From the Department of Biochemistry, Faculty of Medicine, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada
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ABSTRACT |
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We have identified a novel transcriptional
repressor, AEBP2, that binds to a regulatory sequence (termed AE-1)
located in the proximal promoter region of the aP2 gene
that encodes the adipose fatty acid-binding protein. Sequence analysis
of AEBP2 cDNA revealed that it encodes a protein containing three
Gli-Krüppel (Cys2-His2)-type zinc
fingers. Northern blot analysis revealed two transcripts (4.5 and 3.5 kilobases) which were ubiquitously expressed in every mouse tissue
examined. In co-transfection assays, AEBP2 repressed transcription from
the homologous aP2 promoter containing multiple copies of
the AE-1 sequence. Moreover, a chimeric construct encoding a fusion
AEBP2 protein with the Gal4 DNA-binding domain was able to repress the
transcriptional activity of a heterologous promoter containing the
Gal4-binding sequence. The transcriptional repression function of AEBP2
was completely abolished when one of the conserved histidine residues
and a flanking serine residue in the middle zinc finger were replaced
with an arginine residue. The defective transcriptional repression
function of the mutant derivative was due neither to lack of expression
nor to a failure to localize to the nucleus. Moreover, both the
wild-type and mutant derivative of either the histidine-tagged
recombinant AEBP2 proteins or the in vitro translated
Gal4-AEBP2 fusion proteins were equally able to bind to the target DNA.
These results suggest that a portion of the zinc finger structure may
play a direct role in transcriptional repression function, but not in
DNA binding.
The adipose P2 (aP2 or
422)1
gene, which encodes the adipose fatty acid-binding protein, is thought
to be an important gene in triglyceride metabolism during adipocyte
differentiation (1-3). The abundance of aP2 mRNA is
greatly enhanced during adipocyte differentiation (4). The AE-1
sequence (nucleotides Molecular Cloning of AEBP2--
To clone a cDNA encoding a
protein that interacts at the AE-1 site (nucleotides Computer Homology Searches and Sequence Analysis--
The
303-residue AEBP2 sequence was compared with the non-redundant
GenBankTM data base using the National Center for
Biotechnology Information program tblastn. Sequences with significant
matches around the zinc finger domain include the human GLI
protein and other related zinc finger transcription factors (smallest
sum probability values less than 10 Plasmid Constructs--
The AEBP2 cDNA was subcloned into
the mammalian expression vector pRc/CMV (Invitrogen) to construct the
AEBP2 expression plasmid pRc/CMVAEBP2. The Gal4-AEBP2 fusion expression
plasmid was constructed by subcloning the AEBP2 open reading frame
(from amino acids 7 to 247) into the Gal4 fusion vector pG4 as
described (9). For this construction, we utilized another AEBP2
cDNA (clone 20) whose 5' end corresponds to nucleotide 104 of the
full-length AEBP2 cDNA. The AEBP2 coding sequence was subcloned as
a SmaI-XmnI fragment into pG4 in both
orientations to construct pG4-AEBP2 and pG4-AEBP2( Northern (RNA) Blot Analysis--
Total RNA from different mouse
tissues was prepared with the RNA STAT-60 Solution (TEL-TEST "B,"
Inc.) according to the manufacturer's protocol. Twenty micrograms of
total RNA from each tissue were loaded on a formaldehyde denaturating
1% agarose gel and blotted onto MSI nylon transfer membrane (Micron
Separations Inc.) as described (11). The filter was hybridized in the
QuikHyb solution (Stratagene) with 32P-labeled AEBP2
cDNA probe in a hybridization oven (Hybaid) for 3 h at
65 °C. The filter was washed twice with 2 × SSC, 0.1% SDS for
15 min at room temperature and twice with 0.2 × SSC, 0.1% SDS
for 30 min at 65 °C before exposure to x-ray film overnight at
Electrophoretic Mobility Shift Assay (EMSA)--
Recombinant
AEBP2 protein was made by utilizing the expression vector pET-16b
(Novagen) as described previously (9). EMSA was carried out by
incubating the recombinant protein or the in vitro produced
Gal4 fusion proteins, which were made by the TNT-coupled wheat germ
extract system (Promega), with 0.25 fmol of 32P-end-labeled
AE-1 sequence in a buffer containing 100 mM KCl, 10 mM Tris-HCl, pH 7.9, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, and
200 µM ZnCl2. After 30 min incubation at room
temperature, the mixture was loaded on a 4% polyacrylamide nondenaturing gel in 6.75 mM Tris-HCl, pH 7.9, 1 mM EDTA, pH 8.0, 3.3 mM sodium acetate, pH 7.9, and 2.5% glycerol, and electrophoresed at 15 V/cm at 4 °C. Both the
gel and the running buffer contained 200 µM
ZnCl2. For the competition assay, 50 fmol of unlabeled oligonucleotides, either specific AE-1 or nonspecific SP1 and AP3, were
added to the reaction prior to the addition of the probe.
