From the Centro de Biología Molecular "Severo
Ochoa," Facultad de Ciencias, Universidad Autónoma de
Madrid-Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain and the § Laboratory for Developmental Neurobiology,
RIKEN Brain Science Institute, Wako-shi, Saitama 351-0198, Japan
Received for publication, August 3, 2000, and in revised form, October 4, 2000
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ABSTRACT |
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We have used the yeast one-hybrid system to
identify transcription factors that bind to specific sequences in
proximal regions of the apolipoprotein E gene promoter. The sequence
between Apolipoprotein E (apoE)1
is a major component of various classes of plasma lipoproteins. It is a
single-chain polypeptide of 299 amino acids, which plays a prominent
role in transport and metabolism of plasma cholesterol and
triglycerides, resulting from its ability to interact with lipoprotein
receptors (1). The amino acid sequence of the human protein presents
two polymorphic sites that generate three isoforms (apoE2,
apoE3, and apoE4). The major site of synthesis is the liver,
but the protein is also produced in extrahepatic tissues such as
adrenals and nervous system. In the rat brain, the synthesis takes
place in astrocytic cells (2, 3), whereas in peripheral nervous system
apoE is synthesized by nonmyelinating glial cells and resident
macrophages (2). In the human brain, the synthesis occurs both in glial cells and in subpopulations of neurons in the cortex and hippocampus (4). A number of previous in vitro and in vivo
studies as well as recent experiments with apoE-deficient mice and
human APOE transgenic mice reveal that apoE plays an
important role in neuronal maintenance and repair (5-12). Genetic
studies have identified the apoE4 allele as a major risk factor for
developing Alzheimer's disease (AD), both in sporadic and in familial
late onset forms of the disease (13, 14). This allele is also
responsible for poor outcome after acute brain injury (15, 16), stroke
(17), or neurotoxic damage (12).
ApoE synthesis is regulated in hepatic and steroidogenic cells by
complex interaction of developmental, hormonal, and dietary factors
(18-22). The regulatory complexity emerges from interactions of a
number of proteins which bind to proximal regions of the APOE gene promoter, as well as to far downstream elements
involved in its tissue-specific expression (23-30). In brain, the
regulation of this gene remains largely unexplored, despite its
importance in processes of degeneration and regeneration of the nervous
system. The importance of the regulatory region of the APOE
gene in the determination of the apoE levels in the brain is emphasized
by the recent identification of a number of polymorphisms within the
promoter (31, 32). Some of these polymorphic sites are associated with
functional changes in the activity of the promoter and with increased
risk of AD (33-35). We have initiated a search of possible regulatory
proteins that bind to proximal regions of the APOE gene
promoter. Previously, we identified the transcription factor AP-2 as a
mediator of the cAMP stimulation of apoE synthesis in glial cells (36).
In the present report, we analyze the regulatory region that lies
between Reporter Constructs for Library Screen--
The following
oligonucleotides
TCGGGCTCTATGCCCCACCTCCTTCCTCCCTCTGCCCTGCTGTGC and
CCGAGGCACAGCAGGGCAGAGGGAGGAAGGAGGTGGGGCATAGAGC, containing the described previously URE1 region (24) of the APOE promoter were synthesized and annealed. The annealed
oligonucleotide displayed overhanging ends (underlined) to promote
oriented oligomerization upon ligation in the plasmid pAEA (38). A
fragment containing five tandem repeats was subcloned into the yeast
reporter plasmids, pHISi-1 and pLacZi (CLONTECH,
Palo Alto, CA), yielding plasmids APOE1-pHIS-1 and APOE1-pLacZi,
respectively. The reporter constructs were subsequently linearized and
sequentially integrated into the yeast strain YM4271. Yeast were
transformed first with APOE1-pLacZi, followed by APOE1-pHIS-1.
Transformants selected on leu Screening of the cDNA Library--
The host yeast strain was
transformed with a MATCHMAKER human brain cDNA library constructed
in the pGAD10 vector (CLONTECH) by the
LiAc/polyethylene glycol method. Approximately 5 × 104 transformants were plated per 150-mm dish containing
his Purification of Recombinant His-Zic1 and His-Zic2--
To
produce histidine-tagged Zic1 and Zic2 proteins (His-Zic1 and
His-Zic2), we prepared bacterial expression constructs for both
proteins. First, the Zic-encoding cDNA inserts from the pGAD10 cloning vector, isolated after the one-hybrid procedure, were transferred into the BamHI/BglII sites of the
prokaryotic expression vector pTrcHisA (Invitrogen Inc, San Diego, CA).
