Cloning and Characterization of Gonadotropin-Inducible Ovarian Transcription Factors (GIOT1 and -2) That Are Novel Members of the (Cys)2-(His)2-Type Zinc Finger Protein Family
Tetsuya Mizutani,
Kazuya Yamada,
Takashi Yazawa,
Toshinori Okada,
Takashi Minegishi and
Kaoru Miyamoto
Department of Biochemistry (Te.M., K.Y., T.Y., K.M.), Fukui Medical
University, Matsuoka, Fukui 910-1193, Japan; Department of
Obstetrics and Gynecology (T.O., Ta.M.), Gunma University School of
Medicine, Maebashi, Gunma 371-8511 and CREST (Te.M., K.Y., T.Y., Ta.M.,
K.M.), JST (Japan Science and Technology), Japan
Address all correspondence and requests for reprints to: Kaoru Miyamoto, Department of Biochemistry, Fukui Medical University, Shimoaizuki, Matsuoka, Fukui 910-1193, Japan. E-mail:
kmiyamot{at}fmsrsa.fukui-med.ac.jp
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ABSTRACT
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Gonadotropins are essential for ovarian follicular development and
differentiation. To identify genes that are rapidly induced by
gonadotropin in the immature rat ovary, ovarian genes were screened by
a subtraction cloning procedure. cDNA clones encoding novel members of
the (Cys)2-(His)2-type zinc finger protein
family GIOT1 and -2 (gonadotropin-inducible transcription factor 1 and
2), were identified. Two isoforms of GIOT2 (GIOT2
and 2ß), which
are probably produced by alternative splicing, also exist. Nucleotide
sequence analysis revealed that GIOT1, but not GIOT2, contains the
krüppel-associated box-A domain at the NH2 terminus.
RNA analyses revealed that these mRNAs were rapidly and temporarily
induced by gonadotropins in the rat testis as well as in the ovary.
In situ hybridization study revealed that expression of
GIOT1 was induced in theca interna cells in the ovary and Leydig cells
in the testis. Interestingly, the gene expression of GIOT1 is
restricted to the pituitary, adrenal, testis, and ovary, while GIOT2
gene is expressed ubiquitously. A functional analysis of GIOT1 and -2
by a GAL4-based mammalian one-hybrid system revealed that GIOT1, but
not GIOT2, is a transcriptional repressor and that the
krüppel-associated box-A domain of GIOT1 is responsible for the
transcriptional repressor activity. A GAL4-based yeast two-hybrid
system was also used to identify proteins that interact with the rat
GIOT1. We cloned genes encoding rat homologs of human I-mfa domain
containing protein and transcriptional intermediary factor 1 ß, both
of which are transcription-regulatory proteins. Interaction of these
proteins with GIOT1 was directly demonstrated by GST pull-down assay.
Our data strongly suggest that GIOT1 may function as a novel
transcriptional repressor by working with rat homologs of human I-mfa
domain containing protein and transcriptional intermediary factor 1ß
proteins and may play a significant role at the transcription level in
the folliculogenesis.
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INTRODUCTION
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GONADOTROPINS, LH AND FSH, are essential
for folliculogenesis in the ovary, and ovarian development can be
promoted by exogenously administrated gonadotropins
(1, 2, 3). PMSG, which has both LH- and FSH-like activities,
is known to strongly induce follicular growth and maturation
(4). The activation of certain genes by PMSG in ovarian
cells appears to be essential for the initiation of follicular growth.
In earlier studies we reported, using a subtraction cloning method, the
cloning of a number of rat ovarian genes, which are induced within
3 h by treatment with PMSG in immature rat ovaries. Of the
PMSG-inducible genes, we initially characterized those that are
involved in ovarian steroidogenesis, including carbonyl reductase,
scavenger receptor class B type I, and steroidogenic acute
regulatory protein (StAR) genes (5, 6, 7). In the present
study, we report on our efforts to identify PMSG-inducible
transcription factors that may be involved in ovarian
folliculogenesis.
It is well known that transcription factors regulate important cellular
processes, such as cell-lineage determination and cell growth and
differentiation, via the temporal or spatial gene expression of cell
type-specific genes (8, 9, 10). Gene transcription is
primarily regulated by the specific interaction of
trans-acting proteins and cis-acting DNA
sequences (11, 12). Some common structural motifs that are
useful in characterizing the DNA-binding properties of these
transcription factors have been identified, one of which is the
(Cys)2-(His)2-type zinc
finger motif. This motif defines a large superfamily of nuclear binding
proteins which, in a typical vertebrate genome, encompasses several
hundred structurally distinct members (13). Approximately
one-third of these proteins contain an evolutionarily conserved region
in the NH2 terminus, which is referred to as a
krüppel-associated box (KRAB) and which consists of about 75
amino acid residues (14).
In this study, we isolated two novel and closely related genes that
encode proteins with the
(Cys)2-(His)2-type zinc
finger motif. Since they are strongly induced in the ovary by
gonadotropin treatment, we refer to them as GIOT1
(gonadotropin-inducible ovarian transcription factor 1) and GIOT2,
respectively.1 Although both
GIOT1 and -2 belong to the
(Cys)2-(His)2-repeating
zinc finger family, GIOT1, but not GIOT2, contains a KRAB-A domain in
the NH2 terminus. The gene expression of GIOT1 is
restricted to the pituitary, adrenal gland, testis, and ovary, while
GIOT2 is expressed ubiquitously. We also report herein on the
functional analysis of GIOT1 and -2 by a GAL4-based mammalian
one-hybrid system, the findings of which suggest that the KRAB-A domain
of GIOT1 is responsible for the transcriptional repressor activity.
Finally, we cloned GIOT1-interacting proteins using a yeast two-hybrid
system. These clones encoded rat homologs of human I-mfa domain
containing protein (15) and transcriptional intermediary
factor 1ß (TIF1ß) (16, 17, 18, 19), respectively. Human I-mfa
domain containing protein and TIF1ß belong to families of
transcription factors and cofactors, respectively. The present findings
suggest that GIOT1 may play a significant role at the transcription
level on the folliculogenesis by working with the interacting
transcription factors or cofactors mentioned above.
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RESULTS
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Cloning of Rat GIOT1 and -2 cDNAs
PMSG-inducible ovarian genes were isolated via the PCR-based
subtraction cloning procedure as described previously (6, 7). Of the 386 clones that were partially sequenced, two novel
clones were identified as members of the zinc-finger type transcription
factors. To obtain a full-length cDNA that encodes for the novel
transcription factor-like protein, we screened a PMSG-primed immature
rat ovary cDNA library using the insert of the initially isolated
clones as the probe. Four independent clones were isolated. Nucleotide
sequence analysis revealed that all clones encode
(Cys)2-(His)2-type zinc
finger proteins. Two clones are essentially identical to one another
except for the length of the 5'-noncoding region. These clones have the
same open reading frame, which consists of 654 amino acid residues
(Fig. 1A
) and which we refer to as GIOT1.
