From the Institute for Clinical Chemistry, University of Regensburg, Germany and § Novartis Institute of Biomedical Research, Summit, New Jersey, 07901
Received for publication, January 10, 2001
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ABSTRACT |
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The zinc finger gene 202 (ZNF202) located within
a hypoalphalipoproteinemia susceptibility locus on chromosome 11q23 is
a transcriptional repressor of various genes involved in lipid
metabolism. To provide further evidence for a functional linkage
between ZNF202 and hypoalphalipoproteinemia, we investigated the effect
of ZNF202 expression on ATP binding cassette transporter A1 (ABCA1) and ABCG1. ABCA1 is a key regulator of the plasma high density lipoprotein pool size, whereas ABCG1 is another mediator of cellular cholesterol and phospholipid efflux in human macrophage. We demonstrate here that the full-length ZNF202m1 isoform binds to GnT repeats within the
promotors of ABCA1 ( Molecular factors that determine plasma high density
lipoprotein (HDL)1
cholesterol levels include several known genes with defined roles in
reverse cholesterol transport (1) and a variety of susceptibility loci
with to date poorly characterized candidate genes. Mutations in the ATP
binding cassette transporter A1 (ABCA1) gene have recently been
causatively linked to familial HDL-deficiency syndromes (2-4). The
transporter ABCA1 is regulated by cholesterol flux and facilitates the
apoAI-dependent cellular export of cholesterol and
phosholipids, thereby acting as a key regulator of plasma HDL (2, 3,
5). The half-size transporter ABCG1 is another member of the group of
cholesterol-responsive ABC transporters. ABCG1, like ABCA1, has been
shown to regulate cellular cholesterol and phospholipid efflux (2, 3,
6).
In past years, evidence has accumulated to suggest that a number of
transcription factors play critical roles in the coordinate transcriptional regulation of genes involved in lipid metabolism (7,
8). Linkage analysis in large Utah pedigrees led to the identification
of a low HDL-cholesterol locus on chromosome 11q23 that is distinct
from the apoAI/C-III/AIV gene cluster (9). This novel familial
susceptibility locus for hypoalphalipoproteinemia contains the zinc
finger protein 202 (ZNF202) originally described as a testis-specific
transcription factor (9, 10). ZNF202 is expressed in two common splice
variants. The m1 splice form encodes a full-length protein of 648 amino
acids with an amino-terminal SCAN domain, a central KRAB repression
domain, and 8 carboxyl-terminal Cys2-His2 zinc
finger motifs. The m3 splice form encodes a carboxyl-terminal-truncated protein of 133 amino acids that contains only the SCAN domain. The SCAN
domain of ZNF202 has been shown to mediate selective protein
oligomerization and the zinc finger motifs to bind to specific DNA
elements (9, 11). Intriguingly, the ZNF202 DNA binding elements are
present in promotors of various genes involved in lipid metabolism
including apolipoproteins and lipid-processing enyzmes. ZNF202 has been
therefore proposed to function as a transcriptional regulator of lipid
metabolism (9).
Based on this information, we tested the hypothesis of whether the ABC
lipid transporters ABCA1 and ABCG1 are transcriptionally regulated by
ZNF202. Our results provide evidence that ZNF202 acts as a
transcriptional repressor of both ABCA1 and ABCG1 and, thus, establish
a functional link between ZNF202 and hypoalphalipoproteinemia.
Cell Culture--
HepG2 and RAW264.7 cells (American Type
Culture Collection) were cultured in Dulbecco's modified Eagle's
medium (BioWhittaker) supplemented with 10% fetal calf serum (Sigma)
in a 5% CO2 atmosphere at 37 °C. Cells (1 × 106 cells/2-ml medium) were seeded in 6-well plates
overnight before transfection. In some transfection experiments HepG2
cells were washed 4 h after transfection and subsequently
incubated in Dulbecco's modified Eagle's medium containing 5%
lipoprotein-deficient serum, 10 µM
20(S)-OH-cholesterol, and 10 µM
9-cis-retinoic acid (Sigma). In all experiments cells were
harvested after 24 h to measure luciferase activity or to prepare
RNA and nuclear extracts.