Then, Escherichia coli BL21 strain was transformed with the
expression constructs and grown in LB culture medium containing 50 µg/ml ampicillin and 70 µg/ml chloramphenicol. Expression was
induced by addition of 1 mM isopropyl- Electrophoretic Mobility Shift Assay--
Oligonucleotides were
5'-end-labeled with [ Plasmid Constructions--
The luciferase reporter plasmid pXP2
(39) was used to harbor different fragments of the APOE
promoter. The fragments were generated by PCR using oligonucleotides
from the desired regions as primers and the APOE-pCRII construct (36)
as a template. Amplified fragments were ligated to the pCRII vector
(Invitrogen, San Diego, CA), and the identity was confirmed by
sequencing. Fragments were subcloned in the MCS of pXP2, in front of
the luciferase reporter gene. Mutation were introduced by PCR by using
mutant oligonucleotides as described (40). Mouse Zic1 and Zic2
expression vectors were prepared by cloning the full-length genes into
the pEBOS vector as
described.2
Cell Culture and Transfections--
U87 cells were grown in
Dulbecco's modified Eagle's medium containing 10% fetal bovine
serum. The day before transfection, confluent cells were subcultured by
trypsinization and 1-3 × 104 cells/well were plated
in 24-well tissue culture plates. For transient transfections, cells
were transfected with 0.5 µg of DNA/well by the calcium phosphate
method using the CalPhosTM mammalian transfection kit
(CLONTECH) according to the instructions of the manufacturer.
For stable transfection U87 cells were transfected by electroporation
with Zic cDNAs cloned into the expression vector pcDNA3 that
carries the neomycin resistance gene for selection. After selection
with 0.2 g/liter G418 for 12-15 days, single colonies were isolated
with cloning cylinders. Clones stably transfected were maintained in
Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf
serum, 2 mM L-glutamine, and 0.2 g/liter G418.
Luciferase and Analysis of RNA Expression--
Total RNA was extracted from
U87, Zic1-U87, Zic2-U87, SW1088, or HepG2 cell lines as described (42).
Expression of mRNAs for apoE, Zic1, Zic2, and GADPH was analyzed by
RT-PCR by using the GeneAmp RNA PCR kit (Perkin-Elmer) as recommended
by the manufacturer. Oligo(dT)16 was used for the first
strand cDNA syntheses. The following oligonucleotides were
used in the PCR amplification steps: APOE F, GCATGCTCCTGGACGCCGGAC;
APOE R, GGAGCCTGCGACGTGAAGGCTGT; Zic1 F, GCATGCTCCTGGACGCCGGACC; Zic1
R, GGAGCCTGCGACGTGAAGGCTGT; Zic2 F, GCATGCTTCTGGACGCGGGGCC; Zic2 R,
CTCTGACCAGGAGACAGTTCGT; GADPH F, CCACCCATGGCAAATTCCATGGCA; GADPH R,
TCTAGACGGCAGGTCAGGTCCACC. The resultant cDNA fragments were
resolved by electrophoresis on a 2% agarose gel and visualized under
UV illumination.
Analysis of Protein Expression--
Cells grown to confluence
were lysed in 50 mM Tris-HCl, pH 7.5, 150 mM
NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride. The protein concentrations were determined with the Bio-Rad protein assay reagent, and SDS-PAGE was performed in the presence of
2-mercaptoethanol. Samples were transferred by electroblotting onto a
nitrocellulose membrane in a semidry electroblotting system (Life
Technologies, Inc.). Nonspecific protein binding to the blot was
blocked by incubation of the filter with 3% nonfat milk in
phosphate-buffered saline (137 mM NaCl, 2.7 mM
KCl, 4.3 mM
Na2HPO4·7H2O, 1.4 mM
KH2PO4, pH 7.3) containing 0.1% Triton X-100.
