Traffic jam directly regulates the Drosophila piRNA cluster flamenco and the Piwi pathway gene network to ensure transposon silencing and female fertility.

The flamenco (flam) locus is the most prominent piRNA cluster locus expressed in Drosophila ovarian follicle cells that is required for female fertility and silencing of gypsy/mdg4 transposons. Although P-element insertions in the flam promoter region disrupts flam piRNAs and cause female infertility, the flam enhancer architecture is still poorly understood. To illuminate flam’s regulation, we uncovered novel shadow enhancer (SE) sequences within the first exons of flam via promoter-bashing assays in OSS cells. We confirmed the SE relevance in vivo with new Drosophila flam deletion mutants of these regions that compromised flam piRNA expression and lowered female fertility from unleashed transposon expression. In a proteomic analysis of proteins associated with these SE sequences, the MAF transcription factor Traffic jam (Tj) emerged as the most prominent binding candidate. With Tj knockdowns in OSS cells, we see a coordinated loss of flam transcripts and piRNAs as well as a decreased expression of many Piwi pathway genes including piwi, which then also coincided with increased transposon RNA levels. We conducted ChIP-seq of Tj’s interaction at Piwi pathway gene promoters and hundreds of other genes important for follicle cell development. Lastly, we utilized transient Tj knockdown in the fly ovary to support our conclusions of how Tj orchestrates the complex Piwi pathway network in Drosophila follicle cells.