From the Departments of Biological Chemistry and
Medicine and
The Molecular Biology Institute, UCLA, Los Angeles,
California 90095 and ¶ Department of Medicine, Harvard Medical
School, Boston, Massachusetts 02215
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ABSTRACT |
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Ying Yang 1 (YY1) is shown to bind to the proximal promoters of the genes encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, farnesyl diphosphate (FPP) synthase, and the low density lipoprotein (LDL) receptor. To investigate the potential effect of YY1 on the expression of SREBP-responsive genes, HepG2 cells were transiently transfected with luciferase reporter constructs under the control of promoters derived from either HMG-CoA synthase, FPP synthase, or the LDL receptor genes. The luciferase activity of each construct increased when HepG2 cells were incubated in lipid-depleted media or when the cells were cotransfected with a plasmid encoding mature sterol regulatory element-binding protein (SREBP)-1a. In each case, the increase in luciferase activity was attenuated by coexpression of wild-type YY1 but not by coexpression of mutant YY1 proteins that are known to be defective in either DNA binding or in modulating transcription of other known YY1-responsive genes. In contrast, incubation of cells in lipid-depleted media resulted in induction of an HMG-CoA reductase promoter-luciferase construct by a process that was unaffected by coexpression of wild-type YY1.
Electromobility shift assays were used to demonstrate that the proximal
promoters of the HMG-CoA synthase, FPP synthase, and the LDL receptor
contain YY1 binding sites and that YY1 displaced nuclear factor Y from
the promoter of the HMG-CoA synthase gene. We conclude that YY1
inhibits the transcription of specific SREBP-dependent genes
and that, in the case of the HMG-CoA synthase gene, this involves
displacement of nuclear factor Y from the promoter. We hypothesize that
YY1 plays a regulatory role in the transcriptional regulation of
specific SREBP-responsive genes.
Three sterol regulatory element-binding proteins (1) termed
SREBP1-1a, SREBP-1c, also
called ADD-1 (2), and SREBP-2 constitute a unique family of
transcription factors that are synthesized as 125-kDa precursor
proteins that are localized to the endoplasmic reticulum in
sterol-loaded cells. Cellular sterol deprivation results in two
sequential proteolytic cleavages of SREBP and the release of a mature
68-kDa amino-terminal domain of the protein from the endoplasmic
reticulum (1, 3). Mature SREBP enters the nucleus and activates
transcription of target genes by a process that is dependent upon the
binding of SREBP and either NF-Y and/or Sp1 to the proximal promoters
of these genes (1, 4-8). In vitro studies have demonstrated
that SREBP and either NF-Y or Sp1 bind synergistically to DNA derived
from the proximal promoters of the FPP synthase (5, 9) or LDL receptor
(6) genes, respectively. This synergy is thought to account for the
increased transcription of SREBP-responsive genes that include, in
addition to the SREBP-2 gene itself (10), those that control
cholesterol homeostasis (the LDL receptor, HMG-CoA synthase, HMG-CoA
reductase, FPP synthase, and squalene synthase) (1, 7, 11), fatty acid
synthesis (fatty acid synthase and acetyl CoA carboxylase) (12, 13),
fatty acid desaturation (stearoyl-CoA desaturase 2)
(14),2 and triglyceride
synthesis (glycerol-3-phosphate acyltransferase) (15).
In our continued efforts to elucidate the role of the transcriptional
coactivator CREB-binding protein (CBP) in sterol-regulated transcription, we noted that overexpression of the most amino-terminal domain of CBP (amino acids 1-451), as a Gal4 fusion protein, increased the expression of certain sterol-regulated reporter genes under conditions where the cells were incubated in the presence of excess sterols.3 A potential
explanation for these results could be that overexpression of this
domain of CBP interfered with the function of a transcriptional repressor. This prompted us to search for a CBP-dependent
repressor that may regulate the expression of specific sterol-regulated genes. One potential candidate was the transcription factor Ying Yang 1 (YY1), which is known to have repressor functions and to interact with
the amino-terminal domain of CBP (16). A visual inspection of a number
of promoters of sterol-regulated genes indicated that several of these
contained potential YY1 binding sites (CCAT or ACAT), either
overlapping or adjacent to binding sites for NF-Y, Sp1, or SREBP.
