Regulatory elements and transcriptional control of chicken vasa homologue (CVH) promoter in chicken primordial germ cells
© The Author(s). 2017
Received: 5 August 2016
Accepted: 7 December 2016
Published: 13 January 2017
Primordial germ cells (PGCs), the precursors of functional gametes, have distinct characteristics and exhibit several unique molecular mechanisms to maintain pluripotency and germness in comparison to somatic cells. They express germ cell-specific RNA binding proteins (RBPs) by modulating tissue-specific cis- and trans-regulatory elements. Studies on gene structures of chicken vasa homologue (CVH), a chicken RNA binding protein, involved in temporal and spatial regulation are thus important not only for understanding the molecular mechanisms that regulate germ cell fate, but also for practical applications of primordial germ cells. However, very limited studies are available on regulatory elements that control germ cell-specific expression in chicken. Therefore, we investigated the intricate regulatory mechanism(s) that governs transcriptional control of CVH.
We constructed green fluorescence protein (GFP) or luciferase reporter vectors containing the various 5′ flanking regions of CVH gene. From the 5′ deletion and fragmented assays in chicken PGCs, we have identified a CVH promoter that locates at −316 to +275 base pair fragment with the highest luciferase activity. Additionally, we confirmed for the first time that the 5′ untranslated region (UTR) containing intron 1 is required for promoter activity of the CVH gene in chicken PGCs. Furthermore, using a transcription factor binding prediction, transcriptome analysis and siRNA-mediated knockdown, we have identified that a set of transcription factors play a role in the PGC-specific CVH gene expression.
These results demonstrate that cis-elements and transcription factors localizing in the 5′ flanking region including the 5′ UTR and an intron are important for transcriptional regulation of the CVH gene in chicken PGCs. Finally, this information will contribute to research studies in areas of reproductive biology, constructing of germ cell-specific synthetic promoter for tracing primordial germ cells as well as understanding the transcriptional regulation for maintaining germness in PGCs.
KeywordsChicken Chicken vasa homologue Primordial germ cell Regulatory element siRNA-mediated knockdown
Primordial germ cells (PGCs) that emerge during early embryogenesis undergo a series of developmental events, such as specification, migration, and differentiation, to produce a new organism in the next generation [1, 2]. They express RNA binding proteins (RBPs) by modulating tissue-specific cis- and trans-regulatory elements and have specialized genetic programs distinct from those of other somatic cells for maintaining their unique characteristics [3–5]. Significant efforts have been made to elucidate the detailed molecular mechanisms regulating transcriptional control in germ cells [6–8]. At the transcriptional level, certain genes are effectively silenced, whereas other genes are exclusively expressed to maintain the levels of germline-expressed gene products [9, 10].
In chicken, PGCs separate from the epiblast in the blastoderm at Eyal-Giladi and Kochav stage X, which consist of 40,000 to 60,000 undifferentiated embryonic cells, and translocate into the hypoblast area of the pellucida [11, 12]. During gastrulation, they circulate through the vascular system and finally settle in the gonadal anlagen. After the arrival of PGCs, these cells continue to proliferate until they enter meiosis. This development of the PGC lineage is a highly complex process that is controlled by the coordinated action of many key factors, such as the expression and regulation of germline-specific genes [13, 14].
Evolutionarily conserved germ cell-specific vasa has been characterized in germ cells in several organisms, including chicken [15–17], zebrafish , mouse , and human . Several studies have demonstrated that vasa plays critical roles in germ cell specification, supporting germ line development, translational control of transcribed genes, and RNA processes involving the biosynthesis of PIWI-interacting RNAs (piRNAs) in germ cells at the post-transcriptional level [21–26]. However, the intricate regulatory mechanism(s) that governs transcriptional control of vasa expression during chicken germline development has yet to be investigated in detail.
Understanding the cellular and molecular mechanisms that regulate germ cell-specific gene expression during PGC development is critical for the practical use of genetic modifications and germ-cell biology. In the current study, to characterize the promoter of chicken vasa homologue (CVH) for inducing germ cell-specific gene expression, we conducted 5′ deletion and fragment assays using both enhanced green fluorescent protein (eGFP) and NanoLuc luciferase expression vector. Furthermore, we investigated the predicted putative binding of transcription factors (TFs) on the promoter for CVH. Finally, we demonstrated that the transcriptional control of CVH expression through cis-elements and TFs is important for germ cell-specific gene expression in chicken PGCs.
