Regulatory elements and transcriptional control of chicken vasa homologue (CVH) promoter in chicken primordial germ cells

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.

Germline stem cells in human

“…Singlecell transcriptome shows that the germline of Xenopus is evolutionarily closer to that of zebrafish than to humans and mice. 45 In some model organisms, including Xenopus, [46][47][48][49][50][51][52][53][54][55][56][57][58] zebrafish, [59][60][61][62][63][64][65][66][67][68][69] and chicken, [70][71][72][73][74][75][76][77][78][79][80][81][82] their PGCs are specified via the maternally inherited germ plasm, including vasa and nanos, during the first several cleavages. In contrast, in mammals, the PGCs are specified by induction during early embryo development.…”

Nanos2 promotes differentiation of chicken (Gallus gallus) embryonic stem cells to male germ cells

“…17 Vasa are known to be an ideal molecular marker of PGCs and germ cells, and specifically express in germ cells of many species and is an important reference for the research of PGCs generation, migration, and differentiation. cvh is the homologous gene of vasa in chicken, 18 so it is often used as a specific marker gene for chicken PGCs. c-kit specifically binds to stem cell factors and plays an important role in PGCs and SSCs survival and differentiation.…”

A novel F‐box domain containing cyclin F like gene is required for maintaining the genome stability and survival of chicken primordial germ cells

“…Commercially available control siRNA (sense: 5′‐CCU ACG CCA CCA AUU UCG U) was purchased from Bioneer Corp. (Daejeon, Korea). To validate the knockdown efficiency of chicken cyclin F like, the three gene‐specific siRNAs and control siRNA were transfected into PGCs (50 pmol siRNA in Lipofectamine RNAiMAX [Invitrogen] plus Opti‐MEM [Invitrogen] for 3‐5 × 10 5 cells) according to the established procedure . After 0‐48 hours of transfection, total RNA from PGCs was extracted and cDNA was synthesized with the Superscript III First‐Strand Synthesis System, and the knockdown efficiency was calculated by RT‐qPCR from the transcribed cDNAs.…”

“…11,33 After 0-48 hours of transfection, total RNA from PGCs was extracted and cDNA was synthesized with the Superscript III First-Strand Synthesis System, and the knockdown efficiency was calculated by RT-qPCR from the transcribed cDNAs. Three candidate siRNA sequences, siRNA-620 (sense: 5′-AAC AAG GAA GAG AAC AAU UUU-3′), siRNA-802 (sense: 5′-AGA ACU UGC AUC UGG AAA GUU-3′), and siRNA-1096 (sense: 5′-UAA AGA UCU AAU GAA GUU GUU-3′) that targets chicken cyclin F like were selected and synthesized.…”

“…To validate the knockdown efficiency of chicken cyclin F like, the three gene-specific siRNAs and control siRNA were transfected into PGCs (50 pmol siRNA in Lipofectamine RNAiMAX [Invitrogen] plus Opti-MEM [Invitrogen] for 3-5 × 10 5 cells) according to the established procedure. 11,33 After 0-48 hours of transfection, total RNA from PGCs was extracted and cDNA was synthesized with the Superscript III First-Strand Synthesis System, and the knockdown efficiency was calculated by RT-qPCR from the transcribed cDNAs. Of the tested siRNAs, siRNA-620 highly decreased the expression of chicken cyclin F like gene at 48 hours of treatment, and thus this siRNA was selected and further used for all experiments.…”