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The role of ETS transcription factors in transcription and development of mouse preimplantation embryos
BBRC Biochemical and Biophysical Research Communications 344 (2006) 675–679 www.elsevier.com/locate/ybbrc
The role of ETS transcription factors in transcription and development of mouse preimplantation embryos Shun-ichiro Kageyama, Honglin Liu 1, Masao Nagata, Fugaku Aoki
*
Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba 277-8571, Japan Received 20 March 2006 Available online 17 April 2006
Abstract Embryonic transcription is a crucial process for the creation of new life. To clarify the mechanism of embryonic transcription, we investigated the expression and function of the erythroblast transformation specific (ETS) domain containing transcription factors (TFs) during preimplantation development in mice. The expression levels of several ETS TFs, i.e., etsrp71, elf3, and spic, increased after fertilization and remained at a high level until the blastocyst stage. To clarify the function of these TFs, we performed gene suppression using RNA interference, which revealed that they were involved in regulating development to the blastocyst stage. Furthermore, we found that suppression of ETS TFs affected the transcription of eIF-1A and oct3/4 genes whose expression is regulated by TATA-less promoters in the embryos. These results suggest that ETS TFs function in the regulation of transcription with TATA-less promoters in preimplantation embryos, which is essential in preimplantation development. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Embryo; Transcription; Gene expression; Ets; Transcription factor
Alteration of transcriptional regulation from the germ cells to the embryo is a very important process for the creation of new life. Several studies have indicated that a dynamic alteration of transcriptional activity and regulation of gene expression occurs during oogenesis and early development. A growing mouse oocyte transcribes many specific genes, e.g., ZP3 and dnmt1o [1–3]. However, when the oocyte is almost full-sized, transcription stops via an unknown mechanism and a transcriptionally inert state is maintained during meiosis. Fertilization triggers the completion of meiosis and the initiation of transcription from zygotic genome in one-cell embryos [4,5]. Transcriptional regulation is altered markedly during the two-cell stage. In embryos at the late two-cell stage, promoter activity is repressed and an enhancer is necessary in transcription
*
Corresponding author. Fax: +81 471 36 3698. E-mail address: [email protected] (F. Aoki). 1 Present address: College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, Japan. 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.03.192
[6,7]. Thus, following these alterations, differentiated germ cells obtain totipotency. An interesting feature of embryonic transcription is a high utilization of the TATA-less promoter, which is not utilized in growing oocytes. From the two-cell to the blastocyst stage, the TATA-less promoter becomes more active [8,9]. Recently, TBP knockout mice have been reported and it was discovered that TBP-deficient embryos did not show any deficiency in whole RNA polymerase transcription, indicating the importance of TATA-less promoter activity in embryos [10,11]. These changes in transcriptional regulation imply a global shift in the gene expression profile [12–14], which is likely to be regulated by alterations in epigenetic factors, such as transcription factors. To clarify the mechanism that regulates embryonic transcription, we focused on ETS family TFs, which are known to interact with TATA-less promoters and activate gene expression [15,16]. Therefore, we hypothesized that ETS might play important roles in embryonic transcription. In this study, we investigated the expression of ETS TFs and their functions in embryos using RNA interference.
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Materials and methods
Table 2 Sequences of siRNAs
Media. Whitten’s medium contained 109.51 mM NaCl, 4.78 mM KCl, 1.19 mM KH2PO4, 1.19 mM MgSO4, 22.62 mM NaHCO3, 5.55 mM D-glucose, 0.31 mM sodium pyruvate, 1.49 mM calcium lactate, 0.075 mg/ ml sodium penicillin-G, 0.05 mg/ml streptomycin sulfate, and 3 mg/ml BSA [17]. For the culture of embryos, we used KSOM [18], which consisted of 95 mM NaCl, 2.5 mM KCl, 0.3 mM KH2PO4, 0.2 mM MgSO4, 25 mM NaHCO3, 0.2 mM D-glucose, 0.01 mM EDTAÆ2Na, 0.2 mM sodium pyruvate, 10 mM DL-lactic acid (sodium salt, 60% syrup), 0.3 mM KH2PO4, and 3 mg/ml BSA. Collection and culture of oocytes and embryos. Female mice, 21–23 days of age (ddy; SLC, Shizuoka, Japan), were superovulated with 5 IU of pregnant mare’s serum gonadotropin (PMSG; Sankyo Co., Ltd., Tokyo, Japan) and 48 h later with 5 IU of human chorionic gonadotropin (hCG; Sankyo Co.). The oocytes were collected in Whitten’s medium from the ampullae of the oviduct at 15–16 h after hCG injection. Sperm were obtained in Whitten’s medium from the cauda epididymis of a mature male ICR mouse (SLC). The oocytes were inseminated with the sperm that had been incubated for 2 h at 38 °C. The embryos were washed with KSOM 2.5 h