GSE210270
Transcriptional readthrough as a conserved mechanism for piRNA cluster expression in mosquitoes
We performed combined transcriptional and chromatin state analyses and showed that these core piRNA clusters in Aedes mosquitoes do not have their own independent promoters, but depend on readthrough transcription from upstream genes for piRNA biogenesis. We further showed core piRNA clusters biogenesis depends on key factors, the mRNA exporter Nxf1, three Tudor proteins, Veneno, Tejas, Yb, and an evolutionarily conserved cochaperone Shutdown. Overall design: In Aedes aegypti Aag2 cells, we performed ChIP-seq of four histone marks, including H3K27ac, H3K4me3, H3K36me3, H3K9me3. Besides, we generated a series of RNA pol II ChIP-seq,  RNA pol II Ser5P ChIP-seq,  RNA pol II Ser2P ChIP-seq. In addition, we performed ATAC-seq to assay open chromatin regions. Such a comprehensive chromatin profiling data provides precise insights into transcriptional regulation and chromatin states of Aedes mosquitoes. We further explored key factors involoved in piRNA cluster biogenesis. We performed knockdown of the nuclear exporter Nxf1, UAP56 which plays an important role in piRNA cluster export and three Tudor proteins that were among the top enriched hits from Piwi5-IP experiment, Vreteno (AAEL020359), Tejas (AAEL022773), and Yb (AAEL001939). We also included Shutdown (AAEL011632), which is required for the biogenesis of all piRNA populations in D. melanogaster. After knockdown of target genes, RNA was isolated and prepared for small RNA-seq sequencing. dsLuc-1-3 serve as control knockdown for Nxf1 and UAP56, whereas dsLuc4-5 serve as control for knockdown of Vreteno, Tejas, Yb, and Shutdown.