Experimental designs, animals and animal care
This study was designed with the aim of investigating the cis- and trans-regulatory elements for modulating the transcription of CVH gene in chicken PGCs through dual luciferase assay and transcriptome analysis. The care and experimental use of chickens were approved by the Institute of Laboratory Animal Resources, Seoul National University (SNU-150827-1). The chickens were maintained in accordance with a standard management program at the University Animal Farm, Seoul National University, Korea. The procedures for animal management, reproduction, and embryo manipulation adhered to the standard operating protocols of our laboratory.
Construction of eGFP and NanoLuc luciferase expression vectors controlled by CVH promoters of different sizes
List of primer sequences used for cloning of the CVH promoter
Primer sequence (5′ → 3′)
CVH −1,575 bp_F
CVH −1,231 bp_F
CVH −625 bp_F
CVH −316 bp_F
CVH −227 bp_F
CVH −135 bp_F
CVH +25 bp_F
eGFP poly A _F
eGFP poly A_R
Culture of chicken PGCs and DF-1
Chicken PGCs were cultured in accordance with our standard procedure . Briefly, PGCs from White Leghorn embryonic gonads at 6 days old (Hamburger-Hamilton stage 28) were maintained in knockout Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 20% fetal bovine serum (FBS) (Hyclone, South Logan, UT, USA), 2% chicken serum (Sigma-Aldrich, St. Louis, MO, USA), 1× nucleosides (Millipore, Billerica, MA, USA), 2 mmol/L L-glutamine (Gibco), 1× nonessential amino acids (Gibco), β-mercaptoethanol (Gibco), 1 mmol/L sodium pyruvate (Gibco), and 1× antibiotic-antimycotic (Gibco). Human basic fibroblast growth factor (bFGF) (Koma Biotech, Seoul, Korea) at 10 ng/mL was used for PGC self-renewal. The cultured PGCs were subcultured onto mitomycin-inactivated mouse embryonic fibroblasts at 5- to 6-day intervals by gentle pipetting without any enzyme treatment. For DF-1, the cells were maintained in DMEM with high glucose (Hyclone), 10% FBS, and 1× antibiotic-antimycotic. Cultured cells were grown at 37 °C in a 5% CO2 incubator.
In vitro transfection
In vitro transfection was performed using Lipofectamine 2000 in accordance with the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). For expression analysis of eGFP, the constructed CVH promoter vector (1 μg) and 2 μL of Lipofectamine 2000 were separately diluted with 50 μL of Opti-MEM I reduced serum medium (Invitrogen) and incubated at room temperature for 5 min. Liposome-DNA solutions were then mixed and incubated at room temperature for 20 min to form the lipid-DNA complex. Liposome-DNA complex solution was added to 2.5 × 105 cultured PGCs in 500 μL of PGC culture medium. Transfected cells were incubated for 24 h without feeders. After incubation, cells were analyzed using a fluorescence microscope.
Luciferase reporter assay
Nano-Glo Dual-Luciferase Reporter Assay System (Promega) was used to measure the CVH promoter activities. The prepared cells were seeded in a 96-well plate and co-transfected with pGL4.53 firefly luciferase (Fluc) and pNL1.2 (NlucP/CVH RE) NanoLuc luciferase (Nluc) plasmid using Lipofectamine 2000 (Invitrogen). The transfected cells were then lysed with lysis buffer with Fluc substrate and incubated on an orbital shaker for 3 min. Fluc signals were then quenched, followed by reaction with Nluc substrate. The signals in arbitrary unit (AU) from both Nluc and Fluc were measured using a luminometer (Glomax-Multi-Detection System; Promega). The promoter activities were calculated by the ratio of the respective AU values of Nluc/Fluc. pNL1.2, an empty vector, was used as a negative control.
Prediction of putative transcriptional binding elements by in silico sequence analysis
The 591-base pair (bp) fragment (−316/+275) of the CVH promoter that had the highest activity was analyzed for TF binding sites. Such sites were predicted by MatInspector, a Genomatix program (http://www.genomatix.de) using TRANSFAC matrices (vertebrate matrix; core similarity 1.0 and matrix similarity 0.8) PROMO, which uses version 8.3 TRANSFAC (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) and TFBIND, which uses weight matrix in the database TRANSFAC R.3.4 (http://tfbind.hgc.jp).