GIOT1 contains 14 zinc finger motifs, and many residues within the
N-terminal 44 amino acids of GIOT1 are conserved to be characteristic
to KRAB-A, which is the evolutionarily conserved region at the
NH2 terminus of a number of
(Cys)2-(His)2 zinc finger
proteins (Fig. 1B
) (20, 21, 22, 23, 24, 25, 26, 27). The other two clones,
GIOT2
and -2ß, appear to be products from the same gene, which is
closely related to, but distinct from, the GIOT1 gene. GIOT2
contains an open reading frame that consists of 708 amino acid residues
with 18 zinc finger motifs. GIOT2ß lacks amino acid residues
corresponding to residues 268603 of GIOT2
(Fig. 1A
) and contains
six zinc finger motifs. As shown in Fig. 1A
, GIOT1 and -2 are
remarkably homologous, with 94% amino acid identity and an 85%
identity in nucleotide sequences, respectively, when compared with
GIOT1 and 2
.

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Figure 1. Deduced Amino Acid Sequence of GIOT1 and -2
A, Comparison of the deduced amino acid sequences between GIOT1 and
-2 . Asterisks indicate positions of identical amino
acid. A schematic illustration of structures of GIOT1, -2 , and -2ß
are shown on the bottom. The locations of the KRAB-A and
zinc-finger domains are depicted as solid and
striped boxes, respectively. B, Comparison of the KRAB-A
domain of GIOT1 with that of other zinc finger proteins
(20 21 22 23 24 25 26 27 ). The KRAB-A consensus sequence is derived from
Bellefroid et al. (14 ), where
uppercase letters represent highly conserved residues, and
lowercase letters represent moderately conserved residues.
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Tissue Distribution of GIOT1 and -2 mRNAs and Their Induction by
Gonadotropins
The tissue distribution and the hormonal regulation of GIOT1 and
-2 were examined by Northern blot analysis and by an in situ
hybridization study. A single transcript of about 3.2 kb for GIOT1 and
two major transcripts of about 7 kb and 11 kb for GIOT2 were detected
by hybridization with GIOT1- and GIOT2
-specific probes, respectively
(Fig. 2
). With respect to the GIOT2
transcripts, hybridization with the GIOT2ß-specific probe gave
essentially the same results as those by the GIOT2
-specific probe.
Both the GIOT1 and -2 transcripts were temporarily induced in the
ovaries of 21-d-old immature rats by the administration of PMSG. The
GIOT1 and -2 mRNA levels began to increase within 3 h, reaching
maximal levels at 6 h, and then declining at 12 h after PMSG
administration (Fig. 2
, A and C). Essentially identical results were
obtained when the immature rats were treated with hCG instead of PMSG
(Fig. 2
, B and D). In the case of hCG treatment, the mRNA levels of
GIOT1 and -2 were increased slightly more rapidly than that observed in
the case of PMSG treatment.
The in situ hybridization study clearly revealed that GIOT1
gene expression was rapidly and strongly induced practically in all
ovarian theca interna cells surrounding follicles by the administration
of either PMSG or hCG (Fig. 3
). A very
weak staining was also observed in the granulosa cells when stimulated
with PMSG. Before stimulation, a weak staining was observed in theca
interna cells. The cellular localization and induction pattern of GIOT1
was consistent with results obtained from the Northern blot
analysis.

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Figure 3. In situ Hybridization of GIOT1 in
the Immature (21-d-old) Rat Ovaries
Ovaries from 21-d-old rats were dissected and sectioned and hybridized
with a digoxigenin-labeled antisense GIOT1 probe. Sections of ovaries
from 21-d-old immature rats were primed as follows: A, with PMSG (30
IU) for 6 h, x80; B, with hCG (50 IU) for 3 h, x80; C, with
no treatment, x80; D, with PMSG (30 IU) for 6 h hybridized with a
sense strand cRNA probe, x80.
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To determine whether the gene expression of GIOT1 and -2 is also
induced in vitro, rat ovarian granulosa cells were cultured
in the presence or absence of FSH. The level of detectable GIOT1 mRNA
before the FSH treatment was negligible. However, the mRNA levels
rapidly and strongly increased within 1 h, reaching maximal levels
2 h after the addition of FSH to the culture (Fig. 4A
). A similar pattern of induction was
observed in GIOT2 gene expression, although low levels of GIOT2 mRNA
could be detected before FSH treatment (Fig. 4B
). These results
indicate that the expression of GIOT1 and -2 were also induced in
ovarian granulosa cells by FSH in vitro.

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Figure 4. Expression of GIOT1 and -2 in Cultured Rat
Granulosa Cells
A, Northern blot analysis of GIOT1 mRNA in cultured rat granulosa cells
treated with FSH. Total RNA was isolated at the indicated times after
the addition of FSH. Ten micrograms of total RNA in each lane were
hybridized with 32P-labeled GIOT1 cDNA. The blots were then
rehybridized with a radiolabeled rat GAPDH cDNA. B, RT-PCR was
performed using GIOT2 -specific primers. Total RNA was isolated from
the granulosa cells at the indicated times after the addition of FSH.
Two micrograms of total RNA were reverse-transcribed and a portion
(1/100) was subjected to the PCR reaction for the specific
amplification of GIOT2 and GAPDH, respectively. The reaction
mixtures were separated on a 2% agarose gel and visualized by EtBr
staining.
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We also examined the gene expression of GIOT1 and -2 in the male
system. The expression of GIOT1 and -2 were detected by RT-PCR. As
shown in Fig. 5
, the expression of both
GIOT1 and -2 mRNAs were also induced in testes that had been treated
with PMSG or hCG. In addition, the in situ hybridization
study revealed that GIOT1 gene expression was rapidly and strongly
induced in testicular Leydig cells by the administration of either PMSG
or hCG. The expression was not found in Sertoli cells or spermatogenic
cells (Fig. 6
). No staining was observed
in the unstimulated immature rat testis. These results suggest that
GIOT1 may also function in the male gonadal system.

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Figure 5. Expression of GIOT1 and -2 in Immature Rat Testes
RT-PCR was performed using GIOT1- and -2 -specific primers,
respectively. Total RNA was isolated from untreated immature rat
testes, testes primed with PMSG for 6 h or 12 h, or testes
primed with hCG for 3 h. Two micrograms of total, extracted RNA
were reverse-transcribed, and a portion (1/100) was subjected to the
PCR reaction for the specific amplification of GIOT1, -2 , and GAPDH,
respectively. The reaction mixtures were separated on a 2% agarose gel
and visualized with EtBr staining.
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Figure 6. In Situ Hybridization of GIOT1 in
the Immature (21-d-old) Rat Testes
Testes from 21-d-old rats were dissected, sectioned, and hybridized
with a digoxigenin-labeled antisense GIOT1 probe. Sections of testes
from 21-d-old immature rats were primed as follows: A, with PMSG (30
IU) for 6 h, x 80; B, with hCG (50 IU) for 3 h, x80; C,
with no treatment, x 80; D, with PMSG (30 IU) for 6 h, x80,
hybridized with a sense strand cRNA probe. Arrows show
regions of positive hybridization.
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The tissue distributions of GIOT1 and -2 were also determined. RT-PCR
was used for detection, because the sensitivity of Northern blot
analysis was not sufficient for the detection of transcripts of GIOT1
and -2 in most of the rat tissues. The gene expression of GIOT1 is
restricted to the pituitary, adrenal, testis, and ovary, while GIOT2 is
expressed ubiquitously (Fig. 7
). GIOT1
was not present in the hypothalamus, while GIOT2 was present in the
tissue.

