Characterization of the Human elk-1 Promoter
POTENTIAL ROLE OF A DOWNSTREAM INTRONIC SEQUENCE FOR
elk-1 GENE EXPRESSION IN MONOCYTES*
Ulrich
Lehmann
§,
Pascale
Brocke
¶,
Jürgen
Dittmer
, and
Alfred
Nordheim
**
From the
Institut für Molekularbiologie,
Medizinische Hochschule Hannover, 30623 Hannover, and the
Institut für Zellbiologie, Abteilung Molekularbiologie,
Universität Tübingen, Auf der Morgenstelle 15,
72076 Tübingen, Germany
 |
ABSTRACT |
To characterize the human elk-1
promoter, we mapped the transcriptional start site and
isolated elk-1-specific genomic phage clones that contained
extensive upstream and downstream sequences. A TATA-like motif was
identified immediately upstream of the transcriptional start site.
Functional analyses of DNA fragments containing the TATA element and
the identification of a DNase I-hypersensitive chromatin site (HS 1) in
close proximity to the TATA box suggest that the identified TATA motif
is important for elk-1 transcription in vivo.
Sequences upstream and downstream from the TATA box were found to
contribute to elk-1 promoter activity. A second
hypersensitive site (HS 2) was identified within the first intron in
pre-monocytic cells, which express Elk-1 only when differentiating to
monocytes. In a variety of other cell types, which display a
constitutive Elk-1 expression, HS 2 did not exist, suggesting that
inducibility of elk-1 expression is associated with the
presence of HS 2. Egr-1 and the serum response factor were found to
interact specifically with the intronic sequence at +265 and +448,
respectively. Because Egr-1 mRNA and protein levels were observed
to increase significantly before induction of elk-1
expression, we propose that Egr-1 is important for the regulation of
elk-1 transcription in differentiating monocytes.
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INTRODUCTION |
The Ets family of transcription factors is composed of proteins
that share the Ets domain, a DNA binding domain that recognizes a
GGAA/T-based sequence (for review, see Refs. 1-3). Ets proteins are
involved in a variety of cellular activities, including proliferation and differentiation, and are suggested to contribute to the development of certain diseases (for review, see Ref. 4). Based on the homology
within the Ets domain, Ets proteins are divided into several subclasses.
One subclass of Ets factors consists of the ternary complex factors
(TCFs),1 which are able to
form a ternary complex with the serum response factor (SRF) and the
serum response element (SRE) (for review, see Refs. 5-7). TCFs include
the Ets proteins Elk-1, Sap1a, Sap1b, Net/Erp/Sap2, and Netb (8).
Although all TCFs can be activated by different mitogen-activated
protein kinases such as ERK, JNK, and p38/RK (6, 8), there are
differences in the efficiency by which these kinases are able to
phosphorylate the different TCFs (9-14). For instance, in NIH3T3
cells, Elk-1 and Sap1a are similarly well activated by p38, but only
Elk-1 is a good substrate for JNK. Because certain stimuli activate
certain signaling pathways, each TCF may serve as an effector for a
specific set of stimuli. One could speculate, therefore, that in some
cell types, a particular stimulus may exert its effect on the cell
through one, but not another TCF. In such a case, the cell's ability
to respond to that stimulus would depend upon a sufficient expression
level of this TCF, which, on the other hand, would allow this cell to control its sensitivity to certain stimuli by regulating the expression of this TCF.
By screening several cell lines for Elk-1 expression we found that,
compared with a variety of non-monocytic cells, pre-monocytic cells
expressed Elk-1 only at very low levels. However, Elk-1 expression
could be induced when these cells were stimulated to undergo
differentiation toward mature monocytes upon exposure to
12-O-tetradecanoylphorbol-13-acetate (TPA). To understand
the mechanism underlying this monocyte-specific regulation of Elk-1 expression, we cloned and analyzed the human elk-1 promoter.
We were able to determine the transcriptional start site of this promoter, and we further located a TATA-like element immediately upstream of the start site and identified an intronic Egr-1 binding site as a potential TPA-responsive element. The TATA box and the Egr-1
binding site were each found to be located within a hypersensitive area, HS 1 and HS 2, respectively. More importantly, HS 2 was observed
to exist only in monocytic cells and not in cells showing a
constitutive expression of elk-1. We assume that Elk-1
serves a specific function, e.g. as an effector of a
particular stimulus, in differentiating monocytes, which cannot be
replaced by another TCF such as Sap1a, whose expression was found to be
unchanged upon treatment with TPA.
