(Received for publication, August 4, 1994; and in revised form, October 14, 1994)
From the
The molecular basis for the commitment of multipotential myeloid
progenitors to the eosinophil lineage, and the transcriptional
mechanisms by which eosinophil-specific genes are subsequently
expressed and regulated during eosinophil development are currently
unknown. Interleukin-5 (IL-5) is a T cell and mast cell-derived
cytokine with actions restricted to the eosinophil and closely related
basophil lineages in humans. The high affinity receptor for IL-5
(IL-5R) is composed of an subunit (IL-5R
) expressed by the
eosinophil lineage, that associates with a
subunit
shared with the receptors for IL-3 and granulocyte-macrophage colony
stimulating factor (GM-CSF). As a prerequisite to studies of the
transcriptional regulation of the IL-5R
subunit gene, we used
three different methods, including primer extension, RNase protection,
and 5`-RACE to precisely map the transcriptional start site to a
position 15 base pairs (bp) upstream of the 5` end of the published
sequence of IL-5R
exon 1. To initially identify the IL-5R
promoter, 3.5 kilobases (kb) and 561 bp of the 5` sequence flanking the
transcriptional start site were subcloned into the promoterless
pXP2-luciferase vector. Transient transfection of these constructs into
an eosinophil-committed HL-60 subline, clone HL-60-C15, induced the
expression of
240-fold greater luciferase activity than the
promoterless vector, identifying a strong functionally active promoter
region within the 561 bp of sequence proximal to the transcriptional
start site and with activity equivalent to pXP2 constructs containing
the entire 3.5 kb of upstream sequence. To more precisely localize the cis-acting regulatory elements in this region important for
promoter activity, a series of 5` deletion mutants of the 561-bp region
were generated in the pXP2-luciferase vector. Deletion of the region
between bp -432 and -398 reduced promoter activity by more
than 80% in the HL-60-C15 cell line. Further analyses of the activity
of the IL-5R
promoter constructs in various other eosinophil,
myeloid, and non-myeloid cell lines indicated that the promoter was
relatively myeloid and eosinophil lineage-specific in its expression.
Consensus sequences for known transcription factor binding sites were
not present in the 34-bp region of the promoter required for maximal
activity, suggesting unique myeloid- and possibly eosinophil-specific
regulatory elements. Using electrophoretic mobility shift assays, we
have identified a nuclear factor(s) that binds to the 34-bp functional
region of the the promoter and that is expressed in the myeloid and
eosinophilic cell lines in which the promoter is active, but not in
non-myeloid or non-hematopoietic lines. This functional promoter
segment likely serves as the binding site for a myeloid- and possibly
eosinophil-specific transcription factor(s). Further study of the
IL-5R
promoter should elucidate unique transcriptional features of
this gene whose expression is essential to the commitment and
differentiation of multipotential myeloid progenitors to the eosinophil
lineage and to the functional activation of the mature cell.
Interleukin-5 (IL-5), ()produced primarily by
activated T cells (1) and mast cells(2, 3) ,
stimulates the proliferation and differentiation of murine activated B
cells and regulates the production of
eosinophils(4, 5, 6) . The proliferation,
differentiation, and maturation of eosinophils in the bone marrow and
their post-mitotic functional activation in tissues occurs in response
to a number of cytokines in addition to IL-5, including GM-CSF and IL-3 (7, 8, 9) . Both IL-3 and GM-CSF have
activities on other hematopoietic lineages, whereas IL-5 is more
eosinophil-specific and plays a crucial role in regulating the
differentiation and development of the eosinophil lineage(10) .
Although IL-3 and GM-CSF participate in the proliferation and
commitment of progenitors to the eosinophil lineage, IL-5 is both
necessary and sufficient for eosinophil development to
proceed(10, 11) . In humans, the high affinity
receptor for IL-5 is apparently restricted to eosinophils and
hematopoietically related basophils(12) ; in contrast to murine
B cells, the activity of IL-5 on human B cells is controversial (10, 13) and is still being delineated (14) .
Thus, the expression of the high-affinity receptor for IL-5 is an
important prerequisite and very early lineage-specific event in the
hematopoietic program for these granulocytes. Of interest, IL-5 is
active in vitro both in the production of eosinophils from
bone marrow and umbilical cord blood progenitors as well as in the
priming, activation, and enhanced survival of mature eosinophils.
Overexpression of IL-5 is observed in many eosinophil-associated
diseases(15, 16, 17) , and IL-5 transgenic
mice develop profound eosinophilia(18, 19) ,
indicating that IL-5 plays important roles in promoting the production
and function of eosinophils in vivo.