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Figure 7. Tissue Distribution of GIOT1 and -2
RT-PCR was performed using GIOT1- and -2 -specific primers,
respectively. Total RNA was isolated from various tissues of immature
rats. Two micrograms of total, extracted RNA were reverse-transcribed,
and a portion (1/100) was subjected to the PCR reaction for the
specific amplification of GIOT1, -2 , and GAPDH, respectively. The
reaction mixtures were separated on a 2% agarose gel and visualized
with EtBr staining.
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Functional Analysis of GIOT1 and -2
To determine the transcriptional activity of GIOT1 and -2,
cotransfection experiments were performed. The 5x GAL4-GL3 reporter
plasmid, in which five copies of the GAL4-binding site were inserted
upstream of the SV40 promoter in the pGL3-control vector were used
(28). We also prepared constructs, pSG-GIOT1 and
pSG-GIOT2, which express the entire coding regions of GIOT1 and GIOT2
fused to GAL4-DNA-binding domain (DBD), respectively. When the reporter
plasmid and various amounts of pSG-GIOT1 were cotransfected into the
NIH3T3 cells, luciferase activity was decreased in a
concentration-dependent manner (Fig. 8A
).
In contrast, the cotransfection of the pSG-GIOT2 had only a marginal
effect in luciferase activity (Fig. 8A
). Cotransfection of
GIOT1-pBKCMV, which lacks GAL4-DBD, was also ineffective in decreasing
luciferase activity. pSG-GIOT1 was not effective when the pGL3-control
reporter, which lacks 5x GAL4 binding sites, was used (data not
shown). These results indicate that GIOT1, but not GIOT2, serves as a
transcriptional repressor.

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Figure 8. Transcriptional Activity of GIOT1 and -2
A, NIH3T3 cells were transiently transfected with 0.1 µg of 5x
GAL4-GL3 control reporter vector along with 0.001 µg of the pRL-SV40
Renilla luciferase vector. Indicated amounts of the
pSG-GIOT1 or pSG-GIOT2 expression vectors were simultaneously
transfected into the cells, respectively. pSG424, which expresses
GAL4-DBD alone, was added to some samples so that each sample contained
the same amount of DNA. Renilla luciferase activity was
also measured for normalization. The promoter activity from the
reporter vector in the absence of expression plasmids was defined as 1.
Each value represents the mean and SE of four independent
transfection experiments. B, NIH3T3 cells were transiently transfected
with 0.1 µg of 5x GAL4-GL3 control reporter vector along with 0.001
µg of the pRL-SV40 vector. These cells were simultaneously
cotransfected with 0.3 µg of effector plasmids expressing the various
deletion mutants of GIOT1 fused to GAL4-DBD. Renilla
luciferase activity was also measured for normalization. The promoter
activity of the reporter vector in the presence of pSG424 was defined
as 1. Each value represents the mean and SE of four
independent transfection experiments.
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To map the minimal transcriptional repression domain of GIOT1, plasmid
constructs were designed to produce truncated GIOT1 proteins with
GAL4-DBD. As shown in Fig. 8B
, both pSG-GIOT1 (1145) and pSG-GIOT1
(165), which express fusion proteins involving the
NH2-terminal 145 and 65 amino residues of GIOT1,
respectively, showed strong transcriptional repressor activity. The
elimination of the KRAB-A domain from GIOT1, i.e. pSG-GIOT1
(33654) and pSG-GIOT1 (138), led to the abolition of
transcriptional repressor activity. These results indicate that amino
acid sequences 165 of GIOT1, which corresponds to the KRAB-A domain,
represent the minimal domain required for transcriptional repressor
activity.
Isolation of GIOT1-Interacting Proteins Using a Yeast Two-Hybrid
System
To identify proteins that interact with GIOT1, we screened a rat
granulosa cell cDNA library using a GAL4-based yeast two-hybrid system.
A plasmid cDNA library was constructed from cultured rat
granulosa cells. A chimeric construct, which expresses a GIOT1
fusion protein with the GAL4 DBD, was employed as the bait. We screened
approximately 7 x 106 independent clones
from the cDNA library and obtained seven
HIS3+/ADE2+/MEL1+-positive
clones. We characterized all clones by nucleotide sequence analysis and
subsequent homology search on GenBank DNA databases. A database search
revealed that nucleotide sequences of three clones showed high
similarity with that of human I-mfa domain containing protein, which
was originally cloned from MT-2 cells, a T cell line, by yeast
two-hybrid screening (15). Hereafter, we designated this
protein as rat homolog of human I-mfa domain containing protein (RIC).
One clone among the rest was characterized as a rat homolog of TIF1ß,
which is known as the KRAB-A-interacting protein (16, 17, 18).
The other three clones encoded novel proteins.
We then confirmed the specificity of the interaction between GIOT1 and
RIC or TIF1ß using yeast two-hybrid assays (Table 1
). The yeast SFY526 strain harboring
pGBKT7 or pGBKT7-GIOT1 was transformed with pACT2, pRIC, or pTIF1ß,
respectively. As shown in Table 1
, the transformants containing both
pGBKT7-GIOT1 and pRIC or pTIF1ß plasmids expressed much higher
ß-galactosidase activity than the other transformants tested,
indicating that GIOT1 and RIC or TIF1ß actually interacted with each
other in the yeast system. Furthermore, we also determined interacting
domains of GIOT1 with TIF1ß or RIC by the same system. As also shown
in Table 1
, TIF1ß interacted exclusively with the
NH2-terminal KRAB-A domain of GIOT1, whereas RIC
did not interact with the KRAB-A domain but with the GIOT1 segments
lacking its NH2-terminal region.
In addition, we used GST pull-down assays to confirm the specific
interaction between GIOT1 and RIC or TIF1ß in vitro. The
RIC and the TIF1ß proteins were expressed in Escherichia
coli as GST fusion proteins. In vitro-translated,
35S-labeled GIOT1 was incubated with purified
GST, GST-RIC, or GST-TIF1ß proteins bound to glutathione-agarose.
GIOT1 bound to GST-RIC and GST-TIF1ß, but not to GST alone (Fig. 9
). These results indicate that GIOT1
specifically interacts with RIC and TIF1ß both in vivo and
in vitro.

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Figure 9. Interaction of GIOT1 with TIF1ß or RIC in
Vitro
(A) Unprogrammed lysate or (B) in vitro-translated
35S-labeled GIOT1 were incubated with glutathione-agarose
which bound GST, GST-TIF1ß or GST-RIC fusion proteins. The beads were
washed thoroughly and bound proteins were analyzed on SDS-PAGE and by
autoradiography. The signal in the lane marked 1/10 input represents
10% of the protein added to the reactions shown in the other lanes.
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DISCUSSION
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In the present study, we report the identifications of GIOT1 and
-2 as novel gonadotropin-inducible genes. Full-length cDNAs from
PMSG-primed immature rat ovaries were cloned, and their nucleotide
sequences were determined. Sequence analysis reveals that there are two
closely related genes, designated as GIOT1 and GIOT2, and that both
GIOT1 and -2 are members of the krüppel-like
(Cys)2-(His)2-type zinc
finger proteins. The KRAB domain, which consists of two components,
KRAB-A and KRAB-B, is conserved in the
NH2-terminal region of some krüppel-like
zinc finger proteins. GIOT1, but not GIOT2, contains the KRAB-A domain
in the NH2 terminus.