Small interfering RNA (siRNA) transfection in chicken PGCs
List of small interfering RNA sequences used for knockdown analysis
siRNA sequence (5′ → 3′)
RNA isolation and quantitative RT-PCR
List of primer sequences used for quantitative reverse transcription polymerase chain reaction
Primer sequence (5′ → 3′)
Product Size, bp
E1A binding protein p300
GA binding protein transcription factor, alpha subunit 60 kDa
heat shock transcription factor 2
nuclear transcription factor Y, alpha
Sp3 transcription factor
zinc finger protein 143
All data are expressed as means ± standard deviation from three independent experiments. One-way analysis of variance with Bonferroni correction was used to calculate the significance of differences between experimental groups. GraphPad Prism software (ver. 5.0; GraphPad Software, La Jolla, CA, USA) was used to evaluate the data. P < 0.05; was considered statistically significant.
Identification of the minimal promoter region for transcription of the CVH gene
Investigation of the cis-regulatory elements of the CVH gene
Prediction and selection of the TFs involved in transcriptional control of the CVH promoter in chicken PGCs
Predicted TFs affecting the transcriptional activity of germ cell-specific RBPs
The results of the current study suggest that the promoter region of the CVH gene, which extends from −316 to +275 bp and contains the 5′ UTR and intron 1, can control the transcription of the CVH gene in chicken PGCs. They also suggest that significantly upregulated TFs such as HSF2, NFYA, SP3, and ZNF143 in chicken PGCs play a role in expression of the CVH gene by directly interacting with putative binding sites of the CVH gene promoter.
VASA, an evolutionarily conserved RBP that promotes translational control of germ cell-specific genes, is expressed specifically in germ cells during germline development . Several reports have shown that vasa play a critical role in the formation of the germplasm and gametogenesis in invertebrates such as Caenorhabditis elegans and Drosophila melanogaster [29, 30]. In addition, VASA expression in germ cells is essential for their survival and proliferation [31, 32]. In transgenic animals, 5.1-kb, 4.7-kb, 2.4-kb, 5.6-kb, 8-kb and 4.3-kb of vasa promoter have been used for germ cell specific expression of reporter genes in medaka , rainbow trout , zebrafish , mice , cows  and pig , respectively. Moreover, in Drosophila melanogaster, it is reported that germline specific vas gene expression in oogenesis is required for a 40-bp genomic region of the vas gene though interacting specifically with certain ovarian protein . In addition, in the malaria mosquito, vasa-like gene is specifically expressed in both the male and female gonads in adult mosquitoes and is characterized the regulatory regions that are the entire 5’UTR and only 380-bp of upstream sequence for the specific germline expression in the GSCs of both sexes . Although studies on the transcriptional control of CVH for temporal and spatial regulation hold great promise for practical applications, regarding using a germ cell-specific promoter for tracing germ cells as well as understanding the molecular network of transcriptional regulation behind their unique characteristics, very limited information is available on the regulatory elements involved in transcriptional control of the CVH gene in chicken.
Previous study showed that the CVH gene requires a 5′ flanking region of 1,555-bp for higher induction of specific expression in germ cells at the transcriptional level . However, as shown in Fig. 1, we described that the highest promoter activity region of the CVH gene, which is a 591-bp fragment (−316/+275) containing the 5′ UTR, is sufficient for the induction of specific expression in chicken PGCs, as determined by a 5′ deletion assay. Additionally, our findings demonstrate for the first time that the 5′ UTR containing intron 1 is required for promoter activity of the CVH gene in chicken PGCs, as determined through 5′ and 3′ fragmentation assays (Fig. 2). With regard to the roles of introns in transcriptional control in diverse organisms, several reports have shown that introns play a pivotal role in controlling transcription, including that of germline-specific genes, and act as enhancers to control gene expression [41–47]. In search for regulatory elements of vasa promoter in medaka, Li et al. demonstrated that the first intron plays an important role in the VAS activity from total of 11 regions identified within the 5.1-kb vasa promoter , and subsequently found that the first 35-bp of exon 1 of vasa gene is sufficient to increase transcriptional activity as a enhancer . Therefore, it seems likely that the 5′ UTR containing intron 1 of the CVH gene would be valuable for constructing a germ cell-specific CVH promoter vector for the practical utilization of genetic resources.
