ICln Promoter Region
By screening a human genomic P1 library
(Ressourcen-Zentrum/Primär Datenbank RZPD library (10)) with a
700-bp cDNA probe specific to ICln, we isolated a clone termed P1R3
(the corresponding RZPD reference number is ICRFP700E1924Q). The 3'-end
of this clone was sequenced and corresponded to the 5'-end of the human
CLNS1A gene (7, 8). The P1R3 clone was digested with
KpnI and SacI. The restriction enzyme products
were separated on a 2% agarose gel and transferred to a nitrocellulose
membrane for hybridization. They were subsequently probed using the
5'-end of the ICln open reading frame (ORF). This procedure led to the
isolation of a  2.3-kilobase fragment from the P1R3 clone, which was
cloned into a pBlueskript SK II+ vector thereafter. Sequencing analysis
revealed that the clone carried 2151 nucleotides upstream from the
first coding ATG and parts of exon 1 of the ICln gene.
PCR Amplification
Standard PCR protocols with Taq or Pfu
polymerase (Roche Molecular Biochemicals, Stratagene) were used for
parts of the deletion mutations as well as for the site-directed mutations.
Construction of the Vectors for the Promoter-Reporter
System
The 5'-flanking region of the 2151-bp fragment described above
was cloned into the BglII and HindIII restriction
sites of the pGL3-basic vector (Promega) containing the gene for the
firefly luciferase (isolated from Photinus pyralis). A size
reduction of the 2151-bp fragment was obtained by using the restriction enzyme PvuII, the Erase-a-Base kit (Promega) according to
the manufacturer's instructions, or the PCR technique (see above). Site-directed mutagenesis was performed by introducing the respective mutations into the primer used for PCRs. All promoter fragments were
sequenced to test for the correct sequence before applying them to the
reporter gene assays. To normalize the changing transfection efficacy,
the different promoter constructs were cotransfected in HEK 293 T cells
with the pRL-TK vector (Promega), containing the gene for the
Renilla luciferase (isolated from Renilla
reniformis) controlled by the viral TK promoter. All values of the
reporter assay are given as the ratio obtained from the firefly and
Renilla luciferase readings
(luc/ren).
Transient Transfection
The luciferase reporter gene constructs together with the pRL-TK
plasmid were transfected into HEK 293 T cells by calcium phosphate
precipitation (11, 12). For this reason 105 HEK 293 T
cells were spread on cell culture dishes with a diameter of 30 mm the
day before transfection. For the transfection, 150 ng of promoter
plasmid and 75 ng of pRL-TK vector were mixed with buffer A (0.5 M CaCl2, 100 mM HEPES, pH 6.95;
adjusted with NaOH) and incubated for 10 min at room temperature,
before adding buffer B (0.28 M NaCl, 0.75 mM
NaH2PO4, 0.75 mM
Na2HPO4, 5 mM HEPES, pH 6.7;
adjusted with NaOH). After a further incubation period of 10-20 min at
room temperature, the transfection mix was spread over the cells. The
next day the cells were washed twice with culture medium (see below).
On the 3rd day the luciferase assays were carried out.
Luciferase Reporter Gene Assays
To remove traces of culture medium, cells were washed with
ice-cold phosphate-buffered saline, then lysed by adding 250 µl of
lysis buffer (25 mM glycylglycin, 15 mM
MgSO4, 8 mM EGTA, 2% Tween 20, 1 mM dithiothreitol), and the cells were scraped off the
culture dishes. The lysates were analyzed in a luminometer (EG&G
Berthold) for both firefly and Renilla luciferase by mixing 50 µl of lysate with 350 µl of firefly or Renilla assay
buffer and 200 µl of substrate-buffer for the firefly or
Renilla luciferase, respectively.