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MATERIALS AND METHODS |
Accession Number--
The genomic sequence of the human
elk-1 locus provided here (see Fig. 3) is deposited under
Y11432 HSELK1 in the EMBL Nucleotide Sequence Data base. Upon scanning
this sequence against the data base no significant relatedness to other
sequences was observed.
Library Screening, Cloning, and Sequencing--
For screening of
a human genomic
library (CLONTECH), the
1.3-kilobase pair EcoRI/BamHI fragment and the
0.25-kilobase pair EcoRI/NarI fragment of the
human elk-1 cDNA (15) were labeled with 32P
using a random priming kit (Stratagene). After hybridizations in
hybridization buffer (50% formamide, 4 × SSPE, 5 × Denhardt's solution, 100 µg/ml sonicated denatured salmon sperm DNA,
1% SDS, 10% dextran sulfate) at 42 °C for 16 h, filters were
washed once in 2 × SSC and 0.2% SDS for 5 min at room
temperature and twice in 0.5 × SSC and 0.2% SDS for 30 min at
65 °C. Positive plaques were identified by exposure of the filters
to x-ray films (Kodak) for 3 days. The phage DNA of four positive
plaques was isolated using ultracentrifugation (140,000 × g) and exhaustive organic extractions (16). After
restriction analyses of the phage DNA, appropriate fragments were
subcloned into pBluescript (Promega) for further analysis. Both strands
were sequenced by the dideoxy chain termination method, performed after
progressive deletion of the subcloned genomic fragments using
exonuclease III (Nested Deletion Kit; Promega).
Sequence Analysis--
For the identification of putative
regulatory elements the programs PC/Gene (IntelliGenetic, Inc.) and
Transfac (E. Wingender, GBF Braunschweig, Germany) were employed.
Cell Culture--
The cell lines used were ML-1 (myeloblast-like
cells (17)), U-937 (histiocytic lymphoma cells (18)), RK 13 (rabbit
kidney epithelial-like cells; ATCC CCL37), CV-1 (African green monkey kidney-derived cells; ATCC CCL70), and TE85 (osteosarcoma cells; ATCC
CRL 1543). U-937 and ML-1 cells were grown in RPMI 1640 medium (+Glutamax, Life Technologies, Inc.). Cell density was kept below 106/ml. RK 13 cells were grown in Eagle's minimum
essential medium containing Earle's salts and 4.5 g/liter glucose
(Life Technologies, Inc.). TE85 and CV-1 cells were grown in
Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose
(Life Technologies, Inc.). All media contained 10% fetal calf serum
(Biochrom), streptomycin (100 µg/ml), and penicillin (100 units/ml).
Isolation and Analysis of RNA--
For Northern blot analyses,
total RNA was prepared following the one-step protocol of Chomczynski
and Sacchi (19). Electrophoresis of RNA and transfer onto nylon
membranes (GeneScreen Plus, NEN Life Science Products) were performed
essentially as described (20). After UV cross-linking, prehybridization
and hybridization were performed at 42 °C overnight in hybridization
solution (50% formamide, 4 × SSPE, 5 × Denhardt's
solution, 100 µg/ml sonicated denatured salmon sperm DNA, 1% SDS,
10% dextran sulfate). Filters were washed once in 2 × SSC and
0.2% SDS for 5 min at room temperature and twice in 0.2 × SSC
and 0.2% SDS for 30 min at 65 °C. Radioactive signals were detected
by exposing the filter to an x-ray film (Kodak) for 5-10 days or, for
quantitation, to a PhosphorImager screen (Fuji) for overnight.
For RNase protection and primer extension analysis,
poly(A)+-selected RNA from TE85 cells was used. After lysis
and homogenization of cells in 4 M guanidinium
isothiocyanate (25 mM sodium acetate (pH 5.5), 0.1 M 2-mercaptoethanol), total RNA was purified using CsCl
density gradient centrifugation. After butanol/chloroform extraction
and precipitation, the pellet was dissolved in water, and
poly(A)+ RNA was isolated using the mRNA purification
kit from Amersham Pharmacia Biotech.
