From the Department of Biochemistry and Molecular Biology and the University of Southern California/Norris Comprehensive Cancer Center, Keck School of Medicine of the University of Southern California, Los Angeles, California 90089-9176
Received for publication, July 10, 2000, and in revised form, September 15, 2000
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ABSTRACT |
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In analyzing cis-regulatory elements important
for cell cycle control of the replication-dependent hamster
histone H3.2 gene, we discovered a binding site for the transcription
factor YY1 embedded within GC-rich sequences between the two tandem
CCAAT repeats proximal to the TATA element. Base mutations that
specifically eliminated YY1 binding resulted in suppression of the S
phase induction of the H3.2 promoter. In addition, we discovered that YY1 is an interactive partner of AP-2, which also binds the H3.2 promoter and regulates its cell cycle-dependent expression.
The critical domains for YY1 and AP-2A interaction are mapped,
revealing that the N-terminal portion of YY1 (amino acids 1-300) and
the DNA-binding/dimerization region of AP-2A are required. Our results suggest that YY1, acting as a transcription factor binding to its site
on the promoter, or through protein-protein interaction with AP-2, may
be part of a regulatory network including key cell cycle regulators
such as c-Myc and Rb in controlling growth- and differentiation-regulated gene expression.
The sequence motif CCAT for binding of the transcription
factor Yin Yang 1 (YY1)1
occurs in a large number of cellular and viral genes, and YY1 is known
to mediate the response to a variety of cell signal pathways (1, 2).
YY1 is a zinc finger-containing transcription factor highly conserved
among animal species and is ubiquitously expressed in growing,
differentiated, and growth-arrested cells. Although it has been
established that YY1 is a multifunctional protein that can act as a
transcriptional initiator, repressor, and activator, with the ability
to induce DNA-bending and association with the nuclear matrix, its
functional role in cell growth and differentiation is just emerging.
The YY1 protein is phosphorylated with a relatively short half-life
(3). In differentiating F9, 70Z/3, or U937 cells, no changes in the
mRNA or protein levels of YY1 were detected (3, 4). However, a
decrease in YY1 DNA binding capacity was observed during
differentiation of chicken embryonic myoblasts (5). The decrease in YY1
activity during myogenesis correlates with changes in the expression of
two YY1-dependent genes. The gene for Multiple lines of evidence point to the involvement of YY1 in the
regulation of growth-dependent genes. It has been reported that the YY1 level increases rapidly in quiescent NIH 3T3 cells in
response to serum and insulin-like growth factor 1 treatment (9). YY1
binding sites are found in the cell cycle-regulated genes such as those
for E2F1 (10), dihydrofolate reductase (11), and cdc6 (12), known to be
target genes of E2F. However, the presence of the E2F site is necessary
but not sufficient for S phase induction. Utilizing the E2F-1 minimal
promoter to test the contribution of various transactivation domains,
it was found that CCAAT, YY1 sites, and certain GC boxes cloned
upstream of the E2F1 minimal promoter displayed E2F
site-dependent regulation, suggesting these specific
transcription factors are able to cooperate with E2F to activate S
phase transcription (13). For the FO108 human H4 gene, multiple YY1
sites exist within the regulatory elements associated with its
growth-dependent expression and YY1 stimulates
transcription of reporter genes linked to H4 gene segments (14). In
another example, the replication-dependent mouse histone H3.2 gene contains a coding region activating sequence referred to as
the In examining the cell cycle regulation of the hamster H3.2 promoter, we
have previously shown that AP-2, through its binding to the H3.2
promoter and physical interaction with Rb, may contribute to the
transcriptional regulation of the H3.2 gene (17). In addition to the
AP-2 site located at around 230 base pairs upstream of the
transcription initiation site, the H3.2 promoter contains two CCAAT
elements proximal to TATA element (18). We report here that an inverted
CCAT motif, which binds YY1, was located within GC-rich sequence
between the two tandem CCAAT repeats. Mutation of the YY1 binding site
suppressed the cell cycle-dependent activation of the H3.2
gene. In addition, we discovered that YY1 is a novel interactive
partner of AP-2, suggesting that through protein-protein interaction,
YY1 could also affect the activity of cell cycle transcriptional
regulatory complexes.