Cloning of Expression Constructs--
cDNAs encoding either
the open reading frame of the ZNF202m1 isoform (GenBankTM
accession number aF027219, nucleotide positions 8-1960) (2, 3,
10) or a truncated product lacking the SCAN domain (nucleotide
positions 920-1960) were cloned into pcDNA3.1/V5/His-Topo plasmid
(Invitrogen), and the sequence was confirmed by using an automated
fluorescence DNA sequencer and the ALFexpress AutoRead sequencing kit
(Amersham Pharmacia Biotech). The pcDNA3.1 ZNF202 (DV > AA)
construct with a double amino acid substitution (DV > AA) in the
ZNF202 KRAB domain was generated by using a site-directed mutagenesis
kit (CLONTECH). GST fusions constructs with the
ZNF202 SCAN or KRAB domain were generated as previously described (11). A cDNA encoding KAP1 was obtained from Edge Bio Systems and
directionally subcloned into the pcDNA3.1/His expression vector
(Invitrogen). Sequence fidelity was assessed by using an ABI PRISM 377 DNA sequencer (PerkinElmer Life Sciences).
In Vitro Protein Expression--
KAP1 and ZNF202 cDNA
templates were expressed in vitro in rabbit reticulocyte
lysates in the presence of [35S]methionine (Amersham
Pharmacia Biotech) using the TNT T7 Quick coupled
transcription/translation system (Promega) according to the
manufacturer's procedure. The synthesized products were confirmed by
SDS-PAGE. Lysate samples (10 µl) were separated on ready-to-use gels
(Bio-Rad) and analyzed by autoradiography.
Antisera--
The ZNF202[KRAB] antiserum (RF84) was generated
by immunizing rabbits with the GST-ZNF202aa177-327 protein.
Antigen-specific antibodies (N84) were isolated from the polyclonal
antiserum preparation by immunoaffinity purification using UltraLinkTM
immobilization columns (Pierce). Polyclonal antisera against KAP1 were
kindly obtained from Frank J. Rauscher III (Wistar Institute,
Philadelphia, PA) and used as described (12).
Electrophoretic Mobility Shift Assays--
Radiolabeled
double-stranded oligonucleotide probes (equivalent of 30,000 cpm) were
added to ZNF202-expressing rabbit reticulocyte lysates (1-2 µl) in a
buffer containing 50 mM HEPES/HCl, pH 7.9, 6 mM
MgCl2, 50 mM dithiothreitol, 100 µg/ml bovine
serum albumin, 0.01% Nonidet P-40, and 2 µg of poly(dI-dC) (Amersham
Pharmacia Biotech) and incubated for 20 min at room temperature. The
respective promotor sequences encoded by the oligonucleotides are
described in Fig. 1. Supershift experiments resulted from the addition
of KRAB antibodies, N84 (1 µl). In competition experiments, nuclear extracts were preincubated for 10 min with a 50-fold molar excess of
unlabeled oligonucleotides before the addition of radiolabeled probe.
DNA-protein complexes were finally resolved on a native 8%
polyacrylamide gel and analyzed by autoradiography.
Cloning of Reporter Gene Constructs--
Reporter constructs for
the ABCA1 (GenBankTM accession number AJ252201; position Luciferase Reporter Assays--
HepG2 cells were transiently
transfected with Fugene® reagent (Roche Molecular
Biochemicals) as described by the manufacturer. Two µg of the
respective reporter gene constructs were co-transfected with 1 µg of
the pSV Affinity Purification of KAP1--
GST and GST fusion proteins
of ZNF202 (1.0 µg) were incubated overnight at 4 °C with 2.0 µl
of the KAP1 cDNA template expressing rabbit reticulocyte lysate
sample supplemented with HNTG buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol) diluted
to a final volume of 200 µl. 15 µl of glutathione-Sepharose beads (Amersham Pharmacia Biotech) were subsequently added for 40 min
to collect the protein complexes. All samples were washed three times
with ice-cold HNTG buffer, boiled in electrophoresis buffer, and
analyzed by SDS-PAGE and autoradiography using an autoradiographic
image enhancer (National Diagnostics).
Analysis of ZNF202m1 Binding to KAP1--
For each binding
reaction, 1 µl of anti-KAP1 antibody was incubated with 500 µg of
total HeLa lysate proteins for 90 min at 4 °C. After an additional
incubation for 40 min at 4 °C in the presence of 15 µl of protein
G-Sepharose beads (Amersham Pharmacia Biotech), the KAP1
immunocomplexes were washed four times with ice-cold HNTG buffer. The
KAP1 immunocomplexes were thereupon incubated with 2 µl of the
in vitro translated proteins (ZNF202m1 and/or SDP1, mFPM315)
in a final volume of 100 µl for 90 min at 4 °C. Bead-coupled KAP1
complexes were finally regenerated by centrifugation and washed four
times with HNTG buffer. All binding reactions were analyzed by SDS-PAGE
and fluorography.