Earlier studies show (4) that mutation of three of the four nucleotides
in a CCAT motif in the FPP synthase promoter resulted in increased
expression of an FPP synthase promoter-reporter gene. Because this CCAT
motif was adjacent to SRE-3, the SREBP binding site that is important
for sterol-regulated transcription of this gene (4), the result was
consistent with the hypothesis that mutation of the CCAT sequence
disrupted a cis element that normally functions to inhibit
transcription of the FPP synthase gene. These observations prompted us
to analyze the effect of YY1 on the transcription of various
sterol-regulated genes.
YY1, also known as YY1 is expressed constitutively in many growing, differentiated, and
growth-arrested cells (16). However, recent studies demonstrated that
YY1 protein levels decreased during skeletal and cardiac myocyte
differentiation (24), consistent with an earlier proposal that
down-regulation of YY1 was essential for the expression of the
sarcomeric The current studies demonstrate that co-expression of YY1 results in
transcriptional repression of specific SREBP-responsive genes, that the
promoters of these genes contain binding motifs that are recognized by
YY1, and that YY1 and NF-Y bind competitively to overlapping motifs in
the promoter of the HMG-CoA synthase gene. These results are consistent
with the hypothesis that transcription of specific sterol-regulated
genes, including HMG-CoA synthase, FPP synthase, and the LDL receptor,
is affected by YY1.
Materials--
DNA restriction and modification enzymes were
obtained from Life Technologies, Inc. 32P-Labeled
nucleotide triphosphates were obtained from Amersham Pharmacia Biotech.
pRSETB (Invitrogen) containing both a partial sequence of SREBP-1a
(amino acids 1-490) and T7 and polyhistidine tags and pCMV-CSA10,
which encodes amino acids 1-490 of SREBP-1a, were kindly provided by
Dr. T. Osborne (Department of Molecular Biology and Biochemistry,
University of California, Irvine, CA). Constructs encoding wild-type
YY1 or mutant YY1 proteins under control of the cytomegalovirus
promoter, were generous gifts of Drs. B. Lüscher and M. Austen
(Institut fur Molekularbiologie, Hannover, Germany) (16). Antibodies to
YY1 protein and oligonucleotides containing a consensus YY1 binding
site (5'-CGCTCCGCGGCCATCTTGGCGGCTGGT-3') were from Santa
Cruz Biotechnology, Inc. Lipoprotein-deficient fetal calf serum was
purchased from PerImmune. The sources of all other reagents and
plasmids have been given (4, 5, 7, 9).
Cell Culture, Transient Transfections, and Reporter Gene
Assays--
HepG2 cells were cultured as described (4). Plasmids were
transiently transfected into HepG2 cells using the MBS Transfection Kit
(Stratagene) with minor modifications (4, 14). The luciferase reporter
constructs (1 µg/60-mm dish) under the control of promoters derived
from genes encoding FPP synthase (pFPPS0.319L), HMG-CoA synthase
(pSYNSRE), HMG-CoA reductase (pRED), the LDL receptor (pLDLr), and an
expression vector encoding Purification of Recombinant SREBP-1a, YY1, and NF-Y Fusion
Proteins--
Recombinant SREBP-1a containing amino-terminal T7 and
polyhistidine (His6) tags, recombinant histidine-tagged
YY1, and recombinant histidine-tagged NF-Y A, B, and C were purified to
homogeneity from Escherichia coli extracts by nickel
chromatography (5, 9).