Prior studies based on
binding and cross-linking experiments on murine B cell lines suggested
a two-chain model for the IL-5 receptor (IL-5R): a unique subunit
(60-kDa component) corresponding to the low affinity IL-5 binding site (K
= 10
M), and a
subunit (130-kDa component)
that is shared with the IL-3 and GM-CSF receptors and associates with
the
chain to form the high affinity receptor (K
= 5
10
M) (20, 21, 22) . Only the
high affinity binding site is generated upon induction of eosinophilic
sublines of human promyelocytic HL-60 cells with butyric
acid(23, 24) . Recently, it has been found that the
high affinity IL-5R requires both
and
subunits (25) for optimal signaling, and that the intracellular
cytoplasmic portion of the
chain is essential to this process (64) . (
)The isolated
subunit if the GM-CSFR
has likewise been shown to participate in signaling, albeit via a
phosphorylation independent pathway(26) . To clarify the role
of these two components of the IL-5 receptor in eosinophil
differentiation and biologic function, Tavernier et al.(27) and Murata et al.(28) have explored
the characteristics of the human IL-5 receptor
(IL-5R
) gene.
The gene encoding the IL-5R
subunit is located on chromosome 3 in
the region 3p26(29) . The organization of this gene reflects
the functional domains of this protein and shares many characteristics
with other members of the cytokine/hemopoietin receptor gene
family(9, 24, 29) . Several alternatively
spliced transcripts have been identified in the mRNA and reflect the
membrane versus soluble isoforms(30) . Aside from its
ability to bind IL-5 in vitro(31) , the in vivo function(s) of the soluble form of the IL-5R have not been
elucidated.
Like other hematopoietic genes, expression of the
cytokine receptor genes are likely regulated in part at the
transcriptional level in a lineage-specific and temporal, developmental
manner. Regulation of IL-5R expression is extremely pertinent to an
understanding of the processes involved in the commitment and
differentiation of multipotential hematopoietic progenitors to the
eosinophil lineage. However, the mechanisms for the transcriptional
control and tissue-specific expression of human cytokine receptor genes
such as IL-5R are currently unknown. To investigate the regulation
of human IL-5R
expression, we first isolated the 5` upstream
region of the gene and mapped the transcriptional start site. We have
identified a functional promoter region upstream of the transcriptional
start site that is highly active in eosinophil-inducible myeloid
leukemic cell lines and is active in a myeloid- and eosinophil-specific
manner, and we have localized the minimum cis-acting sequence
required for promoter activity to a 34-bp region between bp -432
and -398 of the gene.
Figure 6:
Functional activity of the IL-5R
promoter. A, construction of the IL-5R
promoter
constructs in the promoterless pXP2 luciferase expression vector. A
3.5-kb fragment, comprised of 2.9 and 0.6 kb of the 5`-flanking region
of IL-5R
including all of exon 1, was subcloned into pXP2 and
analyzed for functional activity (see B and Fig. 7). B, functional activity of the longest IL-5R
-pXP2 promoter
constructs. Constructs of IL-5R
upstream sequence (A)
were transiently transfected along with a cytomegalovirus-human growth
hormone plasmid (CMV-hGH) as transfection control into uninduced
HL-60-C15 cells by electroporation. Luciferase activity was measured by
luminometry in cell lysates prepared 5 h post-transfection. Relative
light units (RLU) were corrected based on the concentration of hGH
(ng/ml) released into the culture supernatants. The mean ± S.D.
for three or more replicate experiments is
shown.
Figure 7:
Activity of IL-5R promoter deletion
mutants in uninduced HL-60-C15 cells. 5` deletion mutants of the
IL-5R
promoter in the pXP2-luciferase vector were generated as
shown on the left and co-transfected with a CMV-hGH control
plasmid as in Fig. 6B. Relative promoter activity of
each mutant is shown in comparison to the wild type, -561-bp
IL-5R
/pXP2 plasmid (100%).
Figure 1:
Structure of the 5` upstream region of
the IL-5R gene. Oligonucleotide primers and the primer extension,
RNase protection and 5`-RACE strategies, and anticipated fragment sizes
for mapping of the transcriptional start site are indicated. Exons
1-3 encode the 261-bp 5`-untranslated region. The 15-bp dashed line for the primer extension and 5`-RACE procedures
refers to the additional 15 bp of upstream sequence identified by these
techniques. The ``putative exon 0'' represents the 18 bp of
5`-untranslated sequence published by Murata et
al.(28) , which was not identifiable in the IL-5R
cDNA by RT-PCR using oligonucleotides A and D or by 5`-RACE nor present
in 3.5 kb of upstream genomic DNA by Southern blotting. The 5`-RACE
utilized oligonucleotide D for the initial reverse transcription step
and the internal oligonucleotide C for subsequent amplification of the
resultant cDNAs.