5'-RACE
From the human mRNA, the first strand cDNA was made by
using an ICln-specific primer (5'-TGT AGG GAT GTC CCC CTG TCC TTG-3') and avian myeloblastosis virus reverse transcriptase (Promega). The
reaction was heat inactivated and was then subjected to an RNase A
treatment. Unincorporated nucleotides and enzymes were removed using a
QIAquick PCR purification column (Qiagen). Polyadenosine tails were
added to the 5'-end by using terminal deoxynucleotidyl transferase
(MBI). Second strand synthesis was carried out by PCR using
Taq polymerase (MBI) and the primer RACE-N (5'-GCA TCG ATC
GCG CGA CTC TTT TTT TTT TTT TTT TT(AGC)-3'). For the first PCR, the
gene-specific primer (5'-AGG TTC AAC ATC ATC ATC ACT GTC-3') and the
RACE-N primer were added to the reaction above, and a standard PCR
protocol was carried out. To enhance specificity further, the PCR
products were subjected to a second nested PCR using the primer RACE-N2
(5'-GCA TCG ATC GCG CGA CTC-3', corresponding to the 5'-end of the
primer RACE-N), and a second gene-specific nested primer (5'-AGA GCC
ATC TAA CCA AGA CA-3'). The fragments obtained were subcloned and sequenced.
Primer Extension
Using Radioactive-labeled Primer--
The synthetic
oligonucleotide primer (5'-ACA AAT GCT CTC CTA GAC AGT C-3', starting
within exon 2) was labeled at the 5'-end with
[ -32P]ATP using T4 polynucleotide kinase (MBI). The
labeled primer was purified and annealed with poly(A)+ RNA
from HEK 293 T cells. For the extension, Moloney murine leukemia virus
reverse transcriptase (Promega) was added. After RNase A treatment, the
samples were loaded on a denaturing 6% polyacrylamide gel.
Using Fluorescence-labeled Primer--
A primer starting at
position +87 (5'-CCC GTT CAG CAC AGC CTC-3'; +1 being the adenosine of
the first ATG) labeled with IRD-800 was used for the reverse
transcription, and the products were sequenced by an automatic
sequencer (LiCor Gene ReadIR 4200, MWG Biotech).
Nuclease Protection Assay
The IRD-800-labeled primer mentioned above, starting at +87, was
employed for unidirectional PCR with a template ending at  224. This
product was used as the antisense probe for the nuclease protection
assay (Multi-NPA; Ambion), and the length of the products was analyzed
by an automatic sequencer (LiCor Gene ReadIR 4200).
Identification of the Monkey ICln Promoter Sequence
Monkey genomic DNA was purchased from
CLONTECH. For the PCR two primers were used which
were identical to the human ICln gene. With respect to the human gene,
the primer started at position 783 (forward; 5'-GAA GAT CTT CTG ATT
GGT TGG GTG GGA GAT G-3') and +24 (reverse; 5'-CGG GAA ACT TTT GAG GAA
GCT CAT-3'). The amplified fragment was subcloned and sequenced (see below).
Promoter Efficacy
To test for the promoter efficacy of the 163/74 fragment
compared with the CMV or RSV promoter we analyzed the ICln protein expression in HEK 293 T cells using the corresponding promoter regions.
The pGL3-basic vector was used after modifying the reporter region. The
luc+ gene was replaced by the ICln coding region
after attaching the nucleotide triplets coding for a sequence of five
histidines prior to the starting methionine of ICln. This allowed us to
purify the ICln protein derived from the translation of the
extrachromosomal transcribed plasmid cDNA. The pGL3-basic vector
replaced by His-ICln was termed His-ICln-basic and used for the
subsequent admission of the respective promoter sequences. The
163/74 fragment was cloned into the SmaI site, whereas
the 654-nucleotide fragment of the CMV promoter was cloned into the
NheI/XhoI sites and the 627-nucleotide fragment
of the RSV promoter was cloned into the SacI/XhoI
sites. The respective vectors were transfected into HEK 293 T cells by
calcium phosphate precipitation (11, 12). For this reason
106 HEK 293 T cells were spread on cell culture dishes
with a diameter of 90 mm the day before transfection. For the
transfection, 7 µg of promoter plasmid was mixed with buffer A and
incubated for 10 min at room temperature before adding buffer B. After
a further incubation period of 10-20 min at room temperature, the
transfection mix was spread over the cells. After 18 h the cells
were washed twice with culture medium, and after an additional 6 h
the cells were harvested, and cell lysis was performed using 200 µl
of lysis buffer (1% Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, 20 µg/ml of each leupeptin, pepstatin
A, antipain, and aprotinin, in phosphate-buffered saline at pH 8.00). A
total volume of 200 µl of supernatant was used for the extraction of
His tag-fused ICln protein (Ni-NTA spin columns; Qiagen). Imidazole
(400 mM imidazole, 50 mM
K2HPO4, pH 7.40) was used for eluting the
His-ICln. The extract was subjected to gel electrophoresis, blotted,
and analyzed using antibodies specific for the ICln protein. The
different Western blots were quantified using the ImageQuant (version
1.0) software (Molecular Dynamics), and the values were normalized according the total protein measurements (Bradford).
