RESEARCH ARTICLE Molecular Reproduction & Development 81:871–882 (2014)

The CCAAT Element in the CIWI Promoter Regulates Transcriptional Initiation in Chicken Primordial Germ Cells YOON AH SOHN,1 SANG IN LEE,1 HEE JUNG CHOI,1 HYUN JEONG KIM,1 KI HYUN KIM,1 TAE SUB PARK1,2*, AND JAE YONG HAN1* 1 2

Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul, Korea Institute of Green-Bio Science and Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Korea

SUMMARY The P-element-induced wimpy testis (PIWI) protein, which associates with small noncoding RNAs, is responsible for maintaining the integrity of germ cells and stem cells. Thus, transcriptional regulation of PIWI is critical for its effective functional modulation. In this study, we identified the promoter region of the PIWI homolog in chicken (CIWI), and investigated the transcriptional regulatory elements that control expression of CIWI in chicken primordial germ cells (PGCs). We constructed a vector that included the enhanced green fluorescent protein (eGFP) gene controlled by the 4-kb CIWI promoter. The vector was expressed in chicken PGCs, but not in chicken embryonic fibroblasts. Based on promoter deletion and fragmentation assays, we found that a 252-bp fragment of the CIWI promoter (577 to 326 bp) was crucial for CIWI expression in PGCs. A CCAAT transcriptional regulatory element (498 to 494 bp) was detected in the proximal region from the transcription initiation site of CIWI, and mutational analysis confirmed that this element regulates transcriptional initiation in chicken PGCs. Interestingly, the regions flanking the CCAAT element, which are positioned differently in HIWI (human), Miwi (mouse), and CIWI orthologs, were highly conserved. In addition, we predicted that specificity protein 1 (SP1) motifs modulate the transcriptional initiation of CIWI by binding to the 5’-flanking regions of the CCAAT box. Overall, 252 bp of the CIWI promoter possessing the transcriptional regulatory element CCAAT is crucial for regulating CIWI gene expression in chicken PGCs. This promoter may be applicable for the regulation of CIWI expression during germ-cell development.

Mol. Reprod. Dev. 81: 871882, 2014. ß 2014 Wiley Periodicals, Inc. Received 9 March 2014; Accepted 27 June 2014



Corresponding author: Department of Agricultural Biotechnology College of Agriculture and Life Sciences Seoul National University Seoul 151-921, Korea. E-mail: [email protected] [email protected]

Grant sponsor: Next-Generation BioGreen 21 Program; Grant number: PJ008142012014; Grant sponsor: Bioindustry Technology Development Program; Grant number: IPET-312060-5-2-SB020; Grant sponsor: Basic Science Research Program through the National Research Foundation of Korea (NRF); Grant number: 2012R1A1A2039004 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22356

INTRODUCTION Primordial germ cells (PGCs), the progenitors of germ cells, differentiate into functional gametes that transmit genetic information to the next generation. Understanding the molecular mechanisms that regulate cell-fate decisions during early PGC development is important for research in germ-cell biology. Chicken PGCs first arise from the

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Abbreviations: 5’-/3’-UTR, 5’-/3’-untranslated region; CEF, chicken embryonic fibroblast; CIWI/CILI, chicken PIWI-like protein 1/2; eGFP, enhanced green fluorescent protein; HIWI, Human PIWI-like protein 1; MIWI/MILI, Mouse PIWIlike protein 1/2; NF-Y, nuclear transcription factor Y; piRNA, PIWI-interacting RNA; PIWI, P-element-induced wimpy testis; PGC, primordial germ cell; siRNA, small-interfering RNA; SP1, specificity protein 1; SSEA-1, Stage-specific embryonic antigen 1; TFAP2a, transcription factor activating enhancer-binding protein 2 alpha.