Figure 2:
RT-PCR analysis of mRNA for the IL-5R
subunit for detection of putative exon 0 in mRNA from eosinophils
purified from a patient with HES or from HL-60-C15 cells induced with
butyrate for 5 days. Reverse transcription used oligo(dT) priming and
PCR amplification for 40 cycles with primers identical in sequence with
putative exon 0 or the exon 1/2 boundary and an exon 4/5 boundary
reverse primer (primers A, B, and D, Fig. 1). 10 µl of the
PCR products were electrophoresed on a 2% agarose gel and stained with
ethidium bromide. A 252-bp PCR fragment was obtained with the exon
1-2 boundary primer, but not the putative exon 0
primer.
Figure 3:
Primer extension analysis; IL-5R mRNA
from HL-60-C15 cells induced toward eosinophil differentiation with
butyrate for 5 days. The primer for reverse transcription, located at
the exon 4-5 boundary region of the gene (primer D, Fig. 1), was kinased using [
-
P]dATP.
The primer was hybridized to the RNA samples, and cDNA was synthesized
with avian myeloblastosis virus reverse transcriptase. The major 291-bp
product (arrow) corresponds to a transcriptional start site
15 bp upstream of the published exon 1 sequence (Fig. 1).
Figure 4:
RNase protection to locate the 5`-most end
of the IL-5R transcript. A plasmid clone containing a 325-bp
hybrid cDNA/genomic DNA fragment (Fig. 1) was generated and
transcribed with T7 RNA polymerase and
[
-
P]UTP. The probe was hybridized to total
RNA from butyrate-induced HL-60-C15 cells and HeLa cells and treated
with RNase, and protected fragments were analyzed on a 6%
polyacrylamide sequencing gel. The major, largest protected fragment of
148 bp (arrow) corresponds to a transcriptional start site
consistent with the primer extension analysis ( Fig. 1and
3).
Figure 5:
Nucleotide sequence of the IL-5R
promoter. Open boxes show the positions of exon 1 and part of
exon 2 as previously reported. The transcriptional start sites as
identified by primer extension, RNase protection, and 5`-RACE are
indicated. The consensus start site based on results from all three
methods is labeled as +1. Several consensus sequences are
indicated for putative TFIID/TBP, and other potential transcription
factor binding sites are boxed and shaded.
Functionally active promoter regions ( Fig. 7and Fig. 8)
are boldly underlined. This genomic sequence has been
deposited in the GenBank data base under Accession No.
U18373.
Figure 8:
Lineage specificity of IL-5R promoter
activity. The -3.5-kb and -561-bp IL-5R
pXP2-luciferase constructs were transfected into eosinophil-inducible
(HL-60-C15, AML-14, AML-14.eos), myeloid (HL-60), lymphoid (BJA-B
B-cell, REX T-cell), and non-myeloid (HeLa) cell lines. Luciferase
activities for each construct and cell line from at least two
experiments were measured, and values were corrected using hGH levels
to control for differences in transfection efficiency among the various
cell lines.
Figure 9:
Gel mobility shift analysis of the
IL-5R promoter sequence using eosinophil, myeloid, and non-myeloid
nuclear extracts. A 93-bp PCR-generated probe from bp -469 to
-377 of the IL-5R
promoter was labeled with T4
polynucleotide kinase and used for gel shift analysis. Two µg of
crude nuclear extract from the indicated cell lines were mixed with 4
µg of poly(dI-dC) and 1
10
cpm of
P-labeled DNA fragment and incubated 20 min at room
temperature. Two DNA-protein complexes (C1 and C2, arrows) were identified using nuclear extracts from HL-60-C15,
HL-60, AML14, AML14.eos, and U937 cell lines, but not with extracts
from BJA-B or HeLa cells. A third specific complex (C3, arrow) was identified using nuclear extracts from all cell
lines including HeLa, with the exception of BJA-B. Labeled free DNA
probe is indicated (F). A 50-fold molar excess of the
identical unlabeled DNA probe added as cold competitor completely
inhibited the formation of the C1, C2, and C3
complexes.