Northern Blot Analysis--
HepG2 cells were transfected with
increasing amounts of ZNF202 expression vectors using
Fugene® reagent (Roche Molecular Biochemicals). The
transfection efficiency ranged between 50 and 70%. Total RNA was
isolated 48 h after transfection for Northern blot analysis using
the QIAamp RNA isolation kit (Qiagen). Aliquots of 10 µg of total RNA
were separated on denatured agarose gels and transferred to nylon
membranes (Amersham Pharmacia Biotech). The membranes were hybridized
with a random- prime radiolabeled ABCA1-specific probe that was derived
from the 5' end of the coding sequence as described elsewhere (2, 3,
14). The membranes were rehybridized with a ubiquitin-specific probe to
confirm equal RNA loading. The tissue-specific expression of ZNF202 was
assessed by hybridization of a multiple tissue poly(A)+ RNA
master blot featuring 75 distinct human tissues
(CLONTECH) with a radiolabeled ZNF202-specific
probe. Individual signals on the autoradiogram were measured
densitometrically and expressed as relative mRNA abundance in
comparison with testis tissue (set to 100%).
Efflux Experiments--
RAW264.7 cells were transfected with a
ZNF202m1 encoding pcDNA3.1/V5/His-Topo vector or an empty control
vector, and stable colonies were selected in Dulbecco's modified
Eagle's medium containing 500 µl/ml neomycin according to standard
protocols. ZNF202 protein expression was confirmed by Western blotting.
Efflux assays were performed as recently described with minor
modifications (2, 3, 15). Briefly, stably transfected RAW264.7 cells
were radiolabeled with 1.5 µCi/ml [14C]cholesterol and
10 µCi/ml [3H]choline and loaded with 40 µg/ml
enzymatically modified low density lipoprotein that was prepared as
described elsewhere (16). Cells were incubated in 6-well plates for
24 h in Dulbecco's modified Eagle's medium supplemented with 5%
lipoprotein-deficient serum and 10 µM
20(S)-OH-cholesterol, 10 µM
9-cis-retinoic acid (Sigma), or 0.02% v/v ethanol. The
cells were then washed and chased for 17 h with either 100 µg/ml
HDL3 protein, 10 µg/ml purified apoAI (Sigma), or 0.2%
bovine serum albumin in the described medium in the absence of
radiolabeled lipids and enzymatically modified low density lipoprotein.
Lipids were finally extracted as previously described (17).
Radioactivity was determined by liquid scintillation counting. Lipid
efflux is expressed as the ratio of counts in medium to total counts.
Specific efflux rates were calculated by subtracting efflux rates in
the presence of control bovine serum albumin from the efflux rates in
the presence of the lipid acceptors apoAI or HDL.
ZNF202 Binds Specific ABCA1 and ABCG1 Promotor Sequences--
It
has been proposed that ZNF202 functions as a transcriptional repressor
of various genes related to lipid transport and lipoprotein metabolism
(9). All these genes share in their promotor regions a common
repetitive GnT motif that binds in vitro ZNF202. To identify
additional targets of ZNF202, we analyzed the promotor sequences of the
human ABCA1 and ABCG1 genes for the presence of GnT motifs. Both
proteins act as transmembrane lipid transporters (6, 15). In particular
ABCA1 is a key regulator of the plasma HDL pool size and mediates the
cellular efflux of cholesterol and phospholipids. Mutations of the
ABCA1 gene, which lead to genetic HDL deficiency syndromes
characterized by the almost complete absence of plasma HDL, further
underscore the pivotal role of ABCA1 in reverse cholesterol transport
and, ultimately, hypoalphalipoproteinemia (2-4).
As depicted in Fig. 1, we found GnT
motifs within the promotor region of ABCA1 at positions ZNF202m1 Represses ABCA1 and ABCG1 Promotors--
To investigate
the effect of ZNF202 on the transcriptional activity of both ABC
transporter promotors, luciferase reporter gene assays were performed.