The levels of both GIOT1 and -2 mRNAs could be temporarily induced in
the ovary by treatment with PMSG or hCG. This indicates that PMSG/hCG
activates both the GIOT1 and -2 genes in the ovary via the LH
receptor-mediated signaling pathway. Results from in situ
hybridization study are also consistent with the observation mentioned
above, i.e. the gene expression of GIOT1 was strongly
induced in the ovarian theca interna and interstitial cells where LH
receptors are expressed, but not in granulosa cells where LH receptors
are not expressed in the ovary of immature rat (29, 30).
However, FSH had the ability to temporarily induce the expression of
both GIOT1 and -2 mRNAs in cultured granulosa cells. FSH is known to
act on granulosa cells and to play critical roles in the induction of
various genes through the activation of the cAMP-dependent signaling
pathway. Analysis of the upstream region of rat GIOT1 revealed that
there is a cAMP-responsive element in its promoter region (our
unpublished data), suggesting that any reagents including
forskolin that increase intracellular cAMP may increase the expression
of GIOT1 in these cells. Thus, these results indicate that the
expression of GIOT1 and -2 can be induced in both ovarian cell types,
granulosa cells and theca interna cells, by LH or FSH. However,
expression of GIOT1 was very weak in the granulosa cells by the
in vivo studies. This may reflect differences in GIOT1 gene
expression under in vivo and in vitro conditions.
There are several reports describing that interactions between theca
and granulosa cells gave profound effects on the functions of these
cells (31, 32). It might be possible that interactions
between theca and granulosa cells might suppress GIOT1 gene expression
in granulosa cells in vivo. Similar events are observed in
regulation of other gene expressions in the ovary. For instance,
StAR, which is an acute regulator of the rate-limiting
transfer of cholesterol to the inner mitochondrial membrane, is also
induced in both ovarian cell types by gonadotropins
(33, 34, 35, 36). We also demonstrated that expression of StAR
mRNA was induced at 6 h after the administration of PMSG/hCG in
theca interna cells and interstitial cells but not granulosa cells in
immature rat ovary in vivo (7), and that
expression of StAR mRNA was induced by FSH in cultured granulosa cells
in vitro (37). The mRNA levels of GIOT1 were
also temporarily induced in the testicular Leydig cells by treatment
with PMSG/hCG. The expression was observed neither in Sertoli cells nor
in spermatogenic cells. The mechanism of the induction may be common
between female and male systems.
The gene expression of GIOT1 was only detected in pituitary, adrenal
gland, testis, and ovary, whereas GIOT2 is ubiquitously expressed. The
tissue distribution of GIOT1 completely overlapped with that of
Ad4-binding protein/steroidogenic factor-1 (Ad4BP/SF-1) and Dax-1
genes, which are known to be essential for sex differentiation and
steroidogenesis (38, 39). The limited distribution of
GIOT1 encourages us to further examine the role of GIOT1 in ovarian
steroidogenesis.
It has been reported that transcription factors that contain the KRAB
domain function as transcriptional repressors (40, 41).
The transcriptional activity of both GIOT1 and -2 was investigated
using the GAL4-based mammalian one-hybrid system. As a result, GIOT1
was found to be a transcriptional repressor and the KRAB-A domain of
GIOT1 was responsible for the transcription repressor activity. In
contrast, GIOT2, which lacks the KRAB domain, showed no transcriptional
repressor activity.
Finally, we cloned GIOT1-interacting proteins by using the yeast
two-hybrid system. These clones encoded rat homologs of human I-mfa
domain containing protein (15) and TIF1ß
(16, 17, 18, 19), respectively. Human I-mfa domain containing
protein was recently identified as the CD4-interacting protein and is
thought to function as a transcription regulator (15).
TIF1ß is a transcription coregulator and functions as a universal
corepressor working together with KRAB domain proteins
(16, 17, 18) or as a coactivator working with GR and
CCAAT/enhancer-binding protein ß (19).
Domain mapping analysis by ß-galactosidase assay revealed that
TIF1ß actually interacts with the N-terminal KRAB-A domain
region. This confirmed previous reports and strengthened our
prediction that GIOT1 functions as a transcriptional repressor in the
ovary, since TIF1ß is thought to function as a transcriptional
corepressor by interacting with KRAB-A domain of
(Cys)2-(His)2-type zinc
finger transcription factors (16, 17, 18). On the other hand,
RIC interacts with GIOT1 at a different region than the KRAB-A domain.
Although a detailed fashion of this interaction is not yet clear, these
results suggest that GIOT1 probably interacts with various factors
simultaneously at different domains to exert the actions. These
findings suggest that GIOT1 may function as a novel transcriptional
repressor by working with RIC and TIF1ß proteins and may play a
significant role at the transcription level in folliculogenesis.
In conclusion, we identified a novel transcriptional repressor,
designated as GIOT1, that is strongly induced in the gonadal systems.
Further study is needed to elucidate the detailed mechanisms of actions
of this novel transcriptional repressor.
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MATERIALS AND METHODS
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Animals
Twenty-one-day-old immature Wistar rats were obtained from the
Imai Experimental Animal Farm (Saitama, Japan) and were housed in a
photoperiod of 14 h of light and 10 h of darkness, with food
and water freely available. The experimental protocol was approved by
the committee on animal care and use of Gunma University.
Rat Granulosa Cell Culture
Granulosa cells were prepared from immature female Wistar rats
that had received daily injections of 2 mg diethylstilbestrol in 0.1 ml
sesame oil for a 4-d period. The ovaries were then excised, and
granulosa cells were isolated by puncturing the follicles with a
26-gauge needle. The cells were washed and collected by a brief
centrifugation, and cell viability was determined by trypan blue
staining. The granulosa cells were then cultured in Hams F-12-DMEM
(1:1, vol/vol) supplemented with antibiotics and 0.1% BSA on
collagen-coated plates in a humidified atmosphere containing 5%
CO2 and 95% air at 37 C (42).