The blot was probed with anti-apoE antibody (Calbiochem, Bad Soden,
Germany) diluted 1:5000 for 1 h at room temperature. After
washing, the blot was probed with a peroxidase-linked anti-goat IgG.
Bands were visualized with the ECL detection method (Amersham Pharmacia Biotech).
Isolation of cDNA Clones Encoding APOE Promoter-binding
Proteins--
We are interested in identifying transcription factors
that could regulate the expression of the APOE gene in the
nervous system. The yeast one-hybrid system was used to screen for
human brain cDNAs encoding for proteins that bind to sequences of
the APOE promoter located upstream of the TATA box, a region
of the promoter that has been previously shown to contain a number of functional promoter elements (23, 24, 36, 37). Five tandem copies of
the sequence spanning The Bait Oligonucleotide Forms Specific Complexes with Recombinant
Zic Proteins--
To confirm the presence of a Zic-binding site in the
APOE gene promoter, we first produced His-tagged Zic1 and
Zic2 proteins by transferring the Zic-encoding inserts from the yeast
vector pGAD10 to the prokaryotic expression vector pTrcHisA. The
recombinant proteins were then purified with nickel-containing resins.
The binding activity of the recombinant proteins was assayed by EMSA with the 32P-labeled, double-stranded bait oligonucleotide
(bp Nucleotide Sequences Required for Zic Binding--
To determine
more precisely the nucleotide sequence required for binding of Zic
proteins, a number of double-stranded 20-mers included within the
displacing oligonucleotides were synthesized and assayed in EMSA
experiments. DNA-protein complexes were only observed when we used the
DNA fragments labeled with an asterisk in Fig. 2, and these
were selected for further mutational analyses. Various substitution
mutations were introduced within these oligonucleotides, and then used
in EMSA experiments. Fig. 3 shows results
of mutagenesis in DNA fragment Transcriptional Activation of the APOE Promoter by Zic
Proteins--
To analyze the ability of Zic proteins to stimulate
transcription from the APOE promoter, and to assess the
relative contribution of each binding site to the measured activities,
several APOE promoter-luciferase constructs were prepared
and cotransfected with an expression vector containing the full-length
coding region of the mouse Zic1 or Zic2 genes into the glioblastoma
cell line U87. Along with the test constructions, each plate was
cotransfected with a Zic Proteins Also Stimulate the Endogenous APOE Gene
Expression--
We also investigated the effect of Zic proteins on the
expression of the endogenous APOE gene in the glioblastoma
cell line U87. First we generated stably transfected U87 cell lines
that constitutively expressed either Zic1 or Zic2. The expression of mRNA for Zic1 or Zic2 was analyzed by RT-PCR (Fig.
8A, upper panel). Only stably
transfected U87 cells (Zic-1U87 and Zic2-U87 cell lines) expressed the
corresponding Zic mRNA. The expression of apoE mRNA was also
assayed by RT-PCR in mock-, Zic1-, and Zic2-transfected U87 cells and
compared with that of two other cell lines, the glioblastoma SW1088 and
the hepatoma HepG2. ApoE mRNA was detected in all cell lines except
in the parental mock-transfected U87 (Fig. 8A, middle
panel). These observations were paralleled by protein
expression profile obtained by Western blot analysis with a specific
anti-apoE antibody (Fig. 8B). ApoE appeared in blots as a
band with the same electrophoretic mobility as the recombinant apoE (34 kDa) (Fig. 8B, rApoE). Whereas the expression in
mock-transfected cells was undetectable, the expression in the Zic1-U87
and Zic2-U87 cell lines was greater (Fig. 8, Zic1-U87) or
similar (Fig. 8, Zic2-U87) to the endogenous expression
levels observed in the glioblastoma SW1088, and slightly lower than
that measured in HepG2 cells.
In the present report, we identified transcription factors Zic1
and Zic2 as potent transcriptional activators of the human APOE gene. This regulatory activity is mediated by three
Zic-binding sites located in the proximal region of the APOE
promoter. These sites were identified by screening a human brain
library, by the yeast one-hybrid technique, by band mobility shift
experiments, and by functional assay of luciferase reporter gene
activity in various constructs of the APOE promoter. The
Zic-mediated stimulation not only was observed on artificial
APOE promoter constructs, but also on the endogenous gene in
a human glioblastoma cell line. The determination of the molecular
mechanisms involved in the regulation of apoE synthesis in brain is a
matter of importance, as this protein seems to be involved in processes
of brain repair after traumatic injury or in the pathogenesis of AD
(13, 14, 42).