As shown in Fig. 2, transfection of a
reporter gene construct containing the promotor region of
either the apoAIV, ABCA1, or ABCG1 gene together with various
amounts of ZNF202m1 expression vector led to a
dose-dependent inhibition by up to 80% of all promotor
activities (Fig. 2, black bars). Interestingly, a truncated
ZNF202 construct lacking the SCAN domain lost the ability to repress
the promotor activities (Fig. 2, crossed bars). The SCAN
domain of ZNF202 has been shown to mediate selective oligomerization
with itself or other SCAN domain-containing proteins such as ZNF191 or
SCAN domain protein 1 (SDP1) (11). The state of ZNF202 oligomerization
may therefore impact the transcriptional activity of the ZNF202m1
protein.
To resolve the discrepancy between the relatively low binding affinity
of ZNF202 for the ABCA1 promotor sequence (Fig. 1) and the high
capacity to repress the promotor activity (Fig. 2), we further
investigated the interaction of ZNF202 with the ABCA1 promotor. Besides
its affinity for GnT motifs, ZNF202 shares with other zinc finger
proteins the ability to bind to GC boxes (9). The ABCA1 promotor
harbors two GC boxes at positions
KRAB domain-mediated transcriptional repression has been reported to
result from interference with the TATA box-dependent basal
transcription machinery (18, 19). The ZNF202 Binding to the Transcriptional Corepressor KAP1--
ZNF202
shares a number of similar amino acids with the proposed consensus
sequence for the KRAB homology domain, which was derived from the
KOX1 gene (12). Within the KRAB domain, the amino acid identity
between ZNF202 and KOX1 is 42%, and the similarity is 64%, as aligned
with the BLAST algorithm (20). KOX1 has been shown to associate via its
KRAB domain with KAP1, a 97-kDa nuclear phosphoprotein with all the
hallmarks of a universal corepressor. The RING finger, B boxes (
The apolipoprotein AIV and ABCA1 genes both contain a TATA box that may
be targeted by KRAB domain-mediated signaling (22). The underlying
mechanism of ZNF202-induced transcriptional repression of the ABCG1
gene, which lacks a TATA box, needs to be further elucidated. The
relatively low constitutive activity of the ABCG1 promotor may explain
the possibility that minor ZNF202 alterations affect the
pyrimidine-rich initiator element of ABCG1 (13). It also
remains unclear how specificity of ZNF202 repression is defined.
Pengue and Lania (19) provide evidence that KRAB domain-mediated transcriptional repression is not caused by a general and unspecific inhibition of the RNA polymerase II machinery but strongly depends on
the specific arrangement of basal promotor elements.
The amino-terminal SCAN domain of ZNF202 has been shown to readily
oligomerize with SDP1 (11). We investigated therefore the possibility
that SCAN domain-mediated protein oligomerization may affect the
ability of the juxtaposed KRAB domain to interact with KAP1. ZNF202m1,
SDP1, and mFPM315, a SCAN domain-encoding protein without detectable
affinity for ZNF202 and, therefore, used as negative control (11), were
expressed in vitro in rabbit reticulocyte lysates in the
presence of a sulfur radiolabel. A HeLa cell-isolated KAP1
immunocomplex readily purified ZNF202 in the presence or absence of
mFPM315 (Fig. 4C). However, in the presence of SDP1, ZNF202
lost its ability to bind the KAP1 immunocomplex. The state of SCAN
domain oligomerization may therefore modulate the ability of ZNF202 to
recruit the corepressor KAP1 and to mediate transcriptional repression.
Although zinc finger proteins of the Cys2-His2
class are reported to bind DNA in monomeric form, our results suggest
that SCAN domain-containing zinc finger proteins require specific homo-
or heterodimerization for DNA binding and transcriptional modulation.
The existence of SDP1 as a gene encoding an isolated SCAN domain as
well as the truncated ZNF202m3 splice variant, which also contains only
a SCAN domain, further highlights the modulating impact of SCAN domain
interactions (11, 23).
ZNF202 Affects Induction of ABCA1 and Cellular Lipid
Efflux--
To further confirm the role of ZNF202 as a transcriptional
repressor of ABCA1, we determined the ABCA1 mRNA abundance by
Northern blot analysis using RNA preparations from HepG2 human hepatoma cells transfected with increasing amounts of expression vector encoding
either ZNF202m1 (Fig. 5A,
upper panel) or a ZNF202 fragment lacking the SCAN domain
(Fig. 5A, lower panel). In accordance with the
data from the promotor assays, the endogenous expression of ABCA1
mRNA was significantly decreased in ZNF202m1-transfected HepG2
cells, whereas mRNA levels were unaffected upon transfection with a
ZNF202-truncated protein.
