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INTRODUCTION |
Yin Yang 1 (YY11;
previously referred to as FIII, NF-E1,
, F-ACT1, CF1, or UCRBP) is a
GLI-Kruppel family transcription factor that functions either as a
repressor, activator, or initiator of transcription (reviewed in Ref.
1). It is expressed in a wide variety of different cell types and shows
a high degree of sequence similarity among divergent organisms (2-5).
The mammalian YY1 protein (414 aa) contains four C-terminal zinc
fingers, an N-terminal bipartite transcriptional activation domain, and
a number of domains that function to mediate interactions with other proteins (6). The Xenopus YY1 cDNA is highly conserved,
but the predicted protein is somewhat smaller at 373 aa (4).
Numerous studies have revealed that YY1 is subject to complex
regulatory mechanisms in different cell types. The emerging picture is
that the functional diversity of YY1 as an activator, repressor, or
initiator of transcription is context-specific and is modulated by
interactions with various cellular factors and adaptor proteins. YY1
has been shown to interact physically with a number of proteins
including c-Myc (7, 8), p300/CBP (9), CREB (10), ATF/CBP (11),
poly(A)DP-ribosyl transferase (12), histone deacetylase (13), the E1A
oncoprotein (2), and a number of general transcription factors (14,
15).
A variety of tissue culture studies have shown that YY1 controls
expression of developmentally regulated genes, and so it is thought to
play important roles in development and differentiation (reviewed in
Ref. 1). Recognition sites for YY1 have been identified in the
regulatory regions of transferring (15), dystrophin (16), and actin
(17-23) genes expressed in differentiating and aging muscle, fetal and
embryonic globin genes (24-27), the immunoglobulin
3 gene expressed
in pre-B cells (28), and the pre-T-cell
receptor gene (29). YY1
contributes to the tissue-specific regulation of the prosacrosin gene
in testes (30) and to the osteocalcin gene in bone (31). In bone the
activity of YY1 appears to be controlled by targeting to the nucleolus
and nuclear matrix, (32, 33). In developing muscle, proteolytic
processing of YY1 is thought to be responsible for cell-specific
derepression of sarcomeric
-actin genes (23). Despite its apparent
association with cell growth and differentiation in diverse cell types,
however, very little is known about the expression and regulation of
YY1 in vertebrate oocytes and embryos, and the biological role of YY1 during early vertebrate development is poorly understood. A recent study of YY1 in mouse development revealed that it is abundantly expressed in oocytes and early embryos, displays a dynamic pattern of
subcellular localization in early development, and is selectively expressed in certain tissues later in development (34). Because heterozygous mutation of YY1 in the mouse resulted in growth and neurulation defects, and full knockout caused embryonic lethality around implantation, it appears likely that YY1 functions at multiple stages of mouse development (34).
The objective of the present study was to identify and characterize YY1
in the Xenopus developmental model system. We found that YY1
mRNA and protein were expressed in early oocytes and were present
at relatively constant levels throughout oocyte and embryonic
development. The ability of YY1 to interact with its cis-recognition
site was restricted to very early oocytes and to post-MBT stages in
embryos, suggesting that the DNA binding activity of the maternal
factor is modified specifically during development. Expression assays
showed that YY1 had no effect on transcription from the thymidine
kinase (TK) promoter in oocytes, but we also found a similar lack of
transcriptional activity in post-MBT embryos at a point in development
when its DNA binding is reactivated. Analysis of the subcellular
localization of YY1 showed it is sequestered in the cytoplasm of
oocytes and in cells throughout the embryo up to as late as the neurula
stage of development. We suggest that cytoplasmic sequestration is a
mode of developmental regulation used to inhibit the transcriptional
activity of YY1 during early stages of Xenopus development
and that YY1 probably functions in cell differentiation events that
occur later in development.
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EXPERIMENTAL PROCEDURES |
Oocyte and Embryo Manipulations--
Xenopus laevis
frogs were purchased from Xenopus I (Ann Arbor, MI). Oocytes
were obtained surgically from adult female frogs, and follicle cells
were removed by swirling in calcium-free OR2 (82.5 mM NaCl,
2.5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM
NaH2PO4, 5 mM Hepes (pH 7.8) 10 mg/liter streptomycin sulfate, 10 mg/ liter benzyl penicillin)
containing 2 mg/ml collagenase (type II, Sigma) for 3 h. Oocytes
were washed extensively in OR2 (containing 1 mM
CaCl2 (35) prior to microinjection or preparation of cell extracts. For expression of YY1 in oocytes, 200 pg of in
vitro synthesized mRNA was injected directly into the
cytoplasm, and oocytes were incubated for 16 h prior to
preparation of protein extracts. Expression plasmid encoding the
Xenopus YY1 mRNA (4) was a kind gift from E. Beccari,
(Universita di Roma La Sapienza, Rome, Italy) Embryos were
obtained as described in (36).
