Abstract
BackgroundPrimordial 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.ResultsWe 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.ConclusionsThese 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.
Highlights
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
We identified six Transcription factor (TF) (EP300, GA binding protein transcription factor (GABPA), heat shock factor 2 (HSF2), Nuclear transcription factor Y (NFYA), Stimulating protein 3 (SP3), and Zinc finger protein 143 (ZNF143)) that were expressed at significantly higher levels in PGCs and have putative binding sites in the 591-bp fragment (−316/+275) of the chicken vasa homologue (CVH) promoter
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′ untranslated region (UTR) and intron 1, can control the transcription of the CVH gene in chicken PGCs
Summary
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. 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 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. The intricate regulatory mechanism(s) that governs transcriptional control of vasa expression during chicken germline development has yet to be investigated in detail
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