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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4554-4560
A 5 Regulatory Sequence Containing Two Ets Motifs Controls
the Expression of the Wiskott-Aldrich Syndrome Protein (WASP) Gene
in Human Hematopoietic Cells
By
A. Petrella,
I. Doti,
V. Agosti,
P. Carandente Giarrusso,
D. Vitale,
H.M. Bond,
C. Cuomo,
P. Tassone,
B. Franco,
A. Ballabio,
S. Venuta, and
G. Morrone
From the Department of Experimental and Clinical Medicine, Faculty of
Medicine, Catanzaro, Italy; CEINGE Advanced Biotechnology, Naples,
Italy; the Department of Biochemistry and Medical Biotechnology,
University Federico II, Naples, Italy; the Istituto Nazionale Tumori
Fondazione Pascale, Naples, Italy; and the Telethon Institute for
Genetics and Medicine, Milan, Italy.
 |
ABSTRACT |
The recently-identified Wiskott-Aldrich syndrome protein gene (WASP)
is responsible for the Wiskott-Aldrich X-linked immunodeficiency as
well as for isolated X-linked thrombocytopenia (XLT). To characterize the regulatory sequences of the WASP gene, we have isolated, sequenced and functionally analyzed a 1.6-Kb DNA fragment upstream of the WASP
coding sequence. Transfection experiments showed that this fragment is
capable of directing efficient expression of the reporter chloramphenicol acetyltransferase (CAT) gene in all human
hematopoietic cell lines tested. Progressive 5 deletions showed
that the minimal sequence required for hematopoietic-specific
expression consists of 137 bp upstream of the transcription start site.
This contains potential binding sites for several hematopoietic
transcription factors and, in particular, two Ets-1 consensus that
proved able to specifically bind to proteins present in nuclear
extracts of Jurkat cells. Overexpression of Ets-1 in HeLa resulted in
transactivation of the CAT reporter gene under the control of WASP
regulatory sequences. Disruption of the Ets-binding sequences by
side-directed mutagenesis abolished CAT expression in Jurkat cells,
indicating that transcription factors of the Ets family play a key role
in the control of WASP transcription.
| |
INTRODUCTION |
THE WISKOTT-ALDRICH syndrome (WAS) is a
severe X-linked condition characterized by immunodeficiency,
thrombocytopenia, eczema and a highly increased risk of hematopoietic
tumors, in particular non-Hodgkin's lymphomas. The gene responsible
for this syndrome (termed WASP, for Wiskott-Aldrich syndrome protein)
has been identified by positional mapping,1,2 and a number
of different mutations within its coding sequence have been detected in
patients with WAS and the cognate syndrome, X-linked isolated
thrombocytopenia (XLT).3-5 The WASP gene encodes a 502 amino acid-long protein, extremely rich in prolines, that has been
implicated in the control of cytoskeletal organization6,7
as well as in signal transduction processes.8,9
The WASP gene expression appears to be subjected to a developmental,
tissue-specific and lineage-specific control. High levels of WASP mRNA
are observed in fetal lymphoid organs, lymphocytes, and macrophages
from the peripheral blood of adults, as well as in lymphoid and
megakaryoblastic cell lines.1 This is likely to reflect the
presence of regulatory regions within the gene that ensure tissue
specificity. The characterization of the cis-acting sequences that
govern WASP expression is essential not only for understanding the
molecular mechanisms of its specificity, but also for reasons related
to diagnostic and therapeutic issues: (1) The identification of WASP
makes gene replacement a potentially feasible approach for the
treatment of the Wiskott-Aldrich syndrome; however, a prerequisite for
gene therapy is the availability of vectors capable of ensuring an
expression profile as close as possible to that of the endogenous gene;
and (2) so far, a diversity of mutations in the coding sequence or at
splice sites within the WASP gene have been found in most of the
patients analyzed; however, it is conceivable that also mutations
occurring in regulatory sequences may impair WASP expression thereby
giving rise to the disease; furthermore, cases of WASP-like syndromes
have been reported, in which both family history as well as molecular
data ruled out the involvement of the X chromosome and suggested
autosomal inheritance.10 Thus, it is possible that
molecular defects affecting the structure and/or the abundance
of transcription factors, which are crucial for WASP gene expression,
may be involved in the pathogenesis of these syndromes. For these
reasons we have set out to study the mechanisms and molecules that
control WASP gene expression. In this report, we discuss the
characterization of the regulatory sequence of the WASP gene and the
identification of two Ets-binding sites within this region that are
indispensable for tissue-specific expression in human hematopoietic
cells.