Tissue and Cell Extracts--
For protein extracts, oocytes or
embryos were homogenized in Buffer C (50 mM Tris-Cl, pH
7.9, 20% glycerol, 50 mM KCl, 0.1 mM EDTA, 2 mM dithiothreitol, 10 µg/ml each of aprotinin and
leupeptin (36), cell debris was pelleted for 5 min at 15,000 × g (4 °C), and the supernatants were placed in fresh tubes
and frozen in liquid nitrogen. Embryos or stage V and VI oocytes were
counted and homogenized in a volume of 10 µl/embryo/oocyte. To obtain approximately equal protein concentrations, oocytes at stages III and
IV were homogenized in 4 µl/oocyte, stages II and III in 2.5 µl/oocyte, and stages I and II in 1 µl/oocyte. Nuclear and
cytoplasmic extracts of embryos were performed as described by Lemaitre
et al. (37).
For A6 protein extracts, cells were washed in cold PBS, pelleted, and
homogenized by freeze-thawing in a buffer containing 20 mM
HEPES, 25% (v/v) glycerol, 0.42 M NaCl, 1.5 mM
MgCl2, O.2 mM EDTA, 0.5 mM
phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol. The homogenate was centrifuged at 10,000 × g in
Eppendorf tubes, and supernatants were stored at
80 °C.
Rat liver nuclear extracts were prepared as previously described (38).
Rat livers were chopped into small pieces and homogenized in a buffer
containing 10 mM HEPES, pH 7.6, 25 mM KCl, 2 M sucrose, 10% v/v glycerol, 1 mM EDTA, 0.15 mM spermine, and 0.5 mM spermidine using a
Dounce homogenizer. Homogenates were layered onto sucrose cushions and
centrifuged for 20 min at 24,000 rpm in a SW28 rotor. Nuclear pellets
were resuspended in nuclear resuspension buffer (10 mM
HEPES, pH 7.6, 100 mM KCl, 3 mM
MgCl2 0.1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 10%
glycerol, 1 nM microcystin) supplemented with 0.3 g/ml
(NH4)2SO4, shaken for 30 min, and homogenized
with a Dounce homogenizer; they were then centrifuged at 50,000 rpm in
a Ti70 rotor. Protein pellets were resuspended in nuclear resuspension
buffer and dialyzed to 40 mM KCl.
Nucleic Acid Analyses--
Total nucleic acids were extracted
from oocytes or embryos as described (39), by homogenization in Buffer
A (50 mM Tris-HCl, pH 7.6, 50 mM NaCl, 10 mM EDTA, 0.5% SDS) containing 0.25 mg/ml proteinase K. The
homogenate was incubated at 37 °C for 1 and extracted with an equal
volume of phenol/chloroform, and nucleic acids were recovered by
precipitation after the addition of 0.1 volume of5 M
ammonium acetate and 3 volumes of ethanol. Ethanol precipitates
containing total nucleic acids were resuspended in 400 µl of TE (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). To separate DNA from total RNA, 400 µl of 8 M LiCl was added, and the
mixture was incubated at 4 °C overnight. The RNA was pelleted at
15,000 × g for 10 min, resuspended in TE, and stored
as ethanol precipitates. DNA was recovered by precipitation after the
addition of 3 volumes of ethanol to LiCl supernatants. Northern blots
were performed as described previously (40). Blots were prehybridized
overnight at 42 °C and hybridized overnight at 55 °C in
hybridization fluid (50% formamide, 0.8 M NaCl, 0.8 M sodium citrate, 10 mM sodium phosphate, 2.5×
Denhardts', 250 mg/ml salmon sperm DNA) containing 106 cpm probe. The probe was the cDNA insert of the
plasmid encoding Xenopus YY1 mRNA (4) isolated by
BamHI-HindIII digestion.