Transfection and CAT Assay--
The transient transfection assay
was carried out by the Polybrene procedure (12) as described previously
(9). Transfection of NIH 3T3 cells was performed with 2 µg of the
reporter plasmid paP2(3AE-1/ Isolation and Sequence Analysis of AEBP2 cDNA--
A 3T3-L1
preadipocyte cDNA expression library (Uni-Zap XR vector;
Stratagene) was screened with random concatamers of the AE-1 sequence
by the affinity screening procedure (10) as described previously (9).
Three independent phage plaques (A1, A2, and A8) produced fusion
proteins interacting specifically with the AE-1 sequence. Further
screening of the library with the partial cDNA sequence from A2
resulted in the isolation of a longer cDNA, which we named AEBP2
(adipocyte enhancer-binding protein 2, differing from AEBP1 (9)).
Sequence analysis revealed that this cDNA contains an open reading
frame of 303 amino acid residues from the first ATG codon located at
nucleotide 85 to a termination codon located at nucleotide 994 (Fig.
1A). The polypeptide predicted in the open reading frame from the first ATG codon has a calculated relative molecular mass of 33.3 kDa. Coupled in vitro
transcription and translation of the AEBP2 cDNA produced a protein
with a molecular mass of about 35 kDa (data not shown). Moreover, a
protein with a molecular mass of about 33 kDa was immunoprecipitated
with antibody raised against the recombinant AEBP2 protein (data not
shown). Therefore, based on the molecular weights of the endogenous and in vitro translated products and the Kozak rule for the
translation initiation site (13), the first ATG codon is most likely
the translation start site. The cDNA has a relatively long
3'-untranslated region with a consensus polyadenylation signal (AAUAAA)
and poly(A) tail. A schematic representation of AEBP2 cDNA is shown
in Fig. 1B.
Structural Properties of AEBP2--
The predicted protein contains
three tandemly repeated sequence motifs related to the
Gli-Krüppel (Cys2-His2)-type zinc finger (Fig. 2A). All three zinc
fingers encoded by the cDNA of AEBP2 fit the consensus sequence for
this type of zinc finger (Fig. 2B). Sequence alignment of
the zinc fingers of AEBP2 with those of other zinc finger proteins
containing three zinc finger motifs is shown in Fig. 2C.
Outside the putative zinc finger motif, a single consensus
phosphorylation site ((Ser*/Thr*)-Pro-X-(Lys/Arg)) for Cdc2
kinase, a highly conserved cell cycle regulatory serine/threonine
kinase, exists in the open reading frame (SPSK, Fig. 1). The consensus phosphorylation site is flanked by the zinc finger domain and a region
with high proportion of basic amino acids (KRRKLKNKRRR) that may be a
nuclear localization signal (NLS) (Fig. 1). Phosphorylation at the SPSK
site may affect the function of the NLS and regulate import of the
protein into the nucleus (14).
Tissue Distribution of AEBP2 mRNA--
Northern hybridization
was conducted to determine the expression pattern of AEBP2 in a variety
of mouse tissues. Two transcripts (~4.5 and ~3.5 kilobases) with
different degrees of abundance were detected in all the tissues (Fig.
3). Relatively low levels of AEBP2 RNA
were detected in the liver (lane 3). In contrast to all
other tissues, the brain contained the larger form of AEBP2 RNA more
abundantly (lane 6). In some tissues (e.g.
skeletal muscle, brain, and heart) extra RNA with higher molecular
weights were also detected (lanes 5, 6, and 8).