YY1 was eluted from the nickel affinity column in buffer A (20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 10% glycerol,
1 mM PMSF, 200 mM imidazole) and dialyzed
against buffer B (25 mM HEPES, pH 7.6, 2 mM
MgCl2, 20% glycerol, 1 mM EDTA, 100 mM KCl, 0.1% Nonidet P-40, 1 mM
dithiothreitol, 0.2 mM PMSF, 0.7 µg/ml pepstatin, and 1 µg/ml leupeptin). Under these conditions, YY1 precipitated out of
solution. The denatured protein was collected by centrifugation, and
the pellet dissolved in 2 ml of buffer C (6 M
guanidine-HCl, 40 mM HEPES, pH 7.9, 0.1 mM zinc
acetate, 1 mM PMSF, and 50 mM dithiothreitol)
and heated at 50 °C for 2 h. The soluble protein, obtained
after centrifugation, was dialyzed at 4 °C against 200 ml of buffer
D (40 mM HEPES, pH 7.9, 1 mM EDTA, 2 mM EGTA, 0.2 M NaCl, 0.2 M
arginine-HCl, 0.1 mM zinc acetate, 10% glycerol, and 1 mM PMSF) for 24 h without stirring. YY1 was dialyzed
for an additional 3 h against new buffer D with stirring and then for an additional 12 h using 4 changes of 300 ml of buffer E (40 mM HEPES, pH 7.9, 1 mM EDTA, 2 mM
EGTA, 0.1 M NaCl, 0.1 mM zinc acetate, 1 mM PMSF, and 10% glycerol). The soluble YY1 protein obtained after centrifugation was stored at Immunoblot Analysis--
HepG2 cells were transiently
transfected with plasmids that expressed wild-type or mutant YY1
protein. Whole cell extracts were prepared as described previously (9).
The soluble proteins (25 µg) were mixed with Laemmli sample buffer
(Bio-Rad), heated at 100 °C for 5 min, subjected to
SDS-polyacrylamide gel electrophoresis, and transferred to a
nitrocellulose filter (Amersham Pharmacia Biotech). The filter was
incubated with antibody to YY1 and the YY1-antibody complex identified
using ECL as described by the manufacturer (Amersham Pharmacia Biotech).
Gel Mobility Shift Assays--
Double-stranded DNA corresponding
to nucleotides YY1 Inhibits the Expression of Three Sterol-regulated
Genes--
The experiments of Fig. 1
demonstrate that incubation of cells in sterol-depleted media resulted
in increased expression of an HMG-CoA synthase promoter-reporter gene
(pSYNSRE) (lane 1 versus 2). The
induction was attenuated approximately 80% when cells were
cotransfected with low levels of a plasmid encoding wild-type YY1 (Fig.
1A, lane 1 versus 3, 5, and
7). In contrast, the induction of pSYNSRE, in response to
cellular sterol depletion, was relatively unaffected when cells were
cotransfected with plasmids encoding mutant YY1 proteins
YY1(
The experiments of Fig. 2 demonstrate
that the induction of two other reporter genes (pFPPS and pLDLr), in
response to cellular sterol depletion, was attenuated by coexpressed
wild-type YY1 (Figs. 2, A-B, lanes 3 versus 1). The inhibitory effects of YY1 were
specific because induction of the HMG-CoA reductase promoter-reporter construct (pRED) was unaffected by coexpressed YY1; indeed,
coexpression of YY1 often resulted in a small increase in the
expression of this promoter-reporter gene (Fig. 2C, lane 3 versus 1).
The increased expression of pFPPS, pSYNSRE, and pLDLr that occurred
when cells were incubated in sterol-depleted media was unaffected
following cotransfection of the cells with the YY1 mutant constructs
YY1(
Sterol-regulated induction of the reporter genes utilized in the
current study requires nuclear localization of mature SREBP, a process
that is normally dependent on cellular cholesterol depletion (1). In
the experiment of Fig. 4, cells were
incubated in the presence of excess sterols to repress the proteolytic
cleavage and release of mature endogenous SREBP from the endoplasmic
reticulum. Consequently, luciferase activity measured in cells
transiently transfected with either an HMG-CoA synthase (Fig.