| |
MATERIALS AND METHODS |
Identification of the WASP transcription initiation point.
To identify the transcription start site of the WASP gene, because no
obvious TATA consensus sequence was detected in the 5 flanking region, the polymerase chain reaction (PCR)-based method of 5 RACE11 was used (5
rapid amplification of cDNA ends [RACE] kit, Life Technologies,
Gaithersburg, MD). Briefly, Jurkat RNA was subjected to reverse
transcription by using the oligonucleotide WASP REV 1, complementary to
the WASP transcript (nucleotide [nt] 668 to 647:
5-ATCGTGAACTCGTGATGTCAGG-3). A dC tail was
added to the cDNA thus synthesized, and PCR was performed by using an
oligo(dG)-containing primer and a second, nested primer complementary to WASP mRNA (WASP REV 2:
5-GCCCCGCTTGGCAGTCATT-3; nt 423 to 405). The PCR
product was inserted into the vector pGEM-T Easy Vector (Promega,
Madison, WI) and its sequence was determined, showing that the 5
end of the PCR product extended 13 nt further than the cDNA originally
reported by Derry et al.1
Isolation of the 5 flanking region of WASP and preparation of
WASP-chloramphenicol acetyltransferase (CAT) constructs.
An arrayed cosmid library of the chromosome X was screened by
using a probe obtained by PCR that spans the first exon and part of the
first intron of the WASP gene (nt 1-222). Twenty cosmids hybridizing to
this probe were isolated. Digestion with the restriction enzyme,
Pst I (New England Biolabs, Beverly, MA), yielded a DNA fragment of approximately 2 Kb containing part of the first intron, the
entire first exon, and a region of 1,580 bp upstream of the latter.
This fragment was inserted into the Bluescript II KS plasmid (Stratagene, La Jolla, CA) and sequenced. The presence of a unique Bbs I restriction site located in the 5 untranslated
region allowed removal of the WASP coding sequence. WASP-CAT
plasmids were constructed by insertion of different fragments of
the WASP 5 flanking sequence, obtained by digestion with
suitable restriction enzymes ( 1,580/+33, 1,167/+33,
988/+33, 829/+33, 365/+33, and
137/+33) in front of the bacterial CAT gene in the vector
PBL-CAT3.12
The SVETS-1 expression vector, carrying the Ets-1 cDNA driven by the
SV40 promoter13 was kindly provided by Dr N. Taniguchi (University of Osaka, Osaka, Japan).
Cell cultures, transfections, and luciferase and CAT assays.
All hematopoietic cell lines were cultured in RPMI 1640 medium (ICN
Pharmaceuticals Inc, Costa Mesa, CA) containing 10% fetal calf serum
(FCS; Hyclone, Logan, UT), 100 U/mL of penicillin, 100 µg/mL of
streptomycin, and 2 mmol/L glutamine (ICN). The nonhematopoietic cell
lines HeLa, Hep 3B and CaCo-2 were maintained in
Dulbecco's modified Eagle's medium (DMEM; ICN),
supplemented as above.