Molecular Reproduction & Development

epiblast, and migrate to the hypoblast of the area pellucida in the blastoderm. In Stage 1012, PGCs are carried from the germinal crescent into the blood stream, allowing them to migrate through the circulatory system (Ando and Fujimoto, 1983; Ginsburg and Eyal-Giladi, 1986; Ukeshima et al., 1991) a process that is distinct from mammal PGCs, which migrate through embryonic tissues (Han, 2009). Later in development, PGCs actively leave capillary vessels, finally settling in the developing genital ridges (Hamburger and Hamilton, 1951; Meyer, 1964; Fujimoto et al., 1976). During early embryonic development, the PGC lineage is governed by the orchestrated actions of many transcriptional and post-transcriptional regulators. Several recent studies demonstrated that a large number of small noncoding RNAs act as transcriptional and post-transcriptional regulators. There are three major types of non-coding RNAs: small interfering RNAs (siRNAs), microRNAs (miRNAs), and P-element-induced wimpy testis (PIWI)interacting RNAs (piRNAs). siRNAs and miRNAs bind to the Argonaute subfamily, and are ubiquitously present in somatic cells and germ cells (Sijen et al., 2001; Martinez et al., 2002). piRNAs, on the other hand, bind to the PIWI subfamily and are highly enriched in germ cells (Till et al., 2007; Saxe and Lin, 2011). PIWI proteins are largely restricted to germ cells and stem cells, and are required to maintain fertility (Thomson and Lin, 2009). Recent studies suggest that post-transcriptional regulation and epigenetic programming, which are responsible for germ-line development, may be mediated by PIWI-piRNA complexes (Brower-Toland et al., 2007; Aravin et al., 2008; Juliano et al., 2011). For example, PIWI proteins are implicated in directing heterochromatin formation and transcriptional silencing by promoting euchromatic histone modifications (Yin and Lin, 2007). The PIWI-piRNA pathway also directs retrotransposon silencing both transcriptionally and post-transcriptionally. Mili- and Miwi2-mutant mice lose DNA methylation in the trophectoderm (Aravin et al., 2008; Kuramochi-Miyagawa et al., 2008). In addition, PIWI proteins play an important role in maintaining germ-cell genome integrity in several species (Juliano et al., 2011). In Drosophila, Piwi protein is required for the self-renewal of germ-line stem cells. The gonads of piwi-mutant flies contained fewer egg chambers and sperm bundles, and showed abolished proliferation of germ-line stem cells (Lin and Spradling, 1997; Cox et al., 1998). Thus, the PIWI-piRNA complex controls not only epigenetic processes, but also signaling for germ-line stem cell self-renewal (Brower-Toland et al., 2007; Xie, 2013). In a previous study, we identified Gallus gallus PIWI-like proteins 1 (CIWI) and 2 (CILI) from adult chicken testis, and found that the PIWI domain in the CIWI protein was similar between mammals and birds. While the PIWI domain in CIWI showed higher similarities between mouse PIWI-like protein (MIWI) and human PIWI-like protein (HIWI), the PIWI domain in CILI was less similar between MILI and HILI compared with CIWI. mRNA levels of both CIWI and CILI were highly expressed in germ cells of both testis and ovary.

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siRNA-mediated knockdown analysis of CIWI and CILI in chicken PGCs showed upregulated expression of the chicken repetitive element (CR1) retrotransposons and induced DNA double-strand breakage (Kim et al., 2012). These results indicated that CIWI and CILI play a critical role in maintaining the genome integrity of germ cells in chickens. In the present study, we analyzed the transcriptional regulatory elements in the promoter region of CIWI to assess functional modulation of avian germ cellspecific gene expression. Identifying short promoter sequences or regulatory transcription factors in germ cell-specific promoters like the CIWI gene may be valuable not only for an efficient production of transgenic chickens expressing reporter genes specifically in germ cell lineage, but also for the basic study of reproductive biology of germ-cell development and differentiation during early embryogenesis.

RESULTS Identification and Verification of the CIWI Promoter To identify the promoter region in CIWI, gene sequences from the National Center for Biotechnology Information (NCBI, Gene ID: 416804) and the 50 -flanking sequences from the UCSC Genome Browser were analyzed. We cloned a 4-kb genomic fragment from 3,839 to þ161 bp, which included the predicted transcription start site; the þ1 to þ161 bp fragment represents the 5’-untranslationed region (5’-UTR) without the start codon. To construct expression vectors, enhanced green fluorescent protein (eGFP) coding sequence and a bovine poly(A)tail were ligated downstream of the 4-kb CIWI promoter. This 4-kb expression vector containing the CIWI promoter was specifically expressed in cultured chicken primordial germ cells (PGCs), which were also positive for the PGC-specific marker stage-specific embryonic antigen 1 (SSEA-1) (Fig. 1A). In contrast, this CIWI::eGFP reporter vector was not expressed in chicken embryonic fibroblasts (CEFs) (Fig. 1B).

Promoter Deletion and Fragmentation of the CIWI Promoter For the promoter deletion assay, four expression vectors containing CIWI promoter fragments of different sizes and positions were constructed: a 2,986-bp fragment (2,825 to þ161 bp), 1,985-bp fragment (1,824 to þ161 bp), 1,000-bp fragment (839 to þ161 bp), and 3,000-bp fragment (3,839 to 839 bp) (Fig. 2A). The three smaller constructs were strongly expressed in chicken PGCs; the 3,000-bp fragment (3,839 to 839 bp) was not (Fig. 2B and Fig. S1A). In contrast, none of the constructs were expressed in CEFs (Fig. 2C and Fig. S1B). Based on these results, the transcriptional cis elements located between 839 and þ161 bp appear to be critical for CIWI expression. To further verify the efficiency of expression of each construct, the percentages of eGFP-expressing cells were

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and Fig. S2B). Compared to the other vectors, the 577 to 330-bp fragment of the CIWI promoter was expressed by 1.44% of the PGCs. This result suggested that the elements controlling transcriptional initiation in CIWI are located between 577 and 326 bp.