Gel Mobility Shift Assays--
DNA binding reactions using
embryonic or stages V and VI oocyte extracts contained 1 embryo or
oocyte equivalent. Binding reactions for early stage oocyte samples
contained 2.5 oocyte equivalents of stage III an IV extracts and 10 oocyte equivalents of the batch containing stage I and II oocytes. The
YY1 recognition element probe from the mouse H3.2 gene,
(5'-ggatcCTCGGCCGTCATGGCGCTGCAGGAGGC-3') was end-labeled with
[
-32P]dCTP with the Klenow fragment of DNA polymerase
I. The sequence of AP-1 oligonucleotide probe is
(5'-CCGGAAAGCATGAGTCAGACAC-3'). Each binding reaction contained 0.2 ng
of double-stranded labeled probe, 20 µg of soluble protein from
indicated samples, 0.5 µg of poly(dI-dC), 10 mM Tris (pH
7.8), 50 mM NaCl, 1 mM EDTA, 0.5 mM
dithiothreitol, and 5% glycerol. Binding reactions were incubated on
ice for 20 min and immediately loaded onto 5% nondenaturing polyacrylamide gels containing 6.7 mM Tris-Cl (pH 7.5), 1 mM EDTA, 3.3 mM sodium acetate. Complexes were
electrophoresed for 2 h at 150 V, and gels were dried and exposed
overnight to x-ray film.
Cell Culture, Stable Transfections, and CAT Assays--
A6 cells
were grown in L15 medium (Life Technologies, Inc.) supplemented with
10% fetal bovine serum (Hyclone) as described previously (41). Chinese
hamster ovary (CHO) cells were grown in McCoy's 5A medium supplemented
with 10% calf serum, and stable transfections were performed as
described previously (5). CAT assays for CHO cells were performed with
the CAT enzyme assay system (Promega) using liquid scintillation
counting as described in the Promega technical bulletin, part no.
TB084. Microinjections and CAT assays for expression in oocytes were
performed as described previously (42).
Chromatography--
A6 cell extracts (300 µl) were applied to
2 ml of heparin-agarose columns (Sigma) at 4 °C. Columns were
equilibrated to 50 mM KCl with DC50 buffer (20 mM HEPES, pH 7.9, 0.2 mM EDTA, 0.2 mM EGTA, 50 mM KCl, 20% v/v glycerol, 2 mM dithiothreitol, 0.5 mM ZnCl) and proteins
were eluted with a stepwise gradient of DC buffer at 50, 250, and 400 mM KCl. At each step, 2 ml of buffer was applied to the
column, and 200 µl fractions were collected.
Immunocytochemistry--
Ovary portions were fixed by freeze
substitution with isopentane in liquid nitrogen and transfer to
methanol at
80 °C for 3 days and were then infiltrated with molten
paraffin (60 °C overnight) and embedded at room temperature. Staged
embryos where collected and fixed overnight at 4 °C in 0.1 M phosphate-buffered saline (PBS) containing 2.5% (v/v)
gluteraldehyde. Embryos were dehydrated by 30-min incubations in graded
ethanol solutions (2× 70% ethanol, 2× 95% ethanol, 2× absolute
ethanol, 2× xylene/ethanol (1:1), 2× 100% xylene) at room
temperature. Embryos were infiltrated by incubation (30 min, 60 °C)
in xylene/paraffin (1:1) and were then placed in 100% paraffin (30 min, 60 °C) and embedded at room temperature.
Ovary and embryo sections were cut to 8 µm, and then sections were
placed on glass slides and incubated overnight at 35 °C. Sections
were de-paraffinated and rehydrated by rapid immersions in a series of
xylene/ethanol/PBS solutions (100% xylene, xylene/ethanol (1:1),
absolute ethanol, 95% ethanol, 70% ethanol) and then immersed for 3 × 30 s in 1 M PBS and 30 min in 1 M PBS at 4 °C. All subsequent steps were carried out at
room temperature. Sections were incubated for 30 min in block solution
(3% skim milk, 1% Triton X-100, 1 M PBS), and then
primary antibodies were applied to slides at a dilution of 1:200 in 1%
milk block solution for 1 h. Primary antibodies were rabbit
anti-human YY1 polyclonal (Santa Cruz sc-281) and mouse anti-human
proliferating cell nuclear antigen (PCNA) monoclonal (Santa Cruz
sc-56). Control sections were treated with block solution without
primary antibody. Sections were washed (2 × 10 min) in 1 M PBS, incubated in block solution (30 min) and then
exposed to secondary antibody (1:200, 30 min). Secondary antibodies for
detection of YY1 were biotinylated goat anti-rabbit IgG and for PCNA,
biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame,
CA). Slides were washed in 1 M PBS (2 × 10 min),
methanol, 0.3% H2O2 (1 × 15 min) and
then in 1 M PBS (2 × 10 min), blocked in 3% milk,
PBS (30 min), treated for 1 h with avidin solution (Vector
Laboratories), and washed in 1 M PBS (2 × 10 min).