Zic1 and Zic2 belong to the Cys2-His2 family of
zinc finger transcription factors and are the orthologues of the
Drosophila gene odd-paired (44). They seem to be
important genes in the embryonic development. The expression of both
proteins is observed in the gastrula stage. Later, the expression
becomes restricted to the neural tube, where Zic2 seems to play an
essential role in neurulation (45). Indeed, mutant mice lacking this
gene show incomplete closure of the neural tube, producing
holoprosencephaly and spina bifida as well as alterations in digits and
vertebrae (46). In the human, mutations in ZIC2 gene are
associated with holoprosencephaly (47). Later, the expression in adults
is mainly observed in the cerebellum, although Zic1 is observed in
other areas of the brain, and Zic2 has also been reported in testis (48). Zic proteins are also detected in a number of tumor cells (48,
49). Previous studies have shown that Zic1 is able to bind to the
consensus sequence of the closely related Gli proteins (50). The
present report indicates that the sequence for Zic binding is rather
ambiguous, although there are changes of a few nucleotides that are
deleterious for the binding. The triplet CTG, followed by a GC-rich
region occurs in the three Zic-binding sites of the APOE
promoter. Both proteins bind similarly to these sites, although, in
functional studies, Zic2 behaved as a more potent activator of the
promoter than Zic1. Despite the similarity for the target sequence for
both proteins that would suggest a functional redundancy, the phenotype
of the Zic1 mutant mice differs markedly from that of Zic2 mutant (45,
51). Further clarification of the target genes would be necessary.
The region of the promoter used in this study had been previously
identified as the binding site for a number of proteins in several cell
types, although never in nervous cells. For instance, the region from
We do not know whether Zic proteins will play a functional role in the
expression of apoE in the mature brain. In the adult, both proteins do
not share the expression pattern. However, the situation could be
different in the developing embryo. ApoE is highly expressed in several
developing organs, including brain, eye, or bone (52). ApoE is
expressed during osteogenic and chondrogenic differentiation of murine
mesenchymal progenitor cells, where it seems to be induced by bone
morphogenetic protein-2 (BMP2) (52). Interestingly, a BMP2/4-mediated
regulation has been suggested for Zic2 in the differentiating bone of
the limb buds (45), and thus, the inductive action of BMP2 on the
APOE gene could be mediated by Zic2.
Another relevant relationship between Zic2 and APOE gene
could be found in holoprosencephaly. It is known that some alterations in the metabolism of cholesterol such as inhibition of the
7-dehydrocholesterol reductase (53, 54), or alterations in the apoE
receptor megalin (55) result in holoprosencephaly. Although it is
thought that some of these alteration in the metabolism of cholesterol
result in alterations in the postranscriptional processing of
sonic hedgehog and consequently in holoprosencephaly (56),
we suggest that alterations in apoE could also play a role in those
cases of holoprosencephaly that are mediated by mutations in the Zic2 gene.
ApoE plays a crucial role in a number of physiological processes,
including cholesterol transport in peripheral circulation (57) and
central nervous system (43). ApoE is also involved in the response to
neural injury (43, 58, 59), maintenance of dendritic arborizations
(60), and neuronal remodeling in vitro (61, 62) and in AD
(63). The recent association of different polymorphisms in the promoter
region with AD (32-35) strongly suggests that transcriptional
regulation of APOE gene may play an important role in the
development of this deleterious disease. Thus, identification and
characterization of the transcriptional machinery involved in the
regulation of ApoE expression may be relevant to devise a possible
pharmacological modulation of apoE levels. For instance, recent
observations suggest that the protective effect of estrogens, which
transcriptionally regulate the expression of apoE (64, 65), on AD (66)
could be mediated in part by apoE (67). Here we contribute to the
characterization of the APOE transcriptional machinery by
identifying two members of the Zic family as strong transcriptional
activators of this gene.
