DNA methylation of retrotransposon genes is regulated by Piwi family members MILI and MIWI2 in murine fetal testes

TLDR

During fetal male gametogenesis, transposable elements are silenced by de novo DNA methylation, and loss of the Piwi proteins MILI and MIWI2 disrupts this methylation, leading to retrotransposon expression. The study aimed to determine whether the methylation defects in MILI‑ and MIWI2‑null fetal germ cells arise from impaired de novo methylation by analyzing DNA methylation and piRNA expression in wild‑type and mutant cells. To this end, the authors compared DNA methylation patterns and piRNA profiles in wild‑type, MILI‑null, and MIWI2‑null male fetal germ cells. They discovered that LINE‑1 and IAP retrotransposons in MILI‑ and MIWI2‑null germ cells lack de novo methylation, that fetal piRNAs differ from neonatal and adult profiles, and that reduced piRNA levels in mutants demonstrate that MILI and MIWI2 are essential for establishing de novo methylation of retrotransposons.

Abstract

Silencing of transposable elements occurs during fetal gametogenesis in males via de novo DNA methylation of their regulatory regions. The loss of MILI (miwi-like) and MIWI2 (mouse piwi 2), two mouse homologs of Drosophila Piwi, activates retrotransposon gene expression by impairing DNA methylation in the regulatory regions of the retrotransposons. However, as it is unclear whether the defective DNA methylation in the mutants is due to the impairment of de novo DNA methylation, we analyze DNA methylation and Piwi-interacting small RNA (piRNA) expression in wild-type, MILI-null, and MIWI2-null male fetal germ cells. We reveal that defective DNA methylation of the regulatory regions of the Line-1 (long interspersed nuclear elements) and IAP (intracisternal A particle) retrotransposons in the MILI-null and MIWI2-null male germ cells takes place at the level of de novo methylation. Comprehensive analysis shows that the piRNAs of fetal germ cells are distinct from those previously identified in neonatal and adult germ cells. The expression of piRNAs is reduced under MILI- and MIWI2-null conditions in fetal germ cells, although the extent of the reduction differs significantly between the two mutants. Our data strongly suggest that MILI and MIWI2 play essential roles in establishing de novo DNA methylation of retrotransposons in fetal male germ cells.