In the Drosophila ovary, nurse cells and the oocyte, which constitute the germline, are surrounded by a layer of somatic follicle cells. The defensive system in flies relies on piRNA clusters to generate small RNAs known as piRNAs for gene silencing of transposable elements (TEs). The somatic Flamenco (Flam) piRNA cluster operates as a vital cell protection system for ovarian follicle cells. The inactivation of Flam results in premature TE movement that causes infertility in females.
The study aimed to identify the actual regulator of Flam through advanced methodologies after earlier researchers proposed Cubitus interruptus (Ci) might activate Flam. Researchers used a luciferase reporter system with custom DNA constructs to identify regulatory regions of the Flam piRNA cluster. The reporter system evaluates the activation potential of different DNA segments responsible for gene expression. The scientists modified the pNanoLucTK luciferase plasmid by using KpnI restriction sites to replace its native promoter while creating a system that can easily add sections from the Flam promoter and enhancer regions.
Researchers amplified genomic DNA from ovarian somatic sheet (OSS) cells using PCR, followed by HiFi Assembly plasmid cloning and sequence confirmation. They avoided RNA splicing confusion by adding a synthetic intron prior to the luciferase gene that they obtained from past experiments. They constructed LTR luciferase reporter plasmids containing wild-type and mutant versions of both gypsy and mdg1 retrotransposons. They used nucleofection to deliver siRNAs into OSS and S2 cells through an electrical pulse process following cell culture growth. Two days later, the cells were chemically transfected with luciferase reporter plasmids using FuGENE HD. Lysis of cells occurred two days after varying plasmid DNA doses entered the cells, which were later processed using a luminometer to determine luciferase activity. Research results from luciferase measurements demonstrated the ability of DNA sequences to control gene expression in follicle cells.
Researchers from the study generated deletion mutants of FITS, FPSE1, and FPSE2 by utilizing CRISPR-Cas9 targeted small guide RNA (sgRNAs) and single-stranded oligonucleotides (ssODN) templates on Drosophila melanogaster flies. The FPSE2 deletion showed evidence of duplication mistakes because of the DNAREP1 repeat sequence. They generated more control mutants referred to as FPUN and FPRU to validate the function of alternative regulatory domains. The researchers validated all mutant lines through sequencing while stabilizing each line by integrating balancer chromosomes after backcrossing.
Rainbow Transgenic Flies, Inc. received the components to perform embryo injections during the early developmental stages of the Cas9-expressing special fly strain. They used DNA from the offspring between w1118 flies and the developed adult mutants to verify the expected mutations following their development. Multiple backcrossing procedures with balancing over the FM7a balancer chromosome stabilized the mutant lines to ensure genetic uniformity. Two DNA regulatory sequences were identified in the area downstream of Flam TSS through enhanced promoter-reporter analysis, and they exhibited potent activation behavior exclusively within follicle cells. The scientists named these regulatory elements Flam Promoter Shadow Enhancers 1 and 2 (FPSE1 and FPSE2). Flies lacking FPSE1 or FPSE2 exhibited reduced fertility together with decreased mature egg production, just like known Flam mutants (FlamKG and FlamBG).
The loss of Flam RNA and piRNA expression during embryonic stem cell development in FPSE1 mutants caused strong TE reactivation of gypsy, ZAM, and blood. Deletion of the region surrounding the Flam TSS (FITS) shifted the TSS downstream but did not block the remainder of the Flam transcript or piRNA production, which explains the mild phenotype. The fertility issues observed in FPSE2 mutants did not correspond with the slight changes in RNA expression levels, suggesting that the Flam locus may retain alternative regulatory mechanisms.
Proteomic analysis revealed Traffic Jam (TJ) functions as a protein that connects to FPSE1 and FPSE2 enhancer sequences. TJ exhibits powerful expression in follicle cells while existing as crucial for ovarian developmental processes. Previous studies have shown that Traffic Jam-deficient flies entirely lose their gonads. Plasma membrane depletion of Traffic Jam in OSS cells diminished Flam transcription together with piRNA activity which triggered TEs to recover from suppression with gypsy. The FPSE1 enhancer, together with TJ, directly maintains the active state of the Flam piRNA cluster. TEs make use of TJ to advance their own transcription pattern. The evolution of piRNA defense systems has created a natural competition with transposons that need to maintain active expression. The Drosophila genome has developed a strategic and deliberate deployment of TJ elements at piRNA genetic locations to maintain counterbalance.
This study identifies that Traffic Jam (TJ), a large MAF-family transcription factor, is the primary regulator of the Flamenco piRNA cluster in Drosophila follicle cells. Although earlier speculation centered on Ci, experiments identified that TJ directly binds enhancer sequences and drives Flam expression to produce piRNAs that suppress dangerous TEs.
The loss of FPSE1 enhancer causes Flam RNA and piRNA deficiency that results in TE reactivation and fertility problems like conventional Flam mutants. The FITS and FPSE2 mutants cause moderate changes to Flamenco expression because alternative regulatory mechanisms help maintain some support for expression in distinct conditions. Despite its role in Flam regulation, the TJ factor plays an essential function in Flam development, in addition to other genetic control functions it performs within the cell. Retroviral-like TEs have recognized TJ as an essential force to manipulate due to its dominant function in follicle cell gene regulation, thus making it central to the persistent battle for genetic survival between TEs and the host genome.
The research shows that TJ functions as the main factor responsible for activating the Flamenco locus, which brings new insights into Drosophila-mediated control of TE activity in ovarian somatic cells. The study advances our comprehension of piRNA biology while demonstrating the intricate relationship between hosts’ defensive systems and the parasitic elements in their genomes.
References: Rivera AJ, Lee JH, Gupta S, et al. Traffic Jam activates the Flamenco piRNA cluster locus and the Piwi pathway to ensure transposon silencing and Drosophila fertility. Cell Rep. Published online April 9, 2025. doi:10.1016/j.celrep.2025.113554