Because KRAB domain proteins were shown to repress both basal and
activated promotor activity, we investigated the effect of ZNF202 under
conditions that induce ABCA1 expression (Fig. 5B).
Oxysterols such as 20(S)-OH-cholesterol in combination with 9-cis-retinoic acid have been shown to strongly induce ABCA1
expression by interaction with LXR/RXR heterodimers via a DR4 element
(5); the physiological relevance of these compounds still remains
unclear. Incubation with 10 µM
20(S)-OH-cholesterol and 10 µM
9-cis-retinoic acid led to a 3.5-fold induction of ABCA1
promotor activity in RAW264.7 rat macrophages (Fig. 5B).
Transfection of these cells with 4 µ g of ZNF202m1 expression vector
almost completely abolished oxysteroldependent induction of
ABCA1. These data underscore the strong inhibitory capacity of ZNF202,
which is likely driven by a mechanism that targets crucial elements of
transcriptional activation.
Finally, to demonstrate the functional relevance of ZNF202 on
cellular phospholipid and cholesterol efflux, RAW264.7 cells that
stably overexpress ZNF202m1 were generated. Because ABCA1 and ABCG1
expression are strongly induced by oxysterols (24, 25), we compared
efflux rates under basal conditions and during incubation with
20(S)-OH-cholesterol and 9-cis -retinoic acid. As
shown in Fig. 5C , apoAI and HDL3-mediated lipid
efflux markedly depend on the stimulation with oxysterols.
ZNF202-overexpressing cells revealed significantly reduced specific
phospholipid efflux (upper panel) and cholesterol efflux
(lower panel) rates as compared with mock-transfected cells.
Interestingly, the strongest suppression was observed in
HDL3-mediated cholesterol efflux. Since recent studies
demonstrated that apoAI and not HDL3 is the lipid acceptor for the ABCA1 transporter, one may conclude that ZNF202 modulates additional genes with selective cholesterol transport activity independent of the ABCA1 efflux pathway (26).
ZNF202 Expression Profiling in Various Human Tissues--
The
original description of ZNF202 as a testis-specific zinc finger protein
(10) was based on expression studies with limited tissue samples. Using
an RNA master blot derived from 75 distinct human tissues, we observed
that ZNF202 is highly expressed not only in testis tissue but also in
various other tissues such as uterus, brain, intestine, fetal tissues,
bone marrow, and leukocytes, which support a potential role for ZNF202
in the transcriptional regulation of ABCA1 in vivo (Table
I). Since ABCA1 and ABCG1 are induced in
human monocytes during phagocytic differentiation and subsequent lipid
loading using modified low density lipoprotein (6, 14), we tested under
these conditions the expression of ZNF202 in human monocytes.
Preliminary results indicate that ZNF202 expression is down-regulated
during monocyte differentiation and repressed by cholesterol
loading, thus demonstrating inverse regulation of ABCA1/ABCG1 and
ZNF202 (C. Schumacher; data not shown).
The identification of additional ZNF202-interacting proteins and the
elucidation of the transcriptional regulation of ZNF202 will help to
understand the role of ZNF202 in modulating the expression of ABCA1 and
ABCG1 in vivo. It will be of special interest to identify
genotypic sequence variations in regulatory motifs and functional
domains of the ZNF202 gene. Abnormalities in ZNF202-oligomerizing proteins may also contribute to genetically based
hypoalphalipoproteinemia and, thereby, further confirm the role of
chromosome 11q23 as a HDL susceptibility locus.