Slides where then bathed in 0.175 M sodium acetate (pH 7.8)
for 10 min and stained (5 min) with nickel/diaminobenzidene solution
(1.8 × 10
3 M
diaminobenzidene, 0.085% H2O2, 0.16 M nickel sulfate, 0.175 M sodium acetate, pH
7.8). Sections were washed (10 min) in 0.175 M sodium
acetate (pH 7.8), rinsed (5 min) in H2O, and then
dehydrated using graded solutions of ethanol/xylene (as above).
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RESULTS |
Identification of YY1 and Its DN Binding Activity in
Xenopus--
In a previous study, we reported a DNA binding activity
in Xenopus embryos specific to the internal coding region
activator
element of replication-dependent histone
genes, but the proteins in these complexes were not identified (36).
Subsequently, a yeast one-hybrid screen of a HeLa cell cDNA library
established that the protein interacting with the histone regulatory
sequence in mammalian cells was YY1 (5). To confirm the identity of the
Xenopus factor, we performed gel mobility shift assays using the
probe, which contains the YY1 consensus sequence, in
combination with antibody recognition in vitro using
polyclonal anti-YY1 antibodies (Fig.
1A). The addition of YY1
antibodies (sc-281, Santa Cruz Biotechnology) to binding reactions with
crude whole cell extracts from the A6 Xenopus kidney
epithelial cell line or neurula stage (stage 13) embryos disrupted the
specific complex. Similar YY1 complexes and specific antibody
disruptions were observed with rat liver nuclear extracts (Fig.
1A). The slower migrating nonspecific complexes were not
recognized by YY1 antibodies, and migration of the YY1 complex was not
affected in controls using antibodies against other proteins. Thus, it
appears that YY1 binds to and retards the mobility of the
probe in
Xenopus cell extracts.

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Fig. 1.
Identification of YY1 in Xenopus
cells and embryos. A, upper panel, gel
mobility shift assay of rat liver nuclear (RLN), A6, and
neurula stage embryo (St 13) extracts with the YY1
element. Antibodies were added to binding reactions as indicated above
the panel. The positions of nonspecific (NS) and specific
(YY1) bands are indicated on the left.
lower panels: left, Western immunoblot of YY1 in
extracts used for gel mobility shifts; right, Western blot
of YY1 in oocytes that were not injected or microinjected with mRNA
encoding Xenopus YY1. B, gel mobility shift assay
(upper panel) and immunoblot (lower panel) of A6
cell fractions separated on a heparin-agarose column with increasing
KCl concentrations. Fraction numbers are indicated above
each panel. FT is the flow-through fraction.
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The same cell extracts used for mobility shift assays were analyzed on
Western blots using YY1 antibody (Fig. 1A, lower left panel). The amino acid sequence alignment with peptide used for antibody sc-281 is 100% homologous between the human and frog YY1. A
protein at 59 kDa was detected in A6 cells and neurula stage embryos
(stage 13), and a slightly larger band at 65 kDa was detected in rat
liver nuclei. Microinjection of in vitro synthesized mRNA encoding Xenopus YY1 into oocytes resulted in an
increased detection of the 59 kDa band (Fig. 1A, lower right
panel), evidence that the mammalian antibody actually detected the
frog protein in Western blots. The sizes of both the frog and rat YY1
on the blot are larger than predicted from the number of amino acids (41 and 45.5, respectively). Anomalous migration has also been observed
for the human protein, which migrates as a 65-68-kDa protein
although its predicted size is 44 kDa; this difference is thought
to be due to protein structure (1). The protein expressed in both
embryos and somatic cells of Xenopus was slightly smaller
than mammalian YY1; this is consistent with the relative predicted
protein sizes of 373 aa for Xenopus (4) versus
414 aa for rat (2, 5).
We next analyzed the YY1 factor by fractionation of Xenopus
A6 kidney epithelial cell extracts on heparin-agarose columns. Cell
extracts were loaded onto columns and eluted in a stepwise KCl
gradient. The majority of specific YY1 element binding activity eluted
at 400 mM (Fig. 1B, upper panel). Western blot
analysis of column fractions using the YY1 polyclonal antibody showed
that the 59-kDa polypeptide appeared most prominently in the fractions containing peak DNA binding activity. The correlation between the
appearance of the YY1 signal and specific DNA-binding complexes shown
in fractions (Fig. 1B), as well as the supershift of
DNA-binding complexes (Fig. 1A), confirm the identity of the
YY1 transcription factor in Xenopus extracts. Some YY1 was
detected in fractions eluted at 250 mM salt, but there was
no corresponding gel shift activity. It is possible that cofactors
required for efficient DNA binding were not present in the lower salt
fractions, or it could be that the Western blot detection of YY1
protein is much more sensitive than detection of gel shift activity in
these assays.
