The regulation of gene expression is critical to the development
of cells and organisms. Although the regulation of gene expression
during hematopoeisis has been under intense scrutiny, few studies have
examined the eosinophil lineage directly. Whereas the regulatory
regions of several eosinophil genes have been
characterized(1, 2, 3, 4) , the
specific molecular events underlying commitment to and differentiation
of the eosinophil lineage remain unknown.
The eosinophil-derived
neurotoxin (EDN) (
)is a small, cationic granule protein
synthesized during the promyelocyte stage of eosinophil
development(5, 6) . The cDNA sequence and complete
open reading frame identified EDN as a member of the ribonuclease gene
family(7, 8) . The gene encoding EDN (1.2 kilobases,
designated RNS2) contains two exons separated by a 230-bp
intron, with the entire coding sequence residing on exon 2(9) .
This gene structure (noncoding exon 1/single intron/coding exon 2) is
shared by least three additional ribonuclease genes, including
eosinophil cationic protein (ECP)(9) ,
angiogenin(10, 11) , and pancreatic
ribonuclease(12, 13) , and appears to be a consistent
feature of this gene family.
The studies to be described were
performed by transfection of various segments of the EDN gene into
clone 15 cells; clone 15 is a subline of the human promyelocytic
leukemia cell line, HL-60, which was isolated on the the basis of its
propensity to develop into cells resembling mature
eosinophils(14) . Features of this cell line include formation
of Luxol-fast blue staining granules(14, 15) ,
biosynthesis of eosinophil major basic protein and eosinophil
peroxidase(15) , and the ability to express cell surface
receptors for interleukin-5(16, 17) . Most recently,
we have shown that differentiated clone 15 cells also synthesize
immunoreactive ECP and EDN(18) .
In the work presented here,
we have identified features within the EDN gene that are responsible
for promoting gene expression in human cell lines, including at least
one functional enhancer element within the characteristic single
intron.
EXPERIMENTAL PROCEDURES
Cell Lines
The human cell lines used in this
investigation were obtained from the American Type Tissue Culture
Collection (Rockville, MD) and maintained as indicated. The HL-60,
(promyelocytic leukemia), U937, (histocytic lymphoma), Jurkat, (acute T
cell leukemia), and K562 (chronic myelogenous leukemia) were all grown
in RPMI 1640 medium (Biofluids, Inc., Rockville, MD) supplemented with
10% heat-inactivated fetal bovine serum (Hyclone Laboratories, Inc.
Logan, Utah or Life Technologies, Inc.), 2 mML-glutamine, 100 units/ml penicillin, and 100 µg/ml
streptomycin at pH 7.2. The HL-60 clone 15 promyelocytic leukemia cells
were grown in the same medium supplemented with 25 mM HEPPSO
(Sigma) to maintain the pH at 7.6. The human 293 cells, a transformed
primary embryonal kidney cell line, were grown in Dulbecco's
modified Eagle's medium (Biofluids, Inc.) supplemented as above.
All cells were grown at 37 °C and 5% carbon dioxide in a humidified
incubator.
Cellular Differentiation
The clone 15 cells were
induced to differentiate toward the eosinophil lineage with the
addition of 0.5 mM butyric acid (BA) (Sigma) to freshly
cultured cells at a concentration of 0.5
10
cells/ml as described by Fischkoff and
colleagues(16, 17) .
RNA Isolation and Northern Analysis
Total RNA was
isolated at times indicated using the method of Chirgwin et
al.(19) or using an RNA Isolation Kit (Stratagene, La
Jolla, CA). Isolated RNA (15 µg/lane) was electrophoresed in a 1%
agarose gel containing 2% formaldehyde in 10 mM MOPS buffer, 5
mM sodium acetate, and 1 mM EDTA at pH 7.0. After
electrophoresis, RNA was transferred overnight to a Hybond-N nylon
filter (Amersham Corp.), UV-cross-linked using a Stratalinker
(Stratagene), and probed with the following gene-specific antisense
oligonucleotide sequences: (a) EDN,
5`-GTGACAATTTTTGCGACTTTTGTTACTAGGACAGGTCATATTTGGGT-3`, corresponding to
bases 341-296 of EDN cDNA(7) ; (b) ECP,
5`-TGCCCGCATTGCAATGGTGCATCGAGGGGGGTTCAGACT-3`, corresponding to bases
222-184 of ECP cDNA(20) ; and (c) human
beta-actin, 5`-GCACATGCCGGAGCCGTTGTCGACGACGAGCGCGGCGATATCATCATC-3`,
corresponding to amino acids 16-2 encoded by human beta
actin(21) . Fifty na nograms of each oligonucleotide probe were
radiolabeled using T4 polynucleotide kinase (New England Biolabs, Inc.,
Beverly, MA) and [
-
P]ATP (5000Ci/mmol, ICN,
San Diego, CA) and purifed on size exclusion columns (Stratagene).
