Piwi is linked to heterochromatin formation in the embryo of Drosophila melanogaster. Tingting Gu, Sarah Elgin. Department of Biology, Washington University in St Louis, St Louis, MO.

   A persistent question in epigenetics is how heterochromatin is targeted for assembly at specific domains, and how that chromatin state is faithfully transmitted. Stable heterochromatin is necessary to silence transposable elements (TEs) and maintain genome integrity. Both the RNAi system and heterochromatin components HP1 and H3K9me2/3 are required for initial establishment of heterochromatin structures in fungi and plants. We utilized the newly developed Drosophila melanogaster transgenic shRNA lines to deplete proteins of interest at specific developmental stages to dissect their roles in heterochromatin assembly in early zygotes, and in maintenance of the silent chromatin state during development. Using reporters subject to Position Effect Variegation (PEV), we find that depletion of key proteins in the early embryo can lead to a loss of silencing (suppression of PEV) assayed at adult stages. The piRNA component Piwi is required in the early embryo for reporter silencing, but knock-down during larval stages has no impact. This implies that Piwi is involved in targeting HP1a when heterochromatin is established (late blastoderm), but that the silent chromatin state created is transmitted through cell division independent of the piRNA system. In contrast, HP1a is required for both initial assembly and the mitotic inheritance of heterochromatin. HP1a profiles in piwi mutant animals confirm that Piwi depletion leads to decreased HP1a levels in pericentric heterochromatin, particularly at TEs. Piwi is known to physically interact with HP1a, and is important for recruiting HP1a to some TEs in the female germ line. To establish whether Piwis role is direct or indirect, experiments to tether Piwi adjacent to reporters are underway. The present results indicate that the major role of the piRNA system in targeting heterochromatin formation occurs in the early zygote during initial heterochromatin assembly, and further demonstrate that a failure of heterochromatin formation in the early embryo impacts the phenotype of the adult.