after insemination and then cultured in a humidified 5% CO2/95% air atmosphere at 38 °C [19]. Reverse transcription-PCR (RT-PCR). Total RNA samples were isolated from the oocytes and embryos using ISOGEN (Nippon Gene, Tokyo, Japan), as described previously [20]. The RNA was reverse-transcribed in a 20-ll reaction mixture that contained 5 U ReverScript II (Wako, Osaka, Japan) and 0.5 lg oligo(dT) 12–18 primer (Invitrogen Corp., Carlsbad, CA, USA) at 42 °C for 1 h, followed by 51 °C for 30 min. The template mRNA was digested with 60 U RNase H (TaKaRa, Shiga, Japan) at 37 °C for 20 min. As the external control, 50 pg of rabbit aglobin RNA was added to each tube before the isolation of total RNA. PCR was performed using the iCycler (Bio-Rad, Tokyo, Japan). The reaction mixture consisted of the template cDNA, 0.2 lM of each primer, 300 lM dNTPs, 3 mM MgCl2, and 0.05 U/ll ExTaq DNA polymerase (TaKaRa). The sequences of the PCR primers used are shown in Table 1. PCR was performed 32 cycles for rabbit a-globin and 40 cycles for the other genes. Each cycle consisted of denaturation at 95 °C for 15 s, annealing for 15 s, and extension at 72 °C for 20 s. The PCR products were separated by electrophoresis in a 2% agarose gel and stained with ethidium bromide. The gel image was obtained using a DT-20MP UV illuminator (ATTO, Tokyo, Japan). Transgenic mice. EGFP-transgenic mice, which had been originally produced by Dr. M. Okabe, Osaka University, were obtained from RIKEN BRC (BRC No. C57BL/6-TgN(act-EGFP)OsbC14-YO1FM131). The unfertilized oocytes obtained from normal female mice were inseminated with the sperm from EGFP-transgenic male mice. Preparation of siRNA-targeting ETS family members. siRNAs were chemically synthesized by Invitrogen. The sequences of siRNA for ETS family members, spic, elf3, and etsrp71, were designed using the
Name
siRNA sequence
Spic
1 2 1 2 1 2
Table 1 Details of condition for PCR Name
Primer
Sequence
Temperature (°C) annealing
Spic
Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense
5 0 -acgctaactactatggaatg-3 0 5 0 -tttttatgatctccccagtt-3 0 5 0 -tacagaaaagagcaggagcg-3 0 5 0 -tgtttcctgttctgttcccc-3 0 5 0 -ttcccatgcttctgactccg-3 0 5 0 -cagccccgatccttaattcc-3 0 5 0 -gacacaccgatcacaccaat-3 0 5 0 -taacgtagacctcgactcag-3 0 5 0 -gccgatttactggagcagag-3 0 5 0 -aggagatcggtagcggaaat-3 0
58
Gabbpa Elf3 Etsrp71 Etv6
59 58 59 60
Elf3 Etsrp71 GFP
5 0 -ggagaaagucaauacuccucagaaa-3 0 5 0 -ggagaaagcuucggcuguuugaaua-3 0 5 0 -ggaucaucccuaauuuaugugcuau-3 0 5 0 -gcacgaggguguguucaaguuucuu-3 0 5 0 -gcccuccaaaucgaacaagcagucu-3 0 5 0 -ggaauuugguuuctauuucccugaa-3 0 5 0 -ccacuaccugagcacccaguccgcc-3 0
Invitrogen program BLOCK-iTä RNAi Designer (https://rnaidesigner. invitrogen.com/rnaiexpress/). Two sequences were designed for each gene (Table 2). Microinjection of siRNA into zygotes. One-cell embryos were collected 1 h after insemination and freed from surrounding cumulus cells by treating with hyarulonidase. The embryos were then transferred to KSOM. Microinjection was performed under an inverted microscope (ECLIPSE TE300; Nicon Corporation, Tokyo, Japan) using a micromanipulator (Narishige Co., Ltd., Tokyo, Japan) and microinjector (IM300, Narishige Co., Ltd.). After washing three times with KSOM, 5–10 pl of siRNAs (60 lM) was injected into cytoplasm of one-cell embryos using borosilicate glass capillaries (GC100 TF-10; Harvard Apparatus Ltd., Kent, UK). The mixture of siRNAs with two different sequences designed for a single target gene, as described above, was injected into the embryos. The injected embryos were then cultured with KSOM in a humidified 5% CO2/95% air atmosphere at 38 °C. Expression of the target genes and the developmental ratio to the blastocyst stage were examined at 60 and 92 h after insemination, respectively.
Results Expression of ETS TFs An interesting feature of embryonic transcription in mice is the high utilization of TATA-less promoters [8,9]. In our previous study, we investigated global TF expression from germ cells to preimplantation development using microarray analysis (S.K. and F.A., unpublished data) and found that several ETS family TFs increased their expression levels after fertilization. In the present study, we investigated by RT-PCR the expression of five ETS family genes, i.e., etsrp71, gabbpa, elf3, spic, and etv6, in which a prominent increase had been detected in the microarray analysis. The results of RT-PCR analysis showed that the expression of etsrp71 and spic was at very low or undetectable levels in the MII stage oocytes; after fertilization, their expression levels abruptly increased at the two-cell stage, then slightly decreased at the blastocyst stage (Fig. 1). The expression level of elf3 was also low in MII oocytes. It increased at the one-cell stage, declined at the two-cell stage, and then increased again at the blastocyst stage. Gabbpa was already expressed in MII oocytes and increased slightly at the one-cell stage. This expression level was maintained until the blastocyst stage. The expression of etv6 was not detected in any stage. Thus, we determined that the expression levels of etsrp71, elf3, and spic were low before fertilization and increased prominently after fertilization.