The significance of such differential expression of AEBP2 mRNA in
different tissues is not understood. AEBP2 transcripts were also
detected in embryonic brain and liver tissues, and in the plancenta
(data not shown). The ubiquitous nature of AEBP2 expression raises the
possibility that AEBP2 may have an important function in mouse
development.
AEBP2, a DNA-binding Protein--
We have reported previously that
the AE-1 site serves as a bifunctional element in the regulation of
aP2 gene expression (5, 8, 9). The AE-1 sequence binds
either the transcriptional activator C/EBP Transcriptional Function of AEBP2--
To examine whether AEBP2 is
able to regulate transcription through interaction with the AE-1 site,
we used a reporter construct in which the bacterial chloramphenicol
acetyltransferase (CAT) gene expression is driven by the aP2
promoter containing an AE-1 site (9), along with an AEBP2 expression
plasmid (pRc/CMVAEBP2). In preliminary transient transfection
experiments, we have not observed any effect of AEBP2 on expression of
the CAT gene driven by the native aP2 promoter (
To further characterize the transcriptional function of AEBP2, we used
a AEBP2 fusion protein with an added DNA-binding specificity of the
yeast Gal4 transcription factor as an effector, and the reporter
plasmid pGALTKCAT, which contains five copies of the Gal4-binding
sequence upstream of the TATA box of the thymidine kinase (TK) promoter
driving the CAT gene expression (9), in the transient transfection
assay. After co-transfection with pGALTKCAT, a very low CAT activity
was measured from the cells transfected with pG4-AEBP2, the Gal4-AEBP2
expression plasmid (Fig. 5B, lane 2), in comparison to the
activity in the control cells transfected with the control plasmid
pG4-AEBP2( In this report, we have characterized a novel transcriptional
repressor (termed AEBP2) containing three copies of the
Gli-Krüppel (Cys2-His2)-type zinc finger
motif, and demonstrated that at least one of the zinc fingers is
important for the repression function. The zinc finger structure, which
was originally identified as DNA binding structure in the RNA
polymerase III transcription factor TFIIIA (15, 16), is one of the well
known common motifs among transcription factors. Zinc finger proteins
can function as either activators or repressors, and most of these
proteins, in addition to having the zinc finger motif whose
transcriptional role is not clearly understood, contain other types of
transcriptional domains. Some well defined activation motifs have been
characterized by the presence of serine/threonine-rich, proline-rich,
glutamine-rich, or acidic amino acid-rich domains (17). For repression,
the alanine-rich domain in the Drosophila protein
Krüppel has been shown to be responsible for its repression
function (18-20). The KRAB domain, an evolutionarily conserved
Krüppel-associated box located in the N-terminal regions of more
than one-third of all Krüppel-class zinc finger proteins, also
has been characterized as a repression motif (21, 22). Other domains
which have been implicated as repression motifs include a 34-amino acid
element in the early growth response factor-1 transcription factor
(23), the SCAN box (the term derived from the first letters of the
names of the four proteins initially found to contain a common motif: SRE-ZBP, CT-fin-51, AW-1,
Number 18 cDNA; Ref. 24), the POZ domain in the
proto-oncogene product BCL-6 (25), a small C-terminal domain comprising
the last 24 amino acids and containing several leucine-proline
dipeptide repeats in the Mig1 protein (26), and a glycine-rich region
in the YY1 transcription factor (27). A repression domain was also
localized within the zinc finger region of YY1 (28). Unlike other zinc
finger transcription factors, AEBP2 does not contain any of the
repression motifs described above. Surprisingly, the repressing
function of AEBP2 was totally abolished, without affecting its DNA
binding ability, when the middle zinc finger was destroyed by mutating
one of the conserved histidine residues. That a zinc finger motif is
critical only for a transcription function has never been reported,
although a functional domain which overlaps the DNA-binding domain has been localized in a zinc finger motif (28). The AEBP2 mutation in the
middle zinc finger motif may alter the overall conformation of the
AEBP2 protein, since the band shift of the Gal4-AEBP2·DNA complex was
different between the wild-type and the mutant derivative (Fig.
5D). This apparent change in the conformation did not affect the DNA binding ability (Fig. 5D), but did severely affect
the repression function of AEBP2 (Fig. 5, A and
B).