Construction of Plasmids--
The construction of the wild-type
pH3[ Cell Synchronization--
K12 Chinese hamster fibroblast cells
were grown in DMEM with 10% bovine calf serum (Gemino, Calabasas, CA)
and 1% antibiotics (consisting of 50 µg/ml penicillin and
streptomycin, 100 µg/ml neomycin). For cell synchronization,
subconfluent cultures were growth arrested by serum deprivation in low
glucose DMEM containing 0.5% serum for 48-72 h. The cells were
released from the G0/G1 phase by addition of
fresh high glucose DMEM with 10% serum and harvested at various time
points in the cell cycle, up to 24 h. Progression of synchronized
populations was monitored as a function of DNA synthesis by
fluorescence-activated cell sorting analysis or by
[3H]thymidine incorporation (18).
Selection of Stable Transfectants--
Stable cell lines
harboring the wild-type or mutant H3.2 promoter driving luciferase
reporter gene were established by cotransfection using the calcium
phosphate precipitation method (19). A total of 10 µg of test plasmid
DNAs with 2 µg of neomycin expressing vector under the control of the
SV40 promoter (pSV40-Neo3) were used for both reporter constructs. The
transfectants was selected for resistance to G418 (250 µg/ml) for
about 4 weeks. The resistant colonies harboring the respective
constructs were pooled and maintained as stable cell lines. Cell
lysates were assayed for luciferase activities using a luciferase assay
kit (Promega, Madison, WI) in a TD-20/20 Luminometer (Turner Design,
Sunnyvale, CA) according to the manufacturer's protocol. The
luciferase activities of the stable transfectants were determined from
the same number of cells throughout the cell cycle.
Electrophoretic Mobility Shift Assays
(EMSAs)--
Double-stranded oligonucleotides were end-labeled and
purified, and conditions for EMSAs have been described previously
(17).
Antisera--
The anti-AP-2A N-terminal peptide rabbit
polyclonal antibody is a gift of Dr. M. Tainsky (University of Texas,
Houston, TX). The anti-YY1 monoclonal antibody, anti-ATF-1, and
anti-IRF-1 antibodies were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA).
Glutathione S-Transferase (GST) Pull-down Assays--
GST or
GST-fusion proteins were induced in Escherichia coli with
isopropyl-1-thio- Immunoprecipitation and Immunoblot--
Subconfluent K12 hamster
fibroblasts or 293T human kidney fibroblast cells were transiently
co-transfected with various expression plasmids or their empty vectors
(20 µg total in each transfection mixture) using SuperFect reagent
(Qiagen, Hilden, Germany). The cells were then lysed in ELB buffer (50 mM HEPES, pH 7.0, 150 mM NaCl, and 0.5%
Nonidet P-40) in situ at 4 °C for 10 min, and used for
immunoprecipitation as described previously (17). The immunoprecipitated proteins were resolved by a 10% SDS-PAGE and transblotted to Immunobilon-PVDF membrane. The blots were preincubated in 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and
0.1% Tween 20, containing 2% bovine serum albumin, at 4 °C
overnight. The individual primary antibodies were diluted at 1:500 to
1:2000 and incubated for 2 h at 4 °C. The horseradish
peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary
antibody (Roche Molecular Biochemicals) was diluted at 1:10,000. The
protein band was visualized by chemiluminescence (ECL) detection
(Amersham Pharmacia Biotech).
Identification of a Functional YY1 Binding Site in the H3.2
promoter--
The promoter sequence of the hamster histone H3.2 gene
spanning
The ability of YY1 to bind directly to the H3.2 promoter sequence was
demonstrated by using the bacterially expressed GST-YY1 protein in
EMSAs (Fig. 2B). The YY1 site within the H3.2 sequence was
established to reside within the ATGG sequence since a two-base mutation within the oligomer converting the sequence motif to CTCG
(Table I) resulted in the failure of mutated sequence to compete for
YY1 binding (Fig. 2B, lanes 6 and
7).
The functional importance of the YY1 binding site was tested by
site-directed mutagenesis. The mutant H3.2 promoter is identical to the
wild-type promoter with the exception of the two-base change converting
the ATGG sequence to CTCG, thereby destroying specifically the YY1
binding site (Fig. 1). Both wild-type and mutated promoter sequences
were linked to a luciferase reporter gene, and stably transfected into
K12 cells by cotransfection with a neomycin resistance gene. The
promoter activities during the cell cycle after serum release were
determined by luciferase assays in the synchronized cells established
from several independent pools of stable transfectants (Fig.