S. Kageyama et al. / Biochemical and Biophysical Research Communications 344 (2006) 675–679
MII
1C
2C
4C
677
etsrp71, elf3, or spic, the expression levels of these target genes greatly decreased compared to the control embryos, which had been microinjected with EGFP-siRNA (Fig. 2B).
Bl Etsrp71 Gabbpa
Effect of ETS suppression on preimplantation development Elf3 Spic Etv6 globin Fig. 1. ETS TF mRNA expression in mouse preimplantation embryos. The expression of ETS TFs was examined by RT-PCR in MII stage oocytes (MII) and the embryos at the one-cell (1C), two-cell (2C), four-cell (4C), and blastocyst (Bl) stages. Rabbit a-globin RNA was added to the RNA samples as an external control. The expression of etsrp71, gabbpa, elf3, spic, and etv6 transcripts is shown. Experiments were conducted twice and similar results were obtained each time.
Suppression of ETS TFs by siRNA To examine the role of ETS TFs in early development and gene expression, we attempted to suppress the three aforementioned ETS genes, etsrp71, elf3, and spi, by RNA interference. Before addressing these target genes, we suppressed EGFP expression in EGFP-transgenic mice by siRNA to check the efficiency of this system. As shown in Fig. 2A, microinjection of EGFP-siRNA almost completely suppressed the expression of the EGFP protein in the blastocysts, indicating that this system of RNAi suppression works well in preimplantation embryos. In the embryos that had been microinjected with siRNA of A
To investigate the role of ETS TFs on preimplantation development, we examined the developmental rate of the embryos microinjected with TF siRNA. Prior to carrying out this analysis, we first examined the effect of the microinjection procedure and the non-specific toxicity of siRNA on development by comparing the developmental rate between the embryos injected with EGFP-siRNA and non-injected embryos. The results showed that there was no difference in their developmental rates: 93% and 92% of the injected and non-injected embryos, respectively, developed to the blastocyst stage. Then, we microinjected the siRNA-targeting ETS TFs to suppress development. In each ETS TF suppression, the percentage of embryos that developed to the blastocyst stage significantly decreased in the siRNAs targeting etsrp71, elf3, and spic, compared to the control embryos, which had been microinjected with EGFP-siRNA (Fig. 3). Although the reduction in developmental rate by suppressing each ETS TF was obvious, more than half of the embryos still developed to the blastocyst stage. Therefore, it is likely that these three ETS TFs function redundantly in embryos, and when an ETS TF is suppressed by siRNA, the other two TFs may compensate for it. To address this possibility, we injected three ETS siRNA at the same time. This treatment further decreased the developmental rate, as only around 30% of embryos developed to the blastocyst stage, suggesting that ETS TFs play roles in the regulation of development redundantly, and otherwise cooperatively.
ddw
si-eGFP Epifluorescent
Blight-field
B
si-GFP
Elf3 globin
si-Elf3
si-GFP
Etsrp71 globin
si-Etsrp71
si-GFP
si-Spic
Spic globin
Fig. 2. Suppression of gene expression by siRNA microinjection. (A) The oocytes which had been fertilized with the sperm from EGFP-transgenic mice were microinjected with the siRNA-targeting EGFP gene (si-eGFP) or double-distilled water (DDW) as a control, 1 h after insemination. The fluorescence of EGFP was observed in the embryos under an epifluorescence microscope 92 h after insemination. (B) siRNAs targeting elf3, etsrp71, and spic (si-Elf3, si-Etsrp71, and si-Spic, respectively) were microinjected into the respective embryos, which had been obtained from normal mice, 1 h after insemination, and examined for expressions of corresponding genes 60 h after insemination. si-GFP was microinjected into the control embryos. Rabbit a-globin mRNA (globin) was added to the samples before RNA preparation to control the recovery of total RNA and the efficiency of reverse transcription in each sample. The experiments were conducted twice and similar results were obtained each time.
S. Kageyama et al. / Biochemical and Biophysical Research Communications 344 (2006) 675–679
reduced the expression of eIF-1A and oct3/4, which have ETS regulation sites, but did not reduce the expression of hprt and rel, which have no ETS regulation sites (Fig. 4).
100 80
*
60
*
*
40
*
Discussion
20 al l si -3
si -E lf3
si -S pi c
0 N oin je ct io n si -e G FP si -E ts rp 71
Developmental rate to blastocyst (%)
678
Fig. 3. The effect of ETS-siRNA injection on preimplantation development. Zygotes were microinjected with various ETS-siRNAs and eGFPsiRNA 1 h after insemination. The percentage of embryos that had developed to the blastocyst stage was calculated 92 h after insemination. The experiment was conducted three times, each time with similar results, and the data were combined. The total numbers of embryos examined were 162, 120, 95, 93, 76, and 102 for no injection, eGFP-siRNA, Etsrp71siRNA, Spic-siRNA, Elf3-siRNA, and all three ETS-siRNA injections, respectively. An asterisk (*) indicates a significant difference (P < 0.05) from the value of the eGFP-siRNA injection (v2 test).