Expression of YY1 during Early Development--
Previous
descriptions of YY1 as an important factor in cell growth and
differentiation prompted us to examine the profile of YY1 mRNA
accumulation during oocyte and embryonic development. Northern blots
were performed on total RNA isolated from oocytes and embryos at
various stages of development (Fig.
2A). A 4-kilobase mRNA
species was detected with the YY1 cDNA probe. A relatively constant
amount of YY1 mRNA signal was detected in early and late stage
oocytes, unfertilized eggs, in cleavage, blastula and early neurula
embryos, and in swimming larvae. Because the zygote does not become
transcriptionally active until midblastula stage (43), it appears that
transcription of the maternally encoded YY1 gene occurs very early
during oogenesis and maternal YY1 transcripts are relatively stable
through fertilization and early embryonic development. The YY1 mRNA
species detected in Northern blots migrated just below the 28 S
ribosomal RNA bands. To rule out the possibility of cross-hybridization
to ribosomal RNA, we tested for hybridization of the YY1 probe in
poly(A)+ and poly(A)
fractions isolated over
oligo(dT) columns. The 4-kilobase YY1 mRNA signal was detected in
poly(A)+ samples containing mRNA but not in the
poly(A)
sample containing ribosomal RNA (Fig.
2A).

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Fig. 2.
Expression of YY1 during Xenopus
development. A, Northern blot analysis of YY1
mRNA expression during development. Each lane contains 10 µg of
total RNA isolated from developing oocytes (stages indicated in
Roman numerals), unfertilized eggs (E), and
embryos (Arabic numerals). Positions of RNA markers are
shown on the left. B, Western immunoblot of
staged extracts from eggs (E) and embyros at various stages
of development. C, Western immunoblot showing a comparison
of YY1 expression in early and late oocytes and at stages of
development around fertilization. D, Western immunoblot
showing a comparison of YY1 expression during each stage of oogenesis.
Positions of protein molecular weight markers are indicated at the
left of the panels.
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We next characterized the profile of YY1 protein expression during
development. In the Western blots shown in Fig. 2B,
polyclonal anti-YY1 antibodies were used to detect YY1 in staged whole
cell extracts of oocytes and embryos. A single prominent polypeptide migrating at 59 kDa was detected in unfertilized eggs and in developing embryos from early cleavage through to neurula stages. As with the
profile of mRNA expression, there were no significant changes in
the relative amounts of YY1 protein signal detected throughout these
phases of development. We also examined expression of YY1 during
oogenesis and through oocyte maturation and fertilization; the Western
blot to compare YY1 in oocytes, eggs, and early embryos is shown in
Fig. 2C. Similar quantities of the YY1 signal were seen in
stage VI oocytes, in unfertilized eggs, and at cleavage stage. However,
in early oocytes, YY1 appeared to be significantly smaller at 50 kDa. A
more detailed comparison at each stage of oogenesis showed that YY1 was
predominantly a 500-kDa protein in early oocytes, but by stage V1 the
majority of YY1 migrated at 59 kDa (Fig. 2D).
DNA Binding Activity of YY1 during Oocyte and Embryonic
Development--
To examine the profile of YY1 DNA binding activity
during development, crude whole cell extracts were prepared from staged oocytes, eggs, and embryos and analyzed in gel mobility shift assays
using the
probe with the YY1 recognition sequence (Fig. 3A). The specific YY1 complex
was detected in stage I and stage II oocytes and in embryos after the
MBT, but it was absent in the intervening stages. The addition of YY1
antibodies to DNA-binding reactions disrupted band formation,
confirming the presence of YY1 in shifted complexes in these assays.
The activity of other transcription factors are known to be stable
through this period of development (44), and the DNA binding activity
of AP-1 transcription factor is stable in the same extracts (Fig.
3A, lower panel). In addition, the changes in YY1 DNA
binding activity through early development were not due to fluctuations
in the levels of maternal protein, because the same extracts were used
in the Western blots above (Fig. 2) in which relatively constant
amounts of YY1 were detected. Also of note is that DNA binding activity
was down-regulated in oocytes at stages III-V (Fig. 3A),
and so there did not appear to be a correlation between reduced DNA
binding activity and the change in molecular mass from 50 to 59 kDa that occurred between stages V and VI.

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Fig. 3.