4A) or HMG-CoA reductase (Fig. 4B)
promoter-reporter gene was low but increased dramatically when
sterol-treated cells were cotransfected with a plasmid encoding mature
SREBP-1a (Fig. 4, lanes 2 versus 1). Fig. 4A shows that the SREBP-1a-dependent
induction of the HMG-CoA synthase promoter-reporter gene was attenuated
by coexpression of wild-type YY1 (lane 4 versus
2) but not by coexpression of mutant YY1(
Taken together, the studies described in Figs. 1-4 demonstrate that
wild-type YY1 represses the transcriptional induction of promoter-reporter constructs derived from three genes (FPP synthase, HMG-CoA synthase, and the LDL receptor genes) that are known to be
responsive to changes in nuclear SREBPs or cellular sterol depletion.
This inhibitory effect is specific because the expression of a fourth
sterol- and SREBP-responsive reporter gene (pRED) was not affected by
cotransfected YY1. Based on the studies with mutant YY1 proteins that
are either unable to enter the nucleus or are defective in binding DNA,
we hypothesized that YY1 must bind to the promoters of specific
SREBP-responsive genes to bring about transcriptional repression.
YY1 Binds to the Promoters of the FPP Synthase, HMG-CoA Synthase,
and the LDL Receptor Genes and Displaces NF-Y from an Inverted CCAAT
Box in the HMG-CoA Synthase Promoter--
The proximal promoters of
HMG-CoA reductase, FPP synthase, HMG-CoA synthase, and the LDL receptor
genes that were used in the current study are illustrated in Fig.
5A. Transcriptional activation
of the latter three genes in response to cellular sterol depletion is
dependent on the binding of SREBP and either NF-Y or Sp1 to the
proximal promoters (Fig. 5A) (1, 5-8, 34, 36).
Transcriptional activation of the HMG-CoA reductase gene is less well
understood, although such activation does require SREBP (1, 37). The
proximal promoters of all four SREBP-responsive genes contain putative
binding sites (CCAT or ACAT) for YY1 that are adjacent to or overlap
with binding sites for SREBP or NF-Y (Fig. 5A). We
postulated that the inhibitory effect of YY1 (Figs. 1-4) might result
from YY1-mediated displacement of SREBP, NF-Y, or Sp1 from the
promoters of pFPPS, pSYNSRE, and pLDLr. Alternatively, YY1 might bind
to DNA and sterically interfere with critical interactions between
SREBP, NF-Y, Sp1, and coactivators that are required for transcription.
To test these postulates, we utilized 32P-end-labeled DNA
probes corresponding to the proximal promoters of these genes together with recombinant NF-Y, SREBP-1a, and YY1 proteins in electromobility shift assays (Figs. 5, B and D) and DNase I
footprint assays (Figs. 5C). Fig. 5B shows the
results obtained in EMSAs that utilized recombinant YY1 and
32P-end-labeled DNA, corresponding to the proximal
promoters of either HMG-CoA synthase (
Fig. 5C shows that recombinant YY1 protein bound to the
HMG-CoA synthase promoter and protected nucleotides
Incubation of the FPP synthase probe with YY1 and either NF-Y or SREBP
resulted in the formation of multiple complexes (data not shown). In
contrast to the studies with the HMG-CoA synthase probe, we obtained no
evidence that YY1 displaced either NF-Y or SREBP from the FPP synthase
probe (data not shown).
The current studies have led to the identification of YY1 as a
transcriptional repressor of some, but not all, SREBP/ADD1-regulated genes. We identified cis elements that bind recombinant YY1
protein in the proximal promoters of FPP synthase, HMG-CoA synthase,
the LDL receptor, and HMG-CoA reductase genes (Fig. 5B and
data not shown). However, coexpression of wild-type YY1 inhibited the
sterol-dependent regulation of pFPPS, pSYNSRE, and pLDLr
but not pRED (Figs. 1, 2, and 4), consistent with an inhibitory effect
of YY1 on the transcription of some, but not all, SREBP/ADD1-regulated
genes. Detailed studies demonstrate that YY1 and NF-Y bind to
overlapping sites on the HMG-CoA synthase promoter and that NF-Y is
displaced from the DNA by excess YY1 (Figs. 5, C and
D). Previous studies have demonstrated that elevated
transcription of the HMG-CoA synthase gene is dependent, in part, on
the binding of NF-Y to ATTGG (an inverted CCAAT box) (7-9), a motif
that overlaps the YY1 binding site (Fig. 5C). Thus, it
appears likely that the displacement of NF-Y from the inverted CCAAT
box by YY1 (Fig. 5D) accounts for the attenuated
transcription of the HMG-CoA synthase gene by cotransfected YY1 (Figs.