Detection of DNase I-hypersensitive Sites--
Cells were
collected by centrifugation at 500 × g for 10 min at
4 °C and washed twice in ice-cold phosphate-buffered saline. The
cell pellet was resuspended in approximately 5 volumes of RSB buffer
(10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2) containing 0.02% Nonidet P-40 and
held on ice for 10 min. The nuclei were collected by centrifugation at
700 × g for 10 min at 4 °C and washed twice in
ice-cold RSB buffer without Nonidet P-40. 100 µl of the nuclei
suspension, adjusted to an optical density of 40 (measured at 260 nm as
a 1:100 dilution in 1 N NaOH), was incubated with various
amounts of DNase I (Sigma) for 10 min at 37 °C. The reaction was
terminated by adding 100 µl of stop buffer (20 mM Tris-HCl (pH 8), 10 mM EDTA, 1% (w/v) SDS, 600 mM NaCl, 400 µg/ml proteinase K). After careful mixing,
the solution was rotated overnight at 37 °C. DNA was extracted using
phenol/chloroform and treated with RNase A (0.1 mg/ml) and RNase T1
(1,500 units/ml) for 2 h at 37 °C. After another extraction
with phenol/chloroform, DNA was precipitated by the addition of sodium
acetate and isopropyl alcohol. For Southern analysis (21) 25 µg of
DNA was incubated with 100 units of the appropriate restriction enzyme
at 37 °C for 16 h.
RNase Protection Analysis--
2 µg of
poly(A)+-selected RNA was hybridized with 200,000 cpm of
32P-labeled probe (purified using a denaturing acrylamide
gel) in 30 µl of hybridization buffer (80% formamide, 40 mM PIPES (pH 6.4), 400 mM NaCl, 1 mM EDTA) at 50 °C for 16 h, after heat denaturation at 85 °C for 10 min. Excess probe and unhybridized RNA were digested by the addition of 350 µl of RNase reaction buffer (10 mM
Tris-HCl (pH 7.5), 300 mM NaCl, 5 mM EDTA, 40 µg/ml RNase A, 2 µg/ml RNase T1) and incubation at 30 °C for the
indicated time. The reaction was terminated by the addition of 50 µg
of proteinase K and SDS (final concentration 0.5%), incubation at
37 °C for 15 min, phenol extraction, and ethanol precipitation. The
pellets were dissolved in 5 µl of 90% formamide and 10 mM EDTA loading buffer. Nucleic acids were separated on an
8 M urea, 8% acrylamide gel. Radioactive signals were
detected by autoradiography using intensifying screens.
Primer Extension Analysis--
After heat denaturation at
85 °C for 10 min, 2 µg of poly(A)+ RNA was hybridized
with 200,000 cpm 32P-labeled oligonucleotide (0.5 ng) in 15 µl of hybridization buffer (10 mM Tris-HCl (pH 8.3), 150 mM KCl, 1 mM EDTA) at 42 °C for 16 h.
The primer extension reaction was started by the addition of 45 µl of
reverse transcriptase reaction buffer (23 mM Tris-HCl (pH
8.3), 50 mM KCl, 10 mM MgCl2, 5 mM dithiothreitol, 1 mM dNTP, 0.3 mM EDTA) containing 5 units of murine mammary tumor virus reverse transcriptase (RNase H minus, Promega). After incubation at
42 °C for 60 min, the RNA template was removed by adding 105 µl of
RNase mix. After incubation at 37 °C for 15 min followed by phenol
extraction, the DNA was precipitated with ethanol. The pellets were
dissolved in 5 µl of 90% formamide and 10 mM EDTA loading buffer, and the DNA was separated on an 8 M urea,
8% acrylamide gel. Radioactive signals were detected by
autoradiography using intensifying screens.
Preparation of Cell Extracts--
Nuclear and cytosolic
fractions of cells were prepared following the protocol of Andrews and
Faller (22). Briefly, cells were collected by centrifugation at
500 × g for 10 min at 4 °C and washed twice in
ice-cold phosphate-buffered saline. The cell pellets containing
5-15 × 106 cells were resuspended in 400 µl of
buffer A (10 mM Hepes (pH 7.9), 1.5 mM
MgCl2, 10 mM NaCl, 0.5 mM
dithiothreitol, 1 mM Na3VO4, 10 mM NaF, 20 mM 2-phosphoglycerate, 1 mM p-nitrophenylphosphate, 2.8 µg/ml
aprotinin, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml pepstatin, 0.5 mM benzamidine, 2.5 µg/ml
leupeptin, 5 units/ml
2-macroglobulin) and kept on ice
for 10 min. The cells were homogenized using a syringe. After
centrifugation (500 × g for 10 min at 4 °C), the
supernatant was frozen in liquid nitrogen as "cytosolic fraction."
The nuclear pellet was resuspended in 50 µl of extraction buffer C
(10 mM Hepes (pH 7.9), 25% glycerol, 420 mM
NaCl, 1.5 mM MgCl2, 0.2 mM EDTA,
0.5 mM dithiothreitol, 1 mM
Na3VO4, 10 mM NaF, 20 mM 2-phosphoglycerate, 1 mM
p-nitrophenylphosphate, 2.8 µg/ml aprotinin, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml pepstatin,
0.5 mM benzamidine, 2.5 µg/ml leupeptin, 5 units/ml
2-macroglobulin) and kept on ice for 30 min. After
centrifugation (15,000 × g for 10 min at 4 °C) the
supernatant was frozen in liquid nitrogen as nuclear extract.
