Multiple regulatory regions control the transcription of medaka germ gene vasa

Numerous regulatory DNA regions and trans-acting protein factors controlling transcription have been characterized for many genes that are expressed in somatic cells. Little is known about the transcriptional control of germ genes, and no cell culture system has been explored for quantitative reporter assay of germ gene transcription in vitro. Here we report the development of such an in vitro system and the identification of regulatory regions in the medaka germ gene vasa. We established the medaka germ cell line SG3 as a suitable in vitro system for analyzing germ gene transcription. Transgenic production revealed that VAS, a 5.1-kb genomic fragment of medaka vasa, possessed regulatory regions essential for germ cell-specific transcription. Importantly, reporter assays revealed 11 positive and negative regulatory regions alternatively positioned throughout VAS including the first intron. Strikingly, the regulatory regions may act in additive, non-additive and dependent manners. We show that a 39-bp element within one regulatory region is able to interact with the nuclear factor(s) of vasa-expressing embryos and testes. These results demonstrate the complexity of transcriptional control of medaka vasa and provide important insights into opposing mechanisms underlying germ gene transcription.