Comparative Analysis of Putative Transcriptional Binding Elements in PIWI Promoters

Figure 1. In vitro expression analysis of the eGFP vector containing 4 kb of the chicken CIWI promoter in cultured chicken PGCs (A) and CEFs (B). After transfection of the CIWI-eGFP construct, PGCs were immunostained for the marker protein stage-specific embryonic antigen 1 (SSEA-1). Arrows indicate PGCs expressing eGFP under control of the CIWI promoter, and SSEA-1. eGFP vector containing the CIWI promoter was not expressed in CEFs. Scale bars, 20 mm (A) and 100 mm (B).

measured using flow cytometry in PGCs and CEFs. Compared to the CMV promoter, vectors containing the 2,986-, 1,985-, and 1,000-bp fragments of the CIWI promoter were eGFP-positive in over 2% of the PGCs, and the CIWI promoter showed significant activity in PGCs. In contrast, the flow cytometry results recapitulated the absence of eGFP from the 3,000-bp construct in PGCs and from any construct in CEFs (Fig. 2B and C). To identify the transcriptional elements crucial for CIWI expression, four fragmented promoters were designed and constructed: 839 to 567 bp, 577 to 326 bp, 329 to 90 bp, and 89 to þ161 bp (Fig. 3A). Expression of these four constructs was examined by fluorescence microscopy and flow cytometry. Interestingly, the fragmented promoter from 577 to 326 was expressed only in PGCs (Fig. 3B and Fig. S2A). In contrast, none of the constructs were expressed in CEFs (Fig. 3C

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Putative transcriptional elements in the 252-bp fragment of the CIWI promoter (577 to 326 bp) were predicted using PROMO software. No TATA-box sequence was identified in this 252-bp fragment, although a CCAAT box transcriptional-initiation-element sequence was identified. By comparative analysis, the 4-kb promoter of CIWI did not show significant similarity to the promoters of human HIWI and mouse Miwi (data not shown). Sequence alignment of a 200-bp (100 to þ100 bp) fragment of the HIWI promoter, a 325-bp (143 to þ181 bp) fragment of the Miwi promoter, and a 252-bp (577 to 326 bp) region of the CIWI promoter revealed overall similarities between chicken and human or chicken and mouse sequences of 50%, while the flanking regions of the CCAATelement, which was positioned differently in HIWI (132 to 65 bp), Miwi (76 to 7 bp), and CIWI (537 to 467 bp), showed higher similarity (Fig. 4A). All regions flanking the CCAAT element contained the predicted binding sites for the same transcription factors. In addition, all HIWI, Miwi, and CIWI promoters contained a binding sequence of nuclear transcription factor Y (NF-Y), a ubiquitous factor that acts as a heterotrimeric complex to regulate the transcription initiation and repression of CCAAT box-comprising promoters. Specificity protein 1 (SP1) and transcription factor activating enhancer binding protein 2 alpha (TFAP2a) motifs were also predicted to modulate the transcriptional initiation of HIWI, Miwi, and CIWI by binding to the 5’- or 3’-flanking regions of the CCAAT box (Fig. 4B). SP1, a zinc finger transcription factor that binds to GC-rich motifs of many promoters, is involved in many cellular processes, including cell differentiation, cell growth, and DNA damage. TFAP2a, on the other hand, functions as a sequence-specific DNA-binding transcription factor, which binds to the specific DNA sequence and recruits the transcriptional machinery (Williams and Tjian, 1991).

Validation of Transcript Expression by Mutation Analysis of the CCAAT Box and Point Mutation of the Putative SP1-Binding Site To verify transcriptional activity of the CCAAT element, these sequences were replaced by TTCCT sequences (Fig. 5A). The vector harboring the mutated CCAAT element (TTCCT vector) completely lost transcriptional activity in chicken PGCs, and were still not expressed in CEFs (Fig. 5B and Fig. S3). This result suggested that the CCAAT box within the 577 to 326-bp region of the CIWI promoter is important for specific transcriptional expression of the CIWI gene in chicken PGCs.

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Figure 2. Deletion analysis of the 4-kb chicken CIWI promoter in PGCs and CEFs. (A) Schematic diagram of cloned CIWI promoters. About 4 kb of the CIWI promoter containing 161 bp of the 5’-UTR was cloned, and five constructs containing different lengths and promoter elements were designed for expression analysis using an eGFP vector. The right panel indicates percentages of eGFP-expressing PGCs three days after transfection of each construct. Expression of eGFP under the control of the different constructs in cultured chicken PGCs (B) and CEFs (C) were analyzed by fluorescence microscopy and flow cytometry. eGFP expression under control of the cytomegalovirus (CMV) promoter was used as a positive control. Scale bar, 100 mm.