1 and 4).
We have been unable to demonstrate YY1-dependent
displacement of either NF-Y or SREBP from the FPP synthase or LDL
receptor proximal promoters (data not shown). These results are perhaps not surprising because the putative YY1 binding sites do not overlap with the binding sites for NF-Y, Sp1, or SREBP (Fig. 5A).
Further studies will be necessary to determine the mechanism by which YY1 represses the transcription of the FPP synthase and LDL receptor genes. Repression may result from inappropriate bending of DNA by YY1
(18, 38) or quenching, as the result of the sequestration of, or
interference with, coactivator proteins such as CBP (16, 18, 19). CBP
is known to interact with both SREBP (39, 40) and YY1 (16) and to be
required for SREBP-dependent transcriptional induction of
FPP synthase and the LDL receptor genes (39, 40). Alternatively, YY1
may bind to the promoters and recruit histone deacetylases such as
histone deacetylase 1-3 that are known to interact with YY1 (41).
These histone deacetylases repress transcription when tethered to
promoters through interaction with YY1 (41). Further studies are needed
to determine whether histone deacetylation plays a role in
sterol-regulated transcription.
Regardless of the exact mechanism, we hypothesize that the role of YY1
is to maintain SREBP-regulated genes in a transcriptionally repressed
state in the absence of nuclear SREBP. When the nuclear levels of SREBP
increase, following either cellular cholesterol starvation (1) or
adipocyte differentiation (2, 15, 42), we propose that the YY1-mediated
repression is overcome through synergistic interactions between SREBP
and either NF-Y or Sp1 on the DNA. The finding that the nuclear levels
of YY1 remain constant would ensure that the expression of these genes
is turned off in a timely manner as the nuclear levels of SREBP
decrease. Future studies will be directed at understanding the factors
that modulate the activity of YY1.
Transcription of each SREBP-regulated gene is likely to be complex and
to depend not only on the nuclear localization of both positive and
negative transcription factors but also on the relative affinity of
each factor for specific cis elements, the number of these
elements in each promoter, and the transcriptional strength of the
final complex. The observation that transcriptional induction of
different genes in response to SREBP is markedly different in mice that
overexpress SREBPs (35) is but one example of the complexity of this process.
In preliminary studies, an HMG-CoA synthase promoter-reporter gene was
transiently transfected into HepG2 cells, and the cells were then grown
in media supplemented with sterols to repress the proteolytic release
of SREBPs from the endoplasmic reticulum and the transcription of the
reporter gene. A partial relief of this repression was observed in
cells cotransfected with a plasmid encoding the adenoviral E1A protein
(data not shown). A further increase in the expression of the reporter
gene was observed in cells cotransfected with plasmids encoding both
E1A and CBP (data not shown). This result is consistent with earlier
observations demonstrating that E1A can relieve YY1 transcriptional
repression in a CBP/p300-dependent fashion (29). These
additional results with the HMG-CoA synthase promoter-reporter gene
provide further support for the proposal that endogenous YY1 affects
the transcription of specific sterol- and SREBP-regulated genes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, CF-1, UCRBP, NF-E1, FACT-1, CBF, LBF, NMP, and
MAPF-1 (17-19), binds to a core DNA motif (5'-CCAT-3' or 5'-ACAT-3')
that shows considerable heterogeneity in the flanking nucleotides (17,
18, 20). YY1 contains distinct domains that regulate, either by
transactivation (17-19, 21, 22) or transrepression (17-19, 24-31),
the expression of a number of genes. YY1 may also function as an
initiator-binding protein that activates transcription in the absence
of the TATA box-binding protein (17-19, 32). YY1 is identical to
NMP-1, a nuclear matrix protein (33) that is thought to regulate
histone H4 gene transcription.