Transfections were performed by electroporation with a GenePulser
apparatus (Biorad, Hercules, CA). Briefly, 5 × 106
cells were resuspended in 300 µL of RPMI 20% FCS containing 15 µg
of the relevant WASP-CAT plasmid and 3 µg of pGL2 (Promega, Madison,
WI), a luciferase plasmid used as an internal control, and then
subjected to one pulse of 250 V, 960 µFd (Jurkat), or two pulses of
200 mV, 960 µFd (all other cell lines). The cells were then diluted
into RPMI 10% FCS and cultured for 48 hours before preparation of cell
extracts for luciferase and CAT assays.14
The measurement of luciferase activity was performed as
described15 by using a Lumat LB 9501 luminometer (Berthold,
Bad Wildbod, Germany). CAT assays were performed as described
elsewhere.14
Nuclear extracts.
Nuclear extracts were prepared as described16 from 2 × 107 cells by pellet homogenization in 2 volumes of
10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 0.5 mmol/L phenylmethyl sulfonyl fluoride (PMSF), and 10%
glycerol. Nuclei were centrifuged at 1,000g for 5 minutes,
washed, and resuspended in 2 volumes of the above solution. KCl (3 mol/L) was added to a final concentration of 0.39 mol/L KCl. Nuclei
were extracted at 4°C for 1 hour and centrifugation at
10,000g for 30 minutes. The supernatants were clarified by
centrifugation and stored in aliquots at 80°C. Protein
concentration was determined with the Bradford method17
(Biorad, Hercules, CA).
DNA probes and electrophoretic mobility shift assays (EMSA).
The following oligonucleotides were used (coding strand is
reported; mutations are indicated in lower case): W-EtsP
5-GCTGCTCATTGCGGAAGTTCCT-3; W- EtsP
5-GCTGCTCATTGCGagAGTTCCT-3; W-EtsD
5-TTGCATTTCCTGTTCCCTTGCTGC-3; W-EtsD
5-TTGCATTctCTGTctCCTTGCTGC-3; Ets-118
5-GATCTGCGCGCTTCCGCTCTCCGAGGATC-3; Ets-1
Mut18 5-GATCTGCGCGCTTggcgTCTCCGAGGATC-3. EMSAs were performed as described.19,20
Briefly, double-stranded oligonucleotides were
end-labeled with  -32P-ATP (Amersham International,
Milan, Italy) by using polynucleotide kinase (Promega, Madison WI). A
total of 2 × 104 cpm of each oligonucleotide were
incubated at room temperature for 20 minutes with 5 µg of nuclear
extract in the presence of 3 µg of poly (dI-dC), in 20 µL of a
buffer consisting of 10 mmol/L Tris-HCl, pH 7.5, 50 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L DTT, and 5% glycerol. Protein DNA
complexes were separated from free probe on a 5.5% polyacrylamide gel
run in 0.25× Tris borate buffer at 200 V for 3 hours at room
temperature. The gels were dried and exposed to radiograph film (Kodak
AR, Rochester, NY). For competitions, a 50-fold molar
excess of unlabeled competitor oligonucleotide was incubated with the
nuclear extract for 10 minutes at room temperature before addition of
the labeled oligonucleotide.
Site-directed mutagenesis.
To disrupt the Ets sites in the 137/+33 fragment, site-directed
mutagenesis was performed by PCR with the following primers: Bluescript
MCS FWD: 5-CTCGAGGTCGACGGTATCGATA-3; Bluescript MCS REV:
5-CGACTCACTATAGGGCGAATTGG-3; W-EtsP FWD
5-GCTGCTCATTGCGAGAGTTCCT-3; W-EtsP REV
5-AGGAACTCTCGCAATGAGCAGC-3; W-EtsD FWD
5-TTGCATTCTCTGTCTCCTTGCTGC-3; W-EtsD REV
5-GCAGCAAGGAGACAGAGAATGCAA-3.