Subtraction Cloning
Rat ovarian genes, which are rapidly induced by PMSG
administration, were isolated by a subtraction cloning procedure using
a PCR-based cloning kit (CLONTECH PCR-Selected cDNA Subtraction
Kit, CLONTECH Laboratories, Inc., Palo Alto, CA) (6, 7). Briefly, poly (A)+ RNAs were prepared
from immature rat ovaries that had been primed with or without 30 IU of
PMSG (Teikokuzouki, Inc., Tokyo, Japan) for 3 h, and each
double-stranded cDNA was synthesized as described below. The resulting
cDNAs were digested with RsaI to obtain short and
blunt-ended cDNAs. The cDNAs from the PMSG-primed ovaries were then
subdivided into two portions, and each was ligated with a different
adaptor,
5'-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT
and 5'-TGTAGCGTGAAGACGACAGAAAGGGCGTGGTGCGGAGGGCGGT,
respectively. The resultant cDNAs were separately heat denatured,
and each was hybridized at 72 C for 8 h with 30-fold excess of
heat-denatured cDNAs obtained from control ovaries and which had no
adaptor at their ends. At the end of the first hybridization, the
single-stranded and adaptor-harboring cDNAs remaining in the solution
would be expected to encode specific genes that are present only in the
ovaries that had been primed with PMSG. Each hybridization solution was
then combined and further incubated at 72 C for 16 h to complete a
second hybridization. After the hybridization, a portion of the
single-stranded cDNAs with each adaptor at one end was annealed
together to form double-stranded cDNAs, which had two different
adaptor sequences at each end. The cDNAs with such ends were then
amplified by PCR using primers that are specific for each adaptor
sequence, 5'-CTAATACGACTCACTATAGGGC and 5'-TGTAGCGTGAAGACGACAGAA,
respectively. Specific amplification of the cDNAs was further attained
by a second PCR procedure using nested primers that are also specific
for each adaptor sequence, 5'-TCGAGCGGCCGCCCGGGCAGGT and
5'-AGGGCGTGGTGCGGAGGGCGGT, respectively. The resulting cDNAs were
separated by electrophoresis on a 1% agarose gel, and the cDNAs, which
ranged from 0.5 to 2.0 kb in length, were isolated and cloned into the
pGEM-T vector (Promega Corp., Madison, WI) to construct a
subtracted plasmid cDNA library. Of the clones in the plasmid library,
about 400 were randomly selected and their nucleotide sequences were
partially determined by the dideoxy termination method using an
automated DNA sequencer (model 377, Perkin-Elmer Corp.,
Norwalk, CT). Identities of the clones were analyzed by the BLAST
search program from the DNA data bank of Japan.
Construction of
-Phage cDNA Library and Cloning of Full-Length
Rat GIOT1 and -2 cDNAs
To isolate full-length cDNAs for rat GIOT1 and -2, a rat
ovarian cDNA library was constructed. Total RNA was isolated from
immature rat ovaries that had been primed with 30 IU of PMSG for 3
h by the acid guanidium thiocyanate-phenol-chloroform method
(43), and poly (A)+ RNA was prepared
by using oligo-dT-latex beads (Roche Molecular Biochemicals, Indianapolis, IN). Five micrograms of poly
(A)+ RNA from PMSG-primed immature rat ovaries
were used in the synthesis of double-stranded cDNA using a cDNA
synthesis kit (Life Technologies, Inc., Gaithersburg, MD)
using oligo-dT as a primer. The EcoRI/NotI
adaptor was then ligated to a double-stranded cDNA, and both ends were
phosphorylated with a T4 polynucleotide kinase (Amersham Pharmacia Biotech, Arlington Heights, IL). The cDNA was ligated
to
ZAP Express phage arms (Stratagene, La Jolla, CA),
followed by in vitro packaging using GigapackII gold
(Stratagene), to generate a cDNA library. The cDNA library
contained 1 x 106 independent clones
(6, 7).
To isolate full-length cDNAs corresponding to rat GIOT1 and -2, the
library was screened with a Digoxigenin-labeled 320-bp RsaI
fragment of rat GIOT1 (nt 1834/2153). Four positive clones were
isolated from approximately 10,000 cDNA clones. These clones were
excised in vivo to recover the pBKCMV plasmid vector. The
nucleotide sequences were determined from both ends by means of the dye
terminator cycle sequencing method using an automated DNA
sequencer.
RNA Blot Analysis and RT-PCR
Twenty-one-day-old male and female immature rats were primed
with 30 IU of PMSG or 50 IU of human chorionic gonadotropin (hCG)
(Sankyo Co., Ltd., Tokyo, Japan), and the ovaries and
testes were collected at the indicated times. Total RNA was extracted
from various tissues (brain, pituitary, lung, spleen, kidney, adrenal
gland, and ovary) of immature female rats and from the testis of an
immature male rat by the acid guanidium thiocyanate-phenol-chloroform
method. For Northern blot analysis, 10 µg of total RNA were separated
by electrophoresis on a 1% denaturing agarose gel, transferred to a
nylon membrane (Biodyne, ICN Biomedicals, Inc., Glen Cove,
NY), and cross-linked by UV irradiation. A 398-bp (nt -290/108)
fragment of GIOT1, or a 561-bp (nt -2442/-1882) cDNA fragment of
GIOT2
was radiolabeled by the random primer method and used as
probe. The filter was hybridized at 65 C for 16 h in 6x SSC (0.9
M NaCl, 90 mM sodium citrate, pH 7.0), 5x
Denhardts solution (0.1% BSA, 0.1% Ficoll, 0.1%
polyvinylpyrrolidone) and 0.5% SDS with a
32P-labeled probe (NEN Life Science Products, Boston, MA). The membrane was washed three times at 42
C for 15 min in 0.1x SSC and 0.1% SDS. The blot was rehybridized with
a radiolabeled probe that is specific for rat glyceraldehyde
3-phosphate dehydrogenase (GAPDH). For RT-PCR, 2 µg of total RNA were
reverse-transcribed and a portion (1/100) of the reaction mixture was
subjected to the PCR reaction. Primers for GIOT1 were
5'-CTCTTGTCCCCCATTCTCTT [5'-primer: nucleotide (nt) -272/-253] and
5'-CTTTCCATAGTCCTTATGCT (3'-primer: nt 227/246), respectively. Primers
for GIOT2
were 5'-AGAGCAGAGTTCAGTCAAGA (5'-primer: nt
-1189/-1170) and 5'-AGCATCAGCAGGCATTCACT (3'-primer: nt
-766/-747), respectively. Primers for GAPDH were
5'-ACCACAGTCCATGCCATCAC (5'-primer: nt 520/539) and
5'-TCCACCACCCTGTTGCTGTA (3'-primer: nt 952/971), respectively. Reaction
conditions were 34 cycles for GIOT1, 29 cycles for GIOT2
, 24 cycles
for GAPDH, respectively, by denaturing at 94 C for 20 sec, annealing at
57 C for 30 sec and extending at 72 C for 60 sec using a Thermal Cycler
apparatus (TAKARA, Kyoto, Japan). Ten microliters of the PCR products
were electrophoresed on a 2% agarose gel and subsequently visualized
by ethidium bromide (EtBr) staining.
In Situ Hybridization
In situ hybridization was performed by the methods of
Braissant and Wahli (44) with minor modifications.
Briefly, rat ovaries and testes were embedded in a matrix and frozen in
dry ice. Twelve to 14 µm thick sections were cut by a cryostat and
mounted on gelatin-coated glass slides for in situ
hybridization. The sections were fixed in PBS that contained 4%
paraformaldehyde for 1030 min, washed twice for 15 min in PBS, and
equilibrated in 5x SSC (0.75 M NaCl and 0.075
M sodium citrate) for 15 min. The sections were
then prehybridized at 42 C for 2 h in 500 µl of 5x SSC solution
containing 50% formamide and 40 µg/ml salmon sperm DNA. A 568-bp
fragment of the rat GIOT1 cDNA (nt 130/697) was subcloned into the
pGEM-T easy vector. Antisense or sense digoxigenin-UTP labeled RNA
probes were synthesized using T7 or Sp6 polymerase (RNA labeling kit,
Roche Molecular Biochemicals). The probes (400 ng/ml) were
denatured at 80 C for 5 min and added to the hybridization mix.
