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Molecular Endocrinology 15 (10): 1693-1705
Copyright © 2001 by The Endocrine Society

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha} 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo) 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. 1BGo) (20, 21, 22, 23, 24, 25, 26, 27). The other two clones, GIOT2{alpha} and -2ß, appear to be products from the same gene, which is closely related to, but distinct from, the GIOT1 gene. GIOT2{alpha} 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 268–603 of GIOT2{alpha} (Fig. 1AGo) and contains six zinc finger motifs. As shown in Fig. 1AGo, 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{alpha}.



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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{alpha}. Asterisks indicate positions of identical amino acid. A schematic illustration of structures of GIOT1, -2{alpha}, 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.

 
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{alpha}-specific probes, respectively (Fig. 2Go). With respect to the GIOT2 transcripts, hybridization with the GIOT2ß-specific probe gave essentially the same results as those by the GIOT2 {alpha}-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. 2Go, A and C). Essentially identical results were obtained when the immature rats were treated with hCG instead of PMSG (Fig. 2Go, 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.



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  		Figure 2. Expression of GIOT1 and -2 in the Ovary of Immature Rats

Northern blot analysis of GIOT1 (A and B) and -2{alpha} (C and D) mRNAs in immature rat ovaries primed with PMSG (A and C) or hCG (B and D), respectively. Total RNA was isolated at the indicated times as described in Materials and Methods. All samples contain 10 µg total RNA. A single transcript of about 3.2 kb for GIOT1 and two major transcripts of about 7 kb and 11 kb for GIOT2{alpha} were detected by hybridization with GIOT1- and -2{alpha}-specific probes, respectively. The blots were then rehybridized with a radiolabeled probe that is specific for rat GAPDH.

 
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. 3Go). 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.

 
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. 4AGo). A similar pattern of induction was observed in GIOT2 gene expression, although low levels of GIOT2 mRNA could be detected before FSH treatment (Fig. 4BGo). 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{alpha}-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{alpha} and GAPDH, respectively. The reaction mixtures were separated on a 2% agarose gel and visualized by EtBr staining.

 
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. 5Go, 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. 6Go). 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{alpha}-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{alpha}, 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.

 
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. 7Go). 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{alpha}-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{alpha}, and GAPDH, respectively. The reaction mixtures were separated on a 2% agarose gel and visualized with EtBr staining.

 
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. 8AGo). In contrast, the cotransfection of the pSG-GIOT2 had only a marginal effect in luciferase activity (Fig. 8AGo). 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.

 
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. 8BGo, both pSG-GIOT1 (1–145) and pSG-GIOT1 (1–65), 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 (33–654) and pSG-GIOT1 (1–38), led to the abolition of transcriptional repressor activity. These results indicate that amino acid sequences 1–65 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 1Go). The yeast SFY526 strain harboring pGBKT7 or pGBKT7-GIOT1 was transformed with pACT2, pRIC, or pTIF1ß, respectively. As shown in Table 1Go, 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 1Go, 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.


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  		Table 1. ß-Galactosidase Activities of Yeast Harboring Various Combinations of GAL4 DBD and AD Fusion Plasmids

 
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. 9Go). 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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
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 Ham’s 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 {lambda}-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 {lambda} 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{alpha} 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 Denhardt’s 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{alpha} 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{alpha}, 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 10–30 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 6–36 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 (1–38). To construct pSG-GIOT1 (37–654), pSG-GIOT1 was first digested with BamHI and EcoRV to remove a DNA fragment encoding GIOT1 (1–65). To the digested plasmid, a DNA fragment encoding GIOT1 (37–65), which was synthesized by PCR, was inserted, to produce pSG-GIOT1 (NOREF>37–654). 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 (1–65) and pSG-GIOT1 (1–145), 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 50–70% 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 (33–654), GIOT1 (>NOREF>1–145), and GIOT1 (1–65) were used to observe interaction of these domains with TIF1ß or RIC. For this purpose, cDNA inserts of pSG-GIOT1 (33–654), pSG-GIOT1 (1–65), and pSG-GIOT1 (1–145) 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{alpha} (accession number AB047637), and GIOT2ß (accession number AB047638) cDNA sequences have been deposited in the DDBJ/EMBL/GenBank database. Back

Received for publication April 5, 2001. Accepted for publication June 24, 2001.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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C. Hindmarch, S. Yao, G. Beighton, J. Paton, and D. Murphy
A comprehensive description of the transcriptome of the hypothalamoneurohypophyseal system in euhydrated and dehydrated rats
PNAS, January 31, 2006; 103(5): 1609 - 1614.
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 EndocrinologyHome page
M. Jo, M. C. Gieske, C. E. Payne, S. E. Wheeler-Price, J. B. Gieske, I. V. Ignatius, T. E. Curry Jr., and C. Ko
Development and Application of a Rat Ovarian Gene Expression Database
Endocrinology, November 1, 2004; 145(11): 5384 - 5396.
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T. Kajitani, T. Mizutani, K. Yamada, T. Yazawa, T. Sekiguchi, M. Yoshino, H. Kawata, and K. Miyamoto
Cloning and Characterization of Granulosa Cell High-Mobility Group (HMG)-Box Protein-1, a Novel HMG-Box Transcriptional Regulator Strongly Expressed in Rat Ovarian Granulosa Cells
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