Drosophila PIWI associates with chromatin and interacts directly with HP1a

Abstract

The interface between cellular systems involving small noncoding RNAs and epigenetic change remains largely unexplored in metazoans. RNA-induced silencing systems have the potential to target particular regions of the genome for epigenetic change by locating specific sequences and recruiting chromatin modifiers. Noting that several genes encoding RNA silencing components have been implicated in epigenetic regulation in Drosophila, we sought a direct link between the RNA silencing system and heterochromatin components. Here we show that PIWI, an ARGONAUTE/PIWI protein family member that binds to Piwi-interacting RNAs (piRNAs), strongly and specifically interacts with heterochromatin protein 1a (HP1a), a central player in heterochromatic gene silencing. The HP1a dimer binds a PxVxL-type motif in the N-terminal domain of PIWI. This motif is required in fruit flies for normal silencing of transgenes embedded in heterochromatin. We also demonstrate that PIWI, like HP1a, is itself a chromatin-associated protein whose distribution in polytene chromosomes overlaps with HP1a and appears to be RNA dependent. These findings implicate a direct interaction between the PIWI-mediated small RNA mechanism and heterochromatin-forming pathways in determining the epigenetic state of the fly genome.[Keywords: PIWI; ARGONAUTE; HP1; heterochromatin; epigenetic; RNAi] Supplemental material is available at http://www.genesdev.org. . In TGS, small RNAs are incorporated into specialized effector complexes able to regulate chromatin modification, resulting in reduced access for the transcriptional machinery to chromatin. The TGS system that contributes to initiation and maintenance of heterochromatin formation in Schizosaccharomyces pombe is particularly well characterized (Verdel and Moazed 2005;Grewal and Jia 2007). In S. pombe, the RNA silencing system targets histone 3 Lys 9 (H3K9) methylation and recruits HP1 homolog Swi6 to the MAT locus and to repetitive elements in pericentric heterochromatin, setting up a heterochromatin spreading/maintenance loop that is dependent on the interaction of Swi6 with the Clr4 histone methyltransferase (HMT) (Grewal and Jia 2007). In plants, a specialized small RNA pathway utilizes heterochromatic RNAs to direct DNA methylation and presumably other chromatin modifications to effect silencing of target loci (for review, see Vaucheret 2007). In Caenorhabditis elegans, a single episode of RNA interference (RNAi) exposure can induce silencing inherited over many generations; mutations that abolish inheritance are all involved in chromatin structure, suggesting a chromatin-based mechanism (Vastenhouw et al. 2006). While the work in fission yeast and plants provides a possible paradigm for RNAi-dependent TGS in metazoans, a specialized TGS effector complex has not yet been characterized in an animal.

Drosophila Piwi functions downstream of piRNA production mediating a chromatin-based transposon silencing mechanism in female germ line

“…Previous studies have supported a role for the piRNA pathway in heterochromatin-dependent silencing (18,20,21), in particular implicating a direct interaction between Piwi and HP1a (18,27). Chromatin immunoprecipitation experiments show a significant loss of HP1a after germ-line Piwi knockdown at five transposon sites of seven tested, looking at their promoter region or 5′ end (Fig.…”

“…This result confirms the specificity of the knockdown effect and indicates that some transposons, e.g., Invader1, require Piwi but not AGO3 (and the secondary piRNA it helps produce) for proper regulation, suggesting an additional mechanism. In addition, in the cases tested, a Piwi transgene with a valine to alanine substitution at amino acid 30, Piwi V30A (18), also rescues the overexpression phenotype (SI Appendix, Fig. S3), suggesting that an intact PXVXL motif is not required for Piwi to silence transposons in the germ line.…”

“…The direct interaction between Piwi and HP1a by using a PXVXL motif, observed both in vitro (27) and in a yeast two-hybrid assay (18), provides a possible means to mediate this process. However, a direct test using a V30A mutant piwi transgene showed rescue of transposon silencing in the piwi 1 germ line, indicating that germ-line Piwi does not require an intact PXVXL motif for this silencing function (SI Appendix, Fig.…”

“…Despite recent progress, the mechanisms used by piRNA to promote silencing are not clear; evidence supporting both transcriptional and posttranscriptional silencing mechanisms has been reported (10,(18)(19)(20)(21). The fly genome codes for three PIWI family argonaute proteins used in the piRNA pathways are Piwi, Aub, and AGO3 (22).…”

“…In Schizosaccharomyces pombe, the RITS complex uses both an argonaute protein, Ago1, and an HP1 protein, Chp1, in targeting heterochromatin assembly (26). Drosophila Piwi and HP1a interact directly in the yeast two-hybrid system and coimmunoprecipitate from embryo lysates (18). In vitro studies indicate that the Piwi N-terminal peptide binds to a dimer of the HP1a chromo shadow domain by using a PXVXL motif (27).…”