Human EZF, a Krüppel-like Zinc Finger Protein, Is Expressed
in Vascular Endothelial Cells and Contains Transcriptional Activation
and Repression Domains*
Shaw-Fang
Yet,
Megan M.
McA'Nulty,
Sara C.
Folta,
Hsueh-Wei
Yen,
Masao
Yoshizumi,
Chung-Ming
Hsieh,
Matthew D.
Layne,
Michael T.
Chin
§,
Hong
Wang,
Mark A.
Perrella
¶,
Mukesh K.
Jain
§, and
Mu-En
Lee
§
From the Cardiovascular Biology Laboratory, Harvard School of
Public Health,
Department of Medicine, Harvard Medical
School, ¶ Pulmonary and § Cardiovascular Divisions,
Brigham and Women's Hospital, Boston, Massachusetts 02115
 |
ABSTRACT |
Members of the erythroid Krüppel-like
factor (EKLF) multigene family contain three C-terminal zinc fingers,
and they are typically expressed in a limited number of tissues. EKLF,
the founding member, transactivates the
-globin promoter by binding to the CACCC motif. EKLF is essential for expression of the
-globin gene as demonstrated by gene deletion experiments in mice. Using a DNA
probe from the zinc finger region of EKLF, we cloned a cDNA encoding a member of this family from a human vascular endothelial cell
cDNA library. Sequence analysis indicated that our clone, hEZF, is
the human homologue of the recently reported mouse EZF and GKLF. hEZF
is a single-copy gene that maps to chromosome 9q31. By gel mobility
shift analysis, purified recombinant hEZF protein bound specifically to
a probe containing the CACCC core sequence. In co-transfection
experiments, we found that sense but not antisense hEZF decreased the
activity of a reporter plasmid containing the CACCC sequence upstream
of the thymidine kinase promoter by 6-fold. In contrast, EKLF increased
the activity of the reporter plasmid by 3-fold. By fusing hEZF to the
DNA-binding domain of GAL4, we mapped a repression domain in hEZF to
amino acids 181-388. We also found that amino acids 91-117 of hEZF
confer an activation function on the GAL4 DNA-binding domain.
 |
INTRODUCTION |
It has been estimated that 10% of the proteins within a cell are
DNA-binding transcription factors that regulate important cellular
processes such as cell lineage determination, cell growth and
differentiation, and temporal or cell type-specific gene expression (1-3). After binding to cognate cis-acting elements, these
transcription factors either activate or repress initiation of
transcription (4, 5). Transcription factors are grouped into several
classes, which include the helix-loop-helix, leucine zipper,
homeodomain, and zinc finger protein families (2).
The zinc finger transcription factors can be classified further into
subfamilies on the basis of the sequence and position of amino acid
residues important for zinc binding (Cys2-His2, Cys4, or Cys3-His1), the spacing
between the zinc-binding amino acids, and the transcription activation
or repression domains (glutamine-rich, acidic, or proline-rich domains)
(6-10). A new zinc finger subfamily was identified recently whose
members are characterized by a highly conserved C-terminal region
containing three Cys2-His2 zinc fingers and a
proline rich N-terminal domain (8, 10-13). Members of this subfamily
include the erythroid (EKLF),1 lung (LKLF), and
basic (BKLF) Krüppel-like factors, and BTEB2 (or placental
Krüppel-like factor). All four factors transactivate gene
expression after binding to DNA.
The founder of this family, EKLF, was originally isolated as an
erythroid cell-specific factor by subtractive cloning (8). It binds and
transactivates via the CACCC site of the
-globin gene promoter (8,
9, 14). In vitro, EKLF plays an important role in
human
-globin to
-globin gene switching (11). This observation is
consistent with data showing that disruption of the EKLF gene by
homologous recombination in mice results in defective hematopoiesis in
the fetal liver and lethal
-thalassemia (15, 16).
The other members of the EKLF family, LKLF, BKLF, and BTEB2, were
isolated by homology screening with the zinc finger regions of EKLF,
Sp1, and BTEB (a GC box-binding zinc finger protein) (10, 12). LKLF is
expressed highly in the lung and the spleen and transactivates the
-globin gene via the CACCC site (10). Although BKLF is also
expressed in hematopoietic precursor cells, its expression is less
restricted than that of EKLF (13). Also, even though BTEB2 was isolated
from a placental library with a BTEB probe, the BTEB2 zinc finger
region is more homologous to the zinc finger region of EKLF than it is
to that of BTEB or Sp1 (12).