Effect of ETS suppression on gene expression in preimplantation embryos To clarify the role of ETS TFs in transcription, we investigated genes with ETS regulation sites in their promoters. It has previously been reported that eIF-1A and oct3/4 transcription in embryos are mainly regulated by TATAless promoters and that they have ETS regulation sites in their promoters. However, hprt and rel, which are generally expressed in most types of cells, do not contain ETS regulation sites in their promoters. We investigated the expression of these four genes under a condition whereby three ETS TFs were suppressed by injection with a mixture of three ETS siRNAs. As an experimental control, we injected EGFP-siRNA and compared the expression levels of the four genes with those in ETS-siRNA injected embryos. The results showed that suppression of ETS TFs markedly si-GFP
si-Ets
Hprt rel eIF-1A Oct3/4 globin Fig. 4. The effect of ETS-siRNA injection on gene expression in preimplantation embryos. Zygotes were microinjected with mixtures of siRNA, targeting three ETS family members (si-ETS), elf3, etsrp71, and spic, 1 h after insemination. EGFP-siRNA (si-GFP) was injected into the control embryos. The embryos were recovered 60 h after insemination and the expression of Hprt, rel, eIF-1A, and Oct3/4 was examined by RTPCR. The experiments were conducted twice and similar results were obtained each time.
To clarify the role of ETS TFs in embryonic transcription, we investigated the expression of ETS TFs during preimplantation development. The results showed that the expression of etsrp71, elf3, and spic prominently increased after fertilization (Fig. 1). To clarify their functions, we suppressed them by RNA interference, both individually and simultaneously, and investigated their function in development and gene expression. Suppression of these genes significantly decreased the developmental rate of the embryos (Fig. 3). Interestingly, simultaneous suppression of all three ETS TFs was more effective in reducing the developmental rate than individual suppression. This result suggests that ETS TFs play roles in the regulation of development either redundantly or cooperatively. Reports that some ETS family members interact with each other support this idea. For example, ETS2 is associated with the promoter of IL-8 and myeloid elf-1-like factor (MEF), which also belongs to the ETS family, binds to the IL-8 promoter competitively with ETS2 [21], thus suggesting that various ETS TFs function in embryos by interacting with each other. We examined the role of ETS TFs in transcription in embryos and found that they play important roles in activating the TATA-less promoter. As a feature of transcription after fertilization, high levels of TATA-less promoter activity have been reported. eIF-1A is a well-known gene that is highly transcribed at ZGA [11,22]. This gene has two different transcription initiation sites, one of which is activated by the TATA promoter and the other by the TATA-less promoter located upstream of the TATA promoter [22]. Although eIF-1A is transcribed by the TATA promoter in growing oocytes, its transcriptional regulation is altered to use the TATA-less promoter after fertilization. This high TATA-less promoter activity in eIF-1A is maintained until the blastocyst stage. Oct3/4 is also transcribed by the TATA-less promoter in embryos. Oct3/4 is known as a POU domain containing TFs and it is transcribed after the eight-cell stage, playing an essential role in the formation of ICM [23–25]. It has been reported that TBP (TATA box binding protein)-deficient embryos showed a normal RNA polymerase activity level, suggesting TATA-less promoter dependency in embryonic transcription [11]. Thus, although several reports suggest the importance of TATA-less promoter activity in transcription after fertilization, the mechanism that regulates its activity has not been elucidated. In the present study, we showed that the expression of the genes containing the TATA-less promoter and ETS regulation sites, i.e., eIF-1A and oct3/4, was reduced by the suppression of ETS TFs (Fig. 4). These results strongly suggest that the activation of TATA-less promoters after fertilization is regulated by ETS TFs.
S. Kageyama et al. / Biochemical and Biophysical Research Communications 344 (2006) 675–679
We found that the expression patterns of various ETS TFs differed from each other (Fig. 1), which suggests that different ETS TFs work at different stages. As described above, up-regulation of the TATA-less promoter starts after fertilization and continues to the blastocyst stage. However, the mechanism involved in this high TATA-less promoter activity is not a simple one, but one altered during preimplantation development. In the genes that are regulated by the TATA-less promoter, the transcription of eIF-1A is initiated at the one-cell stage [9], while oct3/4 transcription does not start until the eight-cell stage [23,26]. Thus, different mechanisms seem to regulate the expression of the genes with TATA-less promoters at different stages. The current results show that the expression patterns of various ETS TFs differed from each other during preimplantation development (Fig. 1). Gabba was expressed in MII oocytes and stably expressed at all stages examined. Spic and etrp71 were expressed mainly at the two-cell and four-cell stages and Elf3 was highly expressed at the blastocyst stage. These results suggest that ETS TFs alterations play important roles in the changes of expression in genes that are regulated by TATA-less promoters during preimplantation development. We have shown that the expression of several ETS TFs increased at the one-cell or two-cell stage and their expression levels were maintained at a high level until the blastocyst stage. Suppression of these TFs by RNA interference resulted in a reduction of the developmental rate. The expression of eIF-1A and oct3/4, which are target genes of ETS TFs, also decreased under ETS suppression. These results suggest that ETS TFs are involved in the regulation of transcription and play an important role in preimplantation development.
[7] [8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16] [17] [18]
[19]
Acknowledgments This research was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan to F.A. (HD 14360164 and 16045203).
