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(Received for publication, June 26, 1995; and in revised form, September 20, 1995) From the
YY1 is a multifunctional transcription factor capable of either
activation or repression of transcription. Using a series of mutant
proteins, we have characterized domains responsible for activation or
repression. We found that the YY1 transcriptional activation domain
lies near the amino terminus and requires amino acids 16-29 and
80-100 for maximal activity. The region between residues 16 and
29 has the potential to form an acidic amphipathic helix, whereas
residues between 80 and 100 are rich in proline and glutamine. The YY1
repression domain lies near the carboxyl terminus and is embedded
within the YY1 zinc finger region necessary for binding to DNA.
Deletion of YY1 amino acids, which include zinc fingers 3 and 4,
abolishes repression. However, site-directed mutagenesis, progressive
deletion, and internal deletion mutant analyses indicate that the
normal structures of zinc fingers 3 and 4 are not required for
repression.
YY1 (variously called NF-E1, The
mechanism of YY1 function is presently unclear. In some cases, YY1
binding appears to preclude the binding of activator proteins. For
instance, YY1 binding competes with the binding of NF- YY1
may also contribute to transcriptional regulation by serving as an
initiator binding protein during the formation of the basal
transcription complex. YY1 can activate transcription in vitro in the context of the adeno-associated virus P5 initiator or the
cytochrome c oxidase subunit Vb initiator
elements(23, 24) . YY1 can also support basal
transcription from supercoiled plasmid templates in vitro in
the presence of only TFIIB ( We show here that deletion of
only 43 amino acids from the carboxyl terminus of YY1 can convert the
protein into a strong transcriptional activator. Through progressive
deletion and internal deletion analyses, we show that the
transcriptional activation domain of YY1 is composed of two regions
requiring amino acids 16-29 and 80-100 for maximal
activity. The transcriptional repression domain lies within the zinc
finger region near the carboxyl terminus. We show that YY1 sequences
between 333 and 397 are included in the repression domain. These
sequences include zinc fingers 3 and 4. However, point mutation and
deletion studies show that normal zinc finger structure of fingers 3
and 4 is not necessary for repression.
Figure 1:
YY1 can activate or repress
transcription. Panel A, the YY1 expression vector was
cotransfected at various concentrations into 3T3 cells with a reporter
plasmid containing four copies of the YY1 binding site upstream of
TKCAT. The lower panel shows CAT data with the amount of YY1
expression plasmid transfected indicated above each lane (in
nanograms). The upper panel is a histogram of the data
obtained with -fold activation calculated relative to the activity
obtained with the reporter plasmid and empty expression vector. Panel B, the GAL-YY1 expression plasmid was cotransfected at
various concentrations into 3T3 cells with a reporter plasmid
containing five copies of the GAL4 DNA binding sequence upstream of
TKCAT. The lower panel shows CAT data with the amount of
GAL-YY1 expression plasmid indicated above each lane (in nanograms).
The upper panel is a histogram of the data obtained with -fold
activation calculated relative to the activity obtained with the
reporter plasmid and empty expression
vector.
Previously, we studied the YY1 protein
linked to the DNA binding domain of a heterologous protein (GAL4) in
3T3 or PYS-2 cells(2, 18) . These studies showed that
GALYY1 repressed transcription of a GALTKCAT reporter plasmid in 3T3
cells, while it activated transcription in PYS-2 cells. To determine
whether, like the wild-type YY1 protein, GALYY1 might activate
transcription at certain doses of effector plasmid in 3T3 cells, the
CMV-GALYY1 plasmid was transfected at various doses into 3T3 cells.
Indeed, low doses of GALYY1 effector plasmid activated GALTKCAT
activity (Fig. 1B). Maximal activity (7-fold) occurred
at 100 ng of effector plasmid. The higher quantity of GALYY1 effector
plasmid necessary for maximal activity as compared to wild-type YY1 may
reflect the presence of endogenous YY1 protein within the cells. The
fact that the GALYY1 fusion protein was capable of activating
transcription in 3T3 cells afforded us an assay system for identifying
functional domains of the YY1 protein.
Figure 2:
The
YY1 activation domain resides within the amino-terminal 100 amino
acids. Constructs 1-12 contain the GAL 4 DNA binding domain
(amino acids 1-147; solid rectangles) linked to various
portions of the YY1 sequence (open rectangles). The positions
of the acidic amphipathic helix, the consecutive acid stretch, the
consecutive histidine stretch, and the zinc fingers are indicated. Numbers on the left indicate the YY1 sequences
present in each construct. Each construct was transfected into 3T3
fibroblast and S194 plasmacytoma cells with a reporter plasmid
containing five copies of the GAL4 DNA binding site upstream of TKCAT.
CAT activity in transfected cell extracts is calculated relative to the
reporter plasmid alone. In each case the first number represents relative CAT activity with standard errors shown in parentheses. n numbers represent the number of times
each transfection was performed.