Transcriptional control is required for regulatory elements such as specialized promoter sequences and promoter recognition trans-acting factors . But, there are few reports on transcriptional regulators of vasa gene. A previous study revealed that Mitf acts as transcriptional activator of germ cell-specific genes encoded RNA-binding proteins such as vasa, dazl and dnd in medaka spermatogonial cell line . Therefore, we investigated whether predicted TFs that have putative binding sites in the 591-bp fragment (−316/+275) of the CVH promoter can directly regulate the expression of chicken CVH. Using our previous transcriptome analysis, we identified TFs that were more highly expressed in chicken PGCs than in other cell types. The integrated approaches used in this study were complementary for finding novel TFs with putative binding sites in the CVH promoter. However, we could not find Mitf in the analyzed CVH promoter above mentioned. Finally, we selected six TFs (EP300, GABPA, HSF2, NFYA, SP3, and ZNF143) that have putative binding sites in the 591-bp fragment (−316/+275) of the CVH promoter through a series of experiments. In Fig. 3, all TFs are marked for consensus sequences and positions in the sequence of the CVH promoter, including the TATA-box sequence for transcriptional initiation and the start codon. We also validated their expression levels using quantitative RT-PCR. Based on the results, five TFs (EP300, HSF2, NFYA, SP3, and ZNF143) were significantly expressed in PGCs compared with their levels in other samples, while GABPA were significantly expressed in PGCs compared with CEF, DF-1, and GSC, but showed no significant difference in expression compared with that at Stage X (Fig. 4). We further examined whether these TFs affect the transcription of germ cell-specific RBPs (CVH, cDAZL, CIWI, and CDH) in chicken PGCs through siRNA-mediated knockdown.
With regard to significant expression and functions in germ cells, it has been reported that heat shock factor 2 (HSF2) plays a role during embryonic development and under stress conditions, prevents the formation of damaged gametes, and ensures the integrity of the reproductive process . In addition, knockout mouse models have shown that HSF2 is involved in oogenesis and spermatogenesis and strongly expressed in PGCs [52, 53]. As a general transcription activator, NF-Y binds strongly at CCAAT motifs and consists of NF-YA, −YB, and -YC subunits . In C. elegans, mutations in nfya-1 affect the development of germ cells and also reduce the number of sperm . Moreover, NF-Y is greatly affected by the CCAAT motif in terms of its transcriptional activity regarding Miwi and CIWI gene [56, 57]. Additionally, SP3 is one of the Sp family of TFs, which is characterized by three conserved zinc fingers , and positively or negatively controls the transcriptional activity of numerous genes through binding to the GC box in cis-regulatory elements . Importantly, it has been reported that regulation of Nanog gene expression is required for Sp1 and Sp3 expression, besides Oct4 and Sox2, in mouse . Zinc finger protein 143 (ZNF143) was first identified in Xenopus , and most ZNF143 binding sites are disturbed in promoters associated with CpG islands near the transcription start site in the mammalian genome . Znf143 is particularly expressed in the mouse ICM  and its expression has been implicated in the regulation of mammalian embryonic stem cell survival and renewal . It was also proven that ZNF143, interacting with Oct4, governs Nanog expression through direct binding to the Nanog proximal promoter . In addition, ZNF143 has recently been identified as a new factor connecting promoters and distal regulatory elements as an insulator function for lineage-specific gene expression . With regard to transcriptional regulator of Mouse Vasa Homologue (MVH), Znf143 preferred histone H3 lysine 27 acetylation (H3K27ac)-marked regions associated with the early genes including Kit, Prdm1, and Sox2 rather than the late genes such as Mvh, Piwil1, Piwil2, Tdrd7, and Tdrd9 in germ cells . However, our results revealed that HSF2, NFYA, SP3, and ZNF143 would be expected to function as transcriptional regulators in chicken PGCs. Collectively, our results demonstrate for the first time that these TFs are involved in promoter activity of germ cell-specific RBPs in chicken PGCs; however, it remains to be determined whether these TFs directly act on each gene promoter during chicken PGC development.
In conclusion, we have identified the promoter region of the CVH gene for PGC-specific gene expression and found TFs such as HSF2, NFYA, SP3, and ZNF143 associated with transcriptional control of the CVH gene in chicken PGCs. This information should aid a wide range of studies in constructing germ cell-specific synthetic promoters for tracing germ cells using transgenesis, as well as our understanding of the transcriptional regulation that maintains germness in PGCs.
