Abstract
During the development of mammalian embryonic germ cells, global demethylation and de novo DNA methylation take place. In mouse embryonic germ cells, two PIWI family proteins, MILI and MIWI2, are essential for the de novo DNA methylation of retrotransposons, presumably through PIWI-interacting RNAs (piRNAs). Although piRNA-associated MIWI2 has been reported to play critical roles in the process, its molecular mechanisms have remained unclear. To identify the mechanism, transgenic mice were produced; they contained a fusion protein of MIWI2 and a zinc finger (ZF) that recognized the promoter region of a type A LINE-1 gene. The ZF-MIWI2 fusion protein brought about DNA methylation, suppression of the type A LINE-1 gene, and a partial rescue of the impaired spermatogenesis of MILI-null mice. In addition, ZF-MIWI2 was associated with the proteins involved in DNA methylation. These data indicate that MIWI2 functions as an effector of de novo DNA methylation of the retrotransposon.
Highlights
Half of the mammalian genome is composed of transposable elements (TEs) (Lander et al, 2001)
We demonstrated that the zinc finger (ZF)-MIWI2 fusion protein induced de novo DNA methylation of the type A LINE-1 genes and restored spermatogenesis, at least to some extent, in a P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs)-independent manner
Production of ZF-MIWI2 Transgenic Mice First a chromatin immunoprecipitation (ChIP) assay was performed to confirm that MIWI2 bound to the genomic regions of LINE-1 and the intracisternal A particle (IAP) retrotransposons in the gonocytes
Summary
Half of the mammalian genome is composed of transposable elements (TEs) (Lander et al, 2001). Molecular defense mechanisms involving epigenetic transcriptional regulation were acquired during the evolutionary process to silence TEs (Slotkin and Martienssen, 2007). DNA methylation and histone modification represent two major mechanisms (Walsh et al, 1998). DNA methyltransferase-1 (Dnmt1) and ERG-associated protein with an SET domain (ESET) histone methyltransferase ( known as H3K9me methyltransferase) are required for retrotransposon silencing in embryonic stem cells (ESCs) (Lei et al, 1996; Matsui et al, 2010). DNA methylation levels in the genome, including those of retrotransposons and imprinting genes, change dynamically during germ cell development. The vast majority of DNA methylation is lost in the primordial germ cells by embryonic day (E) 13.5 (Lane et al, 2003). The de novo DNA methyltransferases Dnmt3a and Dnmt3b, and the related Dnmt3L protein, play essential roles in this process, and the establishment of the pattern of DNA methylation is a prerequisite for spermatogenesis and other cell lineages (Bourc’his and Bestor, 2004; Kaneda et al, 2004; Kato et al, 2007; Lees-Murdock et al, 2005; Okano et al, 1999; Sakai et al, 2004)
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