Similar results were obtained in S194
plasmacytoma cells, although there were some quantitative differences (Fig. 2). For instance, the full-length GALYY1 protein was a
more potent activator in S194 cells as compared to 3T3 cells, and the
various deletions did not have as dramatic of an activation over the
full-length protein. This can be seen by comparing the -fold induction
over wild-type YY1 for each construct in 3T3 versus S194 cells (Fig. 3). Whether this represents differences in the cellular
milieu of 3T3 and S194 cells is uncertain. However, others have shown
that YY1 can be a potent activator in plasmacytoma cells(31) .
In any case, similar to the case in 3T3 cells, truncation to amino
acids 69 and 42 reduced activity in S194 cells, and all activation was
lost upon deletion to amino acid 15 (Fig. 2).
Figure 3:
Deletion of YY1 carboxyl-terminal
sequences converts YY1 into a strong activator in 3T3 cells. The data
in Fig. 2are represented as -fold induction over the activity
observed with the full-length YY1 protein. Solid bars are data
from 3T3 cells, whereas open bars represent data obtained with
S194 cells.
To assure that
the protein products of the various deletion constructs were being made
within the transfected cells, nuclear extracts were prepared from 3T3
cells transfected with each deletion construct. These nuclear extracts
were evaluated by EMSA with a GAL4 DNA binding site probe.
Representative EMSAs of the carboxyl-terminal deletion constructs are
shown in Fig. 4A. Carboxyl-terminal deletions
containing YY1 amino acids 15-256 (with the exception of the
GAL-1-42 construct) showed similar amounts of probe shifted and
complexes of the appropriate size based on the predicted size of the
fusion proteins (Fig. 4A, lanes 1-9).
Numerous attempts have failed to demonstrate a complex with nuclear
extracts isolated from 3T3 cells transfected with construct
GAL-1-42 (lane 2). However, this construct must be
expressed within the cells because it consistently yielded modest
activation of transcription (Fig. 2). Somewhat unusual EMSA
complexes were observed with carboxyl-terminal deletion constructs
GAL-1-341, GAL-1-370, and GAL-1-397, as well as the
full-length fusion protein GAL-1-414 (lanes 10 and 12-14). To assure that these fusion proteins were
synthesized in transfected cells, nuclear extracts were subjected to
Western blot analysis with polyclonal YY1 antibody (Fig. 4B). This analysis showed that the
GAL-1-341, GAL-1-370, GAL-1-397, and GAL-1-414
(wild-type) proteins are indeed expressed in transfected cells. The
endogenous YY1 protein was also detected in these extracts and served
as a loading control.
Figure 4:
GAL-YY1 fusion proteins in transfected
cells can be detected by EMSA or Western blot. Panel A,
nuclear extracts were prepared from transfected 3T3 cells and EMSA was
performed with a GAL4 DNA binding site probe. The GAL-YY1 construct
used for transfection is indicated above each lane. The positions of
GAL-YY1 fusion protein-DNA complexes are indicated by the bracket on the right. Panel B, some nuclear extracts
were subjected to Western blot analysis with a YY1-specific antibody.
Above each lane is indicated the GAL-YY1 construct used for
transfection. GAL-DBD represents the GAL4 DNA binding domain
(amino acids 1-147). The position of endogenous YY1 is shown by
the arrow on the right, and the positions of the
GAL-YY1 fusion proteins are indicated by the brackets on the left.
EMSA studies were also performed with nuclear
extracts prepared from transfected S194 cells. However, no complexes
were observed perhaps due to the lower transfection efficiency of
plasmacytoma cells.
Figure 5:
The YY1 activation domain is bipartite.
YY1 residues 1-200 were linked to the GAL DNA binding domain
either intact, or with various internal deletion mutants in the context
of a eukaryotic expression vector. Constructs were transfected into
cells and CAT activities were determined. The lower panel shows the constructs used for transfection. Percent CAT activity
relative to the GAL-1-200 construct (defined as 100%) is shown
for transfections into either 3T3 or S194 cells. In each case the first number represents relative CAT activity with standard
errors shown in parentheses. n numbers represent the
number of times each transfection was performed. The upper panel shows a histogram for the data obtained with solid bars representing data from 3T3 cells and open bars representing data from S194 cells.
To assure that the internal deletion
proteins were synthesized in vivo, nuclear extracts were made
from the transfected cells and subjected to EMSA with the GAL4 DNA
binding probe (Fig. 6). All constructs produced the expected
complexes indicating that a loss in transcriptional activity was not
simply due to a lack of efficient production of mutant proteins (lanes 1-8).
Figure 6:
The amino-terminal internal deletion
mutants are expressed in vivo. Nuclear extracts were prepared
from transfected cells and subjected to EMSA with a GAL4 DNA binding
site oligonucleotide probe. The DNA constructs used for transfection
are shown above each lane and the positions of the GAL-YY1 DNA
complexes are indicated by the bracket on the right.