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Figure 3. Identification of transcription regulatory elements in the 839-bp chicken CIWI promoter containing 161 bp of the 5’ UTR. (A) Schematic diagram of fragmented CIWI promoter constructs. Four constructs of the CIWI promoter of 250273 bp in size were designed for identification of transcription regulatory elements using an eGFP vector. The right panel indicates percentages of eGFP-expressing PGCs three days after transfection of each construct. Expression of eGFP under the control of the different constructs in cultured chicken PGCs (B) and CEFs (C) were analyzed by fluorescence microscopy and flow cytometry. Scale bar, 100 mm.

A putative binding site of transcription factor SP1 was also predicted in the 577 to 326-bp region of the CIWI promoter, as well as in the 100 to þ100-bp region of the HIWI promoter and the 143 to þ181-bp region of the Miwi

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promoter. To test for transcriptional regulation by SP1, point-mutation analysis was performed on the predicted SP1 binding sequences (Fig. 5A). A vector harboring the mutated SP1-binding site was expressed in PGCs, but not

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Figure 4. Multiple alignment of chicken CIWI promoter with human HIWI and mouse Miwi promoters. (A) About 200 bp (-100 to þ100 bp) of the HIWI promoter and 325 bp (-143 to þ181 bp) of the Miwi promoter were aligned with a 252-bp (-577 to -326 bp) fragment of the CIWI promoter. (B) Alignment shows the proximal regions of the CCAATelement that were differently positioned in HIWI (-132 to -65 bp), Miwi (-76 to -7 bp), and CIWI (-537 to -467 bp). The transcription factors NF-Y, SP1, and TFAP2a were predicted to bind to the regions flanking CCAAT in HIWI, Miwi, and CIWI.

in CEFs (Fig. 5B and Fig. S3)although only 0.546% of the population receiving the mutated SP1 sequence was eGFP-positive, versus 2.01% of the chicken PGCs receiving vector with the original CIWI sequence. This result indicated that the transcription factor SP1 participates in transcriptional regulation of the CIWI gene.

Transcription Factors SP1, NF-Y, and TFAP2a Affect Transcriptional Activity of CIWI The binding sites for transcription factors SP1, NF-Y, and TFAP2a were predicted in 252 bps of the CIWI promoter. To examine if these three transcription factors affect transcriptional activity of CIWI and germ cell-specific genes such as DAZL and VASA, we used siRNAs for each transcription factor to knock down their levels within chicken PGCs (Table 2). Each siRNA efficiently decreased the expression of each target gene (Fig. 6A), which affected germ cellspecific gene expression in PGCs (Fig. 6BD). Expression of CIWI was decreased in all of the transcription factorknockdown PGCs. In the case of SP1-knockdown PGCs, the expression level of CIWI was decreased by 41% compared with control (Fig. 6B); similarly, NF-Y and TFAP2aknockdown PGCs decreased CIWI transcription by 29.7% and 24%, respectively. DAZL and VASA were also downregulated in all of transcription factor-knockdown PGCs (Fig. 6C-D). Thus, transcription factors SP1, NF-Y, and TFAP2a affect the regulation of germ cell-specific genes, such as CIWI, DAZL, and VASA, and therefore assist in the transcriptional regulation of germ cell-specific genes in chicken PGCs.

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DISCUSSION Transcription is the initial and a primary control process that modulated gene expression in living cells. Altered rates of protein syntheses in different cell types are firstly regulated by induction and repression of promoter initiation. How genes are regulated helps distinguish different cell types. Germ cells, for example, are distinguished from somatic cells by their modulated expression of genes specific for germ-line development, including those networks associated with the meiotic cell cycle (DeJong, 2006). Germ-cell specification and differentiation involve regulatory networks at the transcriptional initiation as well as translational levels. Development of PGCs also includes post-transcriptional regulatory mechanisms, including complexes of small RNAs and RNA-binding proteins. Post-transcriptional regulation, such as repression of retrotransposons, plays an important role in the development and maintenance of germ-cell integrity in PGCs (Cook and Blelloch, 2013; Mochizuki and Matsui, 2010). The regulatory processes of gene expression, however, remain fairly understudied; this is particularly true for germ-cell specification processes in chicken. In this study, we identified regulatory elements of the CIWI promoter, and validated its utilization in chicken PGCs. PIWI proteins play a crucial role in the epigenetic regulation of transposon repression and post-transcriptional and translational modifications that help maintain genome integrity during germ-line development in different species (Juliano et al., 2011). Three PIWI homologs are present in the mouse MIWI, MILI, and MIWI2; each follows a distinct