Cell Culture
HEK 293 T cells were grown in Dulbecco's modified Eagle's
medium (Sigma) supplemented with 44 mM NaHCO3,
280 µM penicillin, 114 µM streptomycin, and
10% fetal calf serum.
DNA Sequencing
All plasmids used were sequenced using an automatic sequencer
(LiCor Gene ReadIR 4200) with the protocols suggested by the manufacturer.
Chemicals
All chemicals used were of pro analysis grade.
Statistical Analysis
Where applicable, data are expressed as arithmetic means ± S.E. Statistical analysis was made by t test, where
appropriate. Significant difference was assumed at p < 0.05.
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RESULTS |
Cloning of the 5'-Untranslated Region of CLNS1A and Transcription
Start Estimation--
By screening a human genomic P1 library as
described under "Experimental Procedures" with a 700-bp cDNA
probe specific for ICln, we isolated a clone termed P1R3 (the
corresponding RZPD reference number is ICRFP700E1924Q). The 3'-end of
this clone was sequenced and corresponded to the 5'-end of the
human ICln ORF (7, 8). Using fluorescence in situ
hybridization, we located the P1R3 clone at the human chromosomal
position 11q13.5-14.1, as expected. The P1R3 clone carries a
>50-kilobase segment containing the 5'-untranslated region of the
CLNS1A gene. To study the promoter activity of
CLNS1A, we subcloned and sequenced a 2.259-bp
KpnI/SacI fragment from P1R3. This fragment was
identified by hybridization with the 5'-end of the CLNS1A
ORF and corresponded to position 2151 to +108 of the
CLNS1A gene (1 being the first 5'-untranslated nucleotide
of the human ICln cDNA, and +1 being the adenosine in the starting
ATG of the ORF).
Several human ICln cDNA clone sequences have been reported so far.
For the different clones, 5'-untranslated regions of varying lengths
have been described, i.e. 88 (accession number 4502890 (13)), 84 this sequence was submitted by Lamb et
al.),273 (HSU17899 (14)), and 30
(AF005422 (15)). Except for the sequence published by Anguita et
al. (14), all of the other sequences are identical to our
KpnI/SacI fragment. The sequence published by
Anguita et al. (14) is perfectly matched by P1R3 up to
position 41; however, the following 32 nucleotides located further
upstream from this position show no homology to the P1R3 sequence or
the cDNAs published by Buyse et al. (13) or Lamb et al.2 At present we cannot clearly determine
whether the initial 32 nucleotides of the sequence published by Anguita
are related to the ICln mRNA or whether they are the result of the
cloning procedure used.
The different lengths of the 5'-untranslated regions of the reported
ICln cDNAs could indicate that multiple starting sites are used for
the ICln gene transcription. To test this possibility, we performed
several tests including primer extension, nuclease protection, and
5'-RACE using poly(A)+ RNA from HEK 293 T cells as a
template. For the primer extension, the mRNA was annealed with a
5'-end 32P-labeled primer, designed to anneal within exon 2 of ICln, and the primer was extended using a Moloney murine leukemia
virus reverse transcriptase. Autoradiographic analysis of the primer extension products separated by polyacrylamide gel electrophoresis revealed products with a transcription start at positions 78 and
49. Using a primer starting at position +87, labeled with IRD-800, a
predominant start for transcription at position 48 was measured. By
using the 5'-RACE, we obtained three major products, which were
subsequently sequenced. The respective transcripts started at positions
95 (two independent clones were isolated and sequenced), 78, 77,
and 48, respectively. The transcription starts at the 78 and 48
clusters were also verified by using the nuclease protection assay.