Figure 7:
The YY1 repression domain lies near the
carboxyl terminus. Panel A, 3T3 cells were transfected with
DNA constructs containing the GAL4 DNA binding domain either alone, or
linked to various YY1 sequences, in the presence of the GAL4TKCAT
reporter plasmid. The lower panel shows the expression
plasmids used for transfection. The solid rectangle represents
GAL4 sequence 1-147, and the open rectangles represent
various YY1 sequences. CAT activity is shown with the activity of the
reporter plasmid cotransfected with the GAL-DBD expression plasmid
defined as 1.0. Numbers in parentheses represent
standard errors, and n numbers indicate the number of times
the transfections were performed. The data are shown in histogram form
in the upper panel. Panel B, nuclear extracts
isolated from transfected cells were subjected to Western blot analysis
with anti-GAL4 antisera. Arrows show the positions of the
GAL4-YY1 fusion proteins, and the asterisk indicates a
background band recognized by the GAL4 antisera. Panel C,
transfections were performed with either the full-length GAL-YY1
expression plasmid (1-414) or expression plasmids with either
cysteine 360 changed to a serine (C360S) or with the sequence
371-380 deleted (
Our carboxyl-terminal
truncation experiments (Fig. 2) indicated that there was a
transition from a weak transcriptional activator to a strong
transcriptional activator when the third zinc finger was disrupted.
Thus, the GAL-1-397 mutant protein, which has a disrupted fourth
zinc finger, is relatively inactive, while the GAL-1-370 mutant
protein, which has a disrupted third zinc finger, is 4 times more
active when compared to the full-length GAL-YY1 fusion. To determine if
the third zinc finger is critical in possibly masking strong
transcriptional activity mediated by the amino-terminal activation
domain, two additional GAL4-YY1 fusion mutants were prepared. One
mutant has a 10-amino acid deletion removing amino acids 371-380
(GAL- To assure
that the fusion proteins were made in vivo, we performed
Western blots on nuclear extracts prepared from the transfected cells.
The two third zinc finger mutants (GAL-C360S and
GAL-
Our deletion studies showed that the YY1 repression domain includes
sequences in the region of the third and fourth zinc fingers.
Carboxyl-terminal deletion to residue 397, which does not disrupt the
repression domain, interrupts the fourth zinc finger indicating that
function of an intact fourth zinc finger is not necessary for
repression. To determine whether repressor function requires the intact
structure of the third zinc finger, we prepared mutations C360S and
While zinc
fingers are typically considered DNA binding motifs, it is interesting
to note that some zinc finger regions have been implicated in
protein-protein interactions. For instance, the zinc finger of the
adenovirus E1A protein has been implicated in interaction with
transcription factors (36) and is necessary for physical
interaction with TATA-binding protein(37) . Similarly, the zinc
finger region of ATF2 can interact with the leucine zipper region of
cAMP response element-binding protein, whereas the Sp1 zinc fingers can
interact with RelA(38, 39) . A short region containing
a zinc finger in TFIIB is necessary for efficient interaction with
TFIIF(40) , and the zinc finger-containing LIM domain has been
implicated in protein-protein interactions(41) . In addition,
the GATA-1 zinc finger region can self-associate (42) and can
interact with other proteins(43) . The repression domain of
YY1 is unlike other characterized repression domains. A number of
repressor proteins have been identified and include Kruppel, eve,
engrailed, SCIP, Dr1, Msx-1, Tst-1, WT1, AEF1, and Ssn6/Tup6
proteins(44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56) .
Repression domains within these proteins are either
alanine-rich(51, 52, 56) , alanine-,
glycine-, and proline-rich(5) , or rich in glutamine and
proline(53, 54) . Repressor proteins constructed from E. coli sequences are rich in basic amino acids(57) .
The YY1 protein contains a region rich in alanine and glycine (residues
137-201), but this segment is dispensable for repressor function.
Volume 270,
Number 50,
Issue of December 15, 1995 pp. 30213-30220
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, or UCRBP; (1, 2, 3, 4) ), is a multifunctional
transcription factor that can either activate or repress transcription.
Repression has been observed in the context of the immunoglobulin
3` enhancer, the Moloney murine leukemia virus long terminal repeat,
the adeno-associated virus P5 promoter, the skeletal 
-actin
promoter, the 
-casein promoter, - and
-globin genes, the
serum amyloid A1 promoter, the human immunodeficiency virus promoter,
and the human papilloma virus type 18 promoter (1, 4-13; reviewed
in ref. 14). In contrast YY1 can activate the c-Myc promoter, the
ribosomal protein L30 and L32 promoters, and the intracisternal
A-particle upstream promoter
element(15, 16, 17, 18) .
Interestingly, YY1 can either activate or repress some promoters
depending upon either promoter architecture or intracellular milieu.
For instance, YY1 typically represses the adeno-associated virus P5
promoter, but can be converted into a potent transcriptional activator
in the presence of adenovirus E1A protein(1) . YY1 can also
either activate or repress the c-Fos promoter based on the orientation
or the position of a YY1 binding site within the promoter(19) .