[20]
[21]
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The role of ETS transcription factors in transcription and development of mouse preimplantation embryos Shun-ichiro Kageyama, Honglin Liu 1, Masao Nagata, Fugaku Aoki
*
Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba 277-8571, Japan Received 20 March 2006 Available online 17 April 2006
Abstract Embryonic transcription is a crucial process for the creation of new life. To clarify the mechanism of embryonic transcription, we investigated the expression and function of the erythroblast transformation specific (ETS) domain containing transcription factors (TFs) during preimplantation development in mice. The expression levels of several ETS TFs, i.e., etsrp71, elf3, and spic, increased after fertilization and remained at a high level until the blastocyst stage. To clarify the function of these TFs, we performed gene suppression using RNA interference, which revealed that they were involved in regulating development to the blastocyst stage. Furthermore, we found that suppression of ETS TFs affected the transcription of eIF-1A and oct3/4 genes whose expression is regulated by TATA-less promoters in the embryos. These results suggest that ETS TFs function in the regulation of transcription with TATA-less promoters in preimplantation embryos, which is essential in preimplantation development. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Embryo; Transcription; Gene expression; Ets; Transcription factor
Alteration of transcriptional regulation from the germ cells to the embryo is a very important process for the creation of new life. Several studies have indicated that a dynamic alteration of transcriptional activity and regulation of gene expression occurs during oogenesis and early development. A growing mouse oocyte transcribes many specific genes, e.g., ZP3 and dnmt1o [1–3]. However, when the oocyte is almost full-sized, transcription stops via an unknown mechanism and a transcriptionally inert state is maintained during meiosis. Fertilization triggers the completion of meiosis and the initiation of transcription from zygotic genome in one-cell embryos [4,5]. Transcriptional regulation is altered markedly during the two-cell stage. In embryos at the late two-cell stage, promoter activity is repressed and an enhancer is necessary in transcription
*
Corresponding author. Fax: +81 471 36 3698. E-mail address: [email protected] (F. Aoki). 1 Present address: College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, Japan. 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.03.192
[6,7]. Thus, following these alterations, differentiated germ cells obtain totipotency. An interesting feature of embryonic transcription is a high utilization of the TATA-less promoter, which is not utilized in growing oocytes. From the two-cell to the blastocyst stage, the TATA-less promoter becomes more active [8,9]. Recently, TBP knockout mice have been reported and it was discovered that TBP-deficient embryos did not show any deficiency in whole RNA polymerase transcription, indicating the importance of TATA-less promoter activity in embryos [10,11]. These changes in transcriptional regulation imply a global shift in the gene expression profile [12–14], which is likely to be regulated by alterations in epigenetic factors, such as transcription factors. To clarify the mechanism that regulates embryonic transcription, we focused on ETS family TFs, which are known to interact with TATA-less promoters and activate gene expression [15,16]. Therefore, we hypothesized that ETS might play important roles in embryonic transcription. In this study, we investigated the expression of ETS TFs and their functions in embryos using RNA interference.
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S. Kageyama et al. / Biochemical and Biophysical Research Communications 344 (2006) 675–679
Materials and methods
Table 2 Sequences of siRNAs
Media. Whitten’s medium contained 109.51 mM NaCl, 4.78 mM KCl, 1.19 mM KH2PO4, 1.19 mM MgSO4, 22.62 mM NaHCO3, 5.55 mM D-glucose, 0.31 mM sodium pyruvate, 1.49 mM calcium lactate, 0.075 mg/ ml sodium penicillin-G, 0.05 mg/ml streptomycin sulfate, and 3 mg/ml BSA [17]. For the culture of embryos, we used KSOM [18], which consisted of 95 mM NaCl, 2.5 mM KCl, 0.3 mM KH2PO4, 0.2 mM MgSO4, 25 mM NaHCO3, 0.2 mM D-glucose, 0.01 mM EDTAÆ2Na, 0.2 mM sodium pyruvate, 10 mM DL-lactic acid (sodium salt, 60% syrup), 0.3 mM KH2PO4, and 3 mg/ml BSA. Collection and culture of oocytes and embryos. Female mice, 21–23 days of age (ddy; SLC, Shizuoka, Japan), were superovulated with 5 IU of pregnant mare’s serum gonadotropin (PMSG; Sankyo Co., Ltd., Tokyo, Japan) and 48 h later with 5 IU of human chorionic gonadotropin (hCG; Sankyo Co.). The oocytes were collected in Whitten’s medium from the ampullae of the oviduct at 15–16 h after hCG injection. Sperm were obtained in Whitten’s medium from the cauda epididymis of a mature male ICR mouse (SLC). The oocytes were inseminated with the sperm that had been incubated for 2 h at 38 °C. The embryos were washed with KSOM 2.5 h after insemination and then cultured in a humidified 