-actin genes (25, 26). Earlier studies had demonstrated
that YY1 inhibited muscle-restricted expression of the skeletal
-actin gene by excluding serum response factor from the serum
response element in the proximal promoter of the
-actin gene (26).
Thus, differential expression of the YY1 protein in myocytes is thought
to affect both transcription of specific genes and cell differentiation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase (pCMV-
-gal) (0.5 µg/plate) have been previously described (7). Plasmids encoding
SREBP-1a (pCMV-CSA10), YY1, or YY1 mutants (
296-331;
399-414;
334-414;
154-199) were transfected into cells as indicated in
the specific legends. After transfection, cells were incubated for
24 h in media supplemented with either 10% lipoprotein-deficient calf serum in the absence (inducing media) or presence (repressing media) of sterols (10 µg/ml cholesterol and 1 µg/ml
25-hydroxycholesterol) (4) as indicated in the legends. Cells were then
lysed, and the luciferase and
-galactosidase activities were
determined (4). The
-galactosidase activity was used to normalize
for any variations in transfection efficiencies (4). Each experimental point was performed in duplicate (variation <10%), and each
experiment was repeated two or more times with similar results.
70 °C. Equal volumes of recombinant NF-Y A, NF-Y B, and NF-Y C were premixed as described (9) before addition to the radiolabeled DNA.
324 to
225 of HMG-CoA synthase,
293 to
47 of FPP
synthase, and
122 to
23 of the LDL receptor genes were end-labeled
with 32P. The radiolabeled probes (20,000 cpm: 1.5 fmol)
were incubated with the indicated amounts of recombinant YY1 and/or
NF-Y in binding buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM dithiothreitol, and 10%
glycerol) for 30 min at room temperature. One µg of poly(dI-dC) was
included in each reaction to reduce nonspecific binding. In competition
assays, an excess of the indicated double-stranded competitor DNA was
added 5 min before the addition of the radiolabeled probe. The reaction
products were separated on 4% polyacrylamide gel, vacuum-dried, and
exposed to film at
70 C.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
296-331) (Fig. 1B) or YY1(
399-414) (Fig. 1C). YY1(
296-331) and YY1(
399-414), unlike wild-type
YY1, are unable to bind DNA in vitro or to transactivate
other reporter genes in vivo (16). In agreement with the
report by Austen et al. (16), Western blot assays, utilizing
antibody to YY1 and extracts obtained from cells transiently
transfected with either the wild-type or mutant YY1 constructs,
demonstrated that similar levels of YY1 protein were expressed from
these different constructs (Fig. 1D).
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Fig. 1.
YY1 attenuates the induction of an HMG-CoA
synthase promoter-reporter gene. Duplicate dishes of HepG2 cells
were transiently transfected with 1 µg of pSYNSRE together with a
plasmid encoding -galactosidase and either 0.1 µg (lanes
3 and 4), 0.25 µg (lanes 5 and
6), or 0.5 µg (lanes 7 and 8) of a
plasmid encoding wild-type (WT) YY1 (A) or the
indicated mutant YY1 (B and C). The cells were
incubated for 24 h in medium supplemented with 10%
lipoprotein-deficient serum (LPDS) in the absence (
) or
presence (+) of sterols (10 µg/ml cholesterol and 1 µg/ml
25-hydroxycholesterol). The relative luciferase activities (mean
±S.E., n = 4) are shown after normalization to account
for differences in transfection efficiency. In D, HepG2
cells were transiently transfected with 1 µg of the indicated YY1
plasmid, and the cells were then incubated for 24 h in medium
supplemented with 10% lipoprotein-deficient serum in the absence of
sterols. Nuclear extracts were isolated, and 25 µg of protein was
analyzed by a Western blot assay as described under "Experimental
Procedures" using antibody to YY1. The transfection efficiency for
each plasmid was similar, as judged by nearly identical
-galactosidase activities (data not shown). No signal was obtained
from nuclear extracts from nontransfected cells (data not shown).