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Figure 5. Mutation analyses of the CCAAT-box and SP1 binding site in the transcriptional initiation element of the CIWI promoter. (A) Schematic diagram of the construct containing the replacement of the CCAAT box (at -498 bp) with TTCCT sequences in the RNA polymerase binding site (Upper). Diagram of the constructs with wild-type and point-mutated SP1 binding site (C-to-Tsubstitution) at position -534 (Lower). (B) Expression of eGFP under control of the normal CIWI promoter (Upper), CCAAT-mutated sequence (Middle), and point-mutated SP1 binding site (Lower) in cultured chicken PGCs and CEFs, as analyzed by fluorescence microscopy and flow cytometry. Scale bar, 100 mm.

expression pattern and function in germ-cell lineages: In Miwi-mutant male mice, spermatogenesis arrests at the early stages of spermiogenesis (Deng and Lin, 2002). In Mili-mutant mice, spermatogenesis arrests at the midpachytene stage. (Kuramochi-Miyagawa et al., 2004). In contrast, Miwi2-mutant mice showed significantly increased levels of apoptosis in the seminiferous tubules compared to wild-type mice (Carmell et al., 2007). These results indicate that the individual mouse PIWI members have stage-specific roles during germ-cell differentiation. The human HIWI protein possesses significant homology to other members of the PIWI family, e.g., mouse and Drosophila (Qiao et al., 2002), and four different genes have been identified: HIWI (PIWI), HILI (PIWI2), HIWI3 (PIWI3), and HIWI2 (PIWI4). HIWI is expressed in spermatocytes and round spermatids during spermatogenesis, as well as in hematopoietic stem and progenitor cells but not in their differentiated lineages (Sharma et al., 2001). HILI and HIWI3 are mainly expressed in the testes. Interestingly, HIWI2 was ubiquitously observed in all tissues (Sugimoto et al., 2007). In avian species, the biological functions of PIWI family members remain unclear. In recent studies, we characterized the chicken PIWI members CIWI and CILI (Kim et al., 2012), and revealed the complete

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genomic structure of the CIWI gene, including a 161-bp fragment of the 5’-UTR and 660 bp of the 3’-UTR followed by a poly(A) tail (Chen et al., 2013). To evaluate regulatory elements of the CIWI promoter, we first identified 4 kb of the CIWI promoter including þ161 bp of the 5’-UTR. Subsequently, the specific activity of this clone CIWI promoter was assessed in chicken PGCs versus CEFs using an eGFP reporter under its control. Similar promoter analysis of PIWI has been reported: In zebrafish, a 4.8-kb upstream promoter of the ziwi gene induced heterologous gene expression in both male and female germ cells at all developmental stages (Leu and Draper, 2010), whereas a reporter gene controlled by the zebrafish vasa promoter was expressed only in germ cells that had entered meiosis and progressed to the pachytene stage. Thus, the ziwi promoter was more suitable for identifying and detecting the earliest germ cells in zebrafish (Leu and Draper, 2010). Hou and colleagues cloned the functional promoter of the mouse Miwi gene. They identified a 303-bp proximal promoter region of Miwi that controls gene expression from mid-pachytene spermatocytes to round spermatids during meiosis. They also found that CpG islands within the proximal promoter of the Miwi gene showed an inverse correlation between the

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Figure 6. Knockdown analysis of transcription factors in chicken PGCs. siRNAs for SP1, NF-Y, or TFAP2a were transfected into cPGCs. Quantitative reverse-transcriptase PCR was conducted, normalizing data to endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. Knockdown efficacies of each siRNA were analyzed first (A). Expression of CIWI (B), DAZL (C), and VASA (D) was then examined following knockdown of each transcription factor in chicken PGCs. Significant differences between control and treatment groups are indicated as  P < 0.001,  P < 0.01, and  P < 0.05. Error bars indicate the standard error of triplicate analyses.