From these experiments we conclude that there are several starting
points for human ICln gene transcription, thus providing an explanation
for the varying 5'-untranslated sequences published for ICln so far.
Determination of the Promoter Sequences Responsible for Base-line
ICln Expression--
The ICln protein is expressed ubiquitously in
mammals (4, 9, 13). To identify the promoter responsible for this
base-line expression, we used the luciferase reporter system (Promega)
by transfecting HEK 293 T cells with promoter-luciferase vector
constructs together with the Renilla pRL-TK vector for normalization.
The first sequence tested was generated by PCR using the
KpnI/SacI fragment as a template. The sequence
was limited by the positions 2151 and 1 and was cloned into the
luciferase reporter vector pGL3-basic. As shown in Fig.
1, this construct gives a signal that is
more than 40-fold higher compared with the pGL3-basic vector containing
no promoter sequences or foreign DNA (1.011 ± 0.1144 luc/ren, n = 49 and 0.023604 ± 0.0072199, n = 51, respectively). The efficacy of
the 2151/1 fragment in driving expression is close to the efficacy
of the viral TK promoter. This is supported by experiments in which the
pGL3-TK vector (luciferase gene under the control of the TK promoter),
cotransfected with the Renilla pRL-TK vector applying the
same molar ratio as used for the fragment analysis, shows an activity
that is only five times higher (5.291 ± 0.524 luc/ren, n = 6). The cloning
direction of the 2151/1 fragment into the pGL3 vector is, as
expected, important because a reporter construct containing a fragment
(568/+108) in the reverse direction showed no promoter activity
(0.0041 ± 0.0005 luc/ren, n = 9).
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Fig. 1.
Deletion analysis of the 5'-flanking region
of the human CLNS1A gene in HEK 293 T cells.
Various 5'-flanking regions were ligated into the luciferase reporter
vector pGL3-basic and transfected into HEK 293 T cells together with
the Renilla luciferase vector pRL-TK. All values are given
as the means of the ratio of the firefly over Renilla
readings (luc/ren) ± S.E. The number of
experiments is given in parentheses. In the upper
panel, the diagram shows an overview of the topology of the
different deletion mutations. The ATG on the right indicates
the starting ATG of the ICln ORF.
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The reduction of the 2151/1 fragment from the 5'- as well as
from the 3'-ends leads to the fragment 783/35, whose promoter activity is not significantly different from that of the 2151/1 fragment (Fig. 1). Nested deletions from the 5'-end of the 783/35 clone produced further five fragments, all of which display activities comparable to the activity of the 2151/1 fragment (Fig. 1). These
experiments suggest that the base-line promoter activity of ICln has to
be associated with a fragment confined by the positions 167 and 35.
Accordingly, the two fragments starting at position 162 and ending at
position 783 and 2151, respectively, show no activity (Fig. 1).
After the restriction of the 167/35 fragment to 163/56,
there was no significant loss of activity compared with the former level (Fig. 2). However, further
reduction of the 5'-end to position 154, 148, 135, or 126
decreased the activity by half. Therefore, the region between 163 and
150 was examined by site-directed mutagenesis to detect the
nucleotides responsible for the 50% reduction of the promoter
activity. As shown in Fig. 3, the mutants A150G, T154G, and 156CC154/156TT154 show no reduction of the signal, whereas the mutant 159TTT157/159GCG157 is
associated with an activity not significantly different from that of
the 154/56 fragment (0.69 ± 0.1 luc/ren, n = 15 for the mutant
and 0.60 ± 0.1 luc/ren, n = 26 for the 154/56 fragment). These experiments indicate that the
thymidines at positions 157, 158, and 159 are most likely
essential for half of the transcriptional capacity observed. The
remaining half can be annihilated by further reduction of the 5'-end of
the 126/56 clone.
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Fig. 2.
Deletion mutations distal of position
167. Deletion mutations further distal of position 163 are
followed by a reduction of the transcriptional efficacy. A basic
description of the figure is given in the legend of Fig. 1. An
asterisk indicates the values taken from Fig. 1.
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Fig. 3.