Electromobility Shift Assays--
Reaction mixtures contained
the following components: 6.5 µl of binding buffer (10 mM
Hepes (pH 7.9), 5 mM MgCl2, 0.1 mM
EDTA, 10 mM NaCl), 1 µl of 10 × SED (20 mM spermidine, 25 mM EDTA, 133 mM
dithiothreitol), 1 µl of bovine serum albumin (20 mg/ml), 1 µl of
salmon sperm DNA (0.5 µg/ml), 1-2 ng of 32P-labeled
probe (30,000-50,000 cpm) and 1-2 µl of in vitro
translated protein or of a nuclear extract (23).
Western Blotting--
Nuclear extracts or cytosolic fractions
were separated by SDS, 8% polyacrylamide gel electrophoresis before
electrophoretic transfer onto a Hybond C Super membrane (Amersham
Pharmacia Biotech). After blocking with 5% nonfat dry milk, the
membranes were incubated with an anti-EGR-1 antiserum (mouse
anti-p82-EGR-1, Santa Cruz) at a concentration of 0.5 µg/ml in TBS
for 1 h at room temperature. The blots were subsequently incubated
for 1 h at room temperature with an anti-mouse horseradish
peroxidase-conjugated antibody prior to exposure to the ECL substrate
(Amersham Pharmacia Biotech). For detection of the signals x-ray films
(Kodak) were exposed for 10-30 s. All Western blotting reagents were
from Amersham Pharmacia Biotech.
Flow Cytometry--
Approximately 105 cells were
washed with ice-cold phosphate-buffered saline containing 2% fetal
calf serum and incubated at 4 °C for 30 min with the appropriate
fluorescein isothiocyanate-labeled primary antibody
(Coulter-Immunotech). After washing the cells twice with
phosphate-buffered saline and 2% fetal calf serum, fluorescence
intensities were measured in a flow cytometer (Becton Dickinson).
Results were analyzed with the LYSIS software (Becton Dickinson).
Construction of Luciferase Reporter Plasmids--
Using the
subcloned genomic fragments as templates the indicated segments of the
promoter region were prepared by polymerase chain reaction, using
oligonucleotide primers specific for this region and containing
appropriate restriction sites for insertion into the polylinker of the
plasmid pKS+/L (5) upstream of the luciferase gene.
Transient Transfection of Luciferase Reporter Gene
Constructs--
Rabbit kidney epithelial-like cells (RK 13, ATTC
CCL37) were transiently transfected by the calcium phosphate
coprecipitation method essentially as described previously (5). Cells
were harvested 36 h after transfection and lysed. The cleared
lysate was measured for luciferase activity. A
cytomegalovirus-
-galactosidase expression vector served as a measure
for transfection efficiency. All transfection experiments were
performed at least three times.
 |
RESULTS |
Identification and Characterization of Human elk-1 Genomic
Clones--
We screened a human genomic
library with DNA probes
corresponding to the coding sequence and the 5'-untranslated region of
human elk-1. The elk-1-specific sequences of
three phages (B6, B9, B21) were analyzed in detail. B6 and B9 were
overlapping clones that contained the first three translated exons
III-V of the elk-1 gene encoding amino acids 1-337 (Fig.
1). The B21 clone included the
5'-untranslated regions (exons I and II), large parts of the second
intron, and sequences upstream of exon I. Note that the last
untranslated exon, exon II, is separated from the first translated exon, exon III, by a large, approximately 12-kilobase pair-long intron.
Because of the length of this intron, the B21 clone did not share any
sequence with B6 or B9. Using fluorescence in situ hybridization analyses, however, we have reported previously that B6
and B21 hybridize to the same locus on the X chromosome (24). This
indicates that these sequences are genomically linked. This was
confirmed recently by Mills and co-workers who isolated genomic clones
that contained sequences of both B6 and
B21.2 In the process of
screening with the B6 or B21 sequence we also identified an
elk-1 related pseudogene that we mapped to chromosome 14q32.3

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Fig. 1.
Physical maps of the genomic clones (B21, B6,
and B9) and the structural organization of the human elk-1
gene. The position of the first five exons is shown relative
to the transcriptional start site. Selected restriction sites are
indicated.