Finally, YY1 can either repress or activate the human papilloma virus
type 18 promoter depending upon the presence of an adjacent DNA
sequence that binds to a distinct nuclear factor(6) .
B to an
overlapping -sequence in the serum amyloid A1 promoter(13) .
Similarly, YY1 competes with serum response factor binding in the
-actin promoter, GATA-1 binding in the -globin promoter, and
with binding of a lactation-associated factor in the
-casein
promoter(7, 9, 10, 11) . YY1
function may also relate to its ability to bend DNA(19) . In
other cases, the function of YY1 appears to be controlled by
interaction with other proteins such as adenoviral E1A, c-Myc, or
switch binding protein(1, 6, 20) .
Interestingly, loss of YY1 binding sites in the human papillomavirus
type 16 silencer region is implicated in the overexpression of viral
oncogenes and in tumor progression(21, 22) .
)and RNA polymerase II (25) . The multiple functions of YY1 suggest that it can
interact with many proteins and that it contains multiple functional
domains. Given its diverse functional properties and its implication in
oncogenesis, it would be interesting to more precisely characterize the
functional domains of the YY1 protein.
Plasmid Constructions
CMVGAL-DBD was created by
subcloning a BglII-XbaI fragment of pSG424 (26) containing the GAL4 DNA binding domain (amino acids
1-147) into the corresponding sites of CMV expression plasmid
pCB6+. The restriction sites upstream of the GAL4 sequence were
destroyed by digesting with BglII and HindIII,
followed by blunting with Klenow polymerase and religation. The
polylinker for subcloning downstream of the GAL4 sequences is identical
to that of the parent plasmid pSG424 except for a distal BamHI
site contributed by the pCB6+ polylinker. Plasmid CMVGAL-YY1 was
created by subcloning a BglII-XbaI DNA fragment from
plasmid GAL4-E1 (2) containing the GAL4 DNA binding domain
fused to amino acids 2-414 of YY1 into the corresponding sites of
pCB6+. This construct contains pCB6+ restriction sites
upstream of the GAL4 encoding region of the plasmid and a BamHI site downstream of the insert. CMVGAL-1-102,
CMVGAL-1-143, CMVGAL-1-188, CMVGAL-1-256, and
CMVGAL-1-341 were generated by exonuclease III digestion using
the Promega Erase-a-Base system. The parent plasmid, CMVGAL-YY1, was
digested at the BamHI site 3` of the cDNA sequences, and the
recessed 3` end was filled in with 40 mM each dNTP and Klenow
polymerase (50 units/ml) to protect from exonuclease III digestion. A
5` protruding end susceptible to exonuclease III digestion was created
by digestion of the XbaI site just 5` to the BamHI
site. Progressive exonuclease III deletions through the YY1 cDNA
sequence were performed using various incubation times at 30 °C,
followed by treatment with S1 nuclease to remove single-stranded DNA.
Blunt-ended molecules were religated and transformed into competent
JM109 cells. For preparation of CMVGAL-1-200, CMVGAL-1-370,
and CMVGAL-1-397 an EcoRI DNA fragment containing the
YY1 cDNA sequences was cut with SmaI, FspI, or HincII, respectively. The appropriate DNA fragments were
gel-purified and cloned into the EcoRI-XbaI/blunted
sites of plasmid CMVGAL-DBD. To prepare CMVGAL-201-414, a SmaI-EcoRI DNA fragment containing the coding
sequence 201-414 was isolated by gel purification. The fragment
was blunted and cloned in frame into the EcoRI-blunted site of
CMVGAL-DBD. CMVGAL-333-414 was made by digesting CMVGAL-YY1#2
(see below) with HindIII and XbaI. The HindIII-XbaI fragment was gel-purified, blunted, and
cloned in frame into the blunted EcoRI site of CMVGAL-DBD.