229/
210) and ABCG1 (
572/
552). ZNF202m1 expression in HepG2 cells dose-dependently repressed the
promotor activities of ABCA1 and ABCG1. This transcriptional effect
required the presence of the SCAN domain in ZNF202 and the
functional integrity of a TATA box at position
24 of ABCA1, whereas
the presence of GnT binding motifs was nonessential. The state of
ZNF202 SCAN domain oligomerization affected the ability of the adjacent
ZNF202 Krüppel-associated box domain to recruit the
transcriptional corepressor KAP1. Overexpression of ZNF202m1 in
RAW264.7 macrophages prevented the induction of ABCA1 gene expression
by 20(S)OH-cholesterol and 9-cis-retinoic
acid, further substantiating the interference of ZNF202 in critical
elements of transcriptional activation. Finally, HDL and
apoAImediated lipid efflux was significantly reduced in RAW264.7
cells stably expressing ZNF202m1. In conclusion, we have identified
ABCA1 and ABCG1 as target genes for ZNF202-mediated repression and
thus, provide evidence for a functional linkage between ZNF202 and hypoalphalipoproteinemia.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
919 to
+224), ABCG1 (GenBankTM accession number AJ289137; position
2912 to
+50), and apolipoprotein AIV (GenBankTM accession number X13368;
position
718 to +30) promotor sequences as well as reporter
constructs for potential ZNF202 binding sites (GnT tandem repeats) were
cloned into the BglII and NheI restriction sites
of the pGL3-basic vector (Promega) and confirmed by sequence analysis
(2, 3, 9, 13). The functional impact of a TATA box within the proximal
ABCA1 promotor region and of ZNF202 binding to GnT or GnC motifs was
assessed with reporter gene constructs containing mutated binding sites or truncations at position
175,
79, or +12. Constructs with mutated
229 GnT,
91 GnC, and
24 TATA motifs were generated by using a
two-step cloning strategy. Two polymerase chain reaction fragments were
generated that overlap at the DNA motif of interest, replacing it by
either a HindIII (
229 GnT), SacI (
91 GnC), or an EcoRI (
24 TATA) restriction site. After restriction
enzyme digestion, the overlapping fragments were ligated and finally cloned into pGL3 basic vectors. All vectors were sequenced on an
automated fluorescence DNA sequencer using the ALFexpress AutoRead sequencing kit (Amersham Pharmacia Biotech).
-galactosidase vector (Promega) and increasing doses (up to
4 µg) of ZNF202 expression vectors. The total amount of transfected
DNA was set constant by supplementation with empty expression vector. A
promotorless pGL3-basic vector served as control. Cells were lysed in
reporter lysis buffer (Promega), and after centrifugation, luciferase
assay reagent with luciferyl-CoA (Promega) was added to the supernatant
as recommended by the manufacturer. Luciferase activity was finally
determined in a LUMAT LB9501 (Berthold). All data were normalized for
protein concentrations (Bio-Rad protein assay) and
-galactosidase
activity (Promega
-galactosidase enzyme assay). Each experiment was
repeated three times, and measurements were performed in triplicates.
Results are expressed either as relative inhibition of luciferase
activity in cells transfected by ZNF202m1 in comparison to empty
expression vector or as multiples in comparison to promotorless
pGL3-basic vector activity.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
210/
229 and
ABCG1 at positions
552/
572. To examine the ability of ZNF202 to
bind these DNA sequences, gel shift assays were performed with rabbit
reticulocyte lysates expressing in vitro full-length
ZNF202m1 protein and radiolabeled oligonucleotides encoding specific
promotor fragments (Fig. 1, A and B). The
published consensus ZNF202 DNA binding sequence, as found in the apoAIV
promotor at positions
265/
243 and represented by an
oligonucleotide, led to a specific and dose-dependent band shift after incubation with the ZNF202m1 lysate (Fig. 1C,
lane 1-3) (9). Oligonucleotides reflecting the ABCG1 and
ABCA1 ZNF202 DNA binding motifs revealed similar migratory gel
properties after incubation with the ZNF202m1 lysate. The distinct
intensities of the detected DNA-protein complexes may reflect
individual affinities of these promotor fragments for ZNF202m1. The
addition of KRAB polyclonal antibody that recognizes epitopes between
amino acid position 177 and 329 of ZNF202m1 to the incubation mixture
affected the affinity of ZNF202 for the apoAIV, ABCG1, or ABCA1
promotor fragment (Fig. 1C, lane 4). The addition
of unlabeled oligonucleotides in a 50-fold excess competitively
abolished the band shift (Fig. 1, lane 5), providing further
evidence for the specific interaction of ZNF202m1 with its target
sequences.
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Fig. 1.
ZNF202 binds to GnT motifs
within the ABCA1 and ABCG1 gene promotors.
A, domain organization of the ZNF202m1 protein (648 amino
acids). Amino acid positions and described functions of SCAN, KRAB, and
zinc finger domains are indicated. B, potential ZNF202
binding sites (GnT motifs) within the promotor sequences of ABCA1 and
ABCG1 were reproduced as oligonucleotides. The GnT consensus motif was
derived from the apolipoprotein AIV promotor (9). Sequences and
positions within the respective promotor regions are shown.