Single nucleotide mutations made in the
region between positions 159 and 150. Mutation of the
thymidine triplet 159TTT157/159GCG157 leads to a reduction of
the activity similar to that effected by a proximal deletion mutation
to position 154. A basic description of the figure is given in the
legend of Fig. 1. An asterisk indicates the values taken
from Fig. 2.
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As shown in Fig. 4, the reduction of the
5'-end to the positions 113, 99, or 81 leads to values not
significantly different from those of the empty pGL3 vector. Therefore,
the thymidine triplet together with nucleotides comprising the string
from 126 to 113 is necessary for the activity observed. By mutating
the sequence between 126 and 113, further reduction of the
essential sequence to single nucleotides was possible. As shown in Fig. 5, the mutant A119G/T117G was not
followed by a decrease in activity. However, the activity of the mutant
122CA121/122GG121was significantly impaired. Accordingly, the
reduction of the 5'-end to position 120 (fragment 120/56) leads
to an activity that does not deviate significantly from the value
obtained by the transfection of the 122CA121/122GG121 mutant.
The activity produced by the transfection of the 120/56 fragment
can be annihilated by further reduction at the 5'-end to position 117
(Fig. 5).
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Fig. 4.
Deletion mutations distal of position
126. Deletion mutations to position 113 are followed by a
reduction of the transcriptional efficacy not significantly different
from the empty pGL3 vector. A basic description of the figure is given
in the legend of Fig. 1. An asterisk indicates the values
taken from Fig. 2.
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Fig. 5.
Deletion mutations distal of position 126
and single mutations made between positions 122 and 117.
Deletion mutations further distal of position 120 are followed by a
reduction of the transcriptional efficacy not significantly different
from the empty pGL3 vector. A basic description of the figure is given
in the legend of Fig. 1. An asterisk indicates the values
taken from Fig. 2.
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The experiments show that the base-line activity measured after the
transfection of fragment 2151/1 can be obtained when the thymidine
triplet (159TTT157), the cytosine C122 and adenosine A121, and
the guanine G120 and thymidine T118 are present. In addition to
these nucleotides mandatory for the promoter activity, the presence of
starting sites is also necessary because the fragment embraced by the
positions 163 and 113 shows no activity compared with the empty
pGL3 vector (0.0137 ± 0.00056 luc/ren,
n = 6). As mentioned above, major transcription starts
were identified at positions 95, 78/77, and 49/48.
Surprisingly, the restriction of the 3'-end of the tested sequences to
position 74, which therefore excludes a transcription start at
position 49/48, has a promoter activity similar to that of the
163/1 fragment (Fig. 6), indicating that the critical sites for the transcription start are located upstream from position 74. This is also evidenced by the finding that
four out of the five clones we have identified start at the 95
cluster (two clones) and the 78 cluster (two clones). Accordingly, the fragment starting at position 163 and ending at position 95,
i.e. exactly at the most proximal end of the cDNA
identified by 5'-RACE, leads to a signal not different from that of the
empty pGL3 vector (Fig. 6). This indicates that the critical sequence for the transcription start is between positions 95 and 74. This
stretch of nucleotides contains two pyrimidine-rich clusters with the
CTTCC sequence. As shown in Fig. 7, these
pyrimidine-rich clusters are perfectly conserved from humans to monkeys
and mice (16). If the 78 cluster is omitted (fragment 163/88),
the transcription efficacy is half of that of the 163/74 fragment (Fig. 6). The reduction of the same amount can be observed when the
78 cluster is present, and a mutation was introduced in the 95
cluster (96CTTC93/96GGGG93), thus suggesting that both pyrimidine-rich clusters are equally important for an effective transcription.
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Fig. 6.
Deletion mutations made at the 3'-end of the
163/1 fragment and single mutations made between positions 96 and
93. A reduction down to position 74 can be effected without
changing the promoter activity. A basic description of the figure is
given in the legend of Fig. 1.
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Fig. 7.
Comparison of the proximal 5'-genomic
sequences of the genes coding for ICln in human, monkey, and
mouse. The sequence of the mouse was taken from Wickman et
al. (16), the sequences of humans and monkeys were determined in
this study. The framed gray boxes indicate the upstream
nucleotides mandatory for expression. The gray boxes lacking the
frame indicate adjacent nucleotide clusters with high homology
between the different species. The more distal boxes
indicate the putative starting sites identified by 32P
primer extension (#), IRD primer extension ( ), and 5'-RACE (*). The
arrows indicate the respective starting nucleotides. The
numbering of the position is made according to the human
sequence.