Nylon filters containing RNA were prehybridized (40% formamide, 10%
dextran sulfate, 4
SSC, 20 mM Tris, pH 7.5, 1
Denhardt's solution, 0.1% sodium dodecyl sulfate, and 20
µg/ml denatured salmon sperm DNA) at 38 °C for 1 h.
Radiolabeled oligonucleotide was added (2
10
cpm/ml), and the filters were hybridized at 38 °C for 8 h.
The hybridized filters were washed with 2
SSC and 0.5% SDS
twice at 42 °C and twice at 55 °C for 30 min each. Quantitation
was performed by densitometric scanning using a Gel Documentation
System (UVP, San Gabriel, CA) monitor to image autoradiograms from the
Northern blots, followed by analysis with the NIH Image software
package.
Primer Extension
Total RNA harvested from clone 15
cells induced to differentiate with BA for 48 h was used for primer
extension experiments. Messenger RNA was purified by polyadenosine
selection using a Poly(A) Quik Kit (Stratagene) following the
manufacturer's procedure. Poly(A)-selected mRNA was then used in
primer extension experiments with the Primer Extension System (Promega
Corporation, Madison, WI). RNA (EDN mRNA or kanamycin-positive kit
control) was reverse transcribed using avian myeloblastosis
virus-reverse transcriptase and an antisense EDN-specific primer,
EDN-1a (see Fig. 1B), corresponding to nucleotides 192
to 175 (5`-TTGCTGGGAGGTCATATT-3`) of the EDN cDNA(7) . EDN-1a
was end-labeled with [
-
P]ATP (ICN, Costa
Mesa, CA) and annealed to the poly(A) selected mRNA prior to reverse
transcription. The primer extension reaction was electrophoresed in a
denaturing 6% TBE polyacrylamide gel along with known DNA sequence for
size determination. The number of nucleotides from the primer to the 5`
end of the mRNA was determined.
Figure 1:
A, Northern analysis of RNA isolated
from the clone 15 eosinophilic variant of HL-60 grown for 0-5
days in the absence (lanes 1-6) or the presence (lanes 7-12) of 0.5 mM BA. Total RNA (10
µg/lane) was probed with a radiolabeled EDN-specific
oligonucleotide (see ``Experimental Procedures''); identical
samples were probed with a radiolabeled human
-actin-specific
oligonucleotide (see ``Experimental Procedures'') to
demonstrate loading of each lane. B, structural schematic of
the EDN gene(9) . Transcribed sequences are shown as boxes. The black area depicts the open reading frame; the shaded
areas represent untranslated transcript as previously documented
by cDNA cloning(7, 8) ; the unshaded area indicates additional transcript demonstrated by primer extension
and RACE (see C and D). Below, schematic
depicting placement of oligonucleotide primers used in primer extension (EDN-1a) and RACE (EDN-2a and EDN)
experiments as described under ``Experimental Procedures.'' C, primer extension from EDN-1a (see B) from mRNA
isolated from clone 15 cells induced for 2 days with 0.5 mM BA (A, lane 9) (EDN mRNA) and from control RNA; the
+ and - symbols indicate reactions performed with or without
the addition of mRNA prior to reverse transcription. The primer
extension product in the +EDN mRNA lane is indicated by
the arrow; its migration relative to the flanking sequence
standards (Std) indicates a 219-bp product, extending the
known transcript size by 25 bp. D, sequence of the extended
transcript isolated by the RACE procedure. Regions of known sequence
are as designated (Anchor, Exon 1, Exon 2,
and the ATG codon at the start of translation). Short arrows designate the sequence of the 25-bp extension to exon
1.