Hybridization was done at 42 C for 16 h. Subsequent washes were as
follows: at room temperature for 10 min in 2x SSC, at room temperature
for 10 min in 2.5 µg/ml ribonuclease A in 2x SSC, twice at room
temperature for 10 min in 2x SSC, at 65 C for 1 h in 2x SSC, and
finally at 65 C for 1 h in 0.1x SSC. Those sections were
equilibrated for 5 min in buffer A (100 mM
Tris-HCl, pH 7.5, 150 mM NaCl) and then further
incubated at room temperature for 2 h in antidigoxigenin-alkaline
phosphatase antibody diluted to 1:5,000 in buffer A containing 1%
blocking reagent. Excess antibody was removed by washing twice with
buffer A for 15 min. The sections were then equilibrated for 5 min in
buffer B (100 mM Tris-HCl, pH 9.5, 100
mM NaCl, and 50 mM
MgCl2). Alkaline phosphatase activity was
detected in 175 µg/ml 5-bromo-4-chloro-3-indolylphosphate, 337.5
µg/ml 4-nitro blue tetrazolium chloride in buffer B (Digoxigenin
Detection kit; Roche Molecular Biochemicals). Typically,
color development was observed within 636 h.
Plasmids
pSG424 vector, which is expression of GAL4-DBD fusion proteins
in mammalian cells, was kindly provided by Dr. R. Stein (Vanderbilt
University, Memphis, TN) (45). A 5x GAL4-GL3 control
vector was kindly provided by Dr. T. Noguchi (Nagoya University,
Nagoya, Japan) (28). pSG-GIOT1 and pSG-GIOT2
vectors for the mammalian one-hybrid system were generated as follows.
To obtain DNA fragments that encode for the N-terminal regions of the
proteins, PCR reactions were carried out using GIOT1-pBKCMV or
GIOT2ß-pBKCMV as a template with the following combination of
primers: 5'-gaggatccccATGGATGCAGTCACCTATGA (5'-primer, nt 1/20 GIOT1)
and 5'-TGGCAGATGATATCCCTTTC (3'-primer, nt 187/206 GIOT1);
5'-gaggatcccgATGATGCAAACCTGCAGGAA (5'-primer, nt 1/20 GIOT2ß) and
5'-TGGCAGATGATATCCCTTTC (3'-primer, nt 91/110 GIOT2ß),
respectively. The numbering of nucleotides is relative to the
translation start sites of GIOT1 and GIOT2ß. The GIOT1-pBKCMV and the
GIOT2ß -pBKCMV vectors were digested with BamHI and
EcoRV to remove the BamHI and EcoRV
fragments, respectively. Each PCR product was then inserted into the
digested GIOT1-pBKCMV or GIOT2ß -pBKCMV vector at the
BamHI/EcoRV site, after digesting each PCR
product with BamHI and EcoRV. DNA fragments
containing the entire coding sequences with the ATG start codon at
their 5'-ends (BamHI/KpnI fragments from the
respective plasmids) were subsequently ligated into the
BamHI/KpnI sites of the pSG424, respectively.
These constructs were designated as pSG-GIOT1 and pSG-GIOT2, which
express the GAL4-DBD which is fused to the entire coding sequences of
GIOT1 and GIOT2ß, respectively. Plasmids with truncated forms of
GIOT1, which express fusion proteins, were also constructed. The
pSG-GIOT1 was digested with BamHI and PstI, and a
122-bp insert was isolated and then ligated into the
BamHI/KpnI site of the pSG424 to produce
pSG-GIOT1 (138). To construct pSG-GIOT1 (37654), pSG-GIOT1 was
first digested with BamHI and EcoRV to remove a
DNA fragment encoding GIOT1 (165). To the digested plasmid, a DNA
fragment encoding GIOT1 (3765), which was synthesized by PCR, was
inserted, to produce pSG-GIOT1 (NOREF>37654). The PCR reactions were
carried out using GIOT1-pBKCMV as a template and the following
combination of primers: 5'-gaggatcccgATGATGCAAACCTGCAGGAA (5'-primer,
nt 97/116 GIOT1) and 5'-TGGCAGATGATATCCCTTTC (3'-primer, nt 187/206
GIOT1). The PCR product was then digested with BamHI and
EcoR V and ligated into the BamHI/EcoR
V site of the plasmid. To produce pSG-GIOT1 (165) and pSG-GIOT1
(1145), pSG-GIOT1 was digested with EcoRV and
XbaI, or NdeI and XbaI, blunt-ended,
and then self-ligated, respectively. The nucleotide sequences of all
the constructs were confirmed by DNA sequencing.
Cell Culture, Transient Transfections, and Luciferase
Assays
NIH3T3 cells were maintained in DMEM supplemented with 10% FCS
and antibiotics. Cells were dispensed into 24-well plates and cultured
to 5070% confluency. The indicated amounts of reporter plasmid,
pRL-SV40 vector, and GAL4DBD fusion protein expression plasmid were
mixed with 1.5 µl of FuGENE 6 (Roche Molecular Biochemicals), and the resulting mixture was added to the cells.
The amount of total DNA was adjusted by adding the pSG424 plasmid.
Cells were harvested 48 h after transfection, and luciferase
activities were determined using a Dual Luciferase Reporter Assay
System. Measurements were made using a Lumat LB9501 (Berthold). Firefly
luciferase activities (relative light units) were normalized to
Renilla luciferase activities.
Yeast Two-Hybrid Screening
A kit purchased from CLONTECH Laboratories, Inc.
(Palo Alto, CA) was used for screening of the yeast two-hybrid system.
All procedures were performed as described by the manufacturer unless
otherwise stated. The pGBKT7 vector, a parent vector for the yeast
two-hybrid system, expresses GAL4-DBD fusion protein in yeast.
pGBKT7-GIOT1 vector, a bait plasmid, was generated as follows. The
pGBKT7 was digested with SalI, blunt-ended by the Klenow
reaction, and digested with BamHI. The pSG-GIOT1 was
digested with BamHI and SmaI. The insert DNA,
which contained the entire open reading frame of GIOT1, was subcloned
into BamHI/SalI (blunt-ended) site of the pGBKT7
vector. AH109 cells were transformed with the indicated bait plasmid by
a TE/LiAc-based high efficiency transformation method
(46). Construction of a plasmid cDNA library from rat
granulosa cells for yeast two-hybrid screening was described
(47). When a yeast strain harboring the pGBKT7-GIOT1 was
transformed with the library, approximately 7 x
106 primary transformants were obtained.
HIS3+/ADE2+/MEL1+
clones were selected and further characterized.
Liquid ß-Galactosidase Assay
Interaction of GIOT1 with TIF1ß or RIC was examined by using
liquid ß-galactosidase assay. The interaction domain mapping of GIOT1
with TIF1ß or RIC was also performed. SFY526 cells harboring the
pGBKT7 or pGBKT7-GIOT1 were transformed with TIF1ß and RIC expression
plasmids as well as with the control pACT2 vector. For domain mapping,
plasmid constructs containing GIOT1 (33654), GIOT1 (>NOREF>1145), and
GIOT1 (165) were used to observe interaction of these domains with
TIF1ß or RIC. For this purpose, cDNA inserts of pSG-GIOT1 (33654),
pSG-GIOT1 (165), and pSG-GIOT1 (1145) were transferred to a bait
vector for a yeast two- hybrid system (pGBKT7). ß-Galactosidase
activity was measured for the resulting transformants as described
(48, 49). Permeabilized cells were used for quantitative
ß-galactosidase assays, using
o-nitrophenyl-ß-D-galactoside as the
substrate.