Alterations in the DNA binding activity of
YY1 during development. A, upper panel, gel mobility
shift analysis showing relative YY1 activity in staged oocyte and
embryonic extracts. The addition of competitor YY1 antibody to DNA
binding reactions is indicated below the panel. Lower panel,
gel mobility shift showing relative AP-1 binding activity during
development. The addition of unlabeled competitor oligonucleotides
(50-fold molar excess) to DNA binding reactions is shown
below the panel. B, stage VI extracts were
treated with calf intestinal phosphatase, and the size of YY1 was
compared directly by Western immunoblot (left panel) using
YY1 polyclonal antibody. Aliquots of the same stage VI extracts were
analyzed by gel mobility shift assay (right panel) with the
YY1 probe.
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Previous studies have provided evidence suggesting that the
phosphorylation state of YY1 can affect its DNA binding activity (5).
We examined whether phosphorylation might affect YY1 during development. Phosphatase treatment of stage VI extracts resulted in a
partial recovery of DNA binding (Fig. 3B). This suggests that YY1 binds more efficiently in the hypophosphorylated state, and
that the DNA binding activity of YY1 might be differentially regulated
by phosphorylation during oocyte development. We also considered the
possibility that the increase in the apparent molecular weight of YY1
in late stage oocytes could be due to increased phosphorylation. On
Western blots, however, YY1 was detected at 59 kDa in both untreated
and phosphatase-treated extracts (Fig. 3B). This suggests
the increase in apparent molecular weight of YY1 at stage VI is not
attributable to phosphorylation.
Transcriptional Activity of YY1 in Xenopus Embryos--
We next
examined the transcriptional activity of YY1 using a series of
microinjected reporter constructs containing the thymidine kinase (TK)
promoter linked to YY1 binding sites. Reporter constructs (Fig.
4A) contained either 6 consecutive YY1 elements in forward (YY1-TKCAT) or reverse orientation
(YY1R-TKCAT) immediately upstream of the TK promoter, 6 repeats of a
mutant YY1 binding site (YY1M-TKCAT), or a single YY1 binding site from
the histone H2a coding region activator sequence (CRAS-TKCAT). CAT
expression was analyzed in embryos that were allowed to develop to
gastrula stage at 4 h past MBT (Fig. 4B).
Insertion of YY1 elements did not significantly stimulate or repress
activity from the TK promoter in post-MBT embryos. In numerous repeats
of this experiment, there were no significant changes in CAT activity
relative to controls. In control experiments using CHO cells, we
determined that YY1 was able to influence transcription in context of
the intact TK promoter (Fig. 4C). YY1 elements stimulated a
3-fold activation over the basal promoter, indicating that these
constructs respond to YY1. Therefore, in Xenopus it appears
that YY1 is transcriptionally inactive in post-MBT embryos. Similar
results were obtained in expression experiments with stage VI oocytes
(data not shown), which was expected given the low level of YY1 DNA
binding activity at later stages of oocyte development (Fig.
3A). Our findings are in agreement with previously reported
experiments showing the YY1 binding site from L1 and L14 ribosomal
protein genes did not affect transcription in Xenopus
oocytes (45).

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Fig. 4.
Transcriptional activity of YY1 in post-MBT
embryos. A, diagram of plasmid constructs containing
the CAT gene driven by the TK promoter and YY1 elements as described
under "Experimental Procedures." B, embryos were
microinjected after fertilization with the indicated plasmid constructs
and then allowed to develop to gastrula stage, at which point CAT
assays were performed (upper panel). The experiment was
repeated at least five times with different batches of embryos; a
representative CAT assay is shown. CAT activity is expressed as a
percentage of the highest relative activity (TK-CAT), which
was arbitrarily assigned a value of 100 (lower panel).
C, CAT assays of stable transfectant CHO cells. Constructs
are indicated above the panel. Activity is presented as percent
acetylation. The experiment was performed with triplicate sets of
plates.
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YY1 Is Localized in the Cytoplasm of Oocytes and Early
Embryos--
It was recently shown that YY1 is localized in the
cytoplasm of unfertilized mouse oocytes and displays a mosaic pattern
of subcellular localization in E3.5 blastocysts (34). To determine the
subcellular location of YY1 during oogenesis, we enucleated oocytes and
analyzed nuclear and cytoplasmic fractions for the presence of YY1 by
Western blotting. At both stages IV and VI, YY1 was detected in the
cytoplasm but was absent in nuclear extracts (Fig.
5A). This finding clearly
demonstrates that YY1 is cytoplasmic in late stage oocytes and suggests
that the increase in molecular mass from 50 to 59 kDa, which occurs
between stage VI and stage VI, is not associated with nuclear
relocalization.

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Fig. 5.