C, electrophoretic mobility shift assays were performed with
in vitro transcribed and translated ZNF202m1 protein.
Complex formation was inhibited by the addition of polyclonal
antibodies against the KRAB domain of ZNF202 (lane 5).
Specific DNA/ZNF202m1 complexes are indicated with brackets
symbols, and the free probe is indicated with the letter P. Lane 1, free probe; lane 2, 1 µl of ZNF202m1;
lane 3, 2 µl of ZNF202m1; lane 4, 1 µl of
ZNF202m1 + 1 µl of N84 KRAB antibody; lane 5, 1 µl of
ZNF202m1 + 1 µl (50-fold) of unlabeled oligonucleotides (wild-type
competitor).
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Fig. 2.
ZNF202 inhibits ABCA1 and ABCG1
promotor activity. Luciferase assays
performed with ABCA1 ( 919/+224), ABCG1 (
2912/+50), apolipoprotein
AIV (
718 to +30), and promotorless pGL3 reporter gene constructs.
HepG2 cells were co-transfected with 2 µg of reporter gene construct,
1 µg of pSV
-galactosidase vector, and ZNF202m1 expression vector
in various amounts as indicated (black bars) or SCAN
domain-truncated ZNF202 vector (crossed bars). Cells were
analyzed for promotor activity 24 h post-transfection. Results are
presented as inhibition of luciferase activity (mean ± S.D. of
triplicate measurements of three independent experiments) in comparison
to cells transfected with empty control vector. Luciferase activity was
normalized for
-galactosidase activity and protein concentrations.
Absolute luciferase levels were 119-fold for ABCA1, 31-fold for
apolipoprotein AIV, and 6-fold for ABCG1 above control vector
level.
91 and
157 that bind the zinc
finger proteins Sp1 and Sp3 (M. Porsch-Özcürümez, data not shown). We generated reporter gene constructs with truncated ABCA1 promotor inserts and mutated putative ZNF202 binding sites (Fig.
3). Surprisingly, we did not observe any
loss in ZNF202-mediated repression with a reporter construct containing
only the
79/+ 224 ABCA1 region and, thus, lacking all potential
ZNF202 binding sites. ZNF202 may therefore interact with other elements
of the transcriptional complex.
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Fig. 3.
Repression of ABCA1
promotor activity by ZNF202 does not depend on
the specific binding to the ( 229/
210) GnT
motif or GC boxes. 1 × 106 HepG2 cells were
transfected with 1 µg of pSV
-galactosidase and 2 µg of reporter
gene vectors for the ABCA1 promotor encompassing the full-length
(
919/+224) wild-type or the fragmented form. The GnT motif, two GC
boxes, and the TATA box were mutated as indicated in the vector scheme.
Transfected cells were cultured for 24 h before being assayed for
luciferase activity. A representative out of three independent
experiments is shown. Luciferase activity was normalized for
-galactosidase activity and protein concentrations. Results are
expressed as multiples of promotorless control vector and indicated as
the mean ± S.D. of triplicate measurements.
79/+ 224 ABCA1 reporter gene
construct with a mutated TATA box at position
24 markedly decreased
promotor activity but also abolished ZNF202-mediated repression (Fig.
3). Identical results were obtained with the +12/+224 construct
containing only exon 1 of ABCA1. These data indicate that (i) the TATA
box is of functional importance for ABCA1 expression and (ii)
transcriptional repression by ZNF202 is likely mediated by a similar
mechanism as reported for other Krüppel-type zinc finger proteins.
1
and
2) and a coiled coil region at the amino terminus collectively
constitute the KRAB interaction, or RBCC domain (12). We therefore
analyzed the ability of ZNF202 to bind KAP1. Indeed, in
vitro radiosynthesized KAP1 protein associated with the ZNF202
KRAB domain, as shown by affinity purification studies with GST-tagged
ZNF202 fusion proteins (Fig.
4A). To evaluate the
specificity of the ZNF202-KAP1 interaction, a double amino acid
substitution (DV > AA) that has been previously shown to functionally inactivate the KOX1 protein was reproduced in the ZNF202
KRAB domain (12, 21). The wild type and mutated ZNF202m1 cDNA
sequences were radiosynthesized in vitro and analyzed for their ability to bind KAP1. Resin-coupled KAP1 immunocomplexes were isolated from HeLa cells and, after incubation with
ZNF202-expressing rabbit reticulocyte lysates, was analyzed by SDS-PAGE
and fluorography (Fig. 4B). KAP1 associated readily with
wild type ZNF202, whereas the DV > AA amino acid substitution
within the KRAB domain abolished, as anticipated, the association of
ZNF202 with KAP1.