Basic fibroblast growth factor
Chicken dead end homologue
Chicken embryonic fibroblast
Chicken PIWI-like protein 1
Chicken vasa homolog
Deleted in azoospermia-like
Dulbecco’s modified eagle medium
Enhanced green fluorescent protein
E1A binding protein p300
Fetal bovine serum
GA binding protein transcription factor, alpha
Glyceraldehyde 3-phosphate dehydrogenase
Gonadal stromal cell
Histone H3 lysine 27 acetylation
Heat shock transcription factor 2
Inner cell mass
Nuclear transcription factor Y, alpha
Primordial germ cell
P-element induced wimpy testis
Quantitative reverse transcription-polymerase chain reaction
RNA binding protein
Small interfering RNA
Stimulating protein 3
Zinc finger protein 143
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2015R1A3A2033826).
Availability of data and materials
The datasets during and/or analyzed during the current study available from the corresponding authors on reasonable request.
HJY participated in study design and coordination. JSD participated in the design of the study, carried out the experiments, statistical analysis and wrote the first draft of the manuscript. LBR participated in oversaw manuscript preparation. JSD, HYS and LHJ were involved in data interpretation. RJS participated in writing the final versions of the manuscript. All authors have read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
The care and experimental use of chickens were approved by the Institute of Laboratory Animal Resources, Seoul National University.
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- Han JY. Germ cells and transgenesis in chickens. Comp Immunol Microbiol Infect Dis. 2009;32:61–80.View ArticlePubMedGoogle Scholar
- Park TS, Han JY. Piggybac transposition into primordial germ cells is an efficient tool for transgenesis in chickens. Proc Natl Acad Sci U S A. 2012;109:9337–41.View ArticlePubMedPubMed CentralGoogle Scholar
- Kim H, Park TS, Lee WK, Moon S, Kim JN, Shin JH, et al. MPSS profilingof embryonic gonad and primordial germ cells in chicken. Physiol Genomics. 2007;29:253–9.View ArticlePubMedGoogle Scholar
- Han JY, Park TS, Kim JN, Kim MA, Lim D, Lim JM, et al. Gene expressionprofiling of chicken primordial germ cell ESTs. BMC Genomics. 2006;7:220.View ArticlePubMedPubMed CentralGoogle Scholar
- Donovan PJ. The germ cell--the mother of all stem cells. Int J Dev Biol. 1998;42:1043–50.PubMedGoogle Scholar
- Zheng YH, Rengaraj D, Choi JW, Park KJ, Lee SI, Han JY. Expression pattern of meiosis associated SYCP family members during germline development inchickens. Reproduction. 2009;138:483–92.View ArticlePubMedGoogle Scholar
- Rengaraj D, Zheng YH, Kang KS, Park KJ, Lee BR, Lee SI, et al. Conservedexpression pattern of chicken DAZL in primordial germ cells and germ-line cells. Theriogenology. 2010;74:765–76.View ArticlePubMedGoogle Scholar
- Kim TH, Yun TW, Rengaraj D, Lee SI, Lim SM, Seo HW, et al. Conserved functional characteristics of the PIWI family members in chicken germ cell lineage. Theriogenology. 2012;78:1948–59.View ArticlePubMedGoogle Scholar
- Reinke V. Germline genomics. WormBook. 2006. doi:10.1895/wormbook.1.74.1.PubMedPubMed CentralGoogle Scholar
- Seydoux G, Braun RE. Pathway to totipotency: lessons from germ cells. Cell. 2006;127:891–904.View ArticlePubMedGoogle Scholar
- Hamburger V, Hamilton HL. A series of normal stages in the development of the chick embryo. J Morphol. 1951;88:49–92.View ArticlePubMedGoogle Scholar
- Ginsburg M, Eyal-Giladi H. Temporal and spatial aspects of the gradual migration of primordial germ cells from the epiblast into the germinal crescent in the avian embryo. Development. 1986;95:53–71.Google Scholar