Rapid Amplification of cDNA Ends
To determine the
precise transcriptional start site, rapid amplification of cDNA ends
(RACE) was performed using mRNA described above with the
5`-AmpliFINDER(TM) RACE Kit (Clontech Laboratories, Inc., Palo Alto,
CA) following the manufacturer's protocol. The cDNA was first
synthesized from mRNA using avian myeloblastosis virus-reverse
transcriptase and antisense EDN-specific primers. These primers
corresponded to nucleotides 291-274, 5`-ACAAACATTAACTACGTT
(EDN-2a) of the EDN probe, nucleotides 341-296, described above,
of the EDN cDNA sequence(7) . An anchor of known sequence was
ligated to the 5` end of the cDNA. Polymerase chain reaction (PCR)
amplification (described below) of the anchor ligated cDNA was
performed using the supplied anchor primer and a nested primer for each
of the antisense EDN primers used to synthesize the cDNA. The nested
primer used with the antisense oligonucleotide EDN was EDN-2a, and for
EDN-2a the nested primer was EDN-1a, as shown in Fig. 1B. The PCR were performed in a Perkin-Elmer Gene
Amp PCR System 9600 using Taq DNA polymerase (5 units)
(Boehringer Mannheim) in 10 mM Tris, pH 8.3, buffer containing
50 mM KCl, 2.5 mM MgCl
, 2 uM each dATP, dCTP, dGTP, and dTTP, 100 ng of each oligonucleotide
primer, and 10-50 ng of DNA template. The DNA was denatured for 3
min at 95 °C, followed by a three-step PCR reaction: denaturation
at 95 °C for 30 s, annealing at an appropriate temperature for each
primer pair for 30 s, and extension at 72 °C for 1 min. After 35
cycles the reactions were extended for an additional 5 min at 72 °C
followed by cooling to 4 °C. PCR products were subcloned into
pBluescript (Invitrogen, San Diego, CA) for dideoxy sequence analysis.
Isolation of Genomic Clones
EDN genomic clones
were isolated from a human lymphocyte genomic library (Clontech
Laboratories, Inc.) by plaque hybridization with an EDN cDNA
radiolabeled probe (7) and confirmed by dot blotting with the
EDN-1a oligonucleotide (see Fig. 1B) and by dideoxy
sequencing. The nucleotide sequence obtained agreed with previously
published data (9) .
Reporter Gene Constructs
The reporter gene used in
these studies was chloramphenicol acetyltransferase (CAT) as found in
the pCAT-basic expression vector (Promega). Various fragments of the 5`
putative promoter region of EDN both with and without additional intron
and/or exon sequence were amplified using the following primers
(numbered as per (9) ): 1, 5`-CTGCAGGCAGCATATAGTTTTCAT-3`,
nucleotides -312 to -288; 2, 5`-GGTCTCCCCTACTTGGAACT-3`,
nucleotides -26 to -45; 3, 5`-CTGTAAGAAAAGAAGAGAAGTAAC-3`,
nucleotides 172-149; 4, 5`-CCAGTCTCCGCGCTGTAGC-3`, nucleotides
42-23; 5, 5`-GTAAGTCAACGATCCCCA-3`, nucleotides 43-60; 6,
5`-CCCTCAGTTGCTGCCCCATTG-3`, nucleotides 102-82; 7,
5`-CTTTACTTCCTGTCTGCAAAG-3`, nucleotides 162-142; 8,
5`-ACACACACTGTAGTGTCTTAC, nucleotides 222-202. These primers also
contained 5` PstI or XbaI sites as indicated to
facilitate subcloning into the pCAT vectors. For the construct
containing promoter plus intron, without exon 1, the primers used were
1 and 9 (nucleotides -26 to -45 with a 5` XhoI
site to amplify the promoter region). The primers 10 (nucleotides
43-60 with a 5` XhoI site) and 3 were used to amplify
the intron sequence. This produced a promoter construct with a XhoI site at its 3` and an intron construct with the XhoI site at its 5` end after PCR amplification. The products
were gel purified, restriction digested with XhoI, and
ligated; the ligation was followed by a second round of PCR
amplification using primers 1 and 3 described above. The 520-bp
fragment was gel purified and ligated into the pCAT-basic vector. All
constructs were confirmed by dideoxy sequencing.