To identify new members of the EKLF family that may be involved in the
regulation of vascular endothelial cell function, we used the zinc
finger region of EKLF to screen a human vascular endothelial cell
cDNA library. We isolated a member of the EKLF family and found it
to be the human homologue of mouse EZF and GKLF (17, 18). Mouse
EZF/GKLF has been shown to be a nuclear protein. Its mRNA is
expressed highly in quiescent fibroblasts. The growth-arresting nature
of EZF/GKLF was demonstrated by its ability to inhibit DNA synthesis in
cells that overexpress the gene (17). By in situ analysis,
the mouse homologue was shown to be expressed at high levels in
epithelial cells of the epidermis, tongue, palate, esophagus, stomach,
and colon (18).
We show in this report that the human homologue (hEZF) is expressed in
vascular endothelial cells of an endodermal origin, in contrast to the
ectodermal origin of the mouse homologue in epithelial cells. We also
demonstrate that purified, recombinant full-length hEZF protein binds
specifically to a probe containing the CACCC core sequence in gel
mobility shift assays. In contrast to other members of the family,
which are transcriptional activators, hEZF functions as a
transcriptional repressor, as demonstrated by its ability to repress
reporter gene activity in transient transfection assays. By gene fusion
experiments, we identified both the activation domain and the
repression domain within hEZF.
 |
EXPERIMENTAL PROCEDURES |
Cloning of hEZF--
A cDNA probe encoding the C-terminal
zinc finger region of EKLF (bp 895-1146) was generated by reverse
transcription polymerase chain reaction (PCR) (19, 20). The forward
primer (5
-GAACTTTGGCACCTAAGAGGCAG-3
) and reverse primer
(5
-ACGCTTCATGTGCAGAGCTAAGTG-3
) were designed according to the
published sequence (8). The DNA fragment was labeled by random priming
(Stratagene, La Jolla, CA) and used as a probe to screen a human
umbilical vein endothelial cell cDNA library. Approximately 1.6 million phages were plated, transferred to nitrocellulose, and screened
according to standard techniques with minor modification (20). The
filters were washed initially with 0.5 × SSC (75 mM
sodium chloride, 7.5 mM sodium citrate) and 0.1% SDS
(sodium dodecyl sulfate) at 37 °C and then more stringently with
0.2 × SSC and 0.1% SDS at 65 °C. More than 40 clones were obtained that hybridized differentially. Six were isolated, three were
sequenced, and one was characterized further. It included the entire
coding region of hEZF. The cDNA was mapped by restriction digestion
and sequenced from both orientations by the dideoxy chain termination
method with Sequenase version 2 (Amersham, Arlington Heights, IL) or on
an automated DNA Sequencer (Licor, Lincoln, NE) according to the
manufacturer's instructions. Sequence analysis was performed using the
GCG software package (Genetics Computer Group, Madison, WI).
Southern Blot Analysis and Chromosomal Localization of
hEZF--
High molecular weight genomic DNA was prepared from cultured
human aortic endothelial cells (21). Genomic DNA (10 µg) was digested
with several restriction enzymes, fractionated on 0.8% agarose gels,
and transferred to nylon membranes. The membranes were then hybridized
with a random-primed hEZF cDNA probe. The final membrane wash was
in 0.1 × SSC and 0.1% SDS at 65 °C for 30 min, after which
the membranes were exposed to Kodak X-AR film at
80 °C. To
localize the hEZF gene, we performed PCR-based radiation hybrid panel
mapping (Research Genetics, Huntsville, AL). Two oligonucleotide
primers specific to the hEZF cDNA sequence
(5
-CCACCTGGCGAGTCTGACAT-3
and 5
-CACCGTGTCCTCGTCAGCGT-3
) were used
to amplify genomic DNA by PCR. The PCR products were separated on 1.2%
agarose gels, and the results were analyzed on the worldwide web server
at the Whitehead Institute/MIT Center for Genome Research (URL:
http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl).
Cell Culture--
Bovine aortic endothelial cells (BAEC) were
isolated and cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum and antibiotics as described
(22). BAEC were passaged every 2-3 days, and cells from passages 5 to
8 were used in all experiments. We used BAEC because they are easy to transfect.