- Lee SI, Lee BR, Hwang YS, Lee HC, Rengaraj D, Song G, et al. MicroRNA-mediated posttranscriptional regulation is required for maintaining undifferentiated properties of blastoderm and primordial germ cells in chickens. Proc Natl Acad Sci U S A. 2011;108:10426–31.View ArticlePubMedPubMed CentralGoogle Scholar
- Lee BR, Kim H, Park TS, Moon S, Cho S, Park T, et al. A set of stage-specific gene transcripts identified in EK stage X and HH stage 3 chick embryos. BMC Dev Biol. 2007;7:1.View ArticleGoogle Scholar
- Tsunekawa N, Naito M, Sakai Y, Nishida T, Noce T. Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells. Development. 2000;127:2741–50.PubMedGoogle Scholar
- Minematsu T, Harumi T, Naito M. Germ cell-specific expression of GFP gene induced by chicken vasa homologue (Cvh) promoter in early chicken embryos. Mol Reprod Dev. 2008;75:1515–22.View ArticlePubMedGoogle Scholar
- Lavial F, Acloque H, Bachelard E, Nieto MA, Samarut J, Pain B. Ectopic expression of Cvh (Chicken Vasa homologue) mediates the reprogramming of chicken embryonic stem cells to a germ cell fate. Dev Biol. 2009;330:73–82.View ArticlePubMedGoogle Scholar
- Yoon C, Kawakami K, Hopkins N. Zebrafish vasa homologue RNA is localized to the cleavage planes of 2-and 4-cell-stage embryos and is expressed in theprimordial germ cells. Development. 1997;124:3157–65.PubMedGoogle Scholar
- Fujiwara Y, Komiya T, Kawabata H, Sato M, Fujimoto H, Furusawa M, et al. Isolation of a DEAD-family protein gene that encodes a murine homolog of Drosophila vasa and its specific expression in germ cell lineage. Proc Natl Acad Sci U S A. 1994;91:12258–62.View ArticlePubMedPubMed CentralGoogle Scholar
- Castrillon DH, Quade BJ, Wang T, Quigley C, Crum CP. The human VASA gene is specifically expressed in the germ cell lineage. Proc Natl Acad Sci U S A. 2000;97:9585–90.View ArticlePubMedPubMed CentralGoogle Scholar
- Styhler S, Nakamura A, Swan A, Suter B, Lasko P. Vasa is required for GURKEN accumulation in the oocyte, and is involved in oocyte differentiation and germline cyst development. Development. 1998;125:1569–78.PubMedGoogle Scholar
- Carrera P, Johnstone O, Nakamura A, Casanova J, Jäckle H, Lasko P. VASA mediates translation through interaction with a Drosophila yIF2 homolog. Mol Cell. 2000;5:181–7.View ArticlePubMedGoogle Scholar
- Raz E. The function and regulation of vasa-like genes in germ-cell development. Genome Biol. 2000;1:1017.View ArticleGoogle Scholar
- Noce T, Okamoto-Ito S, Tsunekawa N. Vasa homolog genes in mammalian germ cell development. Cell Struct Funct. 2001;26:131–6.View ArticlePubMedGoogle Scholar
- Liu N, Han H, Lasko P. Vasa promotes Drosophila germline stem cell differentiation by activating mei-P26 translation by directly interacting with a (U)-rich motif in its 3′ UTR. Genes Dev. 2009;23:2742–52.View ArticlePubMedPubMed CentralGoogle Scholar
- Xiol J, Spinelli P, Laussmann MA, Homolka D, Yang Z, Cora E, et al. RNA clamping by Vasa assembles a piRNA amplifier complex on transposon transcripts. Cell. 2014;157:1698–711.View ArticlePubMedGoogle Scholar
- Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods. 2001;25:402–8.View ArticlePubMedGoogle Scholar
- Gustafson EA, Wessel GM. Vasa genes: emerging roles in the germ line and in multipotent cells. Bioessays. 2010;32:626–37.View ArticlePubMedPubMed CentralGoogle Scholar
- Spike C, Meyer N, Racen E, Orsborn A, Kirchner J, Kuznicki K, et al. Genetic analysis of the Caenorhabditis elegans GLH family of P-granule proteins. Genetics. 2008;178:1973–87.View ArticlePubMedPubMed CentralGoogle Scholar
- Illmensee K, Mahowald AP. Transplantation of posterior polar plasm in Drosophila. Induction of germ cells at the anterior pole of the egg. Proc Natl Acad Sci U S A. 1974;71:1016–20.View ArticlePubMedPubMed CentralGoogle Scholar
