The Role of Piwi in Transposon Silencing and Heterochromatin Formation. Kiri Ulmschneider, Monica Sentmanat, Sidney Wang, Sarah Elgin. Dept Biol, Washington University, St. Louis, MO.

   Transposable elements (TEs) and their remnants make up a large part of eukaryotic genomes. To avoid genome damage, TEs must be silenced, particularly in the germ line. Both post-transcriptional (PTGS) and transcription-based mechanisms (TGS) are utilized; in Drosophila, the piRNA pathway appears to be involved in both. Both primary piRNAs and the products of the ping pong cycle, driven by Argonaute proteins Aubergine and Argonaute 3, are used to eliminate transposon mRNAs (PTGS). piRNAs can be bound by Piwi, and this enables Piwi to enter the nucleus and silence transposons via a chromatin-based mechanism (TGS). Two proteins, Armitage and Squash, have previously been shown to be involved in the piRNA pathway, interacting with Piwi. Here we examine the effects of germ line specific knockdown (KD) of these proteins on accumulation of heterochromatic marks at transposon loci. Squash KD does not appear to affect heterochromatic marks in the germ line, while Armitage KD does have an affect at a subset of transposon loci, consistent with the idea that Armitage (a putative helicase) helps to load piRNAs onto Piwi. To test the impact of the piRNA system on heterochromatin silencing in somatic cells, we use an hsp70-white reporter, situated adjacent to a heterochromatic block and exhibiting Position Effect Variegation (PEV) in the presence of an added TE. Mutations in components of the piRNA pathway or deletion of the piRNA sequences from the TE (either 1360 or Invader4) both result in a loss of silencing, indicating that the piRNA pathway is playing a role in silencing at this site. Transcription across the reporter-associated TE is observed in the early embryo, when heterochromatin formation occurs, suggesting that piRNA sequences could recognize the site through an RNA-RNA interaction, localizing Piwi. Piwi is known to interact with HP1a in vitro, so might be able to recruit heterochromatic factors. This system enables further tests of this hypothesis.