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Efficacy of the 163/74 Promoter Compared with the Viral
Promoters RSV and CMV--
To estimate the efficacy of expression
driven by the 163/74 ICln promoter fragment, we compared the ICln
fragment with the expression governed by the viral promoters RSV and
CMV. Because the ICln protein is endogenously expressed in HEK 293 T
cells, we made promoter constructs harboring the coding region of an ICln protein fused to a string of five histidines, allowing enrichment of the protein extract for this newly expressed protein. We quantified the expressed ICln protein using a polyclonal antibody made against a
peptide composed of the 24 C-terminal amino acids of the ICln protein.
The 163/74 ICln promoter fragment leads to an expression of the
histidine-tagged ICln protein, which is only four times or three times
lower compared with the CMV or RSV promoter, respectively (163/74
ICln promoter fragment expression = 0.69 ± 0.21, n = 4, RSV = 2.22 ± 0.31, n = 3 and CMV = 2.76 ± 0.26, n = 4).
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DISCUSSION |
The cytoplasmic volume of cells is tightly regulated, and the
mechanisms involved comprise ion transporters and/or channels (2) as
well as cellular osmolyte production, uptake, or exit (17, 18). The
transcriptional regulation of transporters for compatible osmolytes
activated after increasing the extracellular osmolality is well
understood (17, 18). The control of rENaC sodium channel expression,
again in response to hypertonicity, is in the process of investigation
(19). However, little is known regarding the transcription of genes
involved in the ion conductive pathways implicated in volume regulation
after swelling.
When whole-cell patch-clamp experiments are made using symmetrical CsCl
concentrations, a chloride current can be measured after the reduction
of the extracellular osmolality (5, 20, 21). Similar findings have been
reported for a variety of different cells (2). The channels activated
under hypotonic conditions are, however, more permeable to other anions
than to chloride i.e. SCN , Br,
and I. They are also permeable, even though to a lower
degree, to cations and, in addition, to osmolytes (22-25). Therefore
the habit to use terms such as "swelling-induced,"
"volume-regulated," or "volume-sensitive outwardly rectifying"
chloride channels seems inappropriate. It is probably more
suitable to name these ionic pores swelling-activated channels. Several
proteins are thought to be molecular targets for SAC. The list of the
candidates comprises ClC-2 (26), mdr-1 (27), phospholemman (28, 29),
ClC-3 (30), and ICln (4, 31). Because the expression of these different
proteins was done in cells bearing endogenous SAC, the experiments
cannot unambiguously clarify whether these proteins are the channels
responsible for the currents observed after cytoplasmic swelling. When
using antisense oligodeoxynucleotides that specifically impair the ICln
production in NIH 3T3 fibroblasts (5) or epithelial cells (32), the activation of SAC is reduced dramatically after the decrease of extracellular osmolality. This indicates that the ICln protein is
critical to the appearance of the SAC current under hypotonic conditions. Based on experiments using immunohistochemistry and Western
blots, we developed the hypothesis that ICln is a constitutively expressed protein that can be transposed from a water-soluble form in
the cytosol into the membrane, thus leading to an ionic current (31).
The fact that ICln can indeed act as an ion channel was confirmed by
functionally reconstituting the protein into lipid bilayers (31, 33).
In the absence of calcium, the current obtained in bilayers is more
selective for cations than for anions; however, the addition of calcium
shifts the selectivity toward chloride. The reconstituted ICln current
is, just like the current effected by native SAC, rectifying and can be
blocked by nucleotides. Therefore, we conclude that ICln is the
molecular entity of SAC or a substantial part thereof (31).