- Parvinen M. The chromatoid body in spermatogenesis. Int J Androl. 2005;28:189–201.View ArticlePubMedGoogle Scholar
- Medrano JV, Ramathal C, Nguyen HN, Simon C, Reijo Pera RA. Divergent RNA-binding proteins, DAZL and VASA, induce meiotic progression in human germ cells derived in vitro. Stem Cells. 2012;30:441–51.View ArticlePubMedPubMed CentralGoogle Scholar
- Tanaka M, Kinoshita M, Kobayashi D, Nagahama Y. Establishment of medaka (Oryzias latipes) transgenic lines with the expression of green fluorescent protein fluorescence exclusively in germ cells: a useful model to monitor germ cellsin a live vertebrate. Proc Natl Acad Sci U S A. 2001;98:2544–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Yoshizaki G, Takeuchi Y, Tominaga H, Kobayashi T, Takeuchi T. Visualization of primordial germ cells in transgenic rainbow trout carrying green fluorescent protein gene driven by vasa promoter. Fish Sci. 2002;68:1067–70.View ArticleGoogle Scholar
- Krøvel AV, Olsen LC. Expression of a vas: EGFP transgene in primordial germ cells of the zebrafish. Mech Dev. 2002;116:141–50.View ArticlePubMedGoogle Scholar
- Gallardo T, Shirley L, John GB, Castrillon DH. Generation of a germ cell-specific mouse transgenic Cre line. Vasa-Cre Genesis. 2007;45:413–7.PubMedGoogle Scholar
- Luo H, Zhou Y, Li Y, Li Q. Splice variants and promoter methylation status of the Bovine Vasa Homology (Bvh) gene may be involved in bull spermatogenesis. BMC Genet. 2013;14:1.Google Scholar
- Song Y, Lai L, Li L, Huang Y, Wang A, Tang X, et al. Germ cell-specific expression of Cre recombinase using the VASA promoter in the pig. FEBS Open Bio. 2016;6:50–5.View ArticlePubMedGoogle Scholar
- Sano H, Nakamura A, Kobayashi S. Identification of a transcriptional regulatory region for germline-specific expression of vasa gene in Drosophila melanogaster. Mech Dev. 2002;112:129–39.View ArticlePubMedGoogle Scholar
- Papathanos PA, Windbichler N, Menichelli M, Burt A, Crisanti A. The vasa regulatory region mediates germline expression and maternal transmission of proteins in the malaria mosquito Anopheles gambiae: a versatile tool for genetic control strategies. BMC Mol Biol. 2009;10:65.View ArticlePubMedPubMed CentralGoogle Scholar
- Kawamoto T, Makino K, Niwa H, Sugiyama H, Kimura S, Amemura M, et al. Identification of the human beta-actin enhancer and its binding factor. Mol Cell Biol. 1988;8:267–72.View ArticlePubMedPubMed CentralGoogle Scholar
- Liu Z, Moav B, Faras A, Guise K, Kapuscinski A, Hackett P. Functional analysis of elements affecting expression of the beta-actin gene of carp. Mol Cell Biol. 1990;10:3432–40.View ArticlePubMedPubMed CentralGoogle Scholar
- Tomaras GD, Foster DA, Burrer CM, Taffet SM. ETS transcription factors regulate an enhancer activity in the third intron of TNF-alpha. J Leukoc Biol. 1999;66:183–93.PubMedGoogle Scholar
- Henkel G, Weiss DL, McCoy R, Deloughery T, Tara D, Brown MA. A DNase I-hypersensitive site in the second intron of the murine IL-4 gene defines a mast cell-specific enhancer. J Immunol. 1992;149:3239–46.PubMedGoogle Scholar
- Wong TT, Tesfamichael A, Collodi P. Identification of promoter elements responsible for gonad-specific expression of zebrafish Deadend and its application to ovarian germ cell derivation. Int J Dev Biol. 2013;57:767–72.View ArticlePubMedGoogle Scholar
- Mohapatra C, Barman HK. Identification of promoter within the first intron of Plzf gene expressed in carp spermatogonial stem cells. Mol Biol Rep. 2014;41:6433–40.View ArticlePubMedGoogle Scholar
- Li M, Guan G, Hong N, Hong Y. Multiple regulatory regions control the transcription of medaka germ gene vasa. Biochimie. 2013;95:850–7.View ArticlePubMedGoogle Scholar