Little is currently known regarding the mechanisms by which
human cytokine and growth factor receptor genes are expressed and
regulated during the commitment and differentiation of hematopoietic
progenitors to the myeloid lineages in general or the eosinophil
lineage in particular. In addition to genes encoding the murine (52) and human IL-5R subunits, the promoters for a number
of other hematopoietic growth factor receptor genes including the M-CSF
(CSF-1)(53, 55, 65) ,
G-CSF(56, 57, 66) , and GM-CSF
(58, 67) receptors are currently being analyzed. Our
isolation of the 5` upstream region of the human IL-5R
gene and
identification of functional promoter sequences provides an opportunity
to elucidate the cis-acting regulatory elements and possibly
unique trans-acting factors that regulate tissue- and
differentiation-specific transcription in the eosinophil as opposed to
other granulocyte or macrophage/monocyte myeloid lineages.
Prior
studies indicate that IL-5 is a late-acting cytokine that demonstrates
maximum activity on an eosinophil progenitor pool expanded by the
earlier-acting, multipotential cytokines such as IL-3 or GM-CSF (10) . These observations suggest that IL-5, as a
tissue-specific cytokine, plays a crucial role in regulating the
development of the eosinophil lineage, and that the IL-5R subunit,
as an essential and specific component of this differentiation pathway,
is expressed very early in response to GM-CSF, IL-3, or possibly IL-5
itself. (
)For these reasons, analysis of how expression of
the IL-5R
gene is regulated, and what inducible and
tissue-specific transcription factors are involved in this process, is
extremely pertinent to understanding the mechanisms regulating
eosinophil development. Since eosinophil differentiation, maturation,
and activation are all regulated in part by IL-5, regulation of the
gene encoding the IL-5R
subunit is likewise pertinent to the
multiple activities of this and other cytokines on eosinophil function (59) , especially with regard to the mechanisms for specific
receptor-mediated signal transduction pathways in eosinophil activation (9) . Studies of the IL-5R
promoter will hopefully lead to
the identification of transcription factors uniquely expressed by the
eosinophilic granulocyte and should add to our understanding of the
molecular basis for the complex cytokine- and growth factor-mediated
processes that occur during the commitment of multipotential myeloid
progenitors to the eosinophil lineage and subsequent eosinophil
development and maturation. These regulatory mechanisms are also likely
to be important both to the development of eosinophilia and to the
functional activation of eosinophils in tissues in
eosinophil-associated allergic, parasitic, inflammatory, and other
diseases.
The cDNA sequence and genomic structure of the human
IL-5R subunit gene were reported
previously(27, 28, 29) . However, the
complete sequence of the 5`-untranslated region, transcriptional start
site, and promoter region had not been determined. The 5`-untranslated
region of the gene was originally reported to be derived in its
entirety from exons 1-3 (29) . In contrast, Murata et
al.(28) reported an additional 18-bp sequence at the
5`-most end of their eosinophil-derived cDNA that was not present in
our own HL-60-C15-derived cDNA clones(27) , as well as
alternative splicing which removes exon 3 in all cDNA clones we
obtained from RNA of HES patient eosinophils or HL-60-C15 cells. These
conflicting results made the identification of the transcriptional
start site an imperative prior to any attempt to localize and
characterize the promoter region of the gene, especially since there
was a possibility of the existence of an additional exon (putative exon
0). To resolve this issue, we first isolated the IL-5R
5`-untranslated sequence from RNA samples obtained from both HL-60-C15
cells and eosinophils purified from a patient with HES using RT-PCR.
These experiments showed that only an oligonucleotide primer identical
in sequence with the 5`-most end of exon 1 (27, 29) and not putative exon 0 (28) was able
to generate an appropriately sized DNA fragment. In addition, we also
failed to detect any homology of the putative exon 0 within 3.5 kb of
the upstream sequence of the IL-5R
genomic DNA. These findings are
consistent with the conclusion that there is no putative exon 0
sequence in the 5`-untranslated region of the IL-5R
gene as
transcribed in either the butyrate-induced eosinophilic HL-60-C15 cell
line or HES patient eosinophils. Sequence analyses also indicated that
exon 3 was missing entirely from the 5`-untranslated region in mRNA
samples from both of these sources. Tavernier and co-workers (29) also noted a cDNA variant from butyrate-induced HL-60-C15
cells in which there was an absence of exon 3; however, only 1 in 8 of
the fully characterized cDNA clones isolated from their
butyrate-induced HL-60-C15 library was missing this exon(27) .
