Restricted nucleation and piRNA-mediated establishment of H3K9me3-dependent heterochromatin during embryogenesis in Drosophila miranda

Heterochromatin is a key architectural feature of eukaryotic genomes, crucial for silencing of repetitive elements and maintaining genome stability. Heterochromatin shows stereotypical enrichment patterns around centromeres and repetitive sequences, but the molecular details of how heterochromatin is established during embryogenesis are poorly understood. Here, we map the genome-wide distribution of H3K9me3-dependent heterochromatin in individual embryos of D. miranda at precisely staged developmental time points. We find that canonical H3K9me3 enrichment patterns are established early on before cellularization, and mature into stable and broad heterochromatin domains through development. Intriguingly, initial nucleation sites of H3K9me3 enrichment appear as early as embryonic stage3 (nuclear cycle 9) over transposable elements (TE) and progressively broaden, consistent with spreading to neighboring nucleosomes. The earliest nucleation sites are limited to specific regions of a small number of TE families and often appear over promoter regions, while late nucleation develops broadly across most TEs. Early nucleating TEs are highly targeted by maternal piRNAs and show early zygotic transcription, consistent with a model of co-transcriptional silencing of TEs by small RNAs. Interestingly, truncated TE insertions lacking nucleation sites show significantly reduced enrichment across development, suggesting that the underlying sequences play an important role in recruiting histone methyltransferases for heterochromatin establishment.