Recombinant hEZF Protein Expression and Purification--
Amino
acids 2-470 of hEZF were fused in-frame with the N-terminal histidine
residues of the pRSET vector (Invitrogen, Carlsbad, CA). His-tagged
hEZF protein was expressed in the BL21 (DE3) pLysS strain of
Escherichia coli and purified with Ni-NTA resin (Qiagen, Santa Clarita, CA). Recombinant protein was eluted from the resin with
50 mM sodium phosphate buffer, pH 6.0, containing 300 mM NaCl, 10% glycerol, 0.2 mM imidazole, 10%
glycerol, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml
aprotinin, and 100 µg/ml leupeptin. The purified protein was then
dialyzed against 50 mM Tris-HCl buffer, pH 8.0, containing
0.005% Tween 20, 2 mM reduced glutathione, 0.02 mM oxidized glutathione, 10 µM
ZnCl2, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, and 100 µg/ml leupeptin for 16 h at 4 °C and stored at
80 °C.
Gel Mobility Shift Assay--
The sequence of the
double-stranded oligonucleotides containing the core CACCC site
(5
-AGCTAGCCACACCCTGAAGCT-3
) was derived from the sequence of the
-globin promoter. Oligonucleotides were labeled with
[
-32P]ATP by using T4 polynucleotide kinase (New
England Biolabs, Beverly, MA) as described (23). A typical binding
reaction mixture contained 25,000 cpm of probe, 40 ng of purified hEZF
protein, 50 ng of poly(dI-dC)·poly(dI-dC), 25 µM HEPES,
pH 7.5, 16 mM KCl, 50 mM NaCl, 2 µM ZnCl2, 0.6 mM
-mercaptoethanol, and 8% glycerol. The probe and protein were
incubated at room temperature for 20 min and fractionated on 5%
polyacrylamide gels in 0.25 × TBE buffer (22 mM Tris
base, 22 mM boric acid, and 0.5 mM EDTA). The
sequence of the mutant competitor oligonucleotide was
5
-AGCTAGCCACACCGTGAAGCT-3
.
Construction of Plasmids--
A cDNA fragment containing bp
411 to 1873 of hEZF was amplified by PCR with Pfu DNA
polymerase (Stratagene). The product was then digested at the
BamHI sites that had been added to the primers. The fragment
was ligated into the BamHI site of the eukaryotic expression
plasmid pcDNA3 (Invitrogen) in the sense (pcDNA3-hEZF) and
antisense (pcDNA3-hEZF(AS)) orientations. The open reading frame
was confirmed by sequencing and by in vitro transcription and translation in a reticulocyte lysate system (Promega, Madison, WI)
according to the manufacturer's instructions. The pcDNA3-EKLF plasmid was constructed by cloning the EKLF EcoRI (filled
in)-BamHI fragment of pSG5/EKLF (8) into the
HindIII (filled in)-BamHI sites of
pcDNA3.
For the GAL4-hEZF fusion constructs, hEZF fragments were fused in-frame
to the C terminus of the GAL4 DNA-binding domain (amino acids 1-147)
of plasmid pSG424. Segments of hEZF were generated by PCR with
Pfu DNA polymerase and 5
- and 3
-primers containing the
BamHI and XbaI sites, respectively. The PCR
products were digested with BamHI and XbaI and
ligated into the corresponding sites of pSG424. The authenticity of the
fusion constructs was verified by dideoxy chain termination
sequencing.
Transient Transfections--
Transient transfection assays in
BAEC were performed with LipofectAMINE according to the manufacturer's
instructions (Life Technologies). Cells were plated at a density of
300,000 per 60-mm dish on the day before transfection. BAEC were
transfected with a total of 3 µg of reporter plasmid and expression
plasmid. To correct for differences in transfection efficiency, we
cotransfected 0.5 µg of pCMV-
gal in all experiments. Each
construct was transfected at least three times, and each transfection
was performed in triplicate. Cell extracts were prepared by a detergent
lysis method (Promega) 48 h after transfection, and
chloramphenicol acetyltransferase (CAT) activity was assayed by a
modified two-phase fluor diffusion method (22).
-Galactosidase
activity was assayed as described (22). The ratio of CAT activity to
-galactosidase activity in each sample served as a measure of
normalized CAT activity.
 |
RESULTS |
Isolation and Characterization of the hEZF cDNA--
To
identify additional members of the EKLF family that may be involved in
the regulation of vascular endothelial cell function, we screened a
human umbilical vein endothelial cell cDNA library using a DNA
probe containing the zinc finger region of EKLF under low-stringency
conditions. One of the cDNAs isolated contained 1876 nucleotides
and a deduced open reading frame coding for a 470-amino acid protein
with an estimated pI of 9.2. Analysis of the amino acid sequence
revealed three Cys2-His2 Krüppel-type fingers at the C terminus, a proline- and serine-rich N terminus, and a
potential nuclear localization signal at amino acids 371-377 (Fig.
