Safeguarding Drosophila female germ cell identity depends on an H3K9me3 mini domain guided by a ZAD zinc finger protein

Abstract

H3K9me3-based gene silencing is a conserved strategy for securing cell fate, but the mechanisms controlling lineage-specific installation of this epigenetic mark remain unclear. In Drosophila, H3K9 methylation plays an essential role in securing female germ cell fate by silencing lineage inappropriate phf7 transcription. Thus, phf7 regulation in the female germline provides a powerful system to dissect the molecular mechanism underlying H3K9me3 deposition onto protein coding genes. Here we used genetic studies to identify the essential cis-regulatory elements, finding that the sequences required for H3K9me3 deposition are conserved across Drosophila species. Transposable elements are also silenced by an H3K9me3-mediated mechanism. But our finding that phf7 regulation does not require the dedicated piRNA pathway components, piwi, aub, rhino, panx, and nxf2, indicates that the mechanisms of H3K9me3 recruitment are distinct. Lastly, we discovered that an uncharacterized member of the zinc finger associated domain (ZAD) containing C2H2 zinc finger protein family, IDENTITY CRISIS (IDC; CG4936), is necessary for H3K9me3 deposition onto phf7. Loss of idc in germ cells interferes with phf7 transcriptional regulation and H3K9me3 deposition, resulting in ectopic PHF7 protein expression. IDCs role is likely to be direct, as it localizes to a conserved domain within the phf7 gene. Collectively, our findings support a model in which IDC guides sequence-specific establishment of an H3K9me3 mini domain, thereby preventing accidental female-to-male programming. Author SummaryTissue development and function relies on cells remembering their identity. A cells identity is defined by the genes it expresses and those it does not. Recent work has shown that genes can be silenced by trimethylation of histone H3 lysine 9 (H3K9me3) marked chromatin, and that H3K9me3-mediated gene silencing is a vital strategy for securing cell fate. But there is very little information about how the machinery responsible for H3K9 methylation finds its target genes. Here we explore this issue using the Drosophila female germline where a mini domain of repressive H3K9me3 chromatin secures female germ cell fate by silencing phf7, a gene normally expressed in male germ cells. Transposable elements are also silenced by H3K9me3 mini domains, but we find that the proteins involved in this process are not required for phf7 silencing. Instead, we find that silencing requires a previously uncharacterized protein, we have named IDENTITY CRISIS. Our work provides evidence that IDENTITY CRISIS directs the H3K9 methylation machinery to build a mini domain at the phf7 locus. Our results shed new light into how cells safeguard their identity by silencing cell type inappropriate genes, and more specifically how these genes are identified by the silencing machinery.