3). At the time of serum release, the
promoter activity of the mutated construct was only slightly lower than
that of the wild-type promoter, thus the basal promoter activity was
not affected significantly by the mutation. As the cells progressed into late G1 and entered early S phase at 12 h after
serum release, the wild-type H3.2 promoter showed a gradual increase
and reached a 15-fold increase at around 22 h. The kinetics of S
phase induction was considerably delayed when the YY1 binding site was
mutated (Fig. 3). The final induction level was reduced to about
6-fold. In contrast, mutation in a previously identified AP-2 binding site of H3.2 promoter resulted in increased promoter activity several
hours prior to the onset of the S phase (Fig. 3). These results showed
that, although the AP-2 site appears to be required for promoter
repression during G1 phase prior to the G1/S
transition, the YY1 site is required for optimal transcriptional
activation of the H3.2 promoter during G1/S transition.
Physical Association between YY1 and AP-2 in Vitro--
AP-2 has
been shown to be an interactive protein of Rb (17, 22). Further, we
identified AP-2 as a major transcription factor binding to the cell
cycle regulatory domain of the H3.2 promoter referred here as the H3
core (17). The H3 core is located about 70 base pairs upstream of the
YY1 binding site (Fig. 1). Since both YY1 and AP2 appear to be
regulators of the S phase induction of the H3.2 promoter, we tested the
interaction between these two transcription factors. Using GST
pull-down assays with [35S]methionine-labeled AP-2A
protein and various GST fusion proteins, we confirmed that AP-2A can
form homodimeric complexes (Fig.
4A, lane
3). In addition, we showed that AP-2A can interact with both GST-YY1 and GST-YY1
To further map the interaction domains of AP-2A with YY1, full-length
and various truncated forms of AP-2A (N262, In Vivo Association of YY1 with AP-2--
To determine whether YY1
and AP-2 can associate in vivo, two independent approaches
were taken. First, in vivo co-immunoprecipitation experiments were performed. Two mammalian cell lines, K12 and 293T
cells, were used. Because AP-2A was present in low abundance in
mammalian cells, to enhance detection between AP-2A and YY1, expression
vectors for AP-2A and YY1 were transfected either singly or in
combination into K12 and 293T cells. After transfections, whole cell
lysates were prepared and immunoprecipitated with an anti-AP-2
antibody. The immunoprecipitated proteins were resolved by SDS-PAGE and
immunoblotted with an anti-YY1 antibody. For both K12 (Fig.
6A) and 293T (Fig.
6B) cells, YY1 were immunoprecipitated by the anti-AP-2
antibody (Fig. 6, A and B, lanes
2 and 4). The expression of AP-2A in cells
transfected with the AP-2A expression vector was confirmed by
immunoblotting the cell lysates with the anti-AP-2A antibody (Fig. 6,
C and D, lanes 3 and
4).
In the second approach to determine for AP-2 and YY1 association
in vivo, we investigated the composition of a novel
multiprotein complex (X'), which was formed in EMSAs using K12 nuclear
extract and a H3 core ( Through deletion mutation analysis, the critical region for
S phase induction of the hamster H3.2 promoter has been mapped to a
region between
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-actin is
activated and c-myc is repressed when YY1 levels fall,
implying that YY1 represses
-actin transcription and activates
c-myc transcription (5-7). Targeted disruption of mouse YY1
results in embryonic lethality, and some heterozygote embryos exhibit
neurulation defects (8).
element involved in its up-regulation during the
G1/S transition (15). The nuclear activity that interacts
with the
element contained within the protein-encoding sequence of
the mouse H3.2 gene has been identified as YY1 (16). YY1 DNA binding to
this coding regulatory region is cell cycle-regulated with most
pronounced binding observed in late G1 cells (15). Further, cyclin D1-dependent kinases may play a role in the
phosphorylating events that regulate the YY1 DNA binding activity.