Subcellular localization of YY1 in
oocytes. A, Western immunoblot of YY1 and PCNA in stage
IV and stage VI oocytes that were manually dissected into nuclear
(N) and cytoplasmic (C) fractions. W,
indicates whole cell extract. B, gel mobility shift analysis
of YY1 binding activity in whole cell, cytoplasmic, and nuclear
fractions. Phosphatase treatments were done with 1 unit of calf
intestinal alkaline phosphatase for 20 min on ice immediately prior to
DNA binding reactions. The addition of antibodies for supershifts is
indicated below the panel.
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We also tested for DNA binding activity in both cytoplasmic and nuclear
compartments (Fig. 5B). Although YY1 does not normally bind
DNA in late stage oocytes, activity was induced by in vitro phosphatase treatment as demonstrated in Fig. 3B.
Phosphatase treatment of subcellular portions from stage IV and VI
oocytes revealed that YY1 DNA binding could be activated in cytoplasmic extracts but not in nuclear extracts (Fig. 5B). Supershift
controls showed that phosphatase-activated complexes were specifically recognized by YY1 antibodies. These experiments confirm the findings of
Western blots (Fig. 5A) showing that YY1 is sequestered in the cytoplasm in stage VI and VI oocytes.
The DNA binding activity of YY1 is present in stage I oocytes, switched
off, and then reactivated after MBT (Fig. 3A). This pattern prompted us to examine a possible correlation between DNA
binding and nuclear localization during development; i.e. DNA binding might be associated with nuclear localization. We hypothesized that YY1 would be a nuclear protein in early oocytes, relocalize to the cytoplasm in later stage oocytes, remain cytoplasmic in the embryo during cleavage and early blastula stages, and then migrate back into nuclei after MBT. To test this hypothesis, we performed an immunocytochemical analysis of developing oocytes and
embryos (Fig. 6). We used the same YY1
antibody as described above for Western blots and mobility supershifts.
As a positive control we used an anti-PCNA monoclonal antibody, which
resulted in nuclear staining in all sections tested. Negative controls were done in which secondary antibodies alone were applied to oocyte
and embryonic sections for detection of background staining (data not
shown). As a positive control, YY1 antibody was applied to adult
Xenopus liver sections, because we had already observed high
levels of DNA binding activity in nuclear extracts from rat liver (Fig.
1). A prominent signal was detected in liver nuclei, but YY1 appeared
to be localized entirely in the cytoplasm in both early (stage I) and
late (stage VI) stage oocytes. A cytoplasmic staining pattern was also
observed in cells of developing embryos. YY1 did not appear in the
nuclei of cleavage stage embryos, nor in gastrula stage embryos several
hours past the MBT, nor in neurula stage embryos at a point in
development 12 h past the onset of zygotic transcription.

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Fig. 6.
YY1 is a cytoplasmic protein in
oocytes and embryos. An immunohistochemical analysis of YY1 and
PCNA in oocytes and embryos was done. Oocyte and embryo stages
are indicated at the left of each series of panels. In the
stage 9 embryo panels, the dorsal part of the embryo is shown. In the
neurula panels, the dorsolateral part of the embyro is shown. The
bottom panels show YY1 localization in hepatocyte nuclei of
adult frog rat liver.
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The finding that YY1 DNA binding activity appears after the MBT (Fig.
3) but appears to remain cytoplasmic (Fig. 6) was surprising and
interesting. To investigate this conclusion further and to confirm the results of immunostaining, we tested for the presence of
YY1 in biochemically produced nuclear and cytoplasmic fractions from
stage 13 neurula embryos. The Western blots shown in Fig. 7 show that YY1 was entirely cytoplasmic.
In a control blot using the same fractions, the PCNA antigen was
detected only in nuclear and whole cell samples. Thus, it appears that
YY1 remains sequestered in the cytoplasm during early embryonic
development and does not contribute to early developmental gene
expression.

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Fig. 7.
YY1 is detected only in cytoplasmic but not
nuclear extracts of neurula stage embryos. Western immunoblot
analysis of YY1 in whole cell (W), nuclear (N),
and cytoplasmic (C) fractions obtained from stage 13 neurula
embryos. An immunoblot of the same fractions using the PCNA
antibody is shown in the lower panel.
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DISCUSSION |
In this study we have demonstrated that YY1 is a maternally
derived protein that is present throughout early Xenopus
development. YY1 is expressed abundantly in early oocytes and, exists
as a cytoplasmic protein in oocytes and early embryos, and its DNA binding activity is restricted to early oocytes and to post-MBT embryos. At first, the developmental profile of DNA binding prompted us
to hypothesize that maternal YY1 might function to modulate expression
of specific target genes in stage I oocytes and once again after MBT
when expression from the zygotic genome commences in the embryo (46).