Plasmids CMVGAL-1-15, CMVGAL-1-42, and CMVGAL-1-69
were generated by the polymerase chain reaction (PCR) using the GAL
forward sequencing primer and appropriate reverse primers with the
CMVGAL-YY1 plasmid as template. The Invitrogen PCR Optimizer kit was
used to establish optimum PCR buffer conditions for these sets of
primers. Standard assays contained 1x appropriate buffer, template DNA
(10 ng), oligonucleotide primers (500 ng each), 200 µM dNTPs, and 2.5 units of Taq polymerase in a final
reaction volume of 100 µl. The PCR cycle was 95 °C, 1 min; 50
°C, 5 min; and 70 °C, 2 min for 30 cycles. Amplified DNAs were
digested with EcoRI and BglII and subcloned into the EcoRI and BamHI sites of CMVGAL-DBD. YY1 internal
deletions 
16-29, 43-53, 70-80, and
70-99 were generated by triple ligation of PCR fragments
coding 5` and 3` YY1 regions with the CMVGAL-DBD vector. The 5`
fragment was generated as indicated above and digested with EcoRI and BglII. The 3` fragment was generated using
the CMV reverse primer and the appropriate forward primer. Amplified
products were digested with BglII and XbaI and the 5`
and 3` fragments were ligated with EcoRI- and XbaI-digested CMVGAL-DBD. To generate carboxyl-terminal
truncations of the internal deletions, the mutant plasmids were
digested with EcoRI and XbaI, the resulting fragment
was purified by gel electrophoresis, and subsequently digested with SmaI. The 5` fragments containing the YY1 sequence 1-200
with the appropriate deletion mutation were purified and subcloned into
the EcoRI and XbaI/blunted sites of CMVGAL-DBD. To
prepare CMVGAL-YY1#2, an EcoRI fragment containing the
full-length YY1 sequence except the initial starting methionine was
subcloned into the EcoRI site of CMVGAL-DBD producing an
in-frame chimeric fusion protein. This plasmid differs from CMVGAL-YY1
in that it lacks the pCB6+ polylinker sites upstream of the GAL4
coding sequence. CMVGALYY1-C360S and CMVGALYY1371-380 were
generated using the Muta-Gene (Bio-Rad) oligonucleotide-mediated in
vitro mutagenesis kit. The HindIII-XbaI fragment
of CMVGAL-YY1#2 containing sequences encoding the last 83 amino acids
of YY1 was subcloned into the phagemid vector pBluescript II/KS+
(Stratagene). Preparation of uracil containing single-stranded DNA,
annealing of the appropriate mutagenic primer, and subsequent in
vitro second strand synthesis in the presence of dNTPs was
performed according to the manufacturer's specifications. The
resulting double-stranded plasmid DNAs were transformed into Escherichia coli HB101. The HindIII-XbaI
mutagenized fragment was subcloned into the corresponding sites of
plasmid CMVGAL-YY1#2, replacing the original wild-type sequences. The
identities of all constructs were confirmed by dideoxynucleotide
sequence analysis.Oligonucleotides
The sequences for
oligonucleotides were as follows. GAL forward primer,
5`-CATCATCATCGGAAGAG-3`; 15 reverse primer,
5`-GCGAGATCTCTCCGAGCCGTCCGTGG-3`; 42 reverse primer,
5`-GCGAGATCTGCCCACCACTGTGGTCT-3`; 69 reverse primer,
5`-GCGAGATCTGCCGGCGTGCCCGCGGC-3`; 16-29 forward primer,
5`-GCGAGATCTACCATCCCGGTGGAGAC-3`; 43-53 forward primer,
5`-GCGAGATCTTGGCGGCGGTGGCGACCA-3`; 70-80 forward primer,
5`-GCGAGATCTCCGCCCATGATCGCTCT-3`; 70-100 forward primer,
5`-GCGAGATCTCAGGAGGTGATCCTGGT-3`; 3` CMV reverse primer,
5`-CTTCCAAGGCCAGGAGAG-3`; C360S, 5`-TGAAAGCGTTTCCCAGAGCCTTCGAACGTGC-3`;
or 371-380, 5`-GGGGCACACATAGGGCCTCAAATTGAAGTCCAGTGA-3`.Cell Culture, Transfections, and CAT Assays
S194
cells were grown and transfected using the DEAE-dextran method as
described previously(27) . 20 
10
log phase
cells were transfected with 2.5 µg of reporter plasmid, 2.5 µg
of effector plasmid, and 1 µg of a pCB6+-based
-galactosidase expression plasmid. 3T3 cells were grown in
Dulbecco's modified Eagle's high glucose medium (Life
Technologies, Inc.) supplemented with 10% heat inactivated fetal bovine
serum (HyClone), 2 mML-glutamine (Life Technologies,
Inc.), 100 units/ml penicillin, and 0.1 mg/ml streptomycin (Life
Technologies, Inc.). Cells were transfected using the calcium phosphate
coprecipitation method(28) . Unless specified, cells were
transfected with 5 µg of reporter plasmid, 5 µg of effector
plasmid, and 1 µg of -galactosidase expression plasmid. After
48 h cellular extracts were prepared by three freeze-thaw cycles. CAT
assays of these extracts were performed as described by Gorman et
al.(29) , using -galactosidase activity to
standardize for transfection efficiency.Preparation of Nuclear Extracts
Mini-nuclear
extracts were prepared from transfected 3T3 cells by the method of
Schreiber et al.(30) , with minor modifications.
Transfections were done in triplicate using 5-10 µg of
effector plasmid/plate. Cells from three plates were pooled, and
two-thirds of the cells (approximately 5-7 10 cells) were transferred to a separate tube and used to prepare
nuclear extracts. The remaining third was processed for CAT assays.
