Transcriptome Analyses and cis- Regulatory Element Assay of Germ Cell-Specific Genes in Chicken Primordial Germ Cells.

Abstract

Primordial germ cells (PGCs), the only type of cells can transfer genetic information to next generation, are distinct from other types of cells in specialized gene expression for maintaining unique characteristics of germ cells. Successful production of functional gamete that finally generates entire new organism is largely dependent on these germ cell-specific gene expressions in PGCs. In this regards, understanding of molecular mechanism(s) related to germ cell-specific gene expression has been emphasized in germ cell biology. One of the important regulating mechanism(s) in the germ cell is the harmonious expression of germ cell-specific genes regulated by transcriptional regulatory elements. Indeed, architecture of specialized germ cell promoters can contribute to mediate distinct transcriptional signals necessary for germ cell-specific programs of appropriate gene expression. Thus, studying on cis-DNA elements and their unique combination of modules is important for deciphering of regulatory networks of germ cell genes that govern transcriptional control during early PGC development. Despite of the importance of transcriptional regulatory network in PGCs, very limited studies are available on cis- and trans- elements that control germ cell-specific expression in avian species. Therefore, in this study, we investigated promoter structures of PGCs-specific genes (CVH, cDAZL and cNANOG) that includes cis- and trans- regulatory elements. To measure the promoter activity, we constructed eGFP and NanoLuc expression vectors that contain the 5 flanking region of these genes. Furthermore, we elucidated predictive transcription factors (TFs) binding to promoter region refer to RNA-seq databases, and in the case of germ cell-related genes, subsequently verified predictive TFs by knockdown analysis. Firstly, we verified the minimal promoter regions of CVH and cDAZL gene, encoding conserved RNA-binding proteins, mediated by dual luciferase reporter assay. As results, we elucidated the minimal upstream regions (−135/+275 of CVH and −83/+173 of cDAZL) which are required for promoter activity in chicken PGCs. And our results indicated that 5 untranslated region (UTR) and intron-1 is dispensable for cDAZL expression, but have positive effect for CVH expression. Furthermore, we identified that in silico prediction of TFs binding to cis-regulatory element sequences were involved in specific expression of genes in chicken PGCs. Taken together, our results suggested that cis- and trans- regulatory factors of cDAZL and CVH genes affect germness related genes expression in chicken PGCs. Secondly, chicken NANOG promoter region which requires at least the 200-bp fragment (−130/+70) was verified in chicken PGCs. Through the 5 deletion assay, we identified that cNANOG promoter had chicken PGCs specific activity, unlike CVH and cDAZL promoter. In addition, comparing promoter activity with each of fragments gave a clue for finding putative proximal and distal enhancers, and then PGC-specific expressed TFs were predicted by referring to RNA-seq databases. Furthermore, mutation assay revealed that putative binding sites of pluripotency factors affect chicken NANOG promoter activity like as mammalian species. In this study, we elucidated cis-transcriptional regulatory regions of chicken PGCs-specific genes, and several TFs that could bind to cis-regulatory regions. Collectively, our results in this study could contribute to constructing of germ cell-specific synthetic promoter for tracing germ cells as well as understanding the molecular mechanism(s) for maintaining germness in chicken PGCs.