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ABSTRACT
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254]Luc was as follows; a subfragment of H3.2 promoter spanning
254 to
6 was amplified by PCR using specific wild-type primers. The
wild-type 5' primer was overhung with a XhoI site
(5'-ctagctcgAGCAGAACCTTGGCGAGTCAGC-3') and the 3' primer with a
HindIII site (5'-ctagaagcttAGACGGAAGTCACCAAGACG-3'). The
PCR-amplified fragment was digested with XhoI and
HindIII and inserted into a pGL3-basic luciferase vector
(Promega, Madison, WI) to generate the pH3[
254]Luc. The
pH3[
254AP2m]Luc construct was generated by PCR
amplification with an AP-2 mutant primer
(5'-ctagctcgAGCAGAACCTTGGCGAGTCAGaataattataatattaACAAGGGGGCGG-3') and
subcloned into pGL3 vector as the wild-type promoter pH3[
254]Luc. Site-directed mutagenesis to generate pH3[
254YY1m]Luc
was performed using the QuickChange site-directed mutagenesis kit
(Stratagene, La Jolla, CA). Mutagenic oligonucleotide primers, each
complementary to opposite strands of the vector, were designed and
synthesized for introducing specific mutation in YY1 site as shown in
Table I. The orientation and sequence of the wild-type and mutated
promoter constructs were confirmed by DNA sequence analysis.
-D-galactopyranoside and purified by
affinity chromatography. Protein yields were quantitated by Coomassie
Blue staining in comparison with bovine serum albumin after
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Protein-protein
interactions were assayed with equal amounts of in vitro
translated and 35S-labeled proteins and purified GST
proteins bound onto beads as described (17). Proteins bound onto beads
were released and resolved by 10% SDS-PAGE.
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254 to +3 is shown in Fig. 1.
Located within this sequence are the canonical TATA sequence, two CCAAT
binding sites for NF-Y binding, an AP-2 binding site, and an AP-1 like
binding site termed X. An ATGG sequence motif, which represents the
inverted CCAT sequence found to be a predictor of high affinity YY1
binding site (20), was found between the NF-Y binding sites and is
embedded within GC-rich motifs resembling that of the Sp1 binding site. To test for YY1 binding to the ATGG motif, synthetic oligonucleotides encompassing the YY1 site (spanning from
188/
131) of H3.2 promoter were used as probe in EMSAs (Fig. 2). The
sequence and size of the binding sites used in EMSAs are shown in Table
I. With HeLa nuclear extract, two
predominant complexes were observed. Both complexes are specific for
the H3.2 sequence as revealed by self-competition with molar excess of
the unlabeled probe and by the failure of competition with a
heterologous AP-2 sequence (Fig. 2A, lanes 1-5). The slower electrophoretic mobility complex
represented NF-Y binding to the probe, since this complex was
abolished by competition with a NF-Y consensus
site.2 (Wu and Lee, data not
shown). Likewise, the faster electrophoretic mobility complex contained
YY1, whose binding was completely abolished with molar excess of a
consensus YY1 site (Fig. 2A, lanes 6 and 7). Since the consensus YY1 oligomer used also contains
a low affinity NF-Y binding site CGAAT (21), partial competition for the NF-Y complex was also observed.
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Fig. 1.
Regulatory elements of the hamster histone
H3.2 promoters and their interaction with putative transcription
factors. The sequence of the wild-type (wt) H3.2
promoter is shown. The A base in the ATG initiation codon is
numbered as +1. The localization of the major
transcriptional initiation site is indicated by the arrow.
The upstream cis-acting regulatory elements TATA (which
binds TATA-binding protein), two tandem repeat CCAAT motifs (which bind
NF-Y), an inverted YY1 binding site, an AP-2 binding site, and the X
site are boxed. The specific base mutations to generate the
mutant YY1 promoter (YY1m) and the mutant AP-2 promoter
(AP-2m) are indicated by bold italic
letters.
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Fig. 2.
In vitro binding of YY1 to the
H3.2 promoter ( 188 to
131). A, EMSAs were performed
with 2 µg of HeLa nuclear extract, 1 ng of the H3.2 (
188/
131) as
probe (asterisk), and 200 ng of poly(dI·dC) as nonspecific
competitor. Lane 1, no competitor;
lanes 2-7, a 25- or 50-fold molar excess of the
various binding site competitors as indicated on top was
used. The sequences of the competitor oligomers are shown in Table I.
B, EMSAs were performed using 2 ng of bacterially expressed
GST-YY1 protein, 1 ng of H3.2 (
188/
131) as probe, and 200 ng of
poly(dI·dC) as nonspecific competitor. Lane 1,
no competitor; lanes 2-9, the YY1 complex was
competed against a 25- or 50-fold molar excess of the indicated
competitors, the sequences of which are shown in Table I. The positions
of NF-Y and YY1 complexes are indicated.
DNA sequences of the synthetic oligonucleotides
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Fig. 3.