5% CO2/95% air atmosphere at 38 °C [19]. Reverse transcription-PCR (RT-PCR). Total RNA samples were isolated from the oocytes and embryos using ISOGEN (Nippon Gene, Tokyo, Japan), as described previously [20]. The RNA was reverse-transcribed in a 20-ll reaction mixture that contained 5 U ReverScript II (Wako, Osaka, Japan) and 0.5 lg oligo(dT) 12–18 primer (Invitrogen Corp., Carlsbad, CA, USA) at 42 °C for 1 h, followed by 51 °C for 30 min. The template mRNA was digested with 60 U RNase H (TaKaRa, Shiga, Japan) at 37 °C for 20 min. As the external control, 50 pg of rabbit aglobin RNA was added to each tube before the isolation of total RNA. PCR was performed using the iCycler (Bio-Rad, Tokyo, Japan). The reaction mixture consisted of the template cDNA, 0.2 lM of each primer, 300 lM dNTPs, 3 mM MgCl2, and 0.05 U/ll ExTaq DNA polymerase (TaKaRa). The sequences of the PCR primers used are shown in Table 1. PCR was performed 32 cycles for rabbit a-globin and 40 cycles for the other genes. Each cycle consisted of denaturation at 95 °C for 15 s, annealing for 15 s, and extension at 72 °C for 20 s. The PCR products were separated by electrophoresis in a 2% agarose gel and stained with ethidium bromide. The gel image was obtained using a DT-20MP UV illuminator (ATTO, Tokyo, Japan). Transgenic mice. EGFP-transgenic mice, which had been originally produced by Dr. M. Okabe, Osaka University, were obtained from RIKEN BRC (BRC No. C57BL/6-TgN(act-EGFP)OsbC14-YO1FM131). The unfertilized oocytes obtained from normal female mice were inseminated with the sperm from EGFP-transgenic male mice. Preparation of siRNA-targeting ETS family members. siRNAs were chemically synthesized by Invitrogen. The sequences of siRNA for ETS family members, spic, elf3, and etsrp71, were designed using the
Name
siRNA sequence
Spic
1 2 1 2 1 2
Table 1 Details of condition for PCR Name
Primer
Sequence
Temperature (°C) annealing
Spic
Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense
5 0 -acgctaactactatggaatg-3 0 5 0 -tttttatgatctccccagtt-3 0 5 0 -tacagaaaagagcaggagcg-3 0 5 0 -tgtttcctgttctgttcccc-3 0 5 0 -ttcccatgcttctgactccg-3 0 5 0 -cagccccgatccttaattcc-3 0 5 0 -gacacaccgatcacaccaat-3 0 5 0 -taacgtagacctcgactcag-3 0 5 0 -gccgatttactggagcagag-3 0 5 0 -aggagatcggtagcggaaat-3 0
58
Gabbpa Elf3 Etsrp71 Etv6
59 58 59 60
Elf3 Etsrp71 GFP
5 0 -ggagaaagucaauacuccucagaaa-3 0 5 0 -ggagaaagcuucggcuguuugaaua-3 0 5 0 -ggaucaucccuaauuuaugugcuau-3 0 5 0 -gcacgaggguguguucaaguuucuu-3 0 5 0 -gcccuccaaaucgaacaagcagucu-3 0 5 0 -ggaauuugguuuctauuucccugaa-3 0 5 0 -ccacuaccugagcacccaguccgcc-3 0
Invitrogen program BLOCK-iTä RNAi Designer (https://rnaidesigner. invitrogen.com/rnaiexpress/). Two sequences were designed for each gene (Table 2). Microinjection of siRNA into zygotes. One-cell embryos were collected 1 h after insemination and freed from surrounding cumulus cells by treating with hyarulonidase. The embryos were then transferred to KSOM. Microinjection was performed under an inverted microscope (ECLIPSE TE300; Nicon Corporation, Tokyo, Japan) using a micromanipulator (Narishige Co., Ltd., Tokyo, Japan) and microinjector (IM300, Narishige Co., Ltd.). After washing three times with KSOM, 5–10 pl of siRNAs (60 lM) was injected into cytoplasm of one-cell embryos using borosilicate glass capillaries (GC100 TF-10; Harvard Apparatus Ltd., Kent, UK). The mixture of siRNAs with two different sequences designed for a single target gene, as described above, was injected into the embryos. The injected embryos were then cultured with KSOM in a humidified 5% CO2/95% air atmosphere at 38 °C. Expression of the target genes and the developmental ratio to the blastocyst stage were examined at 60 and 92 h after insemination, respectively.
Results Expression of ETS TFs An interesting feature of embryonic transcription in mice is the high utilization of TATA-less promoters [8,9]. In our previous study, we investigated global TF expression from germ cells to preimplantation development using microarray analysis (S.K. and F.A., unpublished data) and found that several ETS family TFs increased their expression levels after fertilization. In the present study, we investigated by RT-PCR the expression of five ETS family genes, i.e., etsrp71, gabbpa, elf3, spic, and etv6, in which a prominent increase had been detected in the microarray analysis. The results of RT-PCR analysis showed that the expression of etsrp71 and spic was at very low or undetectable levels in the MII stage oocytes; after fertilization, their expression levels abruptly increased at the two-cell stage, then slightly decreased at the blastocyst stage (Fig. 1). The expression level of elf3 was also low in MII oocytes. It increased at the one-cell stage, declined at the two-cell stage, and then increased again at the blastocyst stage. Gabbpa was already expressed in MII oocytes and increased slightly at the one-cell stage. This expression level was maintained until the blastocyst stage. The expression of etv6 was not detected in any stage. Thus, we determined that the expression levels of etsrp71, elf3, and spic were low before fertilization and increased prominently after fertilization.