163 and
124, that has been previously defined as a
functional promoter element, was used as a bait to screen a human brain
cDNA library. Ten cDNA clones that encoded portions of the
human Zic1 (five clones) and Zic2 (five clones) transcription factors
were isolated. Electrophoretic mobility shift assays confirmed the
presence of a binding site for Zic1 and Zic2 in the
136/
125 region.
Displacement of binding with oligonucleotides derived from adjacent
sequences within the APOE promoter revealed the existence
of two additional Zic-binding sequences in this promoter. These
sequences were identified by electrophoretic mobility shift assays and
mutational analysis in regions
65/
54 and
185/
174.
Cotransfection of Zic1 and Zic2 expression vector and different
APOE promoter-luciferase reporter constructs in U87
glioblastoma cell line showed that the three binding sites partially
contributed to the trans-stimulation of the luciferase reporter.
Ectopic expression of Zic1 and Zic2 in U87 cells also trans-stimulated
the expression of the endogenous gene, increasing the amount of
apolipoprotein E produced by glial cells. These data indicate that Zic
proteins might contribute to the transcriptional activity of the
apolipoprotein E gene and suggest that apolipoprotein E could mediate
some of the developmental processes in which Zic proteins are involved.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
163 and
124, a region that contains positive regulatory
elements in HELA, HepG2, COS, and U87 cells (23, 24, 36, 37). We
identify transcription factors Zic1 and Zic2 as positive
transcriptional regulatory elements for the APOE gene.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, ura
minimal
medium were used as a dual reporter host yeast strain for the library screen.
leu
minimal selective medium
supplemented with 15 mM 3-aminotriazole. Approximately
2.5 × 106 cDNA plasmids were screened. Based on
large colony size and rapid growth, a total of 19 positive clones were
selected. These clones were tested for
-galactosidase activities
using the filter replica method. Ten clones showed strong blue color
compared with the host strain. Plasmids were recovered from
His+/LacZ+ colonies by transformation into
DH5
cells. The plasmids were partially sequenced and the nucleotide
sequences were compared with sequences in the GenBankTM/EMBL data
bases using the Fasta program.
-thiogalactoside
for 5 h. Bacteria were collected and lysed by sonication in 50 mM NaH2PO4, 10 mM
imidazole, 1 mM phenylmethylsulfonyl fluoride, 0.5 mg/ml
lysozime. Fusion proteins were purified with Ni-NTA resins (Qiagen Inc,
Valencia, CA) using a batch protocol as recommended by the manufacturer.
-32P]ATP using T4 polynucleotide
kinase. Recombinant transcription factors (0.5-1 µg) were incubated
for 15 min at room temperature in 20 µl of binding buffer 5 mM (Tris-HCl, pH 7.6, 100 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 10 mM ZnCl2, 1 mM dithiothreitol,
supplemented with 2 µg/assay poly(dI-dC)·poly(dI-dC)). Where
indicated, competitor oligonucleotides were included during the
preincubation period. Labeled oligonucleotide (1 ng/binding reaction;
100,000-200,000 cpm) was then added, and the mixture was incubated for
30 min at room temperature. The incubation mixture was electrophoresed on 4% polyacrylamide gels containing 0.5 × Tris borate-EDTA
buffer at constant voltage (100 V) for 3-4 h. Gels were dried and autoradiographed.
-Galactosidase--
Cells were harvested on
day 2 following transfection with 150 µl of a lysis buffer containing
25 mM Tris-phosphate, 2 mM dithiothreitol, 2 mM EDTA, 10% glycerol, 1% Triton X-100. Unsolubilized
material was removed by 2 min of centrifugation, and the luciferase and
-galactosidase activities of the extracts were determined.
Luciferase was measured using the Luciferase Assay System (Promega,
Madison, WI) in a Monolight 2010 luminometer (Analytical Luminescence
Laboratory) by incubation of 40 µl of cell extract with 90 µl of
luciferase assay reagent as recommended by the manufacturer.