Effect of YY1 and AP-2 binding site mutations
on the cell cycle induction of the H3.2 promoter. Stable
transfectants containing the wild-type ( ), YY1 mutant (
), and
AP-2 mutant (
) H3.2 promoter driving a luciferase gene were
synchronized by serum starvation. Following serum release, the promoter
activities at various time points were determined by measurement of the
luciferase activity. The level of luciferase activity of the wild-type
H3.2 promoter at time 0 was set as 1. The relative promoter activity at
each time point was derived from equal number of cells and was plotted
as a function of time after serum release.
with C-terminal domain deleted (Fig.
4A, lanes 4 and 5), but not
with GST alone or GST-Ha-Ras (Fig. 4A, lanes
2 and 6). Thus, the YY1 domain interacting with
AP-2 lies in its N-terminal portion outside the C-terminal repression
and zinc finger DNA-binding domain (Fig.
5B).
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Fig. 4.
Mapping the interaction domain of YY1 with
AP-2. A, GST pull-down assays were performed with
in vitro translated 35S-labeled full-length
AP-2A and various GST fusion proteins as indicated on top.
The input lanes represent 10% of the input protein. The
electrophoretic mobility of the protein size markers (in kDa) ran in
parallel is shown on the left. B, schematic
drawing of the functional domains of YY1 including the zinc finger
domain required for DNA binding. The binding properties of YY1 and
YY1 with AP-2A are summarized.
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Fig. 5.
Mapping the interaction domain of AP-2 with
YY1. A, in vitro translated
5S-labeled full-length or truncated AP-2A was used. The
input AP-2A protein samples (10%) were shown on the left
panel. The positions of the protein size markers (in kDa)
ran in parallel are indicated. At the right
panel, GST pull-down assays were performed with the various
35S-labeled AP-2A proteins (full-length or subfragments as
indicated on top) and with bacterial expressed and purified
GST-YY1. The proteins bound onto the GST-YY1 were eluted, applied to a
10% SDS-PAGE, and detected by autoradiography. B, schematic
drawing of the functional domains of AP-2 protein and a summary of the
AP-2 domains required for YY1 binding.
N165,
N227,
C390)
were in vitro transcribed and translated and GST pull-down assays were performed with GST-YY1. The radiolabeled AP-2A proteins, as
shown in Fig. 5A, were mixed with equal amounts of GST-YY1 and subjected to GST pull-down assays as described above. Strong interaction with YY1 was observed with both AP-2A (
N165) and full-length AP-2A. In contrast, little interaction was detected with
N262, which is devoid of the dimerization domain;
N227, which has
deleted part of the basic DNA binding domain; and
C390, which has
lost part of its dimerization domain and is not be able to bind DNA
(Fig. 5B). These results show that AP-2 binding to YY1
requires an intact DNA binding and dimerization domain of AP-2.
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Fig. 6.
Co-immunoprecipitation of AP-2 with YY1
in vivo. Expression vectors for human YY1, human
AP-2A, either alone or in combination as indicated on top,
were transiently transfected into K12 (A) or 293T cells
(B). In lane 1, the cells were
co-transfected with the empty expression vector and served as negative
control. The cell lysates prepared from the transfected cells
(lanes 1-4) were immunoprecipitated with a
rabbit polyclonal anti-AP-2A N-terminal peptide antibody. The
immunoprecipitates were applied onto a 10% SDS-PAGE. The filters were
immunoblotted with an anti-YY1 antibody. In A, a whole cell
lysate (WCL) prepared from the K12 cells was also applied to
the SDS-PAGE to provide a positive control for the electrophoretic
mobility of YY1, as indicated by the closed
arrow. The open arrow indicates the nonspecific IgG band cross-reacting with the antibody. The
positions of the protein size markers (in kDa) are indicated. In
C and D, the same lysates used in A
and B were immunoblotted with anti-AP-2 antibody. The
position of AP-2A is indicated.
253 to
214) as probe (Fig.
7A). The X' complex was
observed when highly concentrated cell lysates were used. The H3 core
(
254 to
214) contains the AP-2 binding site and the X binding site
(Table I). Previously we have shown that mutation of site X and the
first G residue of the AP-2 site severely diminished the ability of
AP-2 to bind to the H3 core (17). Likewise, the H3 core mutation
resulted in the reduction of its ability to compete for X' binding
(Fig. 7A). In determining the components of the X' complex,
the K12 nuclear extract was pre-incubated with antibodies against a
large number of transcription factors prior to addition of the H3 core
probe. As expected, one component of X' is AP-2A (Fig. 7B).