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Identification of the Transcriptional Start Site of the Human elk-1
Gene--
The human osteosarcoma cell line TE85 constitutively
expresses elk-1 at high
levels.4 Poly(A)+
RNA was isolated from these cells and analyzed by the primer extension
technique. Using an oligonucleotide corresponding to a stretch of
nucleotides between positions +166 and +186 of the B21 sequence (see
Fig. 3), a major transcriptional start site was identified (termed
nucleotide position +1) (Fig.
2A, arrow). The
smaller fragment in Fig. 2A may suggest an additional, minor transcriptional start site some 65 bp downstream of the major one.
However, by using RNase protection assays, performed with the
XhoI/SacI fragment as a probe (Fig.
2B), we could only confirm the existence of the major start
site (data not shown). According to these data, the start site is
located 39 nucleotides further upstream of the 5'-end of the previously
published elk-1 cDNA sequence (15). It places the
transcriptional start site approximately 25 bp downstream of a
TATA-like sequence (Fig. 3).

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Fig. 2.
Determination of the elk-1
transcriptional start site. Panel A, primer
extension analysis of the 5'-end of the elk-1 mRNA. The
size of the major extension product (lane 2,
arrow) was found to be 186 nucleotides, as determined by
using a defined sequence ladder as size marker (lanes A,
G, C, T). Lane 1 shows a
control reaction using a tRNA as template. Panel B, graphic
illustration of the position of the elk-1 transcriptional
start site, as determined by primer extension studies and RNase
protection analyses (not shown). The hatched box indicates
the 40 nucleotides, by which the elk-1-specific RNA was
found to be extended at the 5'-end relative to the transcribed
elk-1 sequence as reported previously (15).
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Fig. 3.
Nucleotide sequence of the 5'-region of the
human elk-1 locus. The sequences spanned by exons I
and II are boxed, and binding sites for putative
transcription factors are indicated.
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The TATA-like Element in the elk-1 Gene Supports Transcription in
Transient Transfection Experiments--
Several DNA fragments of human
the elk-1 gene including the TATA motif (Fig.
4A) were cloned upstream of a
luciferase gene and tested for their abilities to support transcription
in transient transfection experiments using RK 13 cells. Transfection
with plasmid e-43, which contained the minimal fragment
43/+63,
resulted in a luciferase activity that was approximately 10-fold higher than that obtained with the control plasmid missing this fragment (Fig.
4B). This suggests that the
43/+63 fragment can function as a promoter. To test whether flanking sequences may affect promoter activity, we first gradually extended the elk-1 promoter 5'
up to nucleotide at position
936. Highest activity was found with the
e-480 promoter construct, suggesting that enhancer element(s) were
located between
43 and
480. This region contains potential binding
sites for several transcription factors, including Sp1, Ap1, cAMP
response element-binding protein, and the CCAAT-binding protein (Fig.
3). Further extension to
711 slightly decreased transcription from
the elk-1 promoter. The sequence between
711 and
936 was
found to have a repressive effect on elk-1 promoter activity, suggesting that this region contains silencer element(s). When 3'-flanking sequences down to +679 were added to the e-711 promoter fragment, elk-1 promoter activity was increased by
2-fold. This implies that this downstream sequence, which contains
binding sites for Egr-1 and SRF/TCF (Fig. 3), may contribute to the
regulation of elk-1 gene transcription. Similar data
regarding the differential activities of the different promoter
fragments were obtained when transfection experiments were performed
with CV-1 cells (data not shown).

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Fig. 4.
Analysis of basal transcription activity
displayed by the human elk-1 promoter. Panel
A, schematic showing the different elk-1 promoter
segments used in transient transfection studies. Panel B,
relative luciferase activities generated by the different
elk-1 promoter constructs in transiently transfected RK 13 epithelial cells.
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A DNase I-hypersensitive Site Is Located around the TATA-like
Element in elk-1-expressing TE85 Cells--
Transcriptionally active
regions in the chromatin are often susceptible to DNase I treatment. To
identify DNase I-hypersensitive site(s) within the human
elk-1 promoter, we subjected nuclei isolated from TE85
cells, which express elk-1 constitutively, to DNase I
digestion. After deproteinization and HindIII treatment, the genomic DNA was analyzed by Southern blot hybridization. Fig. 5A shows a typical DNase I
cleavage pattern obtained from hybridizing the blotted DNA with the
elk-1-specific probe A (Fig. 5B). Only one
fragment (panel A, arrow pointing
leftward) smaller than the full-length HindIII
fragment (arrow pointing rightward) was
visualized under these conditions. Note that even high concentrations
of DNase I, which allowed complete digestion of the HindIII
fragment, did not lead to the generation of any other distinct
fragment. The size of the fragment resulting from DNase I treatment
positions the DNase I-hypersensitive site (HS 1) to an area around the
TATA-like element, in close proximity to the XhoI site shown
in Fig. 5B. Results obtained from reprobing the blots with
elk-1-specific DNA probe B (Fig. 5B) allowed the
same conclusion as to the location of HS 1 (data not shown). As with
probe A, no additional hypersensitive site could be identified with
probe B. These probing strategies also allowed us to estimate HS 1 to
extend over a region of approximately 150-200 bp.