-Galactosidase was determined in a 96-well microtiter plate by
incubating 20 µl of cellular extract with 20 µl of a solution
containing 3 mg/ml o-nitrophenyl-
-D-galactopyranoside as
described (41). Absorbance at 405 nm was determined in a microplate reader.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
163 to
124 were cloned upstream of a minimal
GAL4 promoter either in pHIS1 or in pLacZi reporter plasmids and
integrated into the yeast genome of YM4271 (his3, leu2). A
hybrid expression library (MATCHMAKER) consisting of human brain
cDNAs fused to the GAL4 activation domain was then screened to
identify proteins that bound to the APOE promoter and
activated HIS3 transcription from the reporter construct. Transformants
growing on selective medium were assayed for
-galactosidase expression. After screening 2.5 million independent colonies, 10 transformants produced strong blue color on filter assay after 1 h
of incubation in the presence of
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside. Plasmids of these transformants were then recovered and sequenced. Sequence analysis indicated that five clones corresponded to different portions of the transcription factor Zic1 and five to the closely related Zic2. The longest Zic1 clone lacked regions encoding the 72 amino acids of the amino terminus as well as 10 amino acids in
the carboxyl end of the open reading frame of the human gene. The
longest Zic2 clone spans from amino acid 182 to 530. Both clones
contained the respective zinc finger domains and were in frame with the
activation domain of GAL4.
163 to
124). We detected the formation of stable complexes of
this DNA fragment with both His-Zic1 and His-Zic2, but not with control
bacterial extracts (Fig. 1). The binding
of His-Zic1 was specifically competed by increasing amounts of the
unlabeled bait oligonucleotide (
163/
124) (Fig.
2, lanes 5-7). Interestingly,
the binding was also competed by double-stranded oligonucleotides
derived from adjacent regions of the APOE promoter spanning
bp
70 to
40 and bp
188 to
169 (Fig. 2, lanes 2-4
and 8-10), suggesting the existence of additional Zic1
binding elements in the promoter. The binding was not displaced by an
excess of unlabeled oligonucleotides
39/+1 and
113/
80 (data not
shown) or by an unrelated oligonucleotide (Fig. 2, lane 11).
Identical results were obtained by using His-Zic2 (data not shown). We
did not detect displacement by a number of oligonucleotides derived
from other sequences of the proximal region of the APOE gene
promoter (data not shown).
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Fig. 1.
Binding of recombinant His-Zic1 and His-Zic2
to the bait oligonucleotide. Gel mobility shift assays were
performed with 1 ng of 32P-labeled 163/
124 DNA fragment
incubated with 500 ng of bacterial control extract (Control
Extract), purified His-tagged Zic1 (His-Zic1), or
purified His-tagged Zic2 (His-Zic2).
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Fig. 2.
Displacement of His-Zic1 binding to the bait
oligonucleotide. A, electrophoretic mobility shift
assays were performed with 1 ng of 32P-labeled-163/ 124
DNA fragment incubated with 500 ng of His-Zic1 in the absence
(lane 1) or presence of an excess of the indicated unlabeled
oligonucleotides (lanes 2-11). Competitors were added
10-fold (lanes 2, 5, and 8), 100-fold
(lanes 3, 6, and 9), or 1000-fold
(lanes 4, 7, 10, and 11)
excess in the EMSA. B, diagram of the proximal
APOE promoter showing the location of some putative
regulatory elements. TATA, the TATA box element;
SP1, the GC box element; AP-2, AP2 binding sites;
BEF1, BK virus enhancer factor-1 (27); URE1 and
URE3, upstream response elements 1 and 3 (24, 28);
ERE, estrogen response element. The regions covered by DNA
fragments used in the displacement analysis are also shown. Fragments
labeled with an asterisk formed stable complexes with
His-Zic1 and His-Zic2 in EMSA.