As mentioned above, little is known regarding the transcriptional
regulation of SAC. It has been shown that the transcription of the
ClC-2 protein in rat lung is modulated by Sp1 and Sp3 (34); however,
nothing is known about the transcription regulation of potential SAC
candidates in human cells. This has prompted us to study the
transcription regulation of human ICln, for which an important role in
regulatory volume decrease was defined. These studies might bring about
a better understanding of the cross-talk between plasma membrane and
nucleus. Experiments done so far indicate that the constitutive
expression of ICln might be modulated by the necessity for regulatory
volume decrease (35, 36) and seems to be dependent on cell cycle
progression. A number of different consensus regions for the binding of
a variety of transcription factors can be identified 5' of the first
coding ATG of the CLNS1A gene. Among them also a Sp1 site
identical to the one found in the rat ClC-2 promoter was identified;
however, the role of these sites needs to be scrutinized before a
definitive function can be assigned. The characterization of the
nucleotide elements needed for the constitutive expression of ICln is a
prerequisite for the in-depth analysis of these regulatory mechanisms.
Furthermore, it may also facilitate the definition of a human promoter
sequence that could allow a high copy expression of foreign proteins in human cells without the need for viral promoters. The experiments summarized in this paper were therefore made to define the nucleotides necessary for the constitutive expression of human ICln.
Four different cDNAs coding for the human ICln have been reported
so far, each having a 5'-untranslated region of varying length (88,
84, 73, and 30) and indicating that the transcription of the ICln
gene starts at multiple sites. With the aid of primer extension,
nuclease protection, and 5'-RACE, we were able to define three
different initiator regions (Inr) in close proximity to the
5'-untranslated sequences published. The Inr closest to the ORF of the
ICln gene is similar to the canonical Inr sequence described as
Py2ANT/APy2 (the underlined
A corresponds to position +1 of the transcript (37)). In
the human CLNS1A gene this site is located at position 50
and comprises the nucleotides CGCATTGCT. The identical
sequence is also found in the ICln gene of monkeys (Fig. 7). In mice
this sequence is slightly changed to GCGATTGCG but reveals
a thymidine at the crucial position of +3 (A being at position +1 and
therefore confining the predicted transcription start (37)). Testing
the efficacy of this Inr revealed that this starting point is probably
not mandatory for base-line ICln gene transcription because the
deletion of this nucleotide cluster does not reduce the constitutive
expression of the reporter system we used. In contrast, two clusters at
position 95 and 78 seem to be critical for the transcription of the
human ICln gene. The nucleotide sequences of both clusters are
identical, reading CTTCC, which are in addition completely conserved in
mouse, monkey, and humans. Despite the fact that the first base of the
mRNA tends to be adenosine, weight-matrix analysis of an extensive
number of Inr sequences revealed that the transcription start can be efficiently effected at a cytosine embraced by pyrimidines. An effective Inr tested was built by the sequence GTTCTTCC
(the underlined C would be the predicted start of the transcription
(38)). This consensus region for Inr is identical to the most proximal
cluster we identified as a possible Inr for ICln. 5'-RACE in HEK 293 T cells led to two clones that start within this Inr cluster, suggesting that this sequence is functionally important to the ICln transcription. This hypothesis is supported by the finding that mutations made within
this sequence lead to a reduction of the promoter activity by 50%. The
third Inr cluster (position 78 in the human ICln gene) is similar in
sequence (CCTCTTCC in humans and monkeys and CTGCTTCC in mice) and function because the deletion of this
sequence leads again to a drop in the promoter activity by 50%.