One potential explanation for these observations may be variability in
the copy number of the exon 3-containing RNA isoform originally
reported (27) or differences in the patterns of alternative
splicing in HL-60-C15 cells maintained and induced with butyrate in
different laboratories.
We have carefully mapped the transcriptional
start site of the IL-5R gene using three complementary techniques.
Primer extension produced two cDNA fragments of 291 bp and 332 bp in
length. Both species were consistent with additional sequences 5` of
the published end of exon 1 (27) although the longer 332-bp
fragment was likely a primer extension artifact, perhaps resulting from
the 3` end of the cDNA forming a loop by folding back on its own
template and serving as a primer for (limited) second strand synthesis.
These extra nucleotides were either from an additional short exon
further upstream or from the genomic sequence immediately upstream of
exon 1. To distinguish these possibilities, we performed both RNase
protection and 5`-RACE; RNase protection used an artificial probe
containing half of exon 2, all of exon 1, and the genomic sequence
immediately upstream of exon 1 (Fig. 1). Results from RNase
protection were consistent with an additional 15 bp of sequence
upstream of the published end of exon 1, as suggested by primer
extension. However, these results could not exclude the possibility of
an additional small exon further upstream in the gene. To address this
issue, we used RACE to specifically amplify the 5` ends of IL-5R
cDNAs prepared from RNA of butyrate-induced HL-60-C15 cells. Sequences
from the 4 longest clones obtained by the RACE procedure were identical
with the 15 bp of genomic sequence immediately upstream of the
published end of exon 1, providing confirmation of the major
transcriptional start site suggested by both primer extension and RNase
protection; no longer cDNA species were found. Taken together, these
results map the major transcriptional start site to a position
approximately 15 bp upstream of exon 1, albeit at a site that lacks a
canonical CANYYY CAP site(60) .
Activity of the promoter for
the IL-5R gene was analyzed in the eosinophil-inducible HL-60-C15
cell line. Transient transfections with the IL-5R
promoter
constructs suggested that most of the sequence elements required for
maximum promoter activity were located within a 561-bp region
immediately upstream of the transcriptional start site. A series of
smaller, 5` deletions in this region were generated using the
-561/luc plasmid to more closely map the minimal sequence
elements required for promoter activity. Results from transient
transfection of these mutants in HL-60-C15 cells indicated that the
region between -561 and -398 bp contributed
75% of
maximal promoter activity. Of interest, when a bp -561 to
-377 fragment was cloned into the promoterless pXP2-luciferase
vector, it expressed only 20% of total promoter activity suggesting
that both the distal and proximal regions of the promoter are required
for full promoter activity. Two potential TATA boxes (TFIID/TBP binding
sites) located in the bp -23 to -79 region (Fig. 5)
were identified by searching the transcription factor data
base(48) ; a TFIID site is critical for RNA II polymerase
activity (61, 62) . Since the distal region of the
IL-5R
promoter contains the DNA sequences required for functional
activity, it is likely that the promoter requires transactivation by
transcription factors which bind either directly or indirectly to both
this and the more proximal TFIID binding regions
independently(62) . In this regard, the functional cis-elements in the distal region of the promoter are likely
present within the small 34-bp region from bp -432 to -398 (Fig. 7), for which the nucleotide sequence does not contain any
previously identified transcription factor binding sites(48) .
Using electrophoretic mobility shift assays, we identified a nuclear
factor or factors that bind specifically to this region of the promoter
and that are present in the myeloid and eosinophilic cell lines in
which the promoter was functionally active, but absent in the
non-myeloid and non-hematopoietic lines for which the promoter was
significantly less active. Thus, this functional segment likely serves
as a binding site for lineage-specific transcription factor(s) required
for optimal expression of the IL-5R
gene. Whether the two
DNA-protein complexes consistently observed in electrophoretic mobility
shift experiments with this promoter region (Fig. 9) represent
distinct nuclear factors, proteolytic cleavage or post-translational
modification of a single factor, or binding by a heterodimer is unclear
pending further biochemical characterization or cloning.
An analysis
of the lineage specificity of the IL-5R promoter in eosinophilic
and other myeloid and non-myeloid leukemic cell lines (Fig. 8)
suggests that the 561-bp upstream region of the gene required for
maximal expression of promoter activity possesses both myeloid and
possibly eosinophil specificity. Among the various eosinophil cell
lines tested, the IL-5R
promoter showed greatest activity in
AML14.eos, a subline of AML14 that has been differentiated into
eosinophilic myelocytes and mature eosinophils by induction with a
combination of IL-3, IL-5, and GM-CSF(37) , expresses increased
amounts of IL-5R
mRNA, and continues to proliferate in culture and
maintain the differentiated phenotype with cytokine
supplementation.