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Fig. 2.
YY1 inhibition of SREBP-responsive reporter
genes is specific. Duplicate dishes of HepG2 cells were
transiently transfected with either FPP synthase (pFPPS)
(A), LDL receptor (pLDLr) (B), or HMG-CoA
reductase (pRED) (C) promoter-reporter constructs together
with 0.5 µg of a plasmid encoding either wild type (WT) or
the indicated mutant YY1 proteins. The cells were incubated for 24 h in media supplemented with 10% lipoprotein-deficient serum
(LPDS) in the absence or presence of sterols, as described
in the legend to Fig. 1. The relative luciferase activities (mean ± S.E., n = 4) are shown after normalization to
-galactosidase to account for minor differences in transfection
efficiency.
296-331), YY1(
399-414) (Figs. 1 and 2), YY1(
334-414)
(Fig. 3), or YY1(
154-199) (data not
shown). Deletion of amino acids 334-414 or 154-199 from YY1 prevents
nuclear localization, DNA binding, and transactivation or interaction
with CBP (16), respectively.
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Fig. 3.
Nuclear localization of YY1 is necessary for
inhibition of sterol-regulated reporter constructs. Duplicate
dishes of HepG2 cells were transiently transfected with the indicated
reporter plasmid and 0.5 µg of a plasmid encoding YY1( 334-414).
The cells were then incubated in the presence or absence of sterols, as
described in the legend to Fig. 2. The relative luciferase activities
(mean ±S.E., n = 4) are shown after normalization to
account for differences in transfection efficiency.
296-331)
(lane 6 versus 2) or YY1(
399-414)
(lanes 8 versus 2) proteins. In
contrast, induction of the HMG-CoA reductase promoter-reporter gene by
coexpressed SREBP-1a was unaffected by coexpressed YY1 (Fig.
4B, lane 4 versus 2). Under
sterol-repressed conditions, the expression of pFPPS, pLDLr, pSYNSRE,
and pRED was unaffected by cotransfected wild-type or mutant YY1 (Figs. 1-4; data not shown).
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Fig. 4.
Wild-type (WT) YY1 inhibits
the SREBP-dependent induction of an HMG-CoA synthase but
not an HMG-CoA reductase promoter-reporter gene. HepG2 cells were
transiently transfected with either an HMG-CoA synthase (pSYNSRE)
(A) or HMG-CoA reductase (pRED) (B)
promoter-reporter construct in the absence ( ) or presence (+) of a
plasmid encoding mature SREBP-1a (20 ng plasmid) and the indicated
wild-type or mutant YY1 protein (0.5 µg of plasmid). The cells were
incubated for 24 h in media supplemented with 10%
lipoprotein-deficient serum (LPDS) and sterols (10 µg/ml cholesterol
and 1 µg/ml 25-hydroxycholesterol). The relative luciferase
activities are the means of three experiments, each performed in
duplicate.
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Fig. 5.
YY1 protein binds to the proximal promoters
of a number of SREBP- and sterol-regulated genes. In A,
the proximal promoters of four genes (FPP synthase, HMG-CoA synthase,
LDL receptor, and HMG-CoA reductase) that were used to produce
promoter-reporter genes used in the current study are illustrated. The
binding sites for SREBP, NF-Y, Sp1 are indicated. The putative YY1
binding sites are also shown. In B and D, EMSAs
were performed as described under "Experimental Procedures" using
the 32P-end labeled probes (HMG-CoA synthase, 324 to
225; FPP synthase,
293 to
47; LDL receptor,
122 to
23); and
recombinant YY1 (1 µg) or NF-Y (0.25 µg), as indicated. The shifted
DNA:protein complexes and free probes are indicated. In C, a
32P-end-labeled HMG-CoA synthase probe (-324 to
225)
(10,000 cpm) was preincubated in the absence (
) or presence (+) of 1 µg of recombinant YY1 protein. DNase I digestion was performed, and
the resulting products were separated by denaturing polyacrylamide gel
electrophoresis, as described under "Experimental Procedures." The
DNase I-protected region and the corresponding nucleotide sequence of
the lower DNA strand are shown. A schematic of the probe shows the
putative core binding site for YY1 (5'-CCATT-3' on the complementary
DNA strand (open circle)), the NF-Y binding site
(5'-ATTGG-3' on the complementary DNA strand (open
rectangle)), and the SREBP binding site (solid
rectangle). The Maxam and Gilbert-generated G + A ladder is not
shown.