1). A single transcript of 3.5 kilobases
was detected by Northern blot analysis with this 1876-bp cDNA used
as a probe in total RNA from both human aortic endothelial cells and
human umbilical vein endothelial cells (data not shown). By a
comparison with sequences in the GenBankTM data base, we found that our
cDNA is the human homologue of the recently described mouse EZF and GKLF cDNAs (17, 18). We refer to the human gene as hEZF because of
its expression in endothelial and epithelial cells. A comparison of the
human and mouse EZF sequences revealed 91% identity at the amino acid
level (Fig. 1). The three tandem zinc finger motifs (Fig. 1,
boxed) are conserved completely in the human and mouse sequences.

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Fig. 1.
Comparison of human and mouse EZF amino
acids. hEZF amino acid numbers are indicated at the top
of the sequence. The cysteine and histidine residues of the three zinc
fingers (boxed) are highlighted in white type on
a black ground.
|
|
Chromosomal Localization of the hEZF Gene--
Hybridization of an
hEZF cDNA probe with human genomic DNA that had been digested with
BamHI, EcoRI, and PstI revealed a
simple pattern of hybridization, indicating that hEZF is a single-copy gene in the human genome. To map the chromosomal location of hEZF, we
carried out genomic PCR analysis against a GeneBridge 4 radiation hybrid panel with specific primers from the hEZF cDNA sequence. The
results from the genomic PCR experiments were analyzed against a human
genome data base of sequence-tagged sites at the Whitehead Institute/MIT Center for Genome Research worldwide web site. The human
EZF gene mapped to chromosome 9q31. Thioredoxin and the disease locus
TAL2 (T-cell acute lymphocytic leukemia-2) have been mapped to the same
locus.
Binding of Recombinant hEZF to the CACCC Site of the
-Globin
Gene--
The high degree of sequence conservation among the zinc
finger regions of EZF, EKLF, and LKLF suggests that hEZF may also bind
to the CACCC sequence. Gel mobility shift analysis was performed with
the purified recombinant full-length hEZF protein and an oligonucleotide probe encoding a CACCC site derived from the
-globin gene (8). Incubation of hEZF with the probe resulted in a DNA-protein complex (Fig. 2). This complex was
specific because it was competed away by an unlabeled identical probe
but not by an unrelated probe. Mutation of the core CACCC sequence to
CACCG has been shown to obliterate the binding and transactivation of
EKLF (9). In our analysis (Fig. 2), an unlabeled probe with this single
base mutation failed to compete for binding, indicating that hEZF binds specifically to the CACCC site.

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Fig. 2.
Binding of hEZF protein to the CACCC
site. Gel mobility shift assays were performed with purified hEZF
protein and a 21-bp double-stranded oligonucleotide probe containing a
CACCC site as described under "Experimental Procedures." The
binding reactions were also performed in the presence of an identical CACCC-containing oligonucleotide (I), a mutant
CACCG-containing oligonucleotide (M), or a nonidentical
oligonucleotide (NI) encoding an unrelated sequence.
Unlabeled competitors were added at a 50-fold molar excess.
|
|
hEZF Represses Transcription in Transient Transfection
Experiments--
All members of the EKLF family identified before hEZF
function as transcriptional activators. In particular, EKLF, LKLF, and BKLF have been shown to transactivate reporter plasmids via the CACCC
site (8-10, 13). Because hEZF bound to the CACCC site, we decided to
determine the effect of hEZF on a CAT reporter plasmid (pCAC-tkCAT)
that contains a single copy of the
-globin CACCC site upstream of
the minimal thymidine kinase promoter (8). Cotransfection of
pcDNA3-hEZF decreased the promoter activity of pCAC-tkCAT in a
dose-dependent manner in BAEC (Fig.
3A). A 10 to 1 expression
plasmid to reporter plasmid ratio resulted in a 6-fold repression. This
repression was specific because cotransfection of the antisense plasmid
pcDNA3-hEZF(AS) had no effect on activity. In contrast,
cotransfection of pcDNA3-EKLF increased CAT activity by 3-fold in
BAEC (Fig. 3B). These results demonstrate that hEZF functions as a transcriptional repressor in our transient transfection system.

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Fig. 3.