Similarly, the IL-5R
promoter showed
greater activity in the eosinophil-inducible HL-60-C15 line (in which
5-20% of the cells spontaneously become granulated in
culture (38) ) than the undifferentiated parental HL-60 line (Fig. 8). Of interest, the IL-5R
promoter also showed high
levels of activity in U937, a myelomonocytic cell line, with mean
levels of 13,631 ± 4,700 RLU/ng/ml hGH. Other myeloid promoters
analyzed thus far in our laboratories including those for the
eosinophil peroxidase (35) and CLC protein (34) genes
have likewise shown particularly high levels of activity in the U937
line for reasons that are as yet unclear but may relate to a
particularly high transfection efficiency for this monocytoid leukemic
cell line. Further, the U937 cell line contained the same nuclear
factor(s) that formed specific protein-DNA complexes with the
IL-5R
promoter in the gel shift analyses (Fig. 9).
The
structure of the murine IL-5R subunit gene was recently
published(52) . In contrast to the human IL-5R
subunit's unique expression and activity in only the eosinophil
lineage(10) , the murine gene is also expressed and functional
in B cells(52) . Comparison of the promoter regions and
transcription factors regulating the differential expression of the
human versus murine genes in eosinophil only versus eosinophil and B cell lineages(10, 63) ,
respectively, could provide insights into their respective mechanisms
of expression and regulation. While the upstream, 5`-flanking region of
the murine IL-5R
gene contains consensus sequences for Ap1, AP-1,
GATA-1, and PU.1, these sequences have not as yet been analyzed for
functional activity(52) . In the limited functional analysis of
the murine promoter published thus far(52) , 256 bp of the
5`-flanking region (-96 to +160 bp) exhibited minimal
promoter activity in a pCAT vector in fibroblast (NIH3T3), FDC-P1, and
IL-5-dependent pre-B cell (Y16) lines. Of interest, analysis of a
larger 1.5-kb construct (-1371 to +160) to find a region
that directs B cell-specific expression, failed to detect any promoter
activity in any of the murine lines tested, suggesting suppressive
elements in this region, in marked contrast to the results we have
obtained for the human IL-5R
promoter with up to 3.5 kb of
upstream sequence. The murine gene also contains a 52-bp AC-rich Z-type
DNA sequence (-445 to -394), which is also found in the
IL-2R
upstream region, but is absent in the human IL-5R
gene
in the first 576 bp of upstream DNA we have sequenced thus far.
Alignments we have performed on the human and murine upstream regions
did not show any significant sequence similarities within the
functionally defined region of the human IL-5R
promoter, i.e. bp -561 to +51. A comparison of the human IL-5R
upstream sequence to the 5`-flanking sequences of the human
GM-CSFR
gene (58) also failed to identify any regions of
significant similarity in the functionally active segments of the
IL-5R
promoter. These observations indicate important differences
in the regulatory regions of the human versus murine
IL-5R
and human GM-CSFR
genes that are likely relevant to
their differential expression in eosinophil versus B cell
lineages in the two species and granulocyte/macrophage lineages in
humans, respectively. Finally, the promoters for a number of other
genes preferentially expressed in the myeloid series, including
CD11b(54) , M-CSF(53) , and CD14 (44) have been
shown to require either PU.1 and/or Sp1 (44) binding for
myeloid-specific expression. However, the functionally active region of
the IL-5R
promoter we have identified lacks consensus PU.1 or Sp1
binding sites, and the bp -561 to +51 region of the promoter
does not show any binding of in vitro-transcribed PU.1
transcription factor in electrophoretic mobility shift assays. (
)However, these analyses do not exclude the presence of a
nonconsensus, weak, but functionally significant PU.1 binding site in
the promoter.
Our current studies have served to identify a
functionally active promoter region for the human IL-5R gene that
is myeloid- and relatively eosinophil lineage-specific in its
expression in eosinophil-inducible leukemic cell lines in vitro. In addition, we have shown that this region binds a nuclear
factor(s) expressed in the same myeloid and eosinophilic lines in which
the promoter is functionally active. Studies to more precisely map the
functional elements in this region and to characterize and clone the
cognate transcription factors that bind to this region of the
IL-5R
promoter and are required for optimal and
eosinophil-specific activity in vitro and in vivo are
currently in progress.