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Fig. 5.
Identification of a DNase I-hypersensitive
site (HS 1) in the chromatin of TE85 cells at the human elk-1
locus. Panel A, DNase I hypersensitivity mapping
near the human elk-1 promoter, as determined by Southern
analysis using probe A (position of probe A is indicated in Fig.
4B). The cleavage products generated in the chromatin of
TE85 cells by increasing concentrations of DNase I are shown
(arrow pointing leftward). The full-length
5-kilobase pair HindIII fragment is marked by the
arrow pointing rightward. Panel B, a
schematic showing the position of the identified DNase I-hypersensitive
site (HS 1) at the 5'-end of the first exon. The positions of the two
independent probes A and B, used to map the precise position of the HS,
are shown. The two black boxes indicate the locations of
exons I and II.
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A Second Constitutive DNase I-hypersensitive Site Downstream of the
elk-1 TATA-like Element Was Identified in the Human Pre-monocytic Cell
Line ML-1, Which Displays Inducible elk-1 Expression--
In contrast
to a variety of non-monocytic cell lines, the human pre-monocytic cell
lines ML-1 and U-937 were found to synthesize very low basal levels of
elk-1 mRNA (Fig.
6A and data not shown). Treatment with TPA triggered these cells to differentiate to monocytes (Fig. 6C). This was accompanied by the expression of
monocytic markers such as CD14 and CD11C and by down-regulation of the
expression of pre-monocytic proteins like MPO (Fig. 6, A and
B). In differentiating ML-1 (and U-937) cells,
elk-1 mRNA levels were found to be strongly up-regulated
(Fig. 6A), which resulted in enhanced Elk-1 DNA binding activity (data not shown). Because TPA stimulation did not change the
half-life of elk-1 mRNA (t1/2 = 5 h) (data not shown), it is likely that transcriptional
activation was responsible for the up-regulation of elk-1.
In contrast to elk-1, expression levels of other TCFs
(sap-1a and net) remained unchanged (data not
shown), suggesting that Elk-1 fulfills a specific function in
differentiating monocytes. By analyzing ML-1 chromosomal DNA for
elk-1-specific DNase I-hypersensitive sites, we found an
additional hypersensitive site, HS 2, downstream of the TATA-like
element within intron I (Fig. 7). We
estimate HS 2 to extend over some 150-200 bp in length. HS 2 was also
found in nonstimulated ML-1 cells (data not shown), demonstrating that
HS 2 was present before the elk-1 gene was transcriptionally
activated by TPA. In non-monocytic Jurkat T cells, SKW 6.4 B cells and,
as shown above, TE85 cells, which constitutively express Elk-1, HS 2 was not observed (data not shown). This suggests that a pre-established
open chromatin structure within intron I represents a prerequisite for
inducible elk-1 expression in monocytic cells.

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Fig. 6.
Transcriptional activation of the human
elk-1 gene during monocytic differentiation of ML-1
cells. Panel A, Northern blot analysis of
elk-1 and monocytic marker gene expression (MPO, CD14) in
the course of TPA-induced ML-1 differentiation. Cells were either
untreated (lane C) or treated with TPA for the indicated
number of days (lanes 1, 2, and 3; TPA
concentration: 1.6 × 10 8 M). As a
control, the blot was rehybridized with a GADPH-specific probe.
Panel B, TPA-induced expression of monocyte surface marker
genes (CD11C, CD14), as measured by flow cytometric analysis.
Panel C, the morphology of human pre-monocytic ML-1 cells
observed, when these cells were left untreated (upper
picture) or were treated with TPA for 3 days (lower
picture).
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Fig. 7.
An additional DNase I-hypersensitive site (HS
2) is present in the first intron of the elk-1 gene in
monocytic ML-1 cells. Panel A, nuclei of ML-1 cells,
treated for 24 h with TPA, were analyzed for DNase I
hypersensitivity. Two regions of DNase I hypersensitivity, HS 1 and HS
2, were observed. Panel B, graphic depiction of the
positions of the two HSs relative to the elk-1
transcriptional start site and exons I and II. Putative binding sites
for transcription factors Egr1, Elk-1 and SRF at these HSs are
indicated.