Electroporation of CAT Constructs into Human Cell
Lines
Cell lines as described above were grown to a density of
0.5 to 1.0
10
cells/ml. Cells were harvested by
centrifugation at 2000 rpm for 5 min and resuspended to 30
10
cells/ml in complete medium as described above for each
cell line. Ten to twenty micrograms uncut purified plasmid DNA (using
either Qiagen or Promega Maxiprep kit followed by phenol/chloroform
extraction) were added to an electroporation cuvette with a 0.4 cm
electrode gap (Bio-Rad) with 15
10
cells. The cells
were electroporated at a capacitance of 960 microfarads at 250 volts
using a Gene Pulser (Bio-Rad). The cells were placed on ice for 15 min
prior to the addition to 30 ml of complete medium and incubated for 2
days at 37 °C, 5% CO
in a humidified incubator. Cells
were also electroporated with either 10 µg of uncut control
pSV-
-galactosidase (Promega) or pCMV-
-galactosidase
(Stratagene) vector to control for variation in electroporation
efficiency. The level of
-galactosidase activity was determined
spectrophotometrically (
-galactosidase assay kit, Promega). CAT
activity was normalized to the level of expression of this control
vector.
CAT Assays
CAT assays were performed essentially
as described(22) . Transfected cells were harvested after 2
days of incubation by centrifugation at 2000 rpm for 5 min, washed
twice at room temperature with phosphate-buffered saline, and
resuspended in 0.5 ml of 40 mM Tris, pH 7.4, with 1 mM EDTA and 150 mM NaCl. Cells were then harvested by a
1-min centrifugation in a microfuge and resuspended in 100-150
µl of 25 mM Tris, pH 8, and disrupted by freeze/thaw,
three times on dry ice/37 °C water bath. The debris was removed by
centrifugation; clarified extract was added to the following reaction
mixture: 5-45 µl of extract, 35 µl of 1 M Tris,
pH 7.5, 20 µl of 4 mM acetyl Coenzyme A (Pharmacia Biotech
Inc.), and 3-5 µl of
[
C]chloramphenicol (0.25 µCi, DuPont NEN),
with distilled H
O added to a final volume of 150 µl.
After 6-12 h at 37 °C, the
[
C]chloramphenicol and acetylated products were
extracted by vortexing for 30 s with 1 ml of ethyl acetate. The ethyl
acetate layer (upper) was removed and air dried under a hood or
SpeedVac (Savant), resuspended in 30 µl of ethyl acetate, and
applied to a flexible thin layer chromatography sheet (Baker-flex
silica gel 1B, J. T. Baker Inc., Phillipsburg, NJ). Separation of
acetylated and nonacetylated forms proceeded via a chloroform/methanol
(95:5) ascending mobile phase followed by autoradiography. The signals
in each lane of the autoradiogram, including all acetylated forms of
chloramphenicol, were measured with a System 200 Image Scanner
(Bioscan, Inc., Washington, D. C.).
Analysis of Intronic Region
Computer analysis of
the intronic regions of the EDN gene for consensus transcription factor
binding sites was performed using the MacVector sequence analysis
program. Comparison of intronic regions with known sequences was
performed using the FASTA algorithm of the Wisconsin Genetics Computer
Group program on-line at the National Institutes of Health.
Sequence Mutations
Specific point mutations were
introduced into PrExIn by overlapping PCR mutagenesis (23) using the following oligonucleotide primer pairs along
with primers 1 and 8 as described previously: lfn X (5` primer,
5`-TCAACGATCCCCTGTGGTCGCAGACAAGGGGCAGCAAT-3`, and 3` primer,
5`-ATTGCTGCCCCTTGTCTGCGACCACAGGGGATCGTTGA-3`) and lfn Y (5` primer,
5`-TCAACGATCCCCAGTCGACCCACAGAAGGGGCAGCAAT-3`, and 3` primer,
5`-ATTGCTGCCCCTTCTGTGGGTCGACTGGGGATCGTTGA-3`). The altered sequences
were subcloned into pCAT basic as described. All mutations were
confirmed by dideoxy sequencing.
RESULTS
RNA Expression in the Clone 15 Cell
Line
Expression of mRNA for the EDN in the clone 15 cell line is
demonstrated in Fig. 1A. Messenger RNA encoding EDN was
detected in uninduced clone 15 cells (lanes 1-7); the
level of mRNA accumulating in these cells increased 6-7-fold
within 48 h after the addition of 0.5 mM BA (lane 9)
as determined by densitometric analysis. Hybridization with a
-actin probe (below) demonstrated relative loading of each lane.