143/
124. Mutation in the 5' end of
this fragment produced a marked decrease in His-Zic1 binding with
respect to control, although the retarded DNA-protein complex was
clearly detectable (Fig. 3, lane 2). However, when bases
from
136 to
125 were exchanged in groups of three for adenine,
binding of His-Zic1 was severely impaired and the retarded complexes
were undetectable (lanes 3-6). Similar results were
obtained when His-Zic2 was used in the assays (data not shown). These
experiments define the binding site for Zic proteins within the initial
bait. Binding sequence was also investigated within the DNA fragment
spanning
69 to
50. As shown in Fig.
4, binding of both His-Zic1 and His-Zic2 was disrupted by mutations in bases located between
65 to
54 (lanes 3-6 and 10-13). Mutations in bases
69
to
66 and
50 to
53 had a weaker effect on binding of both
proteins (lanes 2, 7, 9, and
14) as compared with the respective controls (lanes 1 and 8). Finally, another site was defined for both
recombinant proteins within the DNA fragment
188/
169. Mutations in
positions between
185 and
174 completely abolished the formation of
retarded complexes (Fig. 5, lanes
3-6 and 10-13), whereas mutations in bases
173 to
171 produced a weaker reduction of binding (Fig. 5,
lanes 7 and 14). In summary, the three binding sites
described above were termed Zic-BS1 (
65/
54), Zic-BS2 (
136/
125),
and Zic-BS3 (
185/
174), according to their positions with respect to
the transcriptional starting point.
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Fig. 3.
Mutational analysis of 143/
124 region of
the APOE promoter. A, diagram of the
mutations introduced into the
143/
124 double-stranded
oligonucleotide. B, electrophoretic mobility shift assays
were performed with 1 ng of the indicated 32P-labeled DNA
fragments incubated with 500 ng of recombinant His-Zic1.
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Fig. 4.
Mutational analysis of 69/
50 region of
the APOE promoter. A, diagram of the
mutations introduced into the
69/
50 double-stranded
oligonucleotide. B, electrophoretic mobility shift assays
were performed with 1 ng of the indicated 32P-labeled DNA
fragments incubated with 500 ng of recombinant His-Zic1 or
His-Zic2.
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Fig. 5.
Mutational analysis of 188/
169 region of
the APOE promoter. A, diagram of the
mutations introduced into the
188/
169 double-stranded
oligonucleotide. B, electrophoretic mobility shift assays
were performed with 1 ng of the indicated 32P-labeled DNA
fragments incubated with 500 ng of recombinant His-Zic1 or
His-Zic2.
-galactosidase expression vector that served as
an internal reference for transfection efficiency. Luciferase and
galactosidase activities were determined 48 h later. The
luciferase activity was strongly stimulated by both Zic1 and Zic2 in a
construct containing the Zic-BS1 (
70/+1) as compared with controls (6 and 27.9 times, respectively) (Fig. 6).
The stimulatory effect was greatly reduced after mutation of this site
(construct
70/+1 m(
62/
57)), although not completely
abolished (the activity for Zic1 and Zic2 was 4 and 8 times over
controls, respectively) (Fig. 6). However, the promoter activity was
more severely impaired in the deletion mutant
60/+1
(
60/+1 in Fig. 6) (1.4 and 2.7 over controls,
respectively), suggesting that the mutant
70/+1
m(
62/
57) still conserved some binding capability. Both
transcription factors also stimulated the luciferase activity in a
longer construct including also Zic-BS2 (bp
143 to
1) (Fig. 6,
construct
143/+1), although to a lower extent than
construct
70/+1 (4.9 and 19.9 times over controls, respectively).
Mutation in the Zic-BS2 decreased the simulation mediated by Zic1 and
Zic2 by more than one half (2.3 and 6.5 times stimulation over controls
respectively) (Fig. 6, construct
143/+1 m(
133/
129)).
The contribution of the Zic-BS3 was analyzed in construct
189/+1.
Both factors stimulated the luciferase activity by 5.2 and 12.6 times,
respectively, in the wild form of this construct (Fig.
7, construct
189/+1).
Mutations in Zic-BS3 decreased drastically the activity of the promoter
(to 2.3 and 2.5 times over controls, respectively) (Fig. 7, construct
189/+1 m(
182/
177)), indicating that in the context of
this construct the Zic-BS3 seems to be preponderant over Zic-BS2 and
Zic-BS1. Nevertheless, these sites also contributed to the observed
activity as the stimulation of the promoter activity was between 60 and
75% lower in mutants of Zic-BS2 or Zic-BS1 in the context of construct
189/+1 (Fig. 7, constructs
189/+1 m(
133/
130) and
189/+1 m(
55/
50)), when compared with the stimulation
observed in the wild type version of this construct (construct
189/+1). Moreover, the Zic1- and Zic2-mediated stimulation
of the promoter activity was completely abolished in a mutant in which
the three binding sites were eliminated (Fig. 7, construct
189/+1 3 m).