Effects of hEZF or EKLF expression plasmids
on a CACCC-containing reporter. A, hEZF represses
CACCC-containing reporter activity. The pCAC-tkCAT reporter plasmid was
cotransfected into BAEC with empty vector (pcDNA3) or expression
plasmids in the sense (hEZF) and antisense (hEZF (AS)) orientations at
the indicated ratios of expression plasmid to reporter plasmid. The
degree of repression (fold repression) was determined by dividing the
average normalized CAT activities measured from pcDNA3-transfected
samples by those measured from hEZF- or hEZF(AS)-transfected samples. Error bars show standard deviations. B, BAEC were
cotransfected with 0.5 µg of reporter plasmid pCAC-tkCAT and 2.5 µg
of empty vector (pcDNA3), the hEZF expression plasmid
(pcDNA3-hEZF), or the EKLF expression plasmid (pcDNA3-EKLF).
pCMV- gal (0.5 µg) was also cotransfected for each construct to
correct for differences in transfection efficiency. Fold
activation/repression was determined as in A.
|
|
hEZF Contains Transcriptional Activation and Repression
Domains--
To identify domains in hEZF that may mediate its
transcriptional effect, we generated a series of plasmids containing
various fragments of hEZF fused to the DNA-binding domain of the yeast transcription factor GAL4 (Fig.
4A). The fusion plasmids were cotransfected with a reporter construct containing five GAL4-binding sites in front of the thymidine kinase minimal promoter
(pGAL45tkCAT). The GAL4-hEZF plasmid containing hEZF amino
acids 2-470 had little effect on reporter activity. In contrast, the
plasmid coding for amino acids 2-388 (from which the three zinc
fingers had been removed) increased transcription by 25-fold (Fig.
4B). These data indicate the presence of a potent activation
domain between amino acids 2 and 388 of hEZF that is inhibited by the
presence of the zinc finger domain. The ability to transactivate was
retained when the N-terminal 90 amino acids were deleted
(GAL4-hEZF(91-388)). However, the ability to transactivate was lost
with deletion of a further 23 amino acids from the N terminus
(GAL4-hEZF(114-388)). The region containing 97 amino acids N-terminal
of the zinc fingers (GAL4(292-388)) or the zinc finger region alone
(GAL4-hEZF(386-470)) did not affect CAT activity.

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Fig. 4.
hEZF contains a potent, modular activation
domain. A, GAL4-hEZF deletion constructs. , GAL4
DNA-binding domain; , zinc fingers. B, transactivation by
a series of GAL4-hEZF fusion plasmids harboring successive N-terminal
and C-terminal deletions. BAEC were transfected transiently with 2.5 µg of pGAL45tkCAT and 0.5 µg of the indicated
expression plasmid. For each construct, 0.5 µg of pCMV- gal plasmid
was cotransfected to correct for differences in transfection
efficiency. Fold activation data represent the degree of activation
obtained with the hEZF fusion plasmids relative to that obtained with
empty vector (GAL4(1-147)). Error bars indicate standard
deviations.
|
|
These N-terminal deletion experiments mapping the hEZF activation
domain to amino acids 91-114 (Fig. 4B) are supported by a
series of C-terminal deletion experiments (Fig. 4B).
GAL4-hEZF constructs coding for hEZF amino acids 2-117 or 2-180 were
able to increase transcription by more than 40-fold. To determine
whether the hEZF activation domain was modular, we made a fusion
construct containing amino acids 91-117 of hEZF and the GAL4
DNA-binding domain. GAL4-hEZF(91-117) increased transcription by more
than 30-fold (Fig. 4B), indicating that a potent modular
activation domain is located between amino acids 91 and 117.
The diminished transcriptional activity of GAL4-hEZF(114-388) in
comparison with that of GAL4(1-147) (Fig. 4B) suggested the presence of repression domains C-terminal of amino acid 114. Furthermore, the enhanced activation obtained with deletions
GAL4-hEZF(2-180) and GAL4-hEZF(2-117) over that obtained with
GAL4-hEZF(2-388) (Fig. 4B) is consistent with the loss of a
domain important for repression. To identify the repression domain(s)
in hEZF, we generated additional plasmids and assayed their effect on
the pGAL45tkCAT reporter. Plasmids containing hEZF amino
acids 140-388, 163-388, and 181-388 repressed transcription by
4-5-fold (Fig. 5). However, plasmids
containing segments C-terminal of amino acid 240 showed no repression
activity. The region containing the zinc fingers alone
(GAL4-EZF(386-470)) or that containing the fingers in conjunction with
the 47 amino acids N-terminal of them (GAL4-EZF(342-470)) did not
repress transcription. Our data from the N-terminal deletion analysis
suggest that hEZF amino acids 181-240 may contain the repression
domain. To further define the C-terminal border of this repression
domain, we generated GAL4-fusion constructs containing hEZF amino acids
181-325 and 178-244. Neither construct repressed transcription (Fig.