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HS 2 Contains Binding Elements for the Transcription Factors Egr-1
and SRF/TCF, Which Can Both Be Activated by TPA--
The
hypersensitive site HS 2 contains an SRE-like element at position +448
and an Egr-1 consensus binding site at +265. Because both SRF/TCF (25)
and Egr-1 (26, 27) can regulate transcription upon activation by TPA,
we hypothesized that these transcription factors may be involved in
TPA-induced transcriptional activation of the human elk-1
gene. To test this possibility, we first analyzed the abilities of
Egr-1 and SRF/Elk-1 to bind to their consensus binding sites within HS
2. As shown in Fig. 8D,
recombinant Egr-1 protein was able to bind to the putative Egr-1
binding site within intron I (lane 1) but failed to interact
with a mutated version of this sequence (lane 3 and see
legend to Fig. 8). Similarly, we found that SRF could specifically bind
to the intronic SRE-like sequence (cI, Fig. 8E, lanes
1 and 7) and form a ternary complex with Elk-1 (cII,
lane 3).

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Fig. 8.
Expression levels in ML-1 cells and DNA
binding characteristics of the regulatory proteins EGR-1 and SRF, which
may be involved in elk-1 gene regulation. Panel
A, changes in expression of Elk-1, Egr-1, and SRF mRNA during
differentiation of ML-1 cells as determined by Northern blot analysis.
Panel B, Western blot analysis of lysates from ML-1 cells
treated with TPA for 2, 4, 6, or 8 h or from untreated cells for
Egr-1 protein expression. Panel C, EMSA performed with
nuclear extracts from ML-1 cells exposed to TPA for 2, 4, or 6 h
or from untreated cells and an oligonucleotide containing the Egr-1
binding site of the elk-1 promoter (eEgr):
5'-CTAGAGTATCCTTCCCGCCCCCGCGCAGAGGATGA-3' in the presence or absence of
an Egr-1-specific antibody. Panel D, electrophoretic
mobility shift analysis of the binding of recombinant Egr-1 to either
the wild-type (WT) eEgr probe or a mutated version of this
oligonucleotide (M):
5'-CTAGAGTATCCTTCCCGAAACCGCGCAGAGGATGA-3' (the mutated
nucleotides are underlined). Panel E, the ability of
recombinant SRF (5) to bind alone or in association with recombinant
Elk-1(5) to the SRE-like element in the first intron of the
elk-1 promoter, as determined by electrophoretic mobility
shift analysis. SRE/EBS: 5'-CTAGATGTTTTTCCCCACGCCAACTTAGGGTGGA-3'.
mSRE/EBS: 5'-CTAGATGTTTTTCCCCACGCCAACTTATTGTGGA-3'.
SRE/mEBS: 5'-CTAGATGTTTTTAACCACGCCAACTTAGGGTGGA-3'. (The
mutated nucleotides are underlined.)
|
|
We next compared the expression pattern of Egr-1 and SRF with that of
Elk-1 in differentiating ML-1 cells. Egr-1 mRNA was detectable as
early as 1 h of exposure to TPA (data not shown), preceding the
rise in elk-1 expression. Typically for an immediate-early response gene (26, 28), mRNA expression of Egr-1 was found to be
transient, starting to decline rapidly after an additional 2-3 h of
TPA treatment (Fig. 8A). The Egr-1 protein level and DNA
binding activity were found to be strongly increased after 2 h of
exposure to TPA (Fig. 8, B and C, lane
2). However, although Egr-1 protein levels remained unchanged for
at least another 6 h in the presence of TPA (Fig. 8B),
the ability of Egr-1 to bind to the intronic Egr-1 binding site was
found be decreased strongly subsequent to the initial 2-h TPA treatment
(Fig. 8C, lane 3). Thus, Egr-1 may be involved in
the initial TPA-dependent up-regulation of elk-1
expression but is unlikely to contribute to elk-1
transcription at a later stage of the differentiation process. In
contrast to the Egr-1 expression, SRF mRNA levels were not found to
increase significantly before 6 h of TPA treatment, a time at
which elk-1 expression already reached its maximum (Fig.
