HP1 Recruitment in the Absence of Argonaute Proteins in Drosophila

Abstract

Highly repetitive and transposable element rich regions of the genome must be stabilized by the presence of heterochromatin. A direct role for RNA interference in the establishment of heterochromatin has been demonstrated in fission yeast. In metazoans, which possess multiple RNA–silencing pathways that are both functionally distinct and spatially restricted, whether RNA silencing contributes directly to heterochromatin formation is not clear. Previous studies in Drosophila melanogaster have suggested the involvement of both the AGO2-dependent endogenous small interfering RNA (endo-siRNA) as well as Piwi-interacting RNA (piRNA) silencing pathways. In order to determine if these Argonaute genes are required for heterochromatin formation, we utilized transcriptional reporters and chromatin immunoprecipitation of the critical factor Heterochromatin Protein 1 (HP1) to monitor the heterochromatic state of piRNA clusters, which generate both endo-siRNAs and the bulk of piRNAs. Surprisingly, we find that mutation of AGO2 or piwi increases silencing at piRNA clusters corresponding to an increase of HP1 association. Furthermore, loss of piRNA production from a single piRNA cluster results in genome-wide redistribution of HP1 and reduction of silencing at a distant heterochromatic site, suggesting indirect effects on HP1 recruitment. Taken together, these results indicate that heterochromatin forms independently of endo-siRNA and piRNA pathways.

Author Summary

One role for silent heterochromatin is to preserve the integrity of the genome by stabilizing regions rich in repetitive sequence and mobile elements. Compaction of repetitive sequences by heterochromatin is needed to prevent genome rearrangement and loss of genetic material. Furthermore, uncontrolled movement of mobile elements throughout the genome can result in deleterious mutations. In fission yeast, one important mechanism of heterochromatin establishment occurs through RNA interference, an RNA–dependent gene silencing process. However, it is unclear whether a direct role for RNA silencing in heterochromatin formation is conserved throughout evolution. In the fruit fly, Drosophila melanogaster, which harbors multiple RNA–silencing pathways that are both functionally distinct and spatially restricted, previous studies have suggested the involvement of the endogenous small interfering RNA (endo-siRNA) and Piwi-interacting RNA (piRNA) pathways in heterochromatin formation. These small RNA silencing pathways suppress the expression of mobile elements in the soma or in both somatic and germline tissues, respectively. Utilizing complementary genetic and biochemical approaches, we monitored the heterochromatin state at discrete genomic locations from which both types of these small RNAs originate in endo-siRNA or piRNA pathway mutants. Our results indicate that heterochromatin can form independently of these two small RNA silencing pathways.