Primer Extension
Primer extension experiments were
undertaken to determine the precise location of the transcriptional
start site. RNA from clone 15 cells induced for 2 days with 0.5 mM BA was used for this analysis; a control RNA reaction was
included, as was a known DNA sequence for determination of molecular
size (Fig. 1C). The band in the +EDN
mRNA lane indicated by the arrowhead is the
reverse-transcribed product. Comparison of the nucleotides in the known
sequence (Std) with the mobility of the band indicated in the +EDN mRNA lane demonstrates that
the distance from the primer to the 5` end of EDN is 219 nucleotides.
This result suggests that there are 25 additional nucleotides in the
mRNA sequence that had not previously been identified by cDNA
cloning(7, 8) .
Rapid Amplification of cDNA Ends
It was unclear as
to whether these additional 25 nucleotides represented a direct
extension of exon 1 or a completely distinct exon; RACE was undertaken
to make this determination. Using the poly(A)-selected mRNA described
for the primer extension experiments, a cDNA strand complementary to
the 5` region of the EDN mRNA was reverse transcribed using the EDN or
EDN-2a primers (Fig. 1B), PCR amplified, and subcloned.
Numerous colonies were obtained; 18 random colonies were selected for
sequencing. The results from a representative sequence are depicted in Fig. 1D. The additional 25-base pair sequence obtained,
which follows the anchor sequence and precedes the first exon sequence,
is 5`-AGCTGCCCCTGAACCCCAGAACAAC-3`. This sequence matches the published
genomic sequence of Hamann and colleagues (9) that is found
immediately 5` to the previously designated exon 1. These results
suggest that the additional nucleotides represent a direct extension of
exon 1 as opposed to another distinct exon. The transcriptional start
site defined by both primer extension and RACE is located 25
nucleotides 3` from a consensus TATA box(9) . Of the 18 clones,
11 sequences were as described in Fig. 1D, two were 4
nucleotides shorter at the 5` end, and five contained unidentified
sequences between the PCR primers. The five unidentified clones are
assumed to be the result of incorrect priming, because they contained
no sequences representing either exon 1 or exon 2.
CAT Activity in Clone 15 Cells
The sequence of the
EDN gene positioned 5` to CAT used in the following experiments are
shown in Fig. 2A. The EDN promoter region alone (PrCAT)
supported a relatively low level of activity in clone 15 cells,
representing 2-3-fold over that of the promotorless CAT (Fig. 2B). Surprisingly, the EDN promoter-exon-intron
construct (PrExInCAT) supported significantly greater degree of
reporter gene activity: 28-fold over CAT in uninduced clone 15 cells
and 80-fold over CAT in clone 15 cells grown in the presence of BA (Fig. 2, B and C). The EDN promoter-exon
(PrExCAT) construct produced low levels of CAT activity, similar to
those of PrCAT alone, suggesting that the dramatic enhancement seen
with PrExInCAT was due to sequence elements found in the intron. The
intron alone (InCAT) did not demonstrate reporter gene activity above
the basal levels in these cells, whereas the EDN promoter-intron
combination (PrInCAT) without the first exon provided a 12-fold
increase in activity over CAT alone. The increase in activity with
promoter and intron and the absence of activity with the intron alone
suggests that the enhanced activity depends on interactions between
sequence elements in both the promoter and intron.
Figure 2:
A,
segments of the EDN gene positioned 5` to the CAT reporter gene: Pr, 287 bp of the 5` promoter region; PrEx, the
promoter region and exon 1; PrExIn, promoter region, exon 1,
and single intron; Pr+In, promoter and intron without
exon 1; In, intron alone. B, reporter gene activity
of each CAT construct transfected into clone 15 cells, relative to that
of CAT alone (no promoter). The bars represent densitometric
analysis of duplicate samples. C, same as B with the
addition of 0.5 mM BA to cells immediately after transfection. D, sample autoradiogram depicting CAT activity of constructs
transfected into undifferentiated clone 15
cells.