8A). This suggests that SRF could not participate in the
TPA-induced rapid increase in elk-1 transcription but may
have been needed to maintain a high expression level of Elk-1 during
monocytic differentiation. For this potential function, SRF might have
required the newly produced Elk-1 and, in this way, may have initiated
a positive feedback loop for Elk-1 expression.
 |
DISCUSSION |
In an effort to characterize the human elk-1 promoter,
we identified a TATA-like element immediately upstream of a major
transcriptional start site, as determined by primer extension analyses
and RNase protection assays. We could show that a DNase
I-hypersensitive site, termed HS 1, was surrounding the TATA-like
sequence, suggesting that the TATA-containing region was accessible for
DNA-binding factors. HS 1 extended over an approximate region of
150-200 bp and accordingly may have been generated by the removal of
one nucleosome. The presence of HS 1 was independent of whether
expression of elk-1 was constitutive, as in TE85 cells, or
inducible, as in ML-1 cells. We could further demonstrate that a
minimal elk-1 promoter, containing the TATA motif, was able
to support transcription. It is therefore very likely that this TATA
sequence functions as a TATA box for elk-1 transcription
in vivo.
The downstream sequence, including exon I, most of exon II, and the
intron separating theses exons, was found to contribute to
elk-1 promoter activity in transient transfections
experiments. Interestingly, a second DNase I-hypersensitive site, HS 2, was found in the intronic sequence. HS 2 was constitutively present in
the monocytic cell lines ML-1 and U-937, which display inducible elk-1 expression. In contrast, HS 2 was not found in
non-monocytic cells lines TE85, Jurkat, and SKW 6.4, which expressed
elk-1 at constant high levels. This may suggest that the
potential for the inducibility of elk-1 expression is
associated with the constitutive presence of HS 2 and the accessibility
of this area for certain DNA-binding proteins. Two transcription
factors, Egr-1 and SRF, could specifically bind to the HS 2 region and
were found to be up-regulated in differentiating ML-1 cells along with
elk-1. We have also preliminary data suggesting that both
Egr-1 and SRF can increase elk-1 promoter activity through
their cognate binding sites within the HS 2 sequence (data not shown).
Egr-1 is known to be involved in differentiation processes, such as
macrophage development (26, 29) and may therefore be a potential
candidate involved in TPA-mediated induction of elk-1
transcription. On the other hand, elk-1 expression was found to be elevated even after 3 days of TPA treatment (data not shown), whereas Egr-1 was only temporarily expressed, and Egr-1 DNA binding activity was only detectable for a couple of hours. This may suggest that Egr-1 may be important for initiation of elk-1
transcription at early stage of the differentiation process, to be
replaced by other factors that allow long lasting up-regulation of
elk-1 expression. Such a factor could be an Ets protein
because a putative Ets binding site was found to be located immediately
downstream of the Egr-1 site. Several Ets proteins, including PU.1,
Ets1, and GABP
, are expressed in monocytic cells (30-32). Of these, PU.1 was observed to be able to bind to the Ets binding site of the
elk-1 promoter (data not shown) and was reported to be
up-regulated during differentiation of pre-monocytic U-937 cells (32).
U-937 was also found here to show an increased elk-1
expression upon TPA treatment (data not shown). Further investigations
are required to elucidate the regulatory function of the intronic
sequence containing HS 2 and the roles of Egr-1, Ets factors and SRF
for elk-1 transcriptional regulation in monocytic cells.
 |
ACKNOWLEDGEMENTS |
We thank Lars-Gunnar Larsson for providing
initial Northern blots and for teaching us the handling of
pre-monocytic cells; Regine Leo for flow cytometric analyses; Edgar
Wingender for DNA sequence analyses; Bob Hipskind for providing TE85
cells and for many stimulating discussions; and Mike Cahill, Olaf
Heidenreich, Bernhard Lüscher, and Boh Wasylyk for helpful
comments on this manuscript. Various plasmids were provided by R. Rauscher, J. Monroe, R. Janknecht, and V. Sukhatme.
 |
FOOTNOTES |
*
This work was funded by the Deutsche Forschungsgemeinschaft
(No. 120/7-3 and No. 120/10-1), the Dr. Mildred Scheel-Stiftung (10-1058-2), and the Fonds der Chemischen Industrie.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) Y11432 HSELK1.
§
Present address: Institut für Pathologie, Medizinische
Hochschule Hannover, 30623 Hannover, Germany.
¶
Present address: DKFZ, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
**
To whom correspondence should be addressed: Institut für
Zellbiologie, Abteilung Molekularbiologie, Universität
Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany.
Tel.: 49-7071-297-8898, Fax: 49-7071-295-359; E-mail:
alfred.nordheim{at}uni-tuebingen.de.
The abbreviations used are:
TCF(s), ternary
complex factor(s); SRF, serum response factor; SRE, serum response
element; TPA, 12-O-tetradecanoylphorbol-13-acetate; HS, hypersensitive site; PIPES, 1,4-piperazinediethanesulfonic acid; bp, base pairs.
2
F. Mills, personal communication.
3
U. Lehmann and A. Nordheim, unpublished data.
4
R. A. Hipskind, unpublished data.
 |
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