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methylation position and germ cell-specific expression (Hou et al., 2012). Interestingly, the Miwi gene promoter lacks a TATA box, while a CCAAT box was identified within the core promoter region. Thus, the mutated CCAAT box significantly decreased expression activity, indicating that its importance for transcriptional regulation of the Miwi gene (Hou et al., 2012). In this study, by means of promoter deletion and fragmentation assays, we identified the core regulatory element in the CIWI promoter. Similar to the Miwi promoter, there was no TATA box sequence; instead, another CCAAT transcriptional initiation element was identified in the 498 to 494-bp fragment of the CIWI promoter. Mutation assays substituting CCAAT with TTCCT sequences showed complete disruption of transcriptional activity in chicken PGCs. Together, these observations indicate that chickens and mammals utilize a similar set of regulatory processes to ensure genes are transcribed specifically in germ cells. The CCAAT box is one of the most common cis elements found in eukaryotic promoters, and acts as a binding site for the RNA transcription factor NF-Y, which is identical to the CCAAT-binding factor (CBF). The CCAAT box-NF-Y interaction is required for transcriptional activation of several eukaryotic genes (Dolfini et al., 2012). The SP family of zinc-finger transcription factors is an important mediator of selective gene activation during embryonic development and cellular differentiation. Transcripts of Sp1 genes are expressed in stage- and cell type-specific manners in differentiating male mouse germ cells, while SP1 proteins are expressed mainly in the primary spermatocytes (Thomas et al., 2005). In SP1-knockdown chicken PGCs, downregulation of CIWI indicated that the transcription factor SP1 is required for CIWI transcription. In mice, two enhancer boxes (E-box) were identified in a 303-bp proximal promoter region of the Miwi gene (Hou et al., 2012). E-boxes are DNA sequences located upstream of some promoter regions that can initiate gene transcription (Massari and Murre, 2000). In the Miwi gene promoter, methylation at the E2 box in the proximal region inhibited binding of the upstream stimulatory factor (USF), and caused a complete block of Miwi gene expression (Hou et al., 2012). Thus, Hou and colleagues suggest that USF controls Miwi expression from mid-pachytene spermatocytes to round spermatids via methylation-mediated regulation. Another transcription factor, A-MYB, also binds to the Miwi promoter and is associated with production of pachytene piRNA. Induction of A-MYB at the pachytene stage of spermatogenesis initiates production of pachytene piRNAs. A-Mybmutant testes showed reduced levels of Miwi and disrupted transcription of piRNA precursor. We also detected binding sequences for A-MYB at þ3 bp to þ11 bp in the Miwi promoter. No A-MYB binding sites were detected in the CIWI promoter, however, suggesting that the promoter sequences for PIWI have different transcription-factor binding sites that are important at different developmental stages in different species. Mutations that alter the cis-regulatory sequence where transcription factors bind can change gene expression as well as alter the activity of transcription factors. Indeed,

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minor changes to cis-regulatory element sequences affect the binding affinity of transcription factor, triggering the alteration of cis-regulatory activity (Wittkopp, 2010). eGFP expression decreased in chicken PGCs possessing a point-mutated SP1-binding site in the CIWI promoter, suggesting that the reduced affinity between the SP1 transcription factor and this mutated binding sequence could impede the transcriptional activity of CIWI. On the other hand, severely truncated promoters plus a single transcription factor, such as the CCAAT box plus NF-Y, are sufficient to control transcriptional initiation of genes specific for cellular proliferation and cell lineage-specific expression in chicken PGCs (Qyang et al., 1999). In the present study, we identified a 252-bp region of the CIWI promoter that controlled germ-line-specific expression of the CIWI gene; this endogenous region also contains the CCAAT box and SP1 binding site. In conclusion, we characterized a 4-kb fragment of the CIWI promoter. Within this 4-kb promoter region, a small region (252 bp) containing the transcriptional regulatory element CCAAT box is crucial for regulating CIWI expression in chicken PGCs. Thus, the transcription regulatory elements contained within this promoter region may be widely applicable for the regulation of CIWI expression during germ-cell development and differentiation.

MATERIALS AND METHODS Experimental Animals and Animal Care The care and experimental use of chickens was approved by the Institute of Laboratory Animal Resources, Seoul National University, Korea (Approval No: SNU070823-5). Chickens were maintained according to a standard management program at the University Animal Farm, Seoul National University. Procedures for animal management, reproduction, and embryo manipulation adhered to the standard operating protocols of our laboratory.

Construction of Expression Vectors Controlled by the CIWI Promoter For cloning of the CIWI promoter, genomic DNA from adult chicken blood was isolated using the DNeasy Blood & Tissue kit (Qiagen, Valencia, CA) and used as PCR template. Primer sets were used to clone fragments of the CIWI promoter of different sizes (Table 1). PCR products of the

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TABLE 1. List of Primer Sequences for Cloning of the CIWI Promoter Using Genomic PCR Primer set CIWI -3,839bp_F CIWI -2,825bp_F CIWI -1,824bp_F CIWI -839bp_F CIWI promoter_R CIWI -3,839/-839_R CIWI -839/-367_F CIWI -839/-367_R CIWI -577/-326_F CIWI -577/-326_R CIWI -329/-90_F CIWI -329/-90_R CIWI -89/þ161_F CIWI -89/þ161_R EGFP poly A _F EGFP poly A_R