Briefly, to obtain single mutants, two rounds of PCR were performed,
using as a template a Bluescript KS plasmid carrying the
W-137/+33 in the HindIII-5 3-Xba
I orientation. In the first round, PCR reactions were performed by
using the following combinations of amplimers: (1) Bluescript MCS FWD + W-EtsP REV; (2) Bluescript MCS FWD + W-EtsD REV; (3) W-EtsP
FWD + Bluescript MCS REV; and (4) W-EtsD FWD + Bluescript MCS REV.
The PCR products thus obtained were purified from agarose gels and used
as templates in the second round of PCR, in which the two fragments
carrying the proximal or distal Ets site mutation were denatured,
allowed to anneal through the overlapping sequence, and amplified in
the presence of the Bluescript MCS FWD and Bluescript MCS REV primers.
The resulting PCR products were digested with HindIII and
Xba I inserted into Bluescript, sequenced to confirm the
fidelity of amplification, and subcloned into PBL-CAT3. The double
mutant, W-EtsP/EtsD, was generated with the same strategy by
using the single mutant Bluescript W-EtsD as a template and amplifying with the W-EtsP and the Bluescript MCS primers.
| |
RESULTS |
Determination of the sequence of the 5 flanking region of the
WASP gene and identification of the transcription initiation site.
A fragment of genomic DNA spanning approximately 1,600 bp upstream of
the WASP coding sequence was isolated from a cosmid library of
chromosome X and sequenced. The nucleotide sequence data of the entire
fragment will appear in the EMBL, GenBank, and DDBJ Nucleotide Sequence
Databases under the accession number Y16094. In agreement with the data
of Derry et al21 on 500 bp of the 5 flanking
sequence, no obvious TATA box or GC-rich islands were found in the
region close to the coding sequence. To address the question whether an
additional 5 exon(s) may exist, we attempted to map the WASP
transcription start site by the PCR-based technique, 5 RACE. One
major band was amplified, in which the 5 end extended 13 nucleotides upstream of the first nucleotide of the longest WASP cDNA
previously identified.1 This start position will be
henceforth referred to as +1. The presence of an AC at the
transcription initiation point, surrounded by a stretch of pyrimidines,
is reminiscent of the initiator motif observed in the adenovirus
major-late promoter.22
Identification and dissection of the regulatory sequence in the
5 flanking sequence of the WASP gene, which confers
hematopoietic-specific expression.
To identify the minimal WASP promoter, we constructed a
series of plasmids in which different fragments of the WASP 5
flanking region were inserted upstream of the CAT reporter gene and
assayed their expression in a variety of hematopoietic and
nonhematopoietic cell lines. Initial experiments showed that a fragment
of approximately 1.6 Kb of the WASP 5 flanking region
(W-CAT5-1,580) was sufficient to ensure CAT expression in
human hematopoietic cell lines including Jurkat (T-lymphoid), De Few
and MC3 (B-lymphoblastoid), HEL (erythroid/megakaryoblastic), and Dami
(megakaryoblastic; Fig 1A) but not in cell
lines derived from cervical carcinoma (HeLa), hepatocarcinoma (Hep 3B),
or colon carcinoma (CaCo-2; Fig 1B). Primer extension confirmed that
the transcription start site of the CAT mRNA was in the expected region (not shown). Progressive 5 deletions of the 1,580/+33
fragment were obtained exploiting the presence of unique restriction
sites and inserted upstream of the CAT gene. Transfection experiments showed that the fragment 137/+33 was still able to direct
strong, specific CAT expression in human hematopoietic cell lines
(Fig 2A and B). Analysis of this sequence
(Fig 3) highlighted the presence in the
region 20/5 of a cluster of three potential binding
sites, partially overlapping, for the hematopoietic transcription
factors Ets-1, c-Rel, and PU.1, respectively. An additional Ets-1
consensus (henceforth referred to as distal) is located between nt
30 and 45. The ability of the putative Ets-binding
sequences to interact with the corresponding nuclear factors was
investigated by EMSA by using two oligonucleotides spanning the region
between nt 47 and 5 of the WASP promoter (designated
W-EtsP and W-EtsD). As shown in Fig 4A and
B, both the proximal and the distal Ets motifs could bind to (lanes
1-3) and cross-compete for (lane 5) a factor(s) present in the nuclear
extracts of Jurkat (Fig 4) and HEL cells (not shown). An
oligonucleotide carrying the "canonical" wild-type, but not the
mutant, Ets-1 consensus effectively competed for binding with both
W-EtsP and W-EtsD (lane 7-8). As expected, a nuclear factor binding
specifically to the PU.1 site and recognized by an anti-PU.1 antibody
was instead expressed in HEL but not in Jurkat cells, (results not
shown).