Expression of EDN-CAT Constructs in Other Cell
Lines
Analysis of CAT activity in several other cell lines is
shown in Fig. 3. PrExInCAT was capable of promoting reporter
gene activity 20-40-fold over that observed with the CAT in the
K562, Jurkat, U-937, and HL-60 human hematopoietic cell lines (Fig. 3). The EDN promoter alone (PrCAT) supported only the
lower, basal levels of expression in all cell lines tested. To evaluate
regulation in a nonhematopoietic cell line, the PrCAT and PrExInCAT
constructs were introduced into the human transformed 293 kidney cell
line. Similar to the observations made with the hematopoietic cells,
PrCAT induced a basal (2-3-fold) increase in reporter gene
activity over CAT in the human kidney cell line. Interestingly, in
contrast to the 20-40-fold increase in activity observed in
hematopoietic cells, PrExInCAT induced only a 3-fold increase in the
kidney cells. This activity was not significantly greater than that
induced by PrCAT, suggesting that the enhancer activity provided by the
intron may function in a tissue-specific fashion.
Figure 3:
Reporter gene activity of constructs
transfected into four distinct human hematopoietic cell lines (HL-60
(promyelocytic leukemia), K562 (chronic myelogenous leukemia), U-937
(histocytic lymphoma), and Jurkat (T cell leukemia)) and one
nonhematopoietic human cell line (293 kidney cell). The constructs and
quantitation were as described for Fig. 2.
Dissection and Analysis of the Intron
In order to
define the region or regions of the intron that enhance gene
expression, truncated promoter-intron constructs were evaluated in
clone 15 cells (Fig. 4). The PrEx0.25InCAT and PrEx0.5InCAT
constructs, which contain the first 60 and first 120 base pairs of the
intron, respectively, were both found to increase reporter gene
activity 8-fold over CAT alone. These results suggest that there are
one or more functional enhancer elements in the first 60 base pairs of
the intron. In contrast, the PrEx0.75InCAT construct, containing the
first 180 bp of the intron, produces only a 2.5-fold increase in
activity over CAT, suggesting that this region (120 and 180 bp)
contains sequence elements that neutralize the effects of the
aforementioned functional enhancer(s). Full activity (28-fold over CAT)
is restored with the complete intron (PrExInCAT), suggesting the
possibility of additional functional enhancers in this final segment of
the intron.
Figure 4:
A, the EDN promoter, exon 1, and truncated
versions of the single intron to be positioned 5` to CAT: PrExInCAT, complete (230 bp) intron; PrEx0.25In,
first 60 bp of the intron; PrEx0.5In, first 120 bp of the
intron; PrEx0.75In, first 180 bp of the intron. B,
reporter gene activity of each construct transfected into clone 15
cells, relative to that of CAT (no promoter). The bars represent densitometric analysis of duplicate
samples.
A sequence map of the EDN gene with a focus on the
intron is shown in Fig. 5A. The first 60 bp of the
intron contain consensus binding sites for both AP-1 and NF-ATp
transcription factors. In addition, there is a segment of 15 bp that is
identical to a segment found in the promoter of the lactoferrin gene
(see ``Discussion'')(24) . Point mutations were
introduced into the sequence of this segment (Fig. 5B),
and the PrExInCAT constructs both with and without mutations were
evaluated for their ability to support reporter gene activity. Neither
set of mutations (lfn X or lfn Y; Fig. 5, B and C) altered the intron-enhancing activity to any significant
degree (21- and 35-fold over CAT alone, as compared with 28-fold for
the wild type).
Figure 5:
A,
nucleotide sequence of the 5` promoter (-287 to -1), exon 1
(1-67), intron (68-297) and the beginning of exon 2 (298
onward). Consensus CAAT and TATA box promoter sequences are as
indicated. Consensus transcription factor binding sites (AP-1, NF-ATp,
and PU.1) are single overlined; the arrowheads denote
the division points for PrExIn0.25CAT, PrEx0.5InCAT, and PrEx0.75InCAT,
respectively. The 15-bp segment that is double overlined is
identical to a segment found in the 5` promoter region of the
neutrophil specific granule protein, lactoferrin(24) . B, mutations introduced into the 15-bp lactoferrin segment in
the intron to create lfn X and lfn Y in PrExIn. Points at which lfn X
and lfn Y differ from the wild type are indicated over each sequence
with filled circles. C, reporter gene activity of
each construct transfected into clone 15 cells, relative to that of CAT
(no promoter). The bars represent densitometric analysis of
duplicate samples.
