GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE307nnn/GSE307911/primary200OKGenomicsDrosophila melanogasterGenome binding/occupancy profiling by high throughput sequencinghttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE307911GEOGSEfalseHijacking pre-tRNA enables LTR-retrotransposon-initiated constitutive heterochromatin formation [ATAC-Seq]As a fundamental component of eukaryotic chromosomes, pericentric heterochromatin, defined by histone H3K9 methylation (H3K9me), endows specialized genomic architecture with broad functional consequences1-3. Although pericentric DNA sequences diverge among different species, they universally retain the highly conserved H3K9 trimethylation (H3K9me3) mark4-6. While H3K9me3-specific methyltransferase was identified more than 30 years ago7,8, how this enzyme is recruited and nucleated at pericentric heterochromatin during Drosophila somatic development remains unknown. Here, by leveraging a transposition reporter system combined with a genome-wide RNAi screen, we identified the poly-U binding protein Puf68 as an imperative factor in recruiting SUV39H methyltransferase to initiate pericentric heterochromatin formation. The Puf68 protein binds with high affinity to poly-U tracts in pre-tRNAs, forming a Puf68/pre-tRNA complex that subsequently base-pairs with the primer binding site (PBS) of nascent LTR-retrotransposons. Then, Puf68 directly interacts and functionally recruits Su(var)3-9 to LTR-retrotransposon regions to catalyze H3K9 trimethylation. Notably, Puf68 is sufficient to initiate de novo heterochromatin assembly at both pericentric regions and the genomic regions of ectopically integrated LTR-retrotransposons. Given the highly divergent DNA composition in Drosophila pericentric regions, our work resolves a long-standing question: how the fly harnesses nascent LTR-retrotransposon transcripts to establish a specialized mechanism for initiating constitutive heterochromatin assembly.2026/09/04GSE307911GSM9233807GSM9233806GSM9233809GSM923380825244307911Drosophila melanogaster[42685192]