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| Fig 1.
(A) Expression of the W-CAT5 -1,580 construct in
human hematopoietic cell lines. Transfections and CAT assays were
performed as indicated in Materials and Methods. The amounts of
extracts used in CAT assays were normalized based on the values of
luciferase activity. PBL3-CAT was used as a negative
control. (B) Comparison of the expression of W-CAT5-1,580 in
Jurkat cells and nonhematopoietic human cell lines. CAT activity is
expressed as a percentage of the values obtained with the strong
universal CAT vector, RSV-CAT, in each cell line.
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| Fig 2.
Expression of CAT constructs carrying progressive
5 deletion mutants of the flanking region of the WASP gene. (A)
Comparison of the expression of W-CAT5-1,580,
W-CAT5-1,167, W-CAT5-988, W-CAT5-829,
W-CAT5-365, and W-CAT5-137 in Jurkat cells. (B)
Analysis of W-CAT5-137 in Jurkat, HEL, and Dami cells. HeLa cells were used as a negative control.
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| Fig 3.
Putative binding sites detected in the 137-bp upstream of
the transcription start site of the WASP gene. Solid arrows,
Ets-1; dashed arrows, c-Rel and PU.1. The PU.1 consensus was found
by comparing the WASP enhancer region with the list of binding sites for vertebrate transcription factors published by Faisst and
Meyer35; the Ets-1 and c-Rel consensus were identified by
using the TFsearch database
(http://www.genome.ad.jp/SIT/TFSEARCH.html). Gray lines indicate the
EtsP and EtsD oligonucleotides used in the EMSA assays shown in Fig
4.
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| Fig 4.
(A) EMSA assay of wild-type (W-EtsP, lanes 1-8) and
mutant (W-EtsP, lanes 9-10) oligonucleotides for the WASP proximal
Ets binding site with Jurkat nuclear extracts: labeled oligonucleotide was incubated with Jurkat nuclear extracts as detailed in Materials and
Methods and competed with a 50-fold molar excess of the competitors indicated. (B) EMSA assay of wild-type (W-EtsD, lanes 1-8) and mutant
(W-EtsD, lanes 9-10) oligonucleotides for the WASP distal Ets
binding site with Jurkat nuclear extracts: labeled oligonucleotide was
incubated with Jurkat nuclear extracts as detailed in Materials and
Methods and competed with a 50-fold molar excess of the competitors indicated.
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To establish the functional relevance of the Ets-binding sites in the
control of WASP expression, two complementary approaches were pursued:
(1) HeLa cells, which do not express the endogenous WASP gene nor the
CAT reporter gene under the control of the WASP 5 flanking
region, were cotransfected with the construct W-CAT5-1,580 and the Ets-1 expression vector SVETS-1. As shown in
Fig 5, cotransfection with increasing
amounts of SVETS-1 resulted in detectable and increasing expression of
W-CAT5-1,580. Similar results were obtained when the
construct W-CAT-5-137 was used (not shown); (2) the Ets sites
within the WASP promoter were deleted by PCR-based site-directed
mutagenesis. To this end, we designed oligonucleotides identical to
W-EtsP and W-EtsD, in which the GGAA core binding sequences were
altered as shown in Materials and Methods. When tested in EMSA
experiments, both mutant oligonucleotides (W-EtsP and
W-EtsD) failed to form specific complexes with Jurkat
nuclear factors (Fig 4A and B, lanes 9 and 10) and were unable to
compete the binding of the wild-type counterparts (Fig 4A and B, lanes 4 and 6). Thus, W-EtsP and W-EtsD were used to generate by PCR single and double Ets deletion mutants of the construct
W-CAT-5-137, which were termed W-CAT-EtsP, W-CAT-EtsD,
and W-CAT-EtsP/EtsD, respectively. Transfection of Jurkat cells
with these plasmids (Fig 6) showed that
disruption of either the proximal or the distal Ets consensus resulted
in a significant decrease in CAT expression (approximately threefold).
