Effects of transgene insertion loci and copy number on Dnmt3L gene silencing through antisense transgene-derived PIWI-interacting RNAs

Abstract

PIWI-interacting RNAs (piRNAs), which are germ cell-specific small RNAs, are essential for spermatogenesis. In the fetal mouse germ cells, piRNAs are synthesized from sense and antisense RNAs of transposable element sequences for retrotransposon silencing. In a previous study, we reported that transgenic mice expressing antisense-Dnmt3L under the control of the Miwi2 promoter (Tg-Miwi2P-asDnmt3L) exhibited piRNA-mediated DNMT3L downregulation. In this study, two transgene integration loci (B3 and E1) were identified on chromosome 18 of the Tg-Miwi2P-asDnmt3L mice; these loci were weak piRNA clusters. Crossbreeding was performed to segregate the mice with the transgene cassette inserted into a single locus. DNMT3L was silenced, and spermatogenesis was severely impaired in mice with the transgene cassette inserted at the B3 locus (Tg-B mice). In contrast, spermatogenesis in mice bearing the transgene at the E1 locus (Tg-E mice) was normal. The number of piRNAs for Dnmt3L in Tg-B mice was eight times higher than that in Tg-E mice. Gene silencing and impaired spermatogenesis are dependent on the transgene copy number rather than on the insertion loci. Additionally, the endogenous Dnmt3L promoter was not methylated in Tg mice, suggesting that Dnmt3L silencing was caused by post-transcriptional gene silencing. Using these data, we discuss a piRNA-dependent gene silencing mechanism against novel gene insertions.