Structural insights into Rhino-mediated germline piRNA cluster formation

The piRNA pathway silences transposons in animal gonads. Animals lacking piRNA pathway components often show transposon activation in the germline and compromised fertility¹. In Drosophila germ cells, piRNA precursors are transcribed from heterochromatic piRNA cluster regions, and then transported into nuage/mitochondria to be processed into mature piRNAs that are loaded into PIWI clade Argonautes¹. Guided by piRNAs, PIWI proteins silence transposons at either transcriptional or post-transcriptional level². Drosophila ovaries contain two types of piRNA clusters, which are often coated with trimethylated lysine 9 of H3 (H3K9me3). Dual-strand clusters are mainly active in germ cells and produce piRNAs from both genomic strands, while uni-strand clusters are dominant in somatic follicle cells and produce piRNAs from only one genomic strand². However, how piRNA clusters are defined in the genome remains elusive. Recent studies have shown that a Heterochromatin Protein 1 (HP1) homolog Rhino (Rhi), also known as HP1D, is anchored at the dual-strand piRNA clusters to trigger the piRNA production^3,4. Rhi is a germline-specific protein that is under rapid and positive selection during evolution⁵. Like other HP1 family proteins, Rhi exists in all Drosophila species and shares the similar domain architecture, which contains a chromodomain (CD) at the N-terminus, a chromoshadow domain (CSD) at the C-terminal region, and a hinged region between CD and CSD domains⁵.

To examine whether the binding to H3K9me3 is important for Rhi function in vivo, we expressed GFP-tagged wild-type Rhi or Rhi^W45A protein in the rhi^2/KG mutant flies. Under laser scanning confocal microscope, wild-type Rhi formed germline-specific nuclear foci, which may represent piRNA cluster regions in the genome. However, the typical Rhi foci were dramatically reduced in the ovaries from Rhi^W45A flies (Figure 1D and Supplementary information, Figure S1B). Importantly, transgenic expression of wild-type Rhi, but not Rhi^W45A in rhi^2/KG mutant flies successfully rescued sterility and transposon hyper-expression defects in these animals (Figure 1E and 1F). Collectively, these results suggest that the CD-mediated H3 lysine binding is indispensable for Rhi function in vivo.

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