324 to
225), FPP synthase
(
365 to
47) or the LDL receptor (
225 to
116) genes. The results
indicate that recombinant YY1 binds to all three DNA fragments and
results in the formation of multiple-shifted DNA-protein complexes
(Fig. 5B). The shifted DNA-YY1 complexes were specific
because inclusion of excess unlabeled DNA containing a consensus YY1
binding site competed for binding, whereas the addition of excess
unlabeled DNA containing a mutated YY1 binding site did not affect the
formation of the 32P-DNA:YY1 complex (data not shown). YY1
also bound to a 117-base pair fragment of the FPP synthase promoter
(
293 to
177) that includes the binding sites for NF-Y and SREBP
(Fig. 5A) (data not shown). The formation of
multiple-shifted complexes was also observed when a radiolabeled DNA
fragment containing a single consensus YY1 binding motif was incubated
with recombinant YY1 protein (data not shown). The formation of
multiple specific complexes between DNA and recombinant YY1 has been
noted previously (19).
306 to
290 from DNase I digestion. Analysis of the complementary (upper) DNA strand sequence indicates that the protected DNA includes a classical core YY1
binding site (CCAT,
297 to
294) (Fig. 5C, open
circle) that overlaps an NF-Y binding site (ATTGG,
295 to
291)
(Fig. 5C, open rectangle). The binding of NF-Y to
this ATTGG sequence has been shown to be critical and necessary for
elevated transcription of the HMG-CoA synthase promoter-reporter gene
in response to cellular sterol deprivation (7, 8). Additional EMSAs
demonstrated that a 100-base pair fragment of the HMG-CoA synthase
promoter (-324 to
225) bound either recombinant YY1 (Fig.
5D, lane 2) or NF-Y (Fig. 5D,
lane 3). However, the addition of excess YY1 displaced NF-Y
from the DNA, and no DNA:YY1:NF-Y complex was observed (Fig.
5D, lane 4). In addition, when the protein ratio
of YY1:NF-Y was <4:1, the radiolabeled DNA probe was bound by either
YY1 or NF-Y but not by the two proteins at once (data not shown),
consistent with the hypothesis that the two proteins are unable to bind
to the probe at the same time.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Bernard Lee for excellent technical assistance and Drs. B. Lüscher and M. Austen for their generosity in supplying various YY1 plasmids, as indicated in the text.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HL 30568 (to P. A. E.) and a grant from the Laubisch Fund (to P. A. E.).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.
§ Supported in part by an American Heart Association, Greater Los Angeles affiliate, post-doctoral fellowship and a grant-in-aid. Current address: Ludwig Institute for Cancer Research, Biomedical Center, Box 595, S-751 24 Uppsala, Sweden.
** To whom correspondence should be addressed: Dept. of Biological Chemistry, CHS 33-257, UCLA, Los Angeles, CA 90095-1769. Tel.: 310-206-3717; Fax: 310-794-7345; E-mail: pedwards{at}mednet.UCLA.edu.
2 D. T. Tabor and P. A. Edwards, unpublished data.
3 J. Ericsson and P. A. Edwards, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: SREBP, sterol regulatory element-binding protein; FPP, farnesyl diphosphate; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; SRE, sterol regulatory element; YY1, Ying Yang 1; NF-Y, nuclear factor Y; LDL, low density lipoprotein; CBP, cAMP response element-binding protein (CREB) binding protein; PMSF, phenylmethylsulfonyl fluoride.
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REFERENCES |
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