S. Kageyama et al. / Biochemical and Biophysical Research Communications 344 (2006) 675–679
MII
1C
2C
4C
677
etsrp71, elf3, or spic, the expression levels of these target genes greatly decreased compared to the control embryos, which had been microinjected with EGFP-siRNA (Fig. 2B).
Bl Etsrp71 Gabbpa
Effect of ETS suppression on preimplantation development Elf3 Spic Etv6 globin Fig. 1. ETS TF mRNA expression in mouse preimplantation embryos. The expression of ETS TFs was examined by RT-PCR in MII stage oocytes (MII) and the embryos at the one-cell (1C), two-cell (2C), four-cell (4C), and blastocyst (Bl) stages. Rabbit a-globin RNA was added to the RNA samples as an external control. The expression of etsrp71, gabbpa, elf3, spic, and etv6 transcripts is shown. Experiments were conducted twice and similar results were obtained each time.
Suppression of ETS TFs by siRNA To examine the role of ETS TFs in early development and gene expression, we attempted to suppress the three aforementioned ETS genes, etsrp71, elf3, and spi, by RNA interference. Before addressing these target genes, we suppressed EGFP expression in EGFP-transgenic mice by siRNA to check the efficiency of this system. As shown in Fig. 2A, microinjection of EGFP-siRNA almost completely suppressed the expression of the EGFP protein in the blastocysts, indicating that this system of RNAi suppression works well in preimplantation embryos. In the embryos that had been microinjected with siRNA of A
To investigate the role of ETS TFs on preimplantation development, we examined the developmental rate of the embryos microinjected with TF siRNA. Prior to carrying out this analysis, we first examined the effect of the microinjection procedure and the non-specific toxicity of siRNA on development by comparing the developmental rate between the embryos injected with EGFP-siRNA and non-injected embryos. The results showed that there was no difference in their developmental rates: 93% and 92% of the injected and non-injected embryos, respectively, developed to the blastocyst stage. Then, we microinjected the siRNA-targeting ETS TFs to suppress development. In each ETS TF suppression, the percentage of embryos that developed to the blastocyst stage significantly decreased in the siRNAs targeting etsrp71, elf3, and spic, compared to the control embryos, which had been microinjected with EGFP-siRNA (Fig. 3). Although the reduction in developmental rate by suppressing each ETS TF was obvious, more than half of the embryos still developed to the blastocyst stage. Therefore, it is likely that these three ETS TFs function redundantly in embryos, and when an ETS TF is suppressed by siRNA, the other two TFs may compensate for it. To address this possibility, we injected three ETS siRNA at the same time. This treatment further decreased the developmental rate, as only around 30% of embryos developed to the blastocyst stage, suggesting that ETS TFs play roles in the regulation of development redundantly, and otherwise cooperatively.
ddw
si-eGFP Epifluorescent
Blight-field
B
si-GFP
Elf3 globin
si-Elf3
si-GFP
Etsrp71 globin
si-Etsrp71
si-GFP
si-Spic
Spic globin
Fig. 2. Suppression of gene expression by siRNA microinjection. (A) The oocytes which had been fertilized with the sperm from EGFP-transgenic mice were microinjected with the siRNA-targeting EGFP gene (si-eGFP) or double-distilled water (DDW) as a control, 1 h after insemination. The fluorescence of EGFP was observed in the embryos under an epifluorescence microscope 92 h after insemination. (B) siRNAs targeting elf3, etsrp71, and spic (si-Elf3, si-Etsrp71, and si-Spic, respectively) were microinjected into the respective embryos, which had been obtained from normal mice, 1 h after insemination, and examined for expressions of corresponding genes 60 h after insemination. si-GFP was microinjected into the control embryos. Rabbit a-globin mRNA (globin) was added to the samples before RNA preparation to control the recovery of total RNA and the efficiency of reverse transcription in each sample. The experiments were conducted twice and similar results were obtained each time.
S. Kageyama et al. / Biochemical and Biophysical Research Communications 344 (2006) 675–679
reduced the expression of eIF-1A and oct3/4, which have ETS regulation sites, but did not reduce the expression of hprt and rel, which have no ETS regulation sites (Fig. 4).
100 80
*
60
*
*
40
*
Discussion
20 al l si -3
si -E lf3
si -S pi c
0 N oin je ct io n si -e G FP si -E ts rp 71
Developmental rate to blastocyst (%)
678
Fig. 3. The effect of ETS-siRNA injection on preimplantation development. Zygotes were microinjected with various ETS-siRNAs and eGFPsiRNA 1 h after insemination. The percentage of embryos that had developed to the blastocyst stage was calculated 92 h after insemination. The experiment was conducted three times, each time with similar results, and the data were combined. The total numbers of embryos examined were 162, 120, 95, 93, 76, and 102 for no injection, eGFP-siRNA, Etsrp71siRNA, Spic-siRNA, Elf3-siRNA, and all three ETS-siRNA injections, respectively. An asterisk (*) indicates a significant difference (P < 0.05) from the value of the eGFP-siRNA injection (v2 test).