Primer sequence 50 -TGTGGTCAGGTACTCTGCTAAA 50 -CAGGATCCCAAAGTGCTGCTAACT 50 -CCTGCAGGCTACTGAGCAAGG 50 -ATGTCTGGAACAGAGCAACAAGTT 50 -TTCCCTTTGAAGACAAAGCA 50 -GCACAGAATGTGGACAAAAATAGAC 50 -ATGTCTGGAACAGAGCAACAAGT 50 -AGAGCCGAGGGCTGTGT 50 -CCCTCGGCTCTGCCCGCT 50 -GACCGCCGTCTCCACAACGC 50 -GGTCTCCCGTTTGTCGCT 50 -ACACTTACCAACACAATCCCGAC 50 -GTCACTTCAAAGTGGCGTTG 50 -TTCCCTTTGAAGACAAAGCAC 50 -ACTAGTCCGCGGATGGTGAGCAAG 50 -CATATGGACGTCTCCCCAGCATGCC

correct size were cloned into the pGEM T easy vector (Promega, Madison, WI). A 4-kb genomic fragment from 3,839 to þ161 bp, including the predicted transcription start site, was cloned, but the þ1 to þ161-bp region includes the 5’-UTR and does not include the ATG start codon (Chen et al., 2013). The eGFP-coding sequence and polyadenylation (poly-A) tail were inserted into the cloned vectors containing the CIWI promoter using restriction enzymes SpeI and NdeI. For the promoter deletion assays, another four expression vectors containing CIWI promoter fragments of different sizes and positions were constructed: 2,986 bp (2,825 to þ161); 1,985 bp (1,824 to þ161); 1,000 bp (839 to þ161); and 3,000 bp (3,839 to 839). Subsequently, the following fragmented promoters between 839 and þ161 bp were designed and constructed: 839 to 567 bp; 577 to 326 bp; 329 to 90 bp; and 89 to þ161 bp.

Construction of the CCAAT-Mutant Vector CCAAT sequences of the CIWI promoter were substituted by TTCCT sequences, and TTCCT sequences were cloned into the pUC57 vector by Bio Basic Inc. (MO, Canada). The synthesized mutant sequences were cloned into the CIWI promoter expression vector by means of SpeI and SacII digestion and ligation.

TABLE 2. List of siRNA Sequences of Each Transcription Factor for Knockdown Analysis siRNA NF-Y SP1 TFAP2a  Negative control 

Sense

Antisense

50 -GCAAGUCUGUUCUACCUUA 50 -CCAUCAGCUCGUCCAACAU 50 -CCCUCUCCAAGUCUAACAA 50 -GGAUGCGGUGGUUAAAGCA

50 -UAAGGUAGAACAGACUUGC 50 -AUGUUGGACGAGCUGAUGG 50 -UUGUUAGACUUGGAGAGGG 50 -GGAUGCGGUGGUUAAAGCA

Negative control siRNAs have no complementary sequences in the chicken genome.

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Culture of Chicken PGCs

Immunocytochemistry of Transfected PGCs

Chicken PGCs were cultured according to our standard procedure (Park and Han, 2012). Briefly, chicken PGCs from White Leghorn embryonic gonads at Day 6 (stage 28) were maintained and sub-passaged in knockout Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Carlsbad, CA) supplemented with 20% fetal bovine serum (FBS) (Hyclone, Logan, UT), 2% chicken serum (Sigma-Aldrich, St. Louis, MO), 1 nucleosides (Millipore, Billerica, MA), 2 mM L-glutamine (Gibco), 1 nonessential amino acids (Gibco), b-mercaptoethanol (Gibco), 1 mM sodium pyruvate (Gibco), and 1  antibioticantimycotic (Gibco). Human basic fibroblast growth factor (bGF) (Koma Biotech, Korea) at 10 ng/ml was used for PGC self-renewal. Chicken PGCs were maintained in an incubator at 378C with an atmosphere of 5% CO2 and 6070% relative humidity. The cultured PGCs were subcultured onto mitomycin-inactivated mouse embryonic fibroblasts at 5 to 6-day intervals by gentle pipetting without any enzyme treatment.

Cultured chicken PGCs were fixed in 4% paraformaldehyde for one day at room temperature, and incubated with 1:200 diluted anti-mouse stage-specific embryonic antigen 1 (SSEA-1) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 48C overnight. After washing with PBS, PGCs were incubated with a secondary antibody labeled with phycoerythrin (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hr at room temperature. Cells were finally mounted with Vectashield containing 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA) and visualized under a fluorescence microscope.

Culture of CEFs The primary culture of CEFs was prepared from the muscles of 6-day-old White Leghorn chicken embryos. Single-cell populations were obtained using 0.05% trypsin-EDTA (Gibco) treatment to dissociate cells, and were maintained in DMEM with high glucose (Hyclone), 10% FBS, and 1  antibioticantimycotic. The cells were seeded approximately 5  105 cells/well. Cultured cells were grown at 378C in a 5% CO2 incubator.