However, we found that YY1 binding sites had no effect on expression
from the TK promoter in oocytes or in post-MBT embryos, and so we
surmised that endogenous YY1 is transcriptionally inactive during early
development. The transcriptional inactivity of YY1 is consistent with
our finding that YY1 remains in the cytoplasm of oocytes and in cells
throughout the developing embryo well past MBT. Therefore, we suggest
that the potential function of YY1 as a transcriptional regulator is
specifically repressed by cytoplasmic partitioning during both
oogenesis and early embryonic development.
YY1 is a maternally expressed protein in unfertilized oocytes in both
mouse (34) and Xenopus. However, the subcellular
localization pattern of YY1 in Xenopus is somewhat different
from that in mouse development. Donohoe et al. (34) showed
that YY1 is cytoplasmic in unfertilized mouse oocytes and becomes
nuclear in two-cell embryos coincident with the onset of mouse zygotic
transcription, and they suggested that YY1 probably functions to
regulate gene expression early in mammalian embryonic development. No
such nuclear relocalization occurs in Xenopus embryos at
MBT, despite the apparent reactivation of its DNA binding activity at
this stage. Unlike mouse embryos in which YY1 becomes nuclear at the
2-cell stage coincident with the onset of zygotic transcription, there
is no apparent correlation between nuclear localization and the MBT in
Xenopus. Therefore, it would appear that YY1 does not have a
direct transcriptional regulatory function at the initial stages of
zygotic gene expression in Xenopus embryos or in early
development to the neurula stage.
The abundant maternal expression and subcellular localization reported
in this study suggest two hypotheses for the potential developmental
function of YY1. YY1 may function to regulate the transcription of
genes important for differentiation and organ formation, which occur
later in development. This has been suggested by numerous studies in
various tissue culture model systems (1) and is consistent with the
expression pattern of YY1 in mouse embryos and the phenotype of
heterozygous YY1 mutants (34). Alternatively, YY1 may play an important
role in the cytoplasm during oogenesis and early embryogenesis, perhaps
in the transport, storage, or translational regulation of maternal
mRNAs. Such a cytoplasmic function would be similar to that
proposed for the CCAAT box transcription factor (CBTF), which is
retained in the cytoplasm prior to MBT through its RNA binding capacity
and is associated with transcriptionally quiescent mRNP complexes (47). Nuclear relocalization of CBTF is associated with degradation of
maternal transcripts (47). Unlike CBTF, however, YY1 does not localize
to the nucleus after MBT, which would suggest that it might associate
with stable mRNAs. Further analyses will be aimed at determining
whether YY1 has RNA binding activity and whether association with mRNPs
might contribute to its cytoplasmic localization.
It is paradoxical that YY1 remains in the cytoplasm and yet displays
the differential ability to bind DNA through development, with peak
activity in very early oocytes and in post-MBT embryos. Because it is
unlikely that the extract preparation methods selectively activates DNA
binding at these stages, we suggest that maternally derived YY1 is
subject to post-translational modifications that occur in the cytoplasm
in vivo. It could be that YY1 becomes primed to activate
transcription but must receive the appropriate cellular signals to
trigger nuclear translocation in specific tissues sometime during later
in development.
What is responsible for changes in the DNA binding activity of YY1
during development? There was no apparent correlation between nuclear
localization and the ability of YY1 to bind DNA in gel mobility shift
assays. DNA binding is probably not regulated by subcellular
localization, because YY1 was retained in the cytoplasm throughout the
same developmental time frame over which YY1 fluctuates between
DNA-binding and non-DNA-binding forms. The ability to reactivate DNA
binding activity in late stage oocytes by in vitro phosphatase treatments suggests that YY1 may be negatively regulated in vivo by phosphorylation. However, we found no direct
evidence that the phosphorylation state of YY1 changes as oocytes
progress through developmental stages when DNA binding activity is lost or regained, and so the significance and mode of fluctuations in DNA
binding during early development remain unclear.
In summary, the data presented here suggest that the potential
transcriptional activity of YY1 is repressed specifically during oocyte
and embryonic development by sequestration to the cytoplasm. The
functional significance of changes in the DNA binding activity of YY1
is unclear, and the potential role of YY1 the cytoplasm during
development remains to be elucidated. Exclusion of YY1 from nuclei
during early development explains the results of expression analyses in
which YY1 binding sites had neither stimulatory nor repressive effects
on basal transcription from the TK promoter in oocytes and embryos.