Three broad mechanisms of transcriptional repression function are
hypothesized (29). Repressors may directly compete with activators for
binding to a common target DNA, repressors may bind simultaneously with
activators but "quench" their functions, possibly by physically
masking the activation domain, and the repressors may bind DNA and
interact with the general transcription machinery itself, preventing
the attainment of a transcriptionally competent state. For example,
Krüppel-regulated transcription is in large part, through
interactions with TFIIB and TFIIE Previous evidence indicates that members of the
Cys2-His2 zinc finger class of transcription
factors are involved in the transcriptional repression of growth factor
gene expression (24) and regulate cell differentiation, probably
affecting the expression of the differentiation-specific genes (33,
34). At least one positive (C/EBP In summary, the cloning and characterization of the structure,
expression, transcriptional function, and DNA-binding property of the
novel transcription factor AEBP2 have revealed that it is a new zinc
finger transcriptional repressor that is able to bind specifically to
the AE-1 site located in the proximal promoter region of the
aP2 gene, and that the repression function, but not the DNA
binding activity, requires an intact middle zinc finger motif. Two
forms of AEBP2 mRNA are broadly expressed in various mouse tissues.
AEBP2 may serve as a general transcriptional regulator since these
transcripts were detected in both embryonic and adult tissues.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
159 to
125) in the proximal promoter region
of the aP2 gene functions in aP2 gene expression
as either a positive or a negative regulatory element. Mutation at the
AE-1 site affects its ability to bind specific nuclear factors and
diminishes the promoter function of the aP2 gene in
adipocytes (5). At least one protein, C/EBP
, binds to the AE-1
sequence and functions as a transcriptional activator for
aP2 gene expression during adipocyte differentiation (5, 6).
Other AE-1-binding proteins (termed AEBP) in 3T3 preadipocytes have
been implicated as transcriptional repressors in the regulation of
aP2 gene expression (5, 7, 8). To clone a cDNA encoding
a protein that interacts at the AE-1 site, we expressed cDNAs from
a 3T3-L1 preadipocyte cell library with a Uni-Zap XR vector
(Stratagene) and screened for the clones by the Affinity Screening
procedure using random concatamers of the AE-1 sequence. Three
independent phage plaques were isolated that produce fusion proteins
interacting specifically with the AE-1 sequence. Further analysis
revealed that one cDNA clone encodes mRNA whose expression is
down-regulated during adipocyte differentiation. This cDNA and its
encoded protein, AEBP1, has been previously characterized in detail.
AEBP1 is a novel carboxypeptidase, and the carboxypeptidase activity of
AEBP1 is involved in aP2 repression. AEBP1, by binding to
the regulatory AE-1 site, acts as a negative regulator of
aP2 gene expression (9). A new member of the family of
AE-1-binding proteins, being reported here, is a zinc finger protein
which is able to repress reporter gene expression through both
homologous and heterologous promoters. Overexpression of AEBP2 in cells
with a reporter construct, which is driven by the aP2
promoter containing a multiple copies of the AE-1 sequence, resulted in
repression of transcriptional activity. Furthermore, an AEBP2 fusion
protein with the DNA-binding domain of the yeast transcriptional
activator Gal4 was able to repress the expression of a reporter gene
driven by a heterologous promoter with five copies of the Gal4-binding
sequence. Significantly, the repression function of AEBP2 was
completely abolished, without affecting its DNA binding ability and
expression level, when one of the conserved histidine residues in the
middle zinc finger motif was mutated. These results indicate that the
middle zinc finger motif of AEBP2 is critical for the transcriptional
function, and suggest that some of the zinc finger motif may not be
involved in DNA binding.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
159 to
125 of
the aP2 gene, Refs. 8 and 9) in 3T3-L1 preadipocytes,
cDNAs from a 3T3-L1 preadipocytes library were expressed with a
Uni-Zap XR vector (Stratagene) and screened for an expression clone by
the affinity screening procedure (10) with random concatamers of the
AE-1 sequence as described previously (9).
10) with
GenBankTM accession numbers X07385, U60762, U57454,
AF026305, U42462, D14827, AB007297, AB007295, AB007298, AB007296, D14828, and U42461.
), respectively.
70 °C.