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Fig. 4.
KAP1 binding to the KRAB domain of
ZNF202 is modulated by the oligomerization state of the adjacent SCAN
domain. A, affinity purification of KAP1 with ZNF202.
Rabbit reticulocyte lysates expressing in vitro produced
radiolabeled KAP1 were extracted with GST or GST fusion proteins of
ZNF202 containing either the KRAB or the SCAN domain of ZNF202. The
precipitates were subsequently analyzed by SDS-PAGE and
autoradiography. Molecular mass markers are indicated at the right side
in kilodalton units (kDa). B, affinity purification of
ZNF202 with KAP1. Resine-coupled KAP1 immunocomplexes were incubated
with rabbit reticulocyte lysates equally expressing wild-type or
mutated ZNF202 cDNA templates as shown below the gel. A KAP1
pre-bleed immunocomplex (PB) was used as control. The
purified immunocomplexes were subsequently analyzed by SDS-PAGE and
autoradiography. C, effect of SCAN domain oligomerization on
KAP1 binding. KAP1 immunocomplexes were isolated from HeLa cell lysates
and tested for their ability to purify in vitro
radiosynthesized ZNF202m1 from rabbit reticulocyte lysates in the
presence or absence of added SDP1 or mFPM315 protein.
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Fig. 5.
Effects of ZNF202 on cellular ABCA1 induction
and lipid efflux. A, ABCA1 mRNA expression is
repressed by ZNF202. HepG2 cells transfected with increasing amounts of
expression vector for ZNF202m1 (upper panel) or for ZNF202
with a truncated SCAN domain (lower panel) were subjected
after 48 h to Northern blot analysis. The blots were probed for
ABCA1 and ubiquitin expression. Lane 1, 2 µg of
empty control vector; lanes 2, 3, and
4, 0.5, 1, or 2 µg of ZNF202m1 and ZNF202 SCAN vectors.
B, ZNF202 abolishes oxysterol-induced ABCA1 promotor
activity. HepG2 cells were co-transfected with 2 µg (
919/+224)
ABCA1 reporter gene vector, 1 pSV
-galactosidase vector and either 4 µg of ZNF202m1 expression vector or an empty control vector
(Mock). Four hours after transfection, cells were stimulated
with 10 µM 20-OH(S)-cholesterol and 10 µM 9-cis -retinoic acid (9CRA)
dissolved in 0.2% (v/v) ethanol. Cells were harvested 24 h after
transfection and assayed for luciferase activity. Results are given as
×-fold of the promotorless control vector. C, ZNF202
reduces apoAI and HDL3-mediated phospholipid and
cholesterol efflux. Stably transfected RAW264.7 cells were radiolabeled
and loaded for 24 h with 40 µg/ml enzymatically modified low
density lipoprotein in the presence of 10 µM
20-OH-cholesterol and 10 µM 9-cis-retinoic
acid or 0.02% (v/v) ethanol. Subsequently, cells were washed and
chased for 17 h with either 100 µg/ml HDL3 protein,
10 µg/ml purified apoAI, or 0.2% bovine serum albumin. Results are
illustrated as the mean ± S.D. from measurements of four wells.
*, p < 0.05, comparing the specific efflux of ZNF202m1
and mock-transfected cells in independent samples by t
test.
Multiple tissue poly(A+) RNA master blot hybridized with a
specific probe for ZNF202
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grant PO708/1-1 and in part by industrial funds.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.
Both authors contributed equally to the paper.
¶ To whom correspondence should be addressed: Universitätsklinikum Regensburg, Institut für Klinische Chemie und Blutbank, Franz-Josef-Strauss-Allee 11, 93042 Regensburg, Germany. Tel.: 49 941 944 6201; Fax: 49 941 944 6202; E-mail: gerd.schmitz@klinik.uni-regensburg.de.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M100218200
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ABBREVIATIONS |
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The abbreviations used are: HDL, high density lipoprotein; ABC, ATP binding cassette; apo, apolipoprotein; ZNF, zinc finger protein; KRAB, Krüppel-associated box; KAP1, KRAB-associated protein 1; PAGE, polyacrylamide gel electrophoresis; SDP1, SCAN domain protein 1; GST, glutathione S-transferase.
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