Pooled cells were washed once with PBS, and resuspended in 400 µl
of cold Buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1
mM EGTA, 0.1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 1 mM pepstatin, 1 mM leupeptin). Following incubation on ice for 15 min, 25 µl of
10% Nonidet P-40 (Sigma) was added, and each sample was vortexed for 10
s. Each sample was centrifuged for 30 s to 1 min in a microcentrifuge
to collect the nuclear pellet, the supernatant was aspirated, and the
pellet resuspended in 100 µl of cold Buffer C (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 2 mM
phenylmethylsulfonyl fluoride, 2 mM pepstatin, 2 mM leupeptin). Samples were incubated for 15 min on ice with frequent
gentle mixing. The nuclear extracts were centrifuged for 5 min at 4
°C to remove insoluble material, and the supernatant was stored at
-70 °C.Electroporetic Mobility Shift Assays (EMSA)
Each
reaction contained a final volume of 20 µl. Reactions contained 4
µg of poly(dI-dC), 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 50%
glycerol, 5000-6000 cpm P-end-labeled GAL4 DNA
probe, and a maximum of 5 µl of nuclear extract. In some assays,
the binding reaction included 1 mM ZnSO
. After
incubation for 30 min at room temperature, reactions were
electrophoresed on native polyacrylamide gels (4%) prerun for 20 min
using either low ionic strength buffer (6.7 mM Tris, pH 7.5,
3.3 mM sodium acetate, 1 mM EDTA) or 0.25 TBE
buffer (22.5 mM Tris borate, 0.5 mM EDTA).SDS-Polyacrylamide Gel Electrophoresis and Western
Blotting
Nuclear extracts (12 µl) from cells transfected
with various effector plasmids were electrophoresed on 10%
SDS-polyacrylamide gels. Transfer of proteins to nitrocellulose
(Scheicher & Schuell) was performed at 4 °C for 1 h at 100 mV
using a 25 mM Tris, 192 mM glycine, 20% (v/v)
methanol, pH 8.3 transfer buffer (Bio-Rad Mini Trans-Blot). Following
blocking in 5% milk (1-2 h), blots were incubated with
affinity-purified rabbit polyclonal YY1 or GAL4 (Santa Cruz
Biotechnology) antibody (1:500) overnight at 4 °C. After washing,
the blots were incubated 2 h at room temperature with a 1:1500 dilution
of secondary antibody, a donkey anti-rabbit IgG (Amersham). Blots were
developed using the ECL detection system (Amersham). To prepare
affinity-purified rabbit polyclonal YY1 antibody, a Sepharose-YY1
affinity matrix was prepared according to the manufacturer's
protocol (Pharmacia Biotech Inc.). CnBr-activated Sepharose (1 ml) was
coupled to 70 µg of purified bacterially produced histidine-tagged
YY1 (Qiagen). Sera (10 ml) from YY1 immune rabbits was precipitated
with an equal volume of 4 M
(NH)
SO. After 30 min, the
precipitate was recovered by centrifugation (2000 g,
10 min). The pellet was dissolved in 2 ml of PBS and dialyzed against 2
liters of PBS overnight at 4 °C. The dialyzed fraction was brought
up to 10 ml with TBST (.01 M Tris-HCl, 0.15 M NaCl,
0.05% (v/v) Tween) and incubated with the affinity matrix overnight at
4 °C with rocking. The matrix was washed three times for 30 min
with 20 ml of TBST. Bound antibody was eluted with 1 ml of 100 mM glycine, pH 2.5. Buffer exchange was done over a NAP-5 column
(Pharmacia) equilibrated with 10 volumes of PBS (0.1% bovine serum
albumin, 0.02% sodium azide). Purified antibody was stored at -70
°C.
YY1 Can Function as Either an Activator or a Repressor
in 3T3 Cells
Previously, we showed that YY1 can function as
either a repressor or an activator (2, 18) of
transcription, depending upon the cell type in which it is expressed
(3T3 versus PYS-2 cells, respectively). These results
suggested that the function of YY1 may be influenced by protein
interactions within differing cellular environments. To determine
whether YY1 could function under certain conditions as a
transcriptional activator in 3T3 cells, we performed transfection
experiments with increasing amounts of YY1 expression plasmid. We
reasoned that if YY1 activity is influenced by interaction with
cellular proteins, differing quantities of YY1 within the cells might
show different transcriptional responses because protein-protein
interactions may be influenced by the quantity of YY1 within the cell.
The reporter plasmid for these experiments contained four copies of the
YY1 binding site upstream of the herpesvirus thymidine kinase promoter
driving expression of the bacterial chloramphenicol acetyltransferase
gene (YY1TKCAT). The effector plasmid contained the intact YY1 cDNA
sequence driven by the CMV promoter in plasmid pCB6+ (CMV-YY1).
Transfection of various quantities of the YY1 expression plasmid showed
that low doses of YY1 indeed caused transcriptional activation in 3T3
cells, while higher doses repressed transcription (Fig. 1A). Maximal activation was 4.4-fold at 0.5 ng of
effector plasmid. Transfection of 3 and 6 µg of effector plasmid
resulted in repression to a level of 14%. These studies show that YY1
is capable of functioning as either a transcriptional activator or a
repressor in 3T3 cells.