5). Thus, the repression domain of hEZF appears to be contained within
amino acids 181-388.

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Fig. 5.
Identification of a repression domain in
hEZF. A, GAL4-hEZF deletion constructs. , GAL4
DNA-binding domain; , zinc fingers. B, BAEC were
transfected transiently with 2.5 µg of pGAL45tkCAT and
0.5 µg of the indicated expression plasmid. For each construct, 0.5 µg of pCMV- gal plasmid was cotransfected to correct for
differences in transfection efficiency. Fold repression data represent
the degree of repression obtained with the hEZF fusion plasmids
relative to that obtained with empty vector (GAL4(1-147)). Error
bars indicate standard deviations.
|
|
 |
DISCUSSION |
Using the zinc finger region from EKLF as a probe to screen a
human endothelial cell cDNA library, we have isolated hEZF, a new
member of the EKLF multigene family. hEZF maps to chromosome 9q31,
close to the T-cell acute lymphocytic leukemia-2 disease locus. Further
investigation will be required to determine whether hEZF is related to
this disease. Although it has been shown that the zinc finger region of
hEZF binds to DNA fragments containing the CACCC motif (18), our
experiments are the first to show that the full-length hEZF protein
binds to this sequence (Fig. 2). It has been shown that all previously
known members of the EKLF family function as transcriptional
activators: EKLF, LKLF, and BKLF activate transcription via the CACCC
site of the
-globin promoter, and BTEB2 activates transcription via
the promoter's GC box (8, 10, 12, 13). We show here that in contrast to EKLF, hEZF represses transcription when transfected into vascular endothelial cells (Fig. 3A). The ability of hEZF to function
as a transcriptional repressor is similar to that of several other Cys2-His2 zinc finger transcription factors,
such as ZNF 174 (24), ZBP-89 (25), and Gfi-1 (26).
We next wanted to identify the functional domains important for the
transcriptional activity of hEZF. By gene fusion experiments, we mapped
the repression domain of hEZF to amino acids 181-388. Unlike
transactivation domains, repression domains are less well characterized
(4, 27). A few of the known repression domains are rich in alanines
(28), basic residues (29, 30), and prolines (4, 31-33). The repression
domain of hEZF is rich in prolines (18%). Runs of proline residues
adopt a single preferred conformation, known as the polyproline II
helix, that is important for protein-protein interactions (34). The
repression domains of WT-1, Eve (Even-skipped, a Drosophila
homeodomain protein), and Mig1 (a zinc finger protein that mediates
glucose repression in yeast) are also rich in prolines (4, 31-33).
The hEZF zinc fingers had no effect on transcription when fused with
GAL4(1-147). Deletion of the zinc fingers, however, revealed a potent
activation domain in the rest of the hEZF molecule (Fig. 4). Further
mapping localized a 27-amino acid activation domain rich in leucine,
serine/threonine, and acidic residues (with an estimated pI of 3.6).
The acidic nature of this activation domain is similar to that of the
activation domains of GAL4, GCN4 (2), and EKLF (35). Like other members
of the Cys2-His2 zinc finger protein family
(such as Egr-1 (27), WT-1 (36), Krüppel (37), and EKLF (35)),
hEZF contains activation as well as repression domains. The presence of
activation and repression domains may allow
Cys2-His2 zinc finger proteins to alter their
function as the situation dictates (38, 39). A potential switch between a positive and negative transcriptional effect could depend on an
interaction with other factors that may change the conformation of hEZF
to expose either the activation or the repression domain (40-42). For
example, the thyroid hormone receptor binds a corepressor to repress
transcription in the absence of thyroid hormone. Hormone binding alters
the receptor's conformation and leads to the release of the bound
corepressor and recruitment of a coactivator. Thus, the hormone-bound
thyroid receptor acts as a transcriptional activator (41). Under
conditions other than those examined here, hEZF may also act as an
activator depending on its binding to other factors.
 |
ACKNOWLEDGEMENTS |
We thank Dr. E. Haber for his enthusiasm and
support of our work. We are grateful to Dr. J. J. Bieker for
giving us the CACCC-containing reporter construct pCAC-tkCAT and the
pSG5/EKLF plasmid and to Dr. V. P. Sukhatme for the
pGAL45tkCAT plasmid. We thank B. Ith for technical
assistance and T. McVarish for editorial assistance.
 |
FOOTNOTES |
*
This work was supported by a grant from the Bristol-Myers
Squibb Pharmaceutical Research Institute and by National Institutes of
Health Grants HL03194 (to M. A. P.) and GM53249 (to
M.-E. L.).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) AF022184.