In the double mutant the transcriptional activity was completely
abolished.
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| Fig 5.
Induction of WASP-CAT expression by Ets-1 in HeLa cells.
HeLa were cotransfected with W-CAT5-1,580 (15 µg) and with
0, 5, and 15 µg of SVETS-1. Bluescript DNA was added to bring the
final amount of DNA to 30 µg. CAT assays were as detailed in
Materials and Methods.
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| Fig 6.
Transcriptional activity of WASP-CAT mutant constructs
bearing mutations in the proximal (W-CAT-EtsP), distal
(W-CAT-EtsD), or in both Ets binding sites (W-CAT-EtsP/EtsD).
CAT activity is expressed as percentage of the wild-type construct,
W-CAT-5-137.
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| |
DISCUSSION |
WASP is the gene whose alterations are responsible for the occurrence
of WAS and of the related disease, isolated X-linked thrombocytopenia.3-5 Increasing evidence indicates that the
WASP protein is involved in signal transduction as well as in the
control of cytoskeletal organization.6-9 Recently,
additional molecules have been identified that share amino acid
homology with specific domains of WASP and are believed to possess a
similar function,23-25 delineating the existence of a
WASP-related superfamily of proteins. Nevertheless, WASP appears to be
a nonredundant molecule, because lack of functional protein produces
the Wiskott-Aldrich or the XLT phenotype. This may be explained either
by functional differences between WASP and related molecules, or by the
fact that expression of different members of the family may be
restricted to different tissues. Indeed, although one report has
described detection of WASP in nonhematopoietic cells,26 a
vast consensus of findings strongly supports the notion that WASP
expression is regulated in a developmental- and tissue-specific manner
with high levels of its transcript being present only in the
hematopoietic system and particularly in fetal thymus and spleen as
well as in human hematopoietic cell lines (our unpublished results,
Derry et al,1 Stewart et al,27 and Parolini et
al28). WASP appears to be required in early stages of
development of the hematopoietic system because, in female obligate
carriers of WAS, the X chromosome bearing the mutation is nonrandomly
inactivated in all hematopoietic cells, including highly immature
progenitors.29
In this report we have isolated, sequenced, and studied the 5
flanking region of the WASP gene. In keeping with the observation of
Derry et al,21 on the first 500 bp of this region, no
obvious TATA consensus nor SP1-binding sites were detected immediately upstream of the coding sequence, although the region encompassing the
transcription start site shares some similarity with the initiator-like motif of the adenovirus major-late promoter.22. Lack of a
TATA box is a feature common to several genes expressed in
hematopoietic cells; however, the question remained whether an
additional exon may be located further upstream. To address this issue,
we mapped the transcription initiation point of WASP by 5 RACE
and found one major transcription start site at position 13 with
respect to the first nucleotide of the WASP cDNA isolated by Derry et
al.1 It cannot, of course, be excluded that additional transcriptional initiation points could exist; RNA protection assays
and/or S1 mapping experiments may yield further information about the possible presence of alternative start sites.