We discovered X' also contains YY1 since antibody against YY1 was also
able to inhibit the formation of X' (Fig. 7C). Other antibodies such as those directed against ATF-1, IRF-1 and other transcription factors were without effect (Fig. 7, B and
C, and data not shown). These results demonstrated that YY1
and AP-2 can associate in vivo and are components of a
multiprotein complex binding to the cell cycle regulatory domain of the
H3.2 promoter.
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Fig. 7.
Association of the endogenous AP-2 and YY1 in
a protein complex binding to the H3 core. In all EMSA reactions,
K12 simplified nuclear extract was incubated with the radiolabeled H3
core ( 253/
214) probe. A, the first
lane showed probe alone and the second
lane is without competitor (
). The position of complex X'
is indicated. The complexes formed were competed with 25- or 50-fold
molar excess of the unlabeled oligomers as indicated on top.
B and C, affinity-purified antibodies against
AP-2A, ATF-1, IRF-1, or YY1 as indicated on top were added
to the EMSA reaction mixtures prior to the addition of the probe.
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241 and
210, which contains a conserved AP-1-like
site (X) adjacent to an AP-2 binding site (17, 18, 23). Although the
promoter sequence downstream of this region by itself is not sufficient
to confer cell cycle regulation to the H3.2 gene (18), it may contain
sequences that are necessary for optimal induction of the H3.2 gene
during G1/S transition. By DNA sequence comparison between
H3.2 and the replication-dependent human thymidine kinase
(htk) promoter, a YY1 binding site core (5'-ATGG-3') was discovered
between the two tandem CCAAT repeats in both promoters
(15).2 Similar to the H3.2 promoter, the two CCAAT motifs
of the htk promoter are downstream of the cell cycle regulatory region
and bind NF-Y (24, 25). The YY1 binding site within the htk promoter is
also immediately adjacent to GC-rich motifs resembling the Sp1 binding
site. This high degree of conservation within the promoter organization
of H3.2 and htk, both of which are replication-dependent, suggests functional significance. This prediction was borne out through
mutation analysis of the YY1 binding site on the H3.2 promoter. In this
report, we showed that the ATGG motif represents an inverted binding
site for YY1. The binding of YY1 to this sequence motif does not
require other nuclear proteins as purified recombinant YY1 is able to
bind this site. Further, mutation of the YY1 site by two base changes
eliminated YY1 binding and suppressed the S phase induction of the H3.2
promoter. These results suggest that a simple mode of action by YY1
toward regulation of the H3.2 promoter is through direct binding to its
site and activate transcription (Fig.
8A). This mechanism may also
be employed for the activation of the histone H4 promoter by YY1
through its multiple binding sites on the histone H4 regulatory
elements (14).
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Fig. 8.
The regulatory network of YY1 in control of
H3.2 and other growth- or differentiation-regulated target genes.
A, two modes of action of YY1 on the H3.2 promoter. YY1 can
affect H3.2 promoter activity through binding directly to the CCAT core
sequence adjacent to the GC-rich sequence located between the two NF-Y
binding sites. YY1 can also interact with AP-2A at the protein level
and is a component of a multiprotein complex containing AP-2A that
binds to the H3 core. B, schematic drawing of the protein
interaction network among c-Myc, Rb, YY1, and AP-2. Through these
interactions, the pleiotrophic regulatory functions c-Myc and Rb on
cell growth and differentiation can be mediated by transcription
factors such as YY1 and AP-2.
Since the binding motif of YY1 is widely distributed in a large number of genes, specific regulation conferred by YY1 is likely related to its ability to associate with other proteins. The ability of YY1 to interact with components of the basal transcription machinery such as TATA-binding protein, transcription factor IIB, and TATA binding protein associated factor (TAF) II 55 suggest that YY1 could act as a bridging protein between the upstream regulatory elements and the core polymerase complex binding to the TATA element (2). The interactions between YY1, c-myc, and E1A strongly imply the importance of YY1 as a transcriptional regulator in growth and differentiation. It has been proposed that one mechanism for YY1 action is through the recruitment of chromatin-modifying enzymes such as histone deacetylases and histone acetyltransferases, leading to chromatin alteration (26). Both cyclin E/cdk2 kinase and the viral oncoprotein E1A have been shown to associate with and activate the histone acetyltransferase activity of CBP/p300 during the mammalian G1/S transition (27). This could lead to alteration in chromatin topology at specific cell cycle stages. Additionally, YY1 has been shown to associate with the nuclear matrix, which is known to play a role in defining the higher order structure of chromatin (28). Under these conditions, YY1 activity might be altered and the transcription of YY1 target genes could be affected.