To whom correspondence should be addressed: Cardiovascular
Biology Laboratory, Harvard School of Public Health, 677 Huntington Ave., Boston, MA 02115. Tel.: 617-432-4994; Fax: 617-432-0031.
1
The abbreviations used are: EKLF, erythroid
Krüppel-like factor; LKLF, lung Krüppel-like factor; BKLF,
basic Krüppel-like factor; BTEB2, basic transcription
element-binding protein 2; GKLF, gut-enriched Krüppel-like
factor; EZF, epithelial/endothelial zinc finger protein; BAEC, bovine
aortic endothelial cells; PCR, polymerase chain reaction; CAT,
chloramphenicol acetyltransferase;
-gal,
-galactosidase; bp, base
pair(s).
 |
REFERENCES |
-
Maniatis, T.,
Goodbourn, S.,
and Fischer, J. A.
(1987)
Science
236,
1237-1245[Medline]
[Order article via Infotrieve]
-
Mitchell, P. J.,
and Tjian, R.
(1989)
Science
245,
371-378[Medline]
[Order article via Infotrieve]
-
Ptashne, M.,
and Gann, A. A.
(1990)
Nature
346,
329-331[CrossRef][Medline]
[Order article via Infotrieve]
-
Johnson, A. D.
(1995)
Cell
81,
655-658[Medline]
[Order article via Infotrieve]
-
Tjian, R.,
and Maniatis, T.
(1994)
Cell
77,
5-8[Medline]
[Order article via Infotrieve]
-
Berg, J. M.
(1990)
J. Biol. Chem.
265,
6513-6516[Free Full Text]
-
Sanchez-Garcia, I.,
and Rabbitts, T. H.
(1994)
Trends Genet.
10,
315-320[CrossRef][Medline]
[Order article via Infotrieve]
-
Miller, I. J.,
and Bieker, J. J.
(1993)
Mol. Cell. Biol.
13,
2776-2786[Abstract]
-
Feng, W. C.,
Southwood, C. M.,
and Bieker, J. J.
(1994)
J. Biol. Chem.
269,
1493-1500[Abstract/Free Full Text]
-
Anderson, K. P.,
Kern, C. B.,
Crable, S. C.,
Lingrel, J. B.
(1995)
Mol. Cell. Biol.
15,
5957-5965[Abstract]
-
Donze, D.,
Townes, T. M.,
and Bieker, J. J.
(1995)
J. Biol. Chem.
270,
1955-1959[Abstract/Free Full Text]
-
Sogawa, K.,
Imataka, H.,
Yamasaki, Y.,
Kusume, H.,
Abe, H.,
and Fujii-Kuriyama, Y.
(1993)
Nucleic Acids Res.
21,
1527-1532[Abstract]
-
Crossley, M.,
Whitelaw, E.,
Perkins, A.,
Williams, G.,
Fujiwara, Y.,
and Orkin, S. H.
(1996)
Mol. Cell. Biol.
16,
1695-1705[Abstract]
-
Bieker, J. J.,
and Southwood, C. M.
(1995)
Mol. Cell. Biol.
15,
852-860[Abstract]
-
Nuez, B.,
Michalovich, D.,
Bygrave, A.,
Ploemacher, R.,
and Grosveld, F.
(1995)
Nature
375,
316-318[CrossRef][Medline]
[Order article via Infotrieve]
-
Perkins, A. C.,
Sharpe, A. H.,
and Orkin, S. H.
(1995)
Nature
375,
318-322[CrossRef][Medline]
[Order article via Infotrieve]
-
Shields, J. M.,
Christy, R. J.,
and Yang, V. W.
(1996)
J. Biol. Chem.
271,
20009-20017[Abstract/Free Full Text]
-
Garrett-Sinha, L. A.,
Eberspaecher, H.,
Seldin, M. F.,
de Crombrugghe, B.
(1996)
J. Biol. Chem.
271,
31384-31390[Abstract/Free Full Text]
-
Lee, M. E.,
Temizer, D. H.,
Clifford, J. A.,
Quertermous, T.
(1991)
J. Biol. Chem.
266,
16188-16192[Abstract/Free Full Text]
-
Sambrook, J.,
Fritsch, E. F.,
and Maniatis, T.