DISCUSSION
In the initial phase of this study, we determined the
transcriptional start site of the EDN gene. Both primer extension and
RACE extended the length of the mRNA to include an additional 25
nucleotides. This additional sequence matched the region of genomic
sequence that was directly 5` to sequence identified as exon
1(9) . Thus, exon 1 has been enlarged to 67 nucleotides, and
the two-exon structure of this gene is confirmed. The transcriptional
start site is situated appropriately at 23 and 101 base pairs 3` to
consensus TATA and CAAT boxes, respectively.
Although significantly
more active than either PrCAT or PrExCAT, we found that PrInCAT was not
as effective as PrExInCAT in producing reporter gene activity. It is
possible that the noncoding exon 1, in conjunction with the intron,
plays a specific role in regulating activity of the EDN gene. However,
it is also possible that the spatial rearrangement caused by deletion
of this 67-base pair segment affects the ability of promoter and intron
binding proteins to interact with one another as they interact with the
DNA. This point may be clarified once the binding proteins mediating
transcription of the EDN gene have been identified. Furthermore, it is
not clear whether the enhancer elements present in the EDN intron
coordinate specifically with the EDN promoter, or whether they might
function equally effectively with other unrelated gene promoters.
Although not completely independent (InCAT produced little reporter
gene activity in clone 15 cells), the specificity of the interaction
between the intron and the EDN promoter has not been established.
Investigators have been examining the promoter regions of granule
protein genes(1, 2, 3, 4, 24, 25) in hopes
of identifying factors promoting their tissue-specific expression. Our
initial results suggest that the function of one or more of the
intronic enhancer elements may be tissue-specific. In contrast to the
results obtained with the human hematopoietic cell lines, the PrExInCAT
construct supported no additional reporter gene activity over that of
the promoter alone (PrCAT) in the human kidney cell line. Although
preliminary, the increase in activity of PrExInCAT in the hematopoietic
cells but not in the kidney cell line suggests that one or more of the
functional enhancer elements may interact with a transcription factor
expressed in a limited range of human cell types.
Sequence analysis
of the EDN intron revealed the presence of several consensus sequences
for transcription factor binding (Fig. 5). In the first 60 bp of
the intron, the region in which a large portion of the enhancing
activity resides, there are consensus sequences representing binding
sites for transcription factors AP-1 (26) and
NF-ATp(27) . In addition, a 15-base pair segment that is
identical to a sequence found in the 5` promoter region of the
neutrophil-specific granule protein, lactoferrin(24) , is
highlighted. The coordinate control of eosinophil and neutrophil
granule protein biosynthesis was suggested by a series of studies
examining the rare genetic disorder known as neutrophil specific
granule deficiency(28, 29) . Lomax and colleagues (30) determined that the defective biosynthesis of lactoferrin
observed in this disorder resulted from a defect in mRNA transcription
affecting specifically cells of the neutrophil lineage. Rosenberg and
Gallin (31) showed that the biosynthetic defect could extend to
include eosinophils; EDN was among the eosinophil granule proteins
affected. Specific mutations introduced into this ``lactoferrin
site'' resulted in no significant alteration in reporter gene
activity, suggesting that this segment is not crucial to this specific
aspect of EDN gene regulation. Additional investigations using
electrophoretic mobility shift assays coupled with DNA footprinting and
mutational analysis of additional sites will be necessary to determine
the source of the enhancing activity.
In summary, this investigation
has demonstrated that the intron of the EDN gene contains one or more
functional enhancer elements and that interaction between the promoter
and these intronic elements is required for the optimal gene
expression. Although intronic enhancer elements have been described
previously(32, 33, 34, 35, 36, 37, 38, 39) ,
this is the first evidence that this mechanism exists within the
mammalian ribonuclease gene family. This finding takes on considerable
significance because the noncoding exon/single intron/coding exon gene
structure is shared by all members of this gene family whose gene
structures have been
determined(9, 10, 11, 12, 13) .
Although the nucleotide sequences of introns of each of the
characterized ribonuclease genes are not overtly homologous to one
another (save for EDN and ECP, whose introns are virtually
identical(9) ), the potential for this as a shared mechanism of
gene expression is intriguing.