Uncoupling DNA- and RNA-directed DNA methylation at Rasgrf1

ABSTRACT Long noncoding RNAs (lncRNAs) have garnered much attention as possible links between DNA sequence and the protein factors that mediate DNA methylation. However, the mechanisms by which DNA methylation is directed to specific genomic locations remain poorly understood. We previously identified a lncRNA in mouse, the pitRNA, that was implicated in the control of DNA methylation at the imprinted Rasgrf1 locus. The pitRNA is transcribed in the developing male germline antisense to the differentially methylated region (DMR) that harbors paternal allele methylation, and is driven by a series of tandem repeats that are necessary for imprinted methylation. MitoPLD, a factor necessary for piRNA biogenesis, both processes piRNAs from the pitRNA, and is necessary for complete methylation at the locus, along with piRNA binding proteins. Using two independent mouse systems where pitRNA transcription is driven by the doxycycline-inducible Tet Operator, we demonstrate that pitRNA transcription across the DMR is insufficient for imprinted methylation, and that the Rasgrf1 repeats have additional, critical cis -acting roles for imparting DNA methylation to Rasgrf1 , independently of their control of pitRNA transcription. Furthermore, pitRNA overexpression and oocyte loading of pitRNA is insufficient to induce transallelic and transgenerational effects previously reported for Rasgrf1 . Notably, manipulation of the pitRNA with the TetOFF system led to transcriptional perturbations over a broad chromosomal region surrounding the inserted Tet Operator, revealing that the effects of this regulatory tool are not localized to a single target gene. AUTHOR SUMMARY DNA methylation is a heritable genetic modification known to impact vital biological processes. While the proteins that establish, maintain, and remove DNA methylation are well characterized, the mechanisms by which these proteins are directed to specific genetic sequences are poorly understood. We have previously demonstrated that DNA methylation at the imprinted Rasgrf1 locus requires a DNA element with a series of tandem repeats. These repeats act as a promoter for a long noncoding RNA, the pitRNA, which is targeted by a small noncoding RNA pathway known to silence viral elements in the male germline via DNA methylation. We queried the sufficiency of the pitRNA to mediate DNA methylation at Rasgrf1 . We show that, in the absence of the repeats, the pitRNA expression is insufficient to establish imprinted methylation. This work supports a pitRNA-independent mechanism for methylation at Rasgrf1 , and a critical cis -acting role for the tandem repeats separate from their control of pitRNA transcription.