In Vitro Transfection In vitro transfection was performed using the lipofection method with Lipofector EZ (Aptabio Therapeutics, Suwon, Korea), according to the manufacturer’s instructions. The 4-kb CIWI promoter vector (10 mg) and 5 ml of Lipofector EZ were separately diluted with 100 ml of Opti-MEM I reducedserum medium (Invitrogen, Carlsbad, CA) 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, which was added to 1  105 cultured PGCs in 500 ml of PGC culture medium. Transfected cells were cultured for 24 hr without feeders and maintained for another 48 hr on MEFs. After three days of incubation, cells were observed under a fluorescence microscope.

Flow Cytometry Analysis Transfected PGCs were resuspended in phosphatebuffered saline (PBS) containing 1% bovine serum albumin (BSA), and passed through a 40-mm cell strainer (BD Falcon; Becton Dickinson, Canada). Fluorescence levels in transfected PGCs were calculated using FACSCalibur (Becton Dickinson) with excitation at 488 nm for eGFP detection. All subsequent analyses were performed using the FlowJo software (Tree Star, Ashland, OR).

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Comparative Analysis of Putative Transcriptional Binding Elements in PIWI Promoters Between Species Human PIWI like-1 (HIWI) and mouse PIWI like-1 (Miwi) promoter sequences were used for transcription factor prediction and multiple-sequence alignment. About 200 bp (100 to þ100 bp) of HIWI (from the University of California at Santa Cruz Genome Browser, http://www. genome.ucsc.edu) and 325 bp (143 to þ181 bp) of Miwi (from Hou et al., 2012) promoter sequences were compared with 252-bp (577 to 326 bp) fragments of the CIWI promoter. Transcriptional binding elements in these promoters were predicted using the software MatInspector (Genomatix, Munich, Germany) and PROMO (Alggen, Barcelona, Spain). Multiple sequence alignment with HIWI, Miwi, and CIWI promoters was conducted using the Geneious software (ver. 6.0.5, Auckland, New Zealand).

Knockdown Analysis Through siRNA Transfection Chicken PGCs were cultured according to our previous report. Hundred pmole of each siRNAs (ST Pharm, Seoul, Korea) (Table 2) were transfected to 2  105 PGCs with Lipofectamine RNAi Max transfection reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s protocol, and the transfected PGCs were seeded to each well of 24well culture plates. SiRNA-treated PGCs were analyzed after 96 hr.

Sample Collection & Quantitative ReverseTranscriptase-PCR Analysis Total RNA was extracted from each sample with Qiagen RNeasy Mini kit (Qiagen, Valencia, CA), according to the included protocol. The RNA quantity was determined by spectrophotometry at 260 nm, and 0.8 mg of each RNA was reverse-transcribed with the Superscript III First-strand Synthesis System (Invitrogen, Carlsbad, CA). The cDNA was diluted 4-fold, and quantitatively equalized for PCR amplification using specific primer sets (Table 2). Primers were designed using the Bioneer website (http://web.bioneer.co.kr/cgi-bin/primer/primer3.cgi).

Mol. Reprod. Dev. 81:871–882 (2014)

CCAAT ELEMENT

Quantitative real-time polymerase chain reaction was performed using the EvaGreen (Biotium, Hayward, CA). Twenty microliter PCR reactions contained 2 ml of cDNA, 2 ml of PCR buffer, 1.6 ml of 2.5 mM dNTP mixture, 10 pmol of each forward and reverse primer, 1 ml of 20x EvaGreen, and 1 unit of Taq DNA polymerase. The reaction was performed in optical 96-well standard plates (Applied Biosystems Inc., Foster City, CA). Each test sample was performed in triplicate. The PCR conditions were 948C for 3 min, followed by 40 cycles at 948C for 30 sec, 598C for 30 sec, and 728C for 30 sec, using a melting curve program and continuous fluorescence measurement. The results are reported as the relative expression after normalization of the transcript to the level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control, with the non-specific control as a calibrator, using the 2DDCt method (Livak and Schmittgen, 2001).

ACKNOWLEDGMENTS This work was supported by a grant from the NextGeneration BioGreen 21 Program (No.PJ008142012014), Rural Development Administration, Republic of Korea, and by Bio-industry Technology Development Program (IPET-312060-5-2-SB020), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea. This research was also supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2012R1A1A2039004).

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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site.

Mol. Reprod. Dev. 81:871–882 (2014)

The CCAAT element in the CIWI promoter regulates transcriptional initiation in chicken primordial germ cells.

The P-element-induced wimpy testis (PIWI) protein, which associates with small non-coding RNAs, is responsible for maintaining the integrity of germ c...
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