Effect of ETS suppression on gene expression in preimplantation embryos To clarify the role of ETS TFs in transcription, we investigated genes with ETS regulation sites in their promoters. It has previously been reported that eIF-1A and oct3/4 transcription in embryos are mainly regulated by TATAless promoters and that they have ETS regulation sites in their promoters. However, hprt and rel, which are generally expressed in most types of cells, do not contain ETS regulation sites in their promoters. We investigated the expression of these four genes under a condition whereby three ETS TFs were suppressed by injection with a mixture of three ETS siRNAs. As an experimental control, we injected EGFP-siRNA and compared the expression levels of the four genes with those in ETS-siRNA injected embryos. The results showed that suppression of ETS TFs markedly si-GFP
si-Ets
Hprt rel eIF-1A Oct3/4 globin Fig. 4. The effect of ETS-siRNA injection on gene expression in preimplantation embryos. Zygotes were microinjected with mixtures of siRNA, targeting three ETS family members (si-ETS), elf3, etsrp71, and spic, 1 h after insemination. EGFP-siRNA (si-GFP) was injected into the control embryos. The embryos were recovered 60 h after insemination and the expression of Hprt, rel, eIF-1A, and Oct3/4 was examined by RTPCR. The experiments were conducted twice and similar results were obtained each time.
To clarify the role of ETS TFs in embryonic transcription, we investigated the expression of ETS TFs during preimplantation development. The results showed that the expression of etsrp71, elf3, and spic prominently increased after fertilization (Fig. 1). To clarify their functions, we suppressed them by RNA interference, both individually and simultaneously, and investigated their function in development and gene expression. Suppression of these genes significantly decreased the developmental rate of the embryos (Fig. 3). Interestingly, simultaneous suppression of all three ETS TFs was more effective in reducing the developmental rate than individual suppression. This result suggests that ETS TFs play roles in the regulation of development either redundantly or cooperatively. Reports that some ETS family members interact with each other support this idea. For example, ETS2 is associated with the promoter of IL-8 and myeloid elf-1-like factor (MEF), which also belongs to the ETS family, binds to the IL-8 promoter competitively with ETS2 [21], thus suggesting that various ETS TFs function in embryos by interacting with each other. We examined the role of ETS TFs in transcription in embryos and found that they play important roles in activating the TATA-less promoter. As a feature of transcription after fertilization, high levels of TATA-less promoter activity have been reported. eIF-1A is a well-known gene that is highly transcribed at ZGA [11,22]. This gene has two different transcription initiation sites, one of which is activated by the TATA promoter and the other by the TATA-less promoter located upstream of the TATA promoter [22]. Although eIF-1A is transcribed by the TATA promoter in growing oocytes, its transcriptional regulation is altered to use the TATA-less promoter after fertilization. This high TATA-less promoter activity in eIF-1A is maintained until the blastocyst stage. Oct3/4 is also transcribed by the TATA-less promoter in embryos. Oct3/4 is known as a POU domain containing TFs and it is transcribed after the eight-cell stage, playing an essential role in the formation of ICM [23–25]. It has been reported that TBP (TATA box binding protein)-deficient embryos showed a normal RNA polymerase activity level, suggesting TATA-less promoter dependency in embryonic transcription [11]. Thus, although several reports suggest the importance of TATA-less promoter activity in transcription after fertilization, the mechanism that regulates its activity has not been elucidated. In the present study, we showed that the expression of the genes containing the TATA-less promoter and ETS regulation sites, i.e., eIF-1A and oct3/4, was reduced by the suppression of ETS TFs (Fig. 4). These results strongly suggest that the activation of TATA-less promoters after fertilization is regulated by ETS TFs.
S. Kageyama et al. / Biochemical and Biophysical Research Communications 344 (2006) 675–679
We found that the expression patterns of various ETS TFs differed from each other (Fig. 1), which suggests that different ETS TFs work at different stages. As described above, up-regulation of the TATA-less promoter starts after fertilization and continues to the blastocyst stage. However, the mechanism involved in this high TATA-less promoter activity is not a simple one, but one altered during preimplantation development. In the genes that are regulated by the TATA-less promoter, the transcription of eIF-1A is initiated at the one-cell stage [9], while oct3/4 transcription does not start until the eight-cell stage [23,26]. Thus, different mechanisms seem to regulate the expression of the genes with TATA-less promoters at different stages. The current results show that the expression patterns of various ETS TFs differed from each other during preimplantation development (Fig. 1). Gabba was expressed in MII oocytes and stably expressed at all stages examined. Spic and etrp71 were expressed mainly at the two-cell and four-cell stages and Elf3 was highly expressed at the blastocyst stage. These results suggest that ETS TFs alterations play important roles in the changes of expression in genes that are regulated by TATA-less promoters during preimplantation development. We have shown that the expression of several ETS TFs increased at the one-cell or two-cell stage and their expression levels were maintained at a high level until the blastocyst stage. Suppression of these TFs by RNA interference resulted in a reduction of the developmental rate. The expression of eIF-1A and oct3/4, which are target genes of ETS TFs, also decreased under ETS suppression. These results suggest that ETS TFs are involved in the regulation of transcription and play an important role in preimplantation development.
[7] [8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16] [17] [18]
[19]
Acknowledgments This research was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan to F.A. (HD 14360164 and 16045203).
[20]
[21]
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