In this study, we discovered that AP-2 is a novel interactive protein of YY1. Previously, we have identified that AP-2 can bind to the cell cycle control region of H3.2 promoter (17). More interestingly, AP-2 physically interacts with the important growth suppressing region of Rb C-terminal domain (17 and data not shown). In addition, AP-2 has been shown to associate with both c-Myc and E1A oncoproteins. These observations raise a possibility that AP-2 and YY1 function at overlapping regulatory pathways. To explore this possibility, we demonstrated by co-immunoprecipitation and co-existence within a multiprotein complex that AP-2 and YY1 physically associate with each other in vivo. The important domains for their interactions have been mapped to the C-terminal basic and dimerization domain of AP-2 and a larger N-terminal portion of YY1 outside most of the zinc finger regions. Although the exact region of the YY1 N-terminal portion (amino acids 1-330) required for AP-2 interaction remained to be determined, since this portion of YY1 is devoid of repression or DNA binding activities, it is most likely that the modulation of AP-2 activity by YY1 is mediated through physical interaction. In an attempt to analysis the effect of overexpression of YY1, either alone and in combination with AP-2A, on the native H3.2 promoter, we found that the promoter activity was repressed (data not shown). The mechanism for the repression is currently unclear and could be due to direct or indirect effects associated with overexpression, such as squelching of basal factors or interference of other critical factors such as proteins binding to the GC-rich sequence adjacent to the YY1 site (29). Here we show, through direct mutation of the YY1 binding site, that YY1 acts as an activator within the context of the H3.2 promoter. In case of the AP-2 binding site, its mutation resulted in increase in H3.2 promoter activity prior to the G1/S transition, suggesting functional consequence of the interaction between AP-2 and Rb as proposed in Fig. 8B.
In summary, the discovery of a functional YY1 binding site required for
optimal S phase induction of a replication-dependent gene
such as H3.2 provides direct evidence for YY1 involvement in the
regulation of a replication-dependent gene promoter. Our study predicts that other growth-regulated genes bearing YY1 binding site around transcriptional control region (11) may also be targets for
YY1 regulation. The identification of interaction between YY1 and AP-2
and their interactive network with c-Myc and Rb suggests that YY1 and
AP-2 may play important roles in mediating the pleiotrophic effects of
c-Myc and Rb on cell growth and differentiation (Fig. 8B).
In support, targeted disruption of the mouse YY1 gene results in
pre-implantation lethality, strongly implying that YY1 may regulate
genes essential for rapid proliferation and differentiation of mouse
embryos in early development (8). Similar to AP-2 gene disruption (30),
YY1 heterozygotes exhibit severe developmental abnormalities and
neurulation defects, supporting the notion that the two factors through
their functional interaction may converge upon similar developmental
regulatory pathways. Future investigations into the possible role of
YY1 in the control of other growth- and differentiation-regulated genes
will put these hypotheses to test.
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ACKNOWLEDGEMENTS |
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We thank Drs. D. Ann (University of Southern California, Los Angeles, CA), R. Buettner (University of Regensburg, Regensburg, Germany), Y. K. Fung (University of Southern California, Los Angeles, CA), P. Kannan and M. Tainsky (University of Texas, Houston, TX), Y. Shi (Harvard Medical School, Cambridge, MA), and T. Williams (Yale University, New Haven, CT) for generously providing us with plasmids and antisera.
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FOOTNOTES |
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* This work was supported in part by United States Public Health Service Grant GM31138 (to A. S. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of the USC/Norris Cancer Center predoctoral fellowship.
§ To whom correspondence should be addressed. Tel.: 323-865-0507; Fax: 323-865-0094; E-mail: amylee@hsc.usc.edu.
Published, JBC Papers in Press, October 3, 2000, DOI 10.1074/jbc.M006074200
2 F. Wu and A. S. Lee, data not shown.
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ABBREVIATIONS |
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The abbreviations used are: YY1, Yin Yang 1; GST, glutathione S-transferase; EMSA, electrophoretic mobility shift assay; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; htk, human thymidine kinase.
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