120)CAT and 5 µg of the AEBP2
expression plasmid pRc/CMVAEBP2. The transfection was also performed
with 5 µg each of the reporter plasmids pGALTKCAT or pTKCAT and the
fusion Gal-AEBP2 expression plasmid pG4-AEBP2 or the control plasmid
pG4-AEBP2(
). All transfections also included 1 µg of pHermes-lacZ.
-Galactosidase activity was assayed 48 h after transfection to
normalize for transfection efficiency, and CAT activity was assayed as
described (9).
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 1.
Primary structure of mouse AEBP2.
A, nucleotide and predicted amino acid sequence of mouse
AEBP2. A polyadenylation signal (aataaa) in the 3'-untranslated region
is underlined. Conserved histidine and cysteine residues in
the zinc finger motif are also underlined. The NLS
consisting of amino acids from 171 to 181 is also
underlined. A Cdc2 kinase phosphorylation site
(SPSK) located at amino acids 163 to 166 is in
italics and underlined. A restriction enzyme
DraIII site at nucleotides 422-430, which was utilized in
the construction of the mutant derivative of AEBP2 (see text), is
underlined. A restriction enzyme XmnI site at
nucleotides 820-829, which was utilized in the construction of the
Gal4-AEBP2 fusion expression plasmid (see "Experimental
Procedures"), is also underlined. B, schematic
representation of the AEBP2 open reading frame and cDNA. The
location of the three zinc fingers is indicated by diagonally
striped boxes. The solid box represents the putative
NLS. The region indicated by a vertical line adjacent to the
NLS is a putative Cdc2 kinase phosphorylation site (SPSK). The
thin lines depict untranslated regions of the
cDNA.
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Fig. 2.
Zinc finger structure and amino acid
alignment of zinc fingers of AEBP2 with those of other zinc finger
proteins. A, a schematic representation of AEBP2 zinc
fingers. B, alignment with the consensus sequence of the
Gli-Krüppel type zinc finger and TFIIIA. C, alignment
of the zinc fingers of AEBP2 with other proteins containing three zinc
fingers. Conserved amino acids in the sequences of zinc fingers are in
bold.
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Fig. 3.
Expression pattern of the mouse AEBP2
gene. The top panel is a Northern blot containing 20 µg of total RNA/lane from 10 different mouse tissues was hybridized
with an EcoRI fragment of AEBP2 cDNA that contains the
complete coding sequence. Lanes 1-10 contain total RNA from
white adipose tissue (W), brown adipose tissue
(B), liver (L), kidney (K), skeletal
muscle (M), brain (Br), small intestine
(G), heart (H), lung (Lu), and spleen
(S), respectively. The lower panel shows the
ethidium bromide-stained 28 S and 18 S RNAs in each lane, used as
loading controls.
(5) or the
transcriptional repressor AEBP1 (9). AEBP2, as another AE-1-binding
protein, may also participate in this regulation. Histidine-tagged
recombinant AEBP2 protein was used to characterize the binding
properties of AEBP2 as described previously (9). Here we show that
AEBP2 binds to the AE-1 oligonucleotide in a sequence-specific manner
(Fig. 4). The AEBP2·AE-1 complex can be
competed away by adding an excess amount of the unlabeled AE-1
oligonucleotides (Fig. 4, lane 3), but not by SP1
(lane 4) or by AP3 (lane 5) binding sequences.
The binding reaction needs Zn2+, for very weak or no
binding was observed in the absence of Zn2+ (data not
shown). Interestingly, even without an intact second zinc finger motif,
as indicated by mutating one of the conserved histidine residues (AEBP2
cDNA was digested with DraIII at nucleotides 422 to 430 and the newly created ends were blunted by exonuclease activity and
ligated, replacing the histidine 114 and serine 115 residues with an
arginine residue), the binding activity remained intact (data not
shown). These results suggest that the second zinc finger motif is
dispensable for DNA binding, and that the DNA binding activity may be
localized in the other two fingers.
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Fig. 4.
AEBP2 is a DNA-binding protein.
Bacterially synthesized histidine-tagged recombinant AEBP2 protein
binds specifically to the AE-1 sequence. Recombinant AEBP2 protein (500 ng) was incubated with 32P-labeled AE-1 sequence in the
absence (lane 2) or presence of ~200-fold molar excess of
either unlabeled AE-1 (lane 3) or the nonspecific
oligonucleotides SP1 (lane 4) or AP3 (lane 5).