GST Pull-Down Assays
pACT2-TIF1ß and pACT2-RIC were obtained from the yeast
two-hybrid screening. A 2.8-kb EcoRI fragment of
pACT2-TIF1ß was isolated and subcloned into the EcoRI site
of the pGEX-5X-1 (Amersham Pharmacia Biotech) to produce
GST-TIF1ß fusion protein expression vector (pGST-TIF1ß). A 2.2-kb
SmaI/XhoI fragment of pACT2-RIC was isolated and
subcloned into the SmaI/XhoI site of the
pGEX-4T-2 (Amersham Pharmacia Biotech) to produce GST-RIC
fusion protein expression vector (pGST-RIC). TOPP3 cells were
transformed with pGEX-5X-1, pGST-TIF1ß, or pGST-RIC. GST fusion
proteins were extracted and purified from the transformants,
respectively. GIOT1 was labeled with
L-[35S]methionine (37
tera-becquerels/mmol, Amersham Pharmacia Biotech)
by in vitro translation. The GST fusion proteins, in
vitro-translated 35S-labeling GIOT1 and
glutathione-agarose, were mixed together, and pull-down analysis was
performed as previously described (48, 49). Finally,
glutathione-agarose beads were resuspended in an equal volume of 2x
SDS sample buffer, and each supernatant was loaded on a 10% SDS-PAGE
gel, along with a prestained mol wt marker. The gel was dried and
exposed to X-AR film (Eastman Kodak Co, Rochester, NY) at
room temperature. The relative purity and amount of each fusion protein
were determined by gel staining with Coomassie Brilliant Blue
R-250.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Drs. R. Stein and T. Noguchi for providing
the plasmids and Ms. Y. Sonoda for technical assistance.
 |
FOOTNOTES
|
---|
This work was supported by Grants from the Smoking Research Foundation
and Kanzawa Medical Research Foundation and by grants from the Ministry
of Education, Science, Sports and Culture of Japan. Te.M. was supported
by the Fellowship of the Japan Society for the Promotion of Science for
Japanese Junior Scientists.
Abbreviations: DBD, DNA-binding domain; GIOT1 and
-2, gonadotropin-inducible transcription factors 1 and 2; GAPDH,
glyceraldehyde 3-phosphate dehydrogenase; GST,
glutathione-S-transferase; hCG, human CG; KRAB,
krüppel-associated box; nt, nucleotide; RIC, rat homolog of human
I-mfa domain containing protein; StAR, steroidogenic acute regulatory
protein; TIF1ß, transcriptional intermediary factor 1ß.
1 The rat GIOT1 (accession number AB047636),
GIOT2
(accession number AB047637), and GIOT2ß (accession number
AB047638) cDNA sequences have been deposited in the DDBJ/EMBL/GenBank
database. 
Received for publication April 5, 2001.
Accepted for publication June 24, 2001.
 |
REFERENCES
|
---|
-
Richards JS 1980 Maturation of ovarian follicles: actions
and interactions of pituitary and ovarian hormones on follicular cell
differentiation. Physiol Rev 60:5189[Free Full Text]
-
Hsueh AJ, Adashi EY, Jones PB, Welsh Jr TH 1984 Hormonal
regulation of the differentiation of cultured ovarian granulosa cells.
Endocr Rev 5:76127[Medline]
-
Tonetta SA, diZerega GS 1989 Intragonadal regulation of
follicular maturation. Endocr Rev 10:205229[Abstract]
-
Peters H, Byskov AG, Lintern-Moore S, Faber M, Andersen M 1973 The effect of gonadotrophin on follicle growth initiation in the
neonatal mouse ovary. J Reprod Fertil 35:139141[CrossRef][Medline]
-
Aoki H, Okada T, Mizutani T, Numata Y, Minegishi T, Miyamoto
K 1997 Identification of two closely related genes, inducible and
noninducible carbonyl reductases in the rat ovary. Biochem Biophys Res
Commun 230:518523[CrossRef][Medline]
-
Mizutani T, Sonoda Y, Minegishi T, Wakabayashi K, Miyamoto K 1997 Cloning, characterization, and cellular distribution of rat
scavenger receptor class B type I (SRBI) in the ovary. Biochem Biophys
Res Commun 234:499505[CrossRef][Medline]
-
Mizutani T, Sonoda Y, Minegishi T, Wakabayashi K, Miyamoto K 1997 Molecular cloning, characterization and cellular distribution of
rat steroidogenic acute regulatory protein (StAR) in the ovary. Life
Sci 61:14971506[CrossRef][Medline]
-
Maniatis T, Goodbourn S, Fischer JA 1987 Regulation of
inducible and tissue-specific gene expression. Science 236:12371245[Medline]
-
Mitchell PJ, Tjian R 1989 Transcriptional regulation in
mammalian cells by sequence-specific DNA binding proteins. Science 245:371378[Medline]
-
Ptashne M, Gann AA 1990 Activators and targets. Nature 346:329331[CrossRef][Medline]
-
Johnson AD 1995 The price of repression. Cell 81:655658[Medline]
-
Tjian R, Maniatis T 1994 Transcriptional activation: a complex
puzzle with few easy pieces. Cell 77:58[Medline]
-
Pieler T, Bellefroid E 1994 Perspectives on zinc finger
protein function and evolutionan update. Mol Biol Rep 20:18[Medline]
-
Bellefroid EJ, Poncelet DA, Lecocq PJ, Revelant O,
Martial JA 1991 The evolutionarily conserved Kruppel-associated box
domain defines a subfamily of eukaryotic multifingered proteins. Proc
Natl Acad Sci USA 88:36083612[Abstract]
-
Thebault S, Gachon F, Lemasson I, Devaux C, Mesnard JM 2000 Molecular cloning of a novel human I-mfa domain-containing protein that
differently regulates human T-cell leukemia virus type I and HIV-1
expression. J Biol Chem 275:48484857[Abstract/Free Full Text]
-
Kim SS, Chen YM, OLeary E, Witzgall R, Vidal M, Bonventre JV 1996 A novel member of the RING finger family, KRIP-1, associates with
the KRAB-A transcriptional repressor domain of zinc finger proteins.
Proc Natl Acad Sci USA 93:1529915304[Abstract/Free Full Text]
-
Abrink M, Ortiz JA, Mark C, et al. 2001 Conserved interaction
between distinct Kruppel-associated box domains and the transcriptional
intermediary factor 1 ß. Proc Natl Acad Sci USA 98:14221426[Abstract/Free Full Text]
-
Friedman JR, Fredericks WJ, Jensen DE, et al. 1996 KAP-1, a
novel corepressor for the highly conserved KRAB repression domain.