- Li M, Zhao H, Wei J, Zhang J, Hong Y. Medaka vasa gene has an exonic enhancer for germline expression. Gene. 2015;555:403–8.View ArticlePubMedGoogle Scholar
- DeJong J. Basic mechanisms for the control of germ cell gene expression. Gene. 2006;366:39–50.View ArticlePubMedGoogle Scholar
- Zhao H, Li M, Purwanti YI, Liu R, Chen T, Li Z, et al. Mitf is a transcriptional activator of medaka germ genes in culture. Biochimie. 2012;94:759–67.View ArticlePubMedGoogle Scholar
- Abane R, Mezger V. Roles of heat shock factors in gametogenesis and development. FEBS J. 2010;277:4150–72.View ArticlePubMedGoogle Scholar
- Sarge KD, Park-Sarge OK, Kirby JD, Mayo KE, Morimoto RI. Expresion of heat shock factor 2 in mouse testis potential role as a regulator of heat-shock protein gene expression during spermatogenesis. Biol Reprod. 1994;50:1334–43.View ArticlePubMedGoogle Scholar
- Kallio M, Chang Y, Manuel M, Alastalo TP, Rallu M, Gitton Y, et al. Brain abnormalities, defective meiotic chromosome synapsis and female subfertility in HSF2 null mice. EMBO J. 2002;21:2591–601.View ArticlePubMedPubMed CentralGoogle Scholar
- Mantovani R. The molecular biology of the CCAAT-binding factor NF-Y. Gene. 1999;1:15–27.View ArticleGoogle Scholar
- Deng H, Sun Y, Zhang Y, Luo X, Hou W, Yan L, et al. Transcription factorNFY globally represses the expression of the C. elegans Hox gene Abdominal-B homolog egl-5. Dev Biol. 2007;308:583–92.View ArticlePubMedGoogle Scholar
- Hou Y, Yuan J, Zhou X, Fu X, Cheng H, Zhou R. DNA Demethylation and USF Regulate the Meiosis-Specific Expression of the Mouse Miwi. PLoS Genet. 2012;8:e1002716.View ArticlePubMedPubMed CentralGoogle Scholar
- Sohn YA, Lee SI, Choi HJ, Kim HJ, Kim KH, Park TS, et al. The CCAAT element in the CIWI promoter regulates transcriptional initiation in chicken primordial germ cells. Mol Reprod Dev. 2014;81:871–82.View ArticlePubMedGoogle Scholar
- Philipsen S, Suske G. A tale of three fingers: the family of mammalian Sp/XKLF transcription factors. Nucleic Acids Res. 1999;27:2991–3000.View ArticlePubMedPubMed CentralGoogle Scholar
- Suske G. The Sp-family of transcription factors. Gene. 1999;238:291–300.View ArticlePubMedGoogle Scholar
- Wu DY, Yao Z. Functional analysis of two Sp1/Sp3 binding sites in murine Nanog gene promoter. Cell Res. 2006;16:319–22.View ArticlePubMedGoogle Scholar
- Myslinski E, Krol A, Carbon P. ZNF76 and ZNF143 are two human homologsof the transcriptional activator Staf. J Biol Chem. 1998;273:21998–2006.View ArticlePubMedGoogle Scholar
- Myslinski E, Gerard MA, Krol A, Carbon P. A genome scale location analysisof human Staf/ZNF143-binding sites suggests a widespread role for human Staf/ZNF143 in mammalian promoters. J Biol Chem. 2006;281:39953–62.View ArticlePubMedGoogle Scholar
- Yoshikawa T, Piao Y, Zhong J, Matoba R, Carter MG, Wang Y, et al. High-throughput screen for genes predominantly expressed in the ICM of mouse blastocysts by whole mount in situ hybridization. Gene Expr Patterns. 2006;6:213–24.View ArticlePubMedGoogle Scholar
- Chia NY, Chan YS, Feng B, Lu X, Orlov YL, Moreau D, et al. A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity. Nature. 2010;468:316–20.View ArticlePubMedGoogle Scholar
- Chen X, Fang F, Liou YC, Ng HH. Zfp143 regulates Nanog through modulation of Oct4 binding. Stem Cells. 2008;26:2759–67.View ArticlePubMedGoogle Scholar
- Bailey SD, Zhang X, Desai K, Aid M, Corradin O, Cowper-Sal R, et al. ZNF143 provides sequence specificity to secure chromatin interactions at gene promoters. Nat Commun. 2015;2:6186.View ArticlePubMedPubMed CentralGoogle Scholar
- Ng J-H, Kumar V, Muratani M, Kraus P, Yeo J-C, Yaw L-P, et al. In vivo epigenomic profiling of germ cells reveals germ cell molecular signatures. Dev Cell. 2013;24:324–33.View ArticlePubMedGoogle Scholar