piRNA-independent recruitment of Piwi to chromatin leads to piRNA-guided repression of transcription
Authors:Alexei Aravin, Adrien Le Thomas, Susan Liao, Georgi Marinov, Edward Perkins, Alicia Rogers
Mentor:Katalin Fejes Toth, Thomas Hunt Morgan Senior Research Fellow of Biology, California Institute of Technology
Preserving genome integrity in metazoan germlines is crucial to ensuring proper gamete development. Piwi proteins and associated Piwi-interacting RNAs (piRNAs) protect genome integrity in metazoan germlines by providing RNA interference (RNAi) defense against the expression of transposons. In the cytoplasm, Piwi complexes are guided to transposon transcripts by complementary piRNA sequences to destroy them through endonucleolytic cleavage; however, some Piwi family members are nuclear, so we hypothesize alternative pathways for piRNA-mediated regulation of gene expression may exist. To investigate the molecular function of Piwi in the nucleus, we generated transgenic Drosophila expressing GFP-tagged Piwi protein. Through co-immunostaining GFP-Piwi and RNA polymerase II in Drosophila ovaries, we observed the GFP-Piwi signal on polytene chromosomes generally overlaps with the RNA polymerase II signal that marks sites of active transcription. To map genome binding sites of Piwi, we cross-linked and immunoprecipitated Piwi-bound chromatin from Drosophila ovaries. Analysis of the chromatin immunoprecipitation results showed that Piwi associated with chromatin at the promoters of active genes. Small RNA sequencing data revealed that the majority of genes which show strong Piwi binding do not generation piRNAs. Despite Piwi localization on the majority of transcribed genes, genes with Piwi localization do not change expression in Piwi knock-downs. Since no decrease in gene expression is observed in these Piwi knockdowns, Piwi alone is not sufficient to repress transcription. Additionally, artificial tethering of Piwi to the promoter of a reporter gene lacking a cognate piRNA resulted in no difference in gene expression. However, upon Piwi knockdown, transposon levels increase, accompanied by increased RNA polymerase II occupancy on transposon promoters, indicating that piRNA targets are regulated by Piwi at the transcriptional level. Our results indicate that the function of Piwi in repressing transcription requires piRNA complementarity to nascent transcripts, although Piwi is initially recruited to chromatin in a piRNA-independent fashion.