Genes Dev 10:20672078[Abstract]
-
Chang CJ, Chen YL, Lee SC 1998 Coactivator TIF1ß interacts
with transcription factor C/EBPß and glucocorticoid receptor to
induce
1-acid glycoprotein gene expression. Mol Cell Biol 18:58805887[Abstract/Free Full Text]
-
Wick MJ, Ann DK, Lee NM, Loh HH 1995 Isolation of a cDNA
encoding a novel zinc-finger protein from neuroblastoma x glioma
NG10815 cells. Gene 152:227232[CrossRef][Medline]
-
Tommerup N, Vissing H 1995 Isolation and fine mapping of 16
novel human zinc finger-encoding cDNAs identify putative candidate
genes for developmental and malignant disorders. Genomics 27:259264[CrossRef][Medline]
-
Tommerup N, Aagaard L, Lund CL, et al. 1993 A zinc-finger gene
ZNF141 mapping at 4p16.3/D4S90 is a candidate gene for the
Wolf-Hirschhorn (4p-) syndrome. Hum Mol Genet 2:15711575[Abstract]
-
Baban S, Freeman JD, Mager DL 1996 Transcripts from a
novel human KRAB zinc finger gene contain spliced Alu and endogenous
retroviral segments. Genomics 33:463472[CrossRef][Medline]
-
Kim J, Lu X, Stubbs L 1999 Zim1, a maternally expressed mouse
Kruppel-type zinc-finger gene located in proximal chromosome 7. Hum Mol
Genet 8:847854[Abstract/Free Full Text]
-
Gebelein B, Fernandez-Zapico M, Imoto M, Urrutia R 1998 KRAB-independent suppression of neoplastic cell growth by the novel
zinc finger transcription factor KS1. J Clin Invest 102:19111919[Abstract/Free Full Text]
-
Witzgall R, Volk R, Yeung RS, Bonventre JV 1994 Genomic
structure and chromosomal location of the rat gene encoding the zinc
finger transcription factor kid-1. Genomics 20:203209[CrossRef][Medline]
-
Bellefroid EJ, Sahin M, Poncelet DA, et al. 1998 Kzf1a novel
KRAB zinc finger protein encoding gene expressed during rat
spermatogenesis. Biochim Biophys Acta 1398:321329[Medline]
-
Tanaka T, Inazu T, Yamada K, et al. 1999 cDNA cloning and
expression of rat homeobox gene, Hex, and functional characterization
of the protein. Biochem J 339:111117[CrossRef][Medline]
-
Camp TA, Rahal JO, Mayo KE 1991 Cellular localization and
hormonal regulation of follicle-stimulating hormone and luteinizing
hormone receptor messenger RNAs in the rat ovary. Mol Endocrinol 5:14051417[Abstract]
-
Peng XR, Hsueh AJ, LaPolt PS, Bjersing L, Ny T 1991 Localization of luteinizing hormone receptor messenger ribonucleic acid
expression in ovarian cell types during follicle development and
ovulation. Endocrinology 129:32003207[Abstract]
-
Kotsuji F, Kamitani N, Goto K, Tominaga T 1990 Bovine theca
and granulosa cell interactions modulate their growth, morphology, and
function. Biol Reprod 43:726732[Abstract]
-
Yada H, Hosokawa K, Tajima K, Hasegawa Y, Kotsuji F 1999 Role
of ovarian theca and granulosa cell interaction in hormone production
and cell growth during the bovine follicular maturation process. Biol
Reprod 61:14801486[Abstract/Free Full Text]
-
Juengel JL, Meberg BM, Turzillo AM, Nett TM, Niswender GD 1995 Hormonal regulation of messenger ribonucleic acid encoding
steroidogenic acute regulatory protein in ovine corpora lutea.
Endocrinology 136:54235429[Abstract]
-
Zhang G, Garmey JC, Veldhuis JD 2000 Interactive stimulation
by luteinizing hormone and insulin of the steroidogenic acute
regulatory (StAR) protein and 17
-hydroxylase/17,20-lyase (CYP17)
genes in porcine theca cells. Endocrinology 141:27352742[Abstract/Free Full Text]
-
Ivell R, Tillmann G, Wang H, et al. 2000 Acute regulation of
the bovine gene for the steroidogenic acute regulatory protein in
ovarian theca and adrenocortical cells. J Mol Endocrinol 24:109118[Abstract/Free Full Text]
-
Silverman E, Eimerl S, Orly J 1999 CCAAT enhancer-binding
protein ß and GATA-4 binding regions within the promoter of the
steroidogenic acute regulatory protein (StAR) gene are required for
transcription in rat ovarian cells. J Biol Chem 274:1798717996[Abstract/Free Full Text]
-
Minegishi T, Tsuchiya M, Hirakawa T, et al. 2000 Expression of
steroidogenic acute regulatory protein (StAR) in rat granulosa cells.
Life Sci 67:10151024[CrossRef][Medline]
-
Morohashi KI, Omura T 1996 Ad4BP/SF-1, a transcription factor
essential for the transcription of steroidogenic cytochrome P450 genes
and for the establishment of the reproductive function. FASEB J 10:15691577[Abstract/Free Full Text]
-
Ikeda Y, Swain A, Weber TJ, et al. 1996 Steroidogenic factor 1
and Dax-1 colocalize in multiple cell lineages: potential links in
endocrine development. Mol Endocrinol 10:12611272[Abstract]
-
Margolin JF, Friedman JR, Meyer WK, Vissing H, Thiesen HJ,
Rauscher III FJ 1994 Kruppel-associated boxes are potent
transcriptional repression domains. Proc Natl Acad Sci USA 91:45094513[Abstract]
-
Witzgall R, OLeary E, Leaf A, Onaldi D, Bonventre JV 1994 The krüppel-associated box-A (KRAB-A) domain of zinc finger
proteins mediates transcriptional repression. Proc Natl Acad Sci USA 91:45144518[Abstract]
-
Nakamura M, Minegishi T, Hasegawa Y, et al. 1993 Effect of an
activin A on follicle-stimulating hormone (FSH) receptor messenger
ribonucleic acid levels and FSH receptor expressions in cultured rat
granulosa cells. Endocrinology 133:538544[Abstract]
-
Chomczynski P, Sacchi N 1987 Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem 162:156159[CrossRef][Medline]
-
Braissant O, Wahli W 1998 A simplified in situ
hybridization protocol using non-radioactively labeled probes to detect
abundant and rare mRNAs on tissue sections. Biochemica 1:1016
-
Sadowski I, Ptashne M 1989 A vector for expressing
GAL4(1147) fusions in mammalian cells. Nucleic Acids Res 17:7539[Medline]
-
Yamada K, Wang JC, Osawa H, Scott DK, Granner DK 1998 Efficient large-scale transformation of yeast. Biotechniques 24:596600[Medline]
-
Yamada K, Mizutani T, Shou Z, et al. 2001 Cloning and
functional expression of an E box-binding protein from rat granulosa
cells. Biol Reprod 64:13151319[Abstract/Free Full Text]
-
Yamada K, Osawa H, Granner DK 1999 Identification of proteins
that interact with NF-YA. FEBS Lett 460:4145[CrossRef][Medline]
-
Yamada K, Printz RL, Osawa H, Granner DK 1999 Human ZHX1:
cloning, chromosomal location, and interaction with transcription
factor NF-Y. Biochem Biophys Res Commun 261:614621[CrossRef][Medline]