Sequence analysis of the fragments used in this study failed to
identify any obvious TATA box. Similar results were obtained when
analyzing the promoter of the ClC-2 chloride channel (34), which is
also a possible candidate for SAC (26). The products of genes lacking a
TATA box are often involved in housekeeping functions. However,
TATA-less genes could also be identified when tissue-specific
expression is observed, i.e. the ClC-K1 chloride channel
(39), CFTR (40, 41), or the betaine -amino-n-butyric acid
transporter (42). The lack of a TATA box usually results in multiple
starting sites, as also shown in this study, and does not rule out that
sequences upstream from the starting sites are necessary for the
binding of proteins needed for effective transcription. It was shown
for the TATA-less simian virus 40 major late promoter that effective
transcription is only possible when the cloned human TATA box-binding
protein hTFIID functionally binds to an upstream sequence element
reading 5'-TACCT-3'. The mutation of both thymidines reduced
transcription more than 50%, whereas the exchange of the two cytosines
in thymidines enhanced transcription 8-fold (43). We performed similar
mutation experiments in a region of the ICln promoter (5'-TTCCT-3'),
which is closely related to the 5'-TACCT-3' region of the simian
promoter, and we found that the mutation of the first thymidine was
also followed by a reduction of the transcription. The mutation of the
second thymidine, however, showed no effect. Moreover, mutating the two
cytosines in thymidines was not followed by an increased transcription. Therefore it is not certain at the moment whether the binding of
hTFIID is obligatory for the ICln gene transcription. Beside the
described thymidine, which is part of the 159 triplet, a second
nucleotide triplet at position 122 was identified being crucial for
ICln transcription. These triplets have identical sequences in humans
and monkeys and are similar in mice (Figs. 7 and
8). Interestingly enough, these
nucleotide triplets are in close proximity to a sequence stretch with a
length of 11 nucleotides, which is identical in all three organisms.
Further experiments are needed to elucidate whether this region of high
homology is functionally important for transcription.
View larger version (7K):
[in this window]
[in a new window]
|
Fig. 8.
Diagram of the location of the putative Inr
and binding sites for additional factor(s) needed for ICln
transcription. ATG indicates the initial ATG of the ORF coding for
ICln. The minimal nucleotide sequence necessary for constitutive
promoter activity is located between positions 163 and 74.
|
|
The expression driven by the 163/74 ICln promoter fragment is
highly efficient because the viral RSV or CMV promoters lead to an
expression efficacy, which is only three or four times higher compared
with the ICln fragment.
In conclusion, we identified promoter elements and Inr sites, which are
essential for the constitutive expression of the ICln protein (Fig. 8).
The minimal sequence stretch showing full efficacy compared with the
2151/1 fragment has a length of 89 nucleotides and is limited by
the positions 163 and 74, related to the human CLNS1A.
The efficacy of this fragment to express the reporter system is high;
and given the fact that no cell type has been identified so far which
would lack ICln expression, it might appear possible to use this
89-nucleotide minimal promoter sequence for the expression of other
proteins in human cells, when the use of viral promoters is not desired.
| |
ACKNOWLEDGEMENTS |
We acknowledge gratefully the expert
technical assistance by E. Papp, A. Wimmer. and M. Frick. We thank M. Erdel (Department of Medical Biology and Human Genetics, University of
Innsbruck) for performing the fluorescence in situ
hybridization analysis and W. Doppler for introducing us to the firefly
Renilla system.
| |
Note Added in Proof |
Characterization of reconstituted ICln in
lipid bilayers is summarized in Functional Reconstitution of ICln
in Lipid Bilayer by Fuerst et al. (Fuerst, J., Bazzini,
C., Jakab, M., Meyer, G., Koenig, M., Gschwentner, M., Ritter, M.,
Schmarda, A., Botta, G., Benz, R., Deetjen, P., and Paulmichl, M. (2000) Pfluegers Arch., in press).
| |
FOOTNOTES |
*
This work was supported in part by Austrian Science
Foundation Grants P10393-MED, P12337-MED, and P13041-MED, Austrian
National Bank Grant 6994/1, European Commission Grant BMH4-CT96-0602,
Gastein Foundation Grant FP46, and by the Österreichische
Gesellschaft für Lungenerkrankungen und Tuberkulose (to M. P.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF148460 (human) and AF148459
(monkey).
¶
To whom correspondence should be addressed.
Tel.:43-512-507-3756; Fax: 43-512-577-656; E-mail:
markus.paulmichl@uibk.ac.at.
1
The abbreviations used are: SAC,
swelling activated channel(s); ICln, ICln protein as well as the
current induced by expression of ICln protein (3, 4); bp, base pair;
ORF, open reading frame; PCR, polymerase chain reaction; TK, thymidine
kinase; HEK 293 T cells, human epithelial kidney cells; 5'-RACE, rapid
amplification of cDNA ends; CMV, cytomegalovirus; RSV, Rous sarcoma
virus; Inr, initiator.
2
F. S. Lamb, T. Barna, and B. C. Schutte,
GenBankTM accession number HSU53454.
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