Identification of the YY1 Activation Domain
To
determine the region of YY1 responsible for transcriptional activation,
11 effector plasmids were constructed consisting of progressive
carboxyl-terminal truncations of YY1 linked to the GAL4 DNA binding
domain. These constructs were tested for their ability to activate
GALTKCAT in 3T3 cells. Our initial experiments indicated that removal
of the carboxyl-terminal 83 amino acids converted GALYY1 into a
constitutive activator even at high doses of effector plasmid. ()Therefore, we tested function of each GALYY1 deletion
construct at high doses of effector plasmid (5 µg). The CAT
activity for each construct relative to the GAL4 basal activity is
summarized in Fig. 2. Removal of 17 amino acids from the
carboxyl terminus (construct 2; GAL-1-397) did not convert GALYY1
into a constitutive activator. However, removal of an additional 27
amino acids (construct 3; GAL-1-370) showed a 4-fold increase in
CAT activity over the GAL4YY1 full-length construct (GAL-1-414)
and a 9-fold increase over basal CAT activity. Carboxyl-terminal
truncations to amino acids 341, 256, 200, 188, 143, or 102 fused to
GAL4 (constructs 4-9) produced proteins that were potent
activators of CAT transcription, ranging from 12- to 33-fold activity
above basal CAT activity. CAT activity diminished with progressive
deletions to amino acids 69 or 42 and returned to basal level upon
deletion to amino acid 15.
The Activation Domain Contains Multiple Functional
Segments
Results obtained with our progressive carboxyl-terminal
deletion mutants indicated that the transcriptional activation domain
of YY1 resides within its amino terminus, approximately between amino
acids 16 and 100. Several amino acid sequence motifs reside within this
region: a segment with the potential to form an amphipathic acidic
helix between amino acids 16 and 29, a stretch of 11 consecutive acidic
residues between amino acids 43 and 53, a stretch of 11 consecutive
histidines between amino acids 70 and 80, and an area enriched in
proline and glutamine between amino acids 80 and 100. Internal deletion
mutants were prepared that deleted each of these regions either
individually or collectively. These mutants were prepared in the
context of the YY1 protein truncated at amino acid 200 and linked to
the GAL4 DNA binding domain. This truncation increases YY1 activation
potential in 3T3 cells (see Fig. 2) thereby facilitating the
identification of sequences important for activation. Results are shown
in Fig. 5, and values are represented as the percent of CAT
activity observed with GAL-1-200. As expected, deletion of amino
acids 16-99 caused a great decrease in CAT activity (5 and 14% of
maximal activity in 3T3 and S194 cells, respectively). Deletion of
amino acids 16-80 decreased activity as well, but to only 40 and
62% of maximal CAT activity in the two cell lines, respectively. This
suggested that sequences between amino acids 80 and 99 contribute to
activation function. However, deletion of amino acids 70-99 had
minimal effects on activation (97 and 91%, respectively).
Interestingly, deletion of the consecutive histidine (70-80)
or acid (43-53) stretches had no deleterious effect on
transactivation. However, deletion of the amphipathic helix region
between amino acids 16 and 29 reduced activation to 33 and 26% of
maximal CAT activity in 3T3 and S194 cells, respectively. The above
results suggest that the YY1 activation domain is bipartite. Sequences
between amino acids 16 and 29 are clearly important for activity.
Sequences between 80 and 100 contribute to maximal activity since the
16-80 construct shows higher activity than the
16-100 construct. However, this region is somewhat redundant
because deletion of residues 70-99 alone did not decrease
transcriptional activity.
Characterization of the Repression
Domain
Experiments with our carboxyl-terminal mutants indicated
that YY1 is a strong activator of transcription when the zinc finger
region is deleted. This suggested that sequences important for
transcriptional repression reside within the carboxyl-terminal region
of YY1. To determine the effect of the carboxyl-terminal domain alone
on transcription, the GAL4 DNA binding domain was fused to the last 214
amino acids of YY1 to produce GAL-201-414. This construct
contains all four YY1 zinc fingers. This fusion protein repressed
transcription in 3T3 cells to a level of 31% (Fig. 7A).
To more narrowly define the YY1 repression domain, we prepared a
construct containing amino acids 333-414. This construct
repressed expression to 39% (Fig. 7A). Western blots of
tranfected cell nuclear extracts with anti-GAL4 antisera (because of
the limited YY1 sequences in construct GAL-333-414) indicated
that the fusion proteins were expressed in vivo (Fig. 7B). These data indicate that YY1 repressive
sequences lie between amino acids 333 and 414.
371-380). The activity of the
full-length YY1 protein is defined as 1.0, and CAT activities are
calculated relative to this value. The data are shown in histogram form
in the upper panel. Panel D, nuclear extracts
isolated from transfected cells were subjected to Western blot analysis
with YY1-specific antibodies. The source of the nuclear extract is
indicated above each lane. The position of endogenous YY1 is indicated
by the arrow on the right, and the positions of the
various GAL-YY1 fusion proteins are shown by the bracket on
the left.