View larger version (16K):
[in a new window]
Fig. 6.
Promoter activity of Zic-BS1 and
Zic-BS2. U87 cells were transiently transfected with the indicated
apoE-luciferase constructs, a -galactosidase expression vector, and
the empty pEBOS vector (
), or pEBOS-Zic1 (
), or pEBOS-Zic2 (
).
Promoter regions are represented as boxes. Mutations in the
Zic-binding sites are represented as discontinuities in
boxes. Luciferase activities were determined after 48 h
and normalized for transfection efficiency as measured by
-galactosidase. For each construct the results are relative to
values measured in pEBOS-transfected cells. Values represent means ± S.E. of at least two triplicate determinations.
View larger version (17K):
[in a new window]
Fig. 7.
Promoter activity of Zic-BS3. U87 cells
were transiently transfected with the indicated apoE-luciferase
constructs, a -galactosidase expression vector, and the empty pEBOS
vector (
), or pEBOS-Zic1 (
), or pEBOS-Zic2 (
). Promoter
regions are represented as boxes. Mutations in the
Zic-binding sites are represented as discontinuities in
boxes. Luciferase activities were determined after 48 h
and normalized for transfection efficiency as measured by
-galactosidase. For each construct the results are relative to
values measured in pEBOS-transfected cells. Values represent means ± S.E. of at least two triplicate determinations.
View larger version (42K):
[in a new window]
Fig. 8.
Induction of endogenous APOE
gene by Zic1 and Zic2. A, total cellular RNA was
isolated from the indicated cell lines and the expression of Zic1,
Zic2, (upper panel), apoE (middle panel), or
human GADPH (lower panel) was determined by RT-PCR. Zic1 and
Zic2 mRNAs were determined simultaneously. B, proteins
in cellular extracts from the indicated cell lines were separated by
PAGE and blotted onto nitrocellulose membranes. The expression of apoE
was determined with a specific antibody. ApoEr corresponds
to 50 ng of human recombinant apoE electrophoresed in parallel (34 kDa).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
161 to
141, included in our original bait and upstream to the
Zic-BS2 (bp
136 to
125), was protected in DNase I footprinting
experiments by nuclear extracts of HepG-2, HeLa, and Chinese hamster
ovary cells, defining the regulatory element URE1 (24). This sequence
was able to bind two transcription factors in a competitive fashion,
one of which was identified as Sp1, the other remaining unidentified
(37). Additionally, the Zic-BS1 is flanked by a Sp1-binding site and
the Zic-BS3 overlaps with a third Sp1-binding site (37). In extracts of
HeLa and HepG2 cells, the protein BEF-1 has been identified as a
negative regulator that binds to the
94/
84 sequence (27), and an
unidentified protein was reported to bind to URE3 between
89 and
101 (28). Finally, a binding site for AP2 overlaps with Zic-BS1 and
is responsible for the cAMP-mediated induction of apoE in glial cells
(36). These results indicate that this region proximal to the
APOE gene promoter must play a critical role in controlling
the expression of apoE. Whether Zic proteins interact with some of
these factors remains to be determined.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. G. J. Liaw for generously providing pAEA plasmid. We are grateful to E. Nuñez for expert technical assistance.
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FOOTNOTES |
---|
* This work was supported by Spanish Dirección General de Enseñanza Superior e Investigación Científica Grant PM98-0006 and an institutional grant from the Fundación Ramón Areces.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.
¶ To whom correspondence should be addressed. Tel.: 34-913978720; Fax: 34-913974799; E-mail: fzafra@cbm.uam.es.
Published, JBC Papers in Press, October 18, 2000, DOI 10.1074/jbc.M007008200
2 K. Mizugishi, J. Aruga, K. Nakata, and K. Mikoshiba, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are: apoE, apolipoprotein E; APOE, human apolipoprotein E gene; EMSA, electrophoretic mobility shift assay; AD, Alzheimer's disease; Zic-BS, Zic-binding site; PAGE, polyacrylamide gel electrophoresis; GADPH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction; RT, reverse transcription; bp, base pair(s); BMP, bone morphogenetic protein.
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