The AEBP2·AE-1 complex and free probe are indicated.
168 to
+21), which contains the AE-1 site at nucleotides
159 to
125. This
lack of effect may be due to a positive element (AP1) at nucleotide
125 to
118 (5). Since transcriptional activity can be influenced by
increasing the extent of binding site of a transcription factor, we
have analyzed the transcription function of AEBP2 with a reporter
containing a multiple copies of the AE-1 sequence. Transient
transfection analysis with the CAT reporter plasmid
paP2(3AE-1/
120)CAT, in which the CAT gene is driven by the
aP2 promoter (
120 to +21) with three copies of the AE-1
sequence inserted upstream of the promoter, demonstrated that AEBP2 has
repression activity. The CAT activity from cells co-transfected with
pRc/CMVAEBP2 was significantly decreased compared with the CAT activity
in cells co-transfected with the control plasmid pRc/CMV (Fig.
5A). From the in
vitro binding assay, we concluded that the middle zinc finger is
dispensable for binding to the AE-1 sequence. We therefore asked
whether the mutation in the middle zinc finger has any affect on the
repression function of AEBP2. We constructed an expression plasmid that
expresses the mutant derivative of AEBP2, and analyzed the
transcriptional repression function using the reporter plasmid
paP2(3AE-1/
120)CAT. As shown in Fig. 5A, the repression
activity of this mutant version of AEBP2 was significantly diminished,
suggesting that the middle zinc finger may be critical for the
repression function.
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Fig. 5.
AEBP2 is a transcriptional repressor.
NIH 3T3 cells were plated in 60-mm dishes and transfected with the
indicated plasmids by the Polybrene method (12). A, CAT
activity from cells transfected with the reporter plasmid
paP2(3AE1/ 120)CAT, which contains three copies of the AE-1 sequence
upstream of the aP2 promoter (
120 to +21). In each
transfection 5 µg of effector plasmid (pRC/CMV, pRC/CMVAEBP2 or the
mutant derivative pRC/CMVAEBP2(HS/R)) and 2 µg of paP2(3AE1/
120)CAT
along with 1 µg of pHermes-lacZ, a CMV-driven
-galactosidase
expressing plasmid, were used.
-Galactosidase activity was used to
normalize the transfection efficiency and CAT assays was performed as
described (9). The values shown represent three separate transfection
experiments. B, transient transfection analysis was carried
out with 5 µg each of the reporter plasmids pGALTKCAT (lanes
1-3) or pTKCAT (lanes 4-6) and the Gal4-fusion
plasmids pG4-AEBP2, the mutant derivative pG4-AEBP2(HS/R) and the
control plasmid pG4-AEBP2(
). AC and C indicate
acetylated chloramphenicol and chloramphenicol, respectively. Similar
results were obtained in three different transfection experiments.
C, both the wild-type and the mutant derivative of the
Gal4-AEBP2 fusion protein were equally expressed. Protein extracts from
cells transfected with either pG4-AEBP2 or pG4-AEBP2(HS/R) were
prepared, and 20 µg of the protein extracts were loaded on a 10%
SDS-polyacrylamide gel electrophoresis, then Western immunoblot
analysis was performed according to the standard protocol using
anti-Gal4 (DNA-binding domain) antibody (Santa Cruz Biotechnology) and
ECL detection reagents (Amersham). The fusion proteins Gal4-AEBP1 (9)
and Gal4-AEBP2 or Gal4-AEBP2(HS/R) are indicated. D, both
the wild-type and the mutant derivative of the Gal4-AEBP2 fusion
protein were equally able to bind the Gal4-binding sequence. The
TNT-coupled wheat germ extract system (Promega) was used to synthesize
the Gal4-AEBP2 fusion proteins. EMSA was carried out with the extract
from the in vitro translation reactions with pG4-AEBP2
(lane 1), pG4-AEBP2(HS/R) (lane 2) or the
parental vector pG4 (lane 3). The probe was a DNA fragment
containing five copies of the Gal4-binding sequence (9). The
protein-DNA complex and the free probe are indicated.
), in which the AEBP2 sequence is inserted in the opposite
orientation in relation to the Gal4 DNA-binding domain (Fig. 5B,
lane 1). No difference in the CAT activity was observed when
pTKCAT, a reporter plasmid lacking a Gal4-binding site, was used (Fig.