371-380), which encompasses the second half of the
third zinc finger including the histidines. The other mutant creates a
point mutation at amino acid 360 (GAL-C360S), changing the second
cysteine of the third zinc finger to a serine. A stable third zinc
finger is not formed by either of these mutations, because these mutant
proteins are incapable of binding to DNA (data not shown). Neither
internal zinc finger mutation mimicked the strong transcriptional
activity of the carboxyl truncated third zinc finger mutant
GAL-1-370 (Fig. 7C). The latter is on average
4-fold more active as a transcriptional activator than the full-length
GAL-1-414 (Fig. 2). The serine-360 mutant was
approximately half as active as full-length YY1, while the 10-amino
acid deletion activated transcription 1.5-2-fold.371-380) were readily detectable on Western blots with
YY1 antibody and were expressed at an abundance similar to the
GAL-1-370 truncation (Fig. 7D).
The YY1 Activation Domain
We have used a series
of progressive carboxyl-terminal deletions and a series of internal
deletions to characterize the YY1 transcriptional activation domain.
The progressive deletions showed that the activation domain resides
within the amino-terminal 100 amino acids of YY1, consistent with the
results of Lee et al.(32, 33) . This region
of YY1 contains sequences with the potential to form an acidic
amphipathic helix (residues 16-29), as well as 11 consecutive
acid residues (43-53), 11 consecutive histidines (70-80),
and a region rich in glutamine and proline (81-100).
Interestingly, deletion of the consecutive acid stretch or the
consecutive histidine stretch had no affect on transcriptional
activation by YY1 (Fig. 5), indicating that these sequences are
not required for transcription. On the contrary, deletion of amino
acids 16-29 caused significant decreases in transcription and
residues 80-100 contribute to maximal activation potential.
Therefore, the YY1 transcriptional activation domain appears to be
bipartite and includes sequences with the potential to form an acidic
amphipathic helix and sequences rich in proline and glutamine. These
features are commonly observed in activation domains of other
transcription factors (34) . It is interesting that the Xenopus laevis YY1 protein lacks the consecutive histidine
stretch but contains both amino acid segments that we determined to be
involved in transcriptional activation(35) . It will be
interesting to determine whether the Xenopus YY1 homologue can
function as a transcriptional activator similar to the human form.The YY1 Repression Domain
Previously, the YY1
repression domain was shown to reside within the carboxyl-terminal
region of the protein(1, 32, 33) . Our
studies in 3T3 fibroblasts are consistent with these results and more
precisely identify sequences important for repression. We found that
deletion of the carboxyl-terminal 17 amino acids (to residue 397) was
not sufficient to convert YY1 into a strong activator. Deletion of an
additional 27 amino acids to residue 370 resulted in strong activation
in 3T3 cells. These results indicate that sequences within the 27 amino
acids between residues 370 and 397 inhibit YY1 activation potential.
Consistent with these results, fusion of YY1 sequences 201-414 or
333-414 to the GAL4 DNA binding domain resulted in repression of
a GAL4 responsive reporter. These results suggest that the repression
domain lies within the 65 amino acids between residues 333 and 397. 371-380, which disrupt the structure of the third zinc
finger. Neither the C360S nor the 371-380 mutants mimicked
the strong transcriptional activity of the 370 deletion mutant. These
results indicate that repression does not absolutely require the normal
structure of either the third or the fourth zinc fingers.Protein Interactions with YY1
Our effector plasmid
titration experiments (Fig. 1) suggest that YY1 function may be
influenced by protein interactions. Similar concentration-dependent
function of transcription factors has been observed (58, 59) and is believed to result from
protein-protein interactions (reviewed in (60) ). YY1 has been
observed to physically interact with Sp1, and this interaction
apparently is important for stimulation of transcription (61, 62) . In addition, a two-hybrid screen in yeast
showed that c-Myc can interact with YY1 and that repressive affects of
YY1 can be relieved by c-Myc expression. Interestingly, a similar
two-hybrid screen identified the nucleolar phosphoprotein B23, which
can also relieve YY1-induced transcriptional repression(63) .
The physical interaction between YY1 and Sp1 requires YY1 amino acid
residues 260-331, and YY1 residues 201-343 are required for
interaction with c-Myc(20) . Neither of these protein
interaction sequences maps to the YY1 repression domain that we have
characterized here. However, it is interesting that the adenovirus E1A
protein, which can relieve repression mediated by YY1 (1) can
physically interact with YY1 residues 332-414(64) .
Recently, it was also determined that E1A-mediated relief of YY1
repression requires the cAMP response element-binding protein-related
factor, p300(65) . This protein is also capable of physically
interacting with YY1(65) . Further studies will be necessary to
identify additional cellular proteins that interact with YY1 and
influence the mechanisms of YY1 repression and activation.
))
We are grateful to F. Rauscher for plasmid pCB6+,
to members of the laboratory for helpful discussions, and to N. G.
Avadhani, R. Perry, and T. Kadesch for useful comments.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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