Transfection experiments with constructs carrying the whole
1,580/+33 fragment or progressive 5 deletions showed that
the region between nt 137 and +33 was necessary and sufficient
to confer strong, hematopoietic-specific expression on the CAT reporter gene. Sequence analysis showed the presence within this fragment of
potential binding sites for transcription factors known to be expressed
in hematopoietic cells (Fig 3). In particular, three partially
overlapping sequences are located next to the initiation of
transcription, which match the consensus for Ets-1, c-Rel, and PU.1 (in
inverted orientation), respectively. An additional putative Ets-1
binding site (in inverted orientation) was detected approximately 20 bp
upstream. EMSA experiments showed that a protein(s) capable of binding
to both Ets sites within the WASP promoter was present in Jurkat
nuclear extracts (Fig 4A and B). In HeLa cells (which do not express
WASP) the WASP-CAT construct was strongly transactivated by
cotransfection with an Ets-1 expression vector (Fig 5). Finally,
mutagenesis of either Ets motif within the WASP promoter, which
abolished the binding, was accompanied by a significant decrease in CAT
expression in Jurkat cells, and disruption of both sites resulted in
complete loss of expression (Fig 6). These data clearly show that the
Ets-binding sites in the WASP promoter are indispensable for expression
in hematopoietic cells. The identity of the factor(s) that bind to the
two Ets consensus in the WASP regulatory region remains to be
determined. Ets-1, widely expressed within the hematopoietic system,
abundant in T cells,30 and implicated in the
transcriptional control of several lymphoid and myeloid
genes,31,32 induces transactivation of WASP-CAT constructs
in HeLa cells, and it would be tempting to speculate that this may
indeed be the factor required for WASP transcription in hematopoietic
cells in vivo. However, the data so far available are not sufficient to
support this hypothesis, because the ETS family includes a variety of
transcription factors that interact with similar or identical sequences
on target genes.33 Studies with recombinant Ets proteins
and transfection experiments in cell lines selectively expressing
distinct factors will help clarify this issue. Another open question
regards the possible role of PU.1, itself a component of the Ets
family, which recognizes a motif slightly divergent from that of most
cognate factors.33 PU.1 has been implicated in the
regulation of hematopoietic genes, in particular in megakaryocytic and
B-lymphoid cells.34 In EMSA experiments on HEL nuclear
extracts (not shown), it specifically binds to its consensus sequence
within the WASP promoter. However, PU.1 is absent in T lymphocytes and
Jurkat cells, which strongly express WASP; therefore its role, if any,
must be accessory and restricted to PU.1-producing cell lineages.
Preliminary data (not shown) suggest that in megakaryocytic cells PU.1
may act as a negative regulator of WASP expression.
In conclusion, in this report we have identified a
hematopoietic-specific WASP promoter and established the essential
importance of Ets factors in the transcriptional regulation of this
gene. The information gained in this work may contribute to the
recognition of molecular lesions responsible for cases of WAS
and/or XLT without apparent mutations within the WASP coding
sequence. In addition, the knowledge of the cis-acting sequences and
trans-acting factors, which control the transcription of WASP, will be
of value for the development of expression vectors capable of directing
the expression of WASP in a physiological manner and suitable for a
rational gene therapy approach for WAS and XLT.
| |
FOOTNOTES |
Submitted August 7, 1997;
accepted February 10, 1998.
Supported by funds from Telethon, from the Italian Association for
Cancer Research (AIRC), and from the Consiglio Nazionale delle Recerche
(CNR).
Address reprint requests to G. Morrone, MD, CEINGE Advanced
Biotechnology, c/o Department of Biochemistry and Medical
Biotechnology, Via S Pansini, 5, 80131 Napoli, Italy.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The skillful technical assistance of Mrs Rita Bisogni and of Mr Carmine
Del Gaudio is gratefully acknowledged. We are grateful to Dr N. Taniguchi for providing the vector SVETS-1. We are indebted to Drs
Luigi Notarangelo and F. Costanzo for discussion of the data and for
their valuable suggestions and criticisms.
| |
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Abstract
Introduction
Abstract