(1989)
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor, NY
-
Laird, P. W.,
Zijderveld, A.,
Linders, K.,
Rudnicki, M. A.,
Jaenisch, R.,
Berns, A.
(1991)
Nucleic Acids Res.
19,
4293[Medline]
[Order article via Infotrieve]
-
Lee, M. E.,
Bloch, K. D.,
Clifford, J. A.,
Quertermous, T.
(1990)
J. Biol. Chem.
265,
10446-10450[Abstract/Free Full Text]
-
Yoshizumi, M.,
Hsieh, C.-M.,
Zhou, F.,
Tsai, J.-C.,
Patterson, C.,
Perrella, M. A.,
Lee, M.-E.
(1995)
Mol. Cell. Biol.
15,
3266-3272[Abstract]
-
Williams, A. J.,
Khachigian, L. M.,
Shows, T.,
Collins, T.
(1995)
J. Biol. Chem.
270,
22143-22152[Abstract/Free Full Text]
-
Merchant, J. L.,
Iyer, G. R.,
Taylor, B. R.,
Kitchen, J. R.,
Mortensen, E. R.,
Wang, Z.,
Flintoft, R. J.,
Michel, J. B.,
Bassel-Duby, R.
(1996)
Mol. Cell. Biol.
16,
6644-6653[Abstract]
-
Zweidler-Mckay, P. A.,
Grimes, H. L.,
Flubacher, M. M.,
Tsichlis, P. N.
(1996)
Mol. Cell. Biol.
16,
4024-4034[Abstract]
-
Gashler, A. L.,
Swaminathan, S.,
and Sukhatme, V. P.
(1993)
Mol. Cell. Biol.
13,
4556-4571[Abstract]
-
Licht, J. D.,
Grossel, M. J.,
Figge, J.,
and Hansen, U. M.
(1990)
Nature
346,
76-79[CrossRef][Medline]
[Order article via Infotrieve]
-
Baniahmad, A.,
Kohne, A. C.,
and Renkawitz, R.
(1992)
EMBO J.
11,
1015-1023[Abstract]
-
Saha, S.,
Brickman, J. M.,
Lehming, N.,
and Ptashne, M.
(1993)
Nature
363,
648-652[CrossRef][Medline]
[Order article via Infotrieve]
-
Han, K.,
and Manley, J.
(1993)
Genes Dev.
7,
491-503[Abstract]
-
Ostling, J.,
Carlberg, M.,
and Ronne, H.
(1996)
Mol. Cell. Biol.
16,
753-761[Abstract]
-
Madden, S. L.,
Cook, D. M.,
Morris, J. F.,
Gashler, A.,
Sukhatme, V. P.,
Rauscher, F. J., III
(1991)
Science
253,
1550-1553[Medline]
[Order article via Infotrieve]
-
Williamson, M. P.
(1994)
Biochem. J.
297,
249-260[Medline]
[Order article via Infotrieve]
-
Chen, X.,
and Bieker, J. J.
(1996)
EMBO J.
15,
5888-5896[Abstract]
-
Wang, Z.-Y.,
Qui, Q.-Q.,
and Deuel, T. F.
(1993)
J. Biol. Chem.
268,
9172-9175[Abstract/Free Full Text]
-
Zuo, P.,
Stanojevie, D.,
Colgan, J.,
Han, K.,
Levine, M.,
and Manley, J. L.
(1991)
Genes Dev.
5,
254-264[Abstract]
-
Sauer, F.,
and Jackle, H.
(1991)
Nature
353,
563-566[CrossRef][Medline]
[Order article via Infotrieve]
-
Natesan, S.,
and Gilman, M.
(1993)
Genes Dev.
7,
2497-2509[Abstract]
-
Glass, C. K.,
Rose, D. W.,
and Rosenfeld, M. G.
(1997)
Curr. Opin. Cell Biol.
9,
222-232[CrossRef][Medline]
[Order article via Infotrieve]
-
Horlein, A. J.,
Naar, A. M.,
Heinzel, T.,
Torchia, J.,
Gloss, B.,
Kurokawa, R.,
Ryan, A.,
Kamei, Y.,
Soderstrom, M.,
Glass, C. K.,
Rosenfeld, M.
(1995)
Nature
377,
397-404[CrossRef][Medline]
[Order article via Infotrieve]
-
Lehming, N.,
Thanos, D.,
Brickman, J. M.,
Ma, J.,
Maniatis, T.,
Ptashne, M.
(1994)
Nature
371,
175-179[CrossRef][Medline]
[Order article via Infotrieve]
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