A dual histone code specifies the binding of heterochromatin protein Rhino to a subset of piRNA source loci [CUT&RUN histone modifications in S2 cells]
Animal germ cells deploy a specialized small RNA-based silencing system, called the PIWI-interacting RNA (piRNA) pathway, to prevent aberrant expression of transposable elements and maintain genome integrity.  In Drosophila germ cells, the majority of piRNA populations originate from dual-strand piRNA clusters, genomic regions highly enriched in transposon fragments, via an elaborate protein machinery centred on the heterochromatin protein 1 homolog, Rhino.  Although Rhino binds to peptides carrying trimethylated H3K9 in vitro, it is not fully understood why it only occupies a fraction of H3K9me3-decorated heterochromatin in vivo.  Recent work uncovered that Rhino is recruited to a subset of piRNA clusters by the zinc finger protein Kipferl.  Here we identify a Kipferl-independent mode of Rhino targeting that is dependent on the histone H3 lysine 27 methyltransferase Enhancer of Zeste and the presence of H3K9me3 and H3K27me3 marks.  At Kipferl-independent sites, we find that Rhino, through its dimeric chromodomain, specifically binds to loci marked by both H3K9me3 and H3K27me3.  These results expand our understanding of the characteristic binding profile of the heterochromatin protein Rhino and reveal a role for dual histone modifications in defining the specificity of a chromatin binding protein. Overall design: CUT&RUN from Drosophila melanogaster S2 cells characterising H2AK119ub, H3K27me3, H3K9me3 or IgG in wildtype cells, or cells depleted for E(z) or Renilla.