5B, lanes 4 and 5). Therefore, repression by
AEBP2 requires localization to the promoter region, and the repression
activity is specific and is not due to transcriptional "squelching"
or to a general nonspecific shutdown of the RNA polymerase II
machinery. Furthermore, these results suggest that the repression function of AEBP2 may be mediated by an active repression mechanism, rather than by a passive repression mechanism which usually involves by
competition with a positive factor(s) in the binding to the common DNA
site. As in the case of the homologous promoter assay, the mutation in
the middle zinc finger of AEBP2 resulted in decreased repression
activity when Gal4-AEBP2 was tested using a heterologous promoter with
the Gal4-binding site (Fig. 5B, lane 3). To examine whether
the wild-type and the mutant derivative of the Gal4-AEBP2 fusion
proteins were equally expressed in the transfected cells, the fusion
proteins were analyzed by Western blotting with the anti-Gal4
antibodies. As shown in Fig. 5C, both Gal4-AEBP2 (lane 1) and Gal4-AEBP2(HS/R) (lane 2) fusion proteins were
equally detectable. The lack of repression function for the mutant
derivative is not due to its failure to localize to the nucleus.
Similar levels of wild-type and mutant derivative of the Gal4-AEBP2
fusion proteins were detected in the Western blot analysis of a nuclear fraction isolated from the transfected cells (data not shown). Moreover, this mutation did not affect the binding of the Gal4-AEBP2 fusion protein to the Gal4 binding sequence: both the wild-type and the
mutant derivative were equally able to bind to the Gal4 binding
sequence in the in vitro binding assay using the in
vitro translated fusion proteins (Fig. 5D). These
results further indicate that the middle zinc finger motif of AEBP2 is
important for its repression function.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
, members of the general
transcription machinery (30). The repression by transcription enhancer
factor-1 of human chorionic somatomammotropin promoter activity is
thought to be mediated by direct interaction with the TATA-binding
protein (31). To interact with the general transcription machinery,
this type of transcriptional repressor binds to specific sites in the
promoter (30, 32). In some cases, the repressor may recruit a global
regulator that functions as a co-repressor (27). AEBP2 may regulate
aP2 gene expression similarly to one of those transcription
factors that directly affect the general transcription machinery
through an active repression mechanism. Alternatively, AEBP2 may
recruit a co-repressor in the regulation of aP2 gene
expression. The mutation in the middle zinc finger may cause an
alteration of the overall conformation of the protein to prevent
interaction with either the general transcription machinery or a
co-repressor.
, Refs. 5 and 6) and two negative
(AEBP1, Ref. 9; and AEBP2) transcription factors are involved in the
regulation of aP2 gene expression through the AE-1 site. In
contrast to AEBP2, which is expressed both in preadipocytes and
adipocytes,2 C/EBP
is
expressed only in the late stage of adipocyte differentiation (1-3)
and AEBP1 expression is abolished in the late stage of adipocyte
differentiation.3 Among these
factors, specific interactions may take place to set the proper stage
in the controlling of gene expression, by either competitively or
synergistically, at different stage of physiological condition.
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ACKNOWLEDGEMENTS |
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We thank T. Hollenberg and R. A. Singer for discussion and comments on the manuscript; A. Li for help in the initial screening and sequencing of the cDNA; J. Shi for technical assistance.
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FOOTNOTES |
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* This work was supported by grants from the Heart and Stroke Foundation (Nova Scotia) of Canada, the Canadian Diabetes Association, and NSERC (to H.-S. R.).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) AF090326.
To whom correspondence should be addressed. Tel.: 902-494-2367;
Fax: 902-494-1355; E-mail: hsro{at}is.dal.ca.
2 G.-P. He and H.-S. Ro, unpublished data.
3 S.-W. Kim, A. Muise, and H.-S. Ro, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
aP2, adipose P2;
AE-1, adipocyte enhancer 1;
C/EBP, CCAAT/enhancer-binding protein
;
AEBP, adipocyte enhancer-binding protein;
CAT, chloramphenicol
acetyltransferase;
EMSA, electrophoretic mobility shift assay;
NLS, nuclear localization signal;
CMV, cytomegalovirus;
TK, thymidine
kinase.
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REFERENCES |
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