Genome-wide distribution analysis of RNA Helicase Maleless (MLE) in Drosophila Melanogaster early male embryo, before (0-2 Hr) and after (2-4 Hr) maternal to zygotic transition (MZT) and in absence of CLAMP

Purpose: Study how maternal Pioneer Transcription factor CLAMP affects sex-specifc splicing during initial embryonic development by regulating occupancy of spliceosome component Maleless (MLE), a RNA helicase on chromatin Method: Cut and Run (CUT&RUN) DNA libraries (Uyehara and McKay 2019) using anti-MLE antibody were generated from male embryos at 0-2 Hr pre-MZT and 2-4 hr post-MZT stages in presence and absence of maternal CLAMP. Maternal CLAMP was depleted by driving UAS-CLAMPRNAi in the ovaries using MTD-GAL4 maternal triple driver. As a control UAS-GFPRNAI was driven using the MTD-GAL4. Embryos were sexed using the meiotic drive system that produces sperm with only Y chromosomes (Reider et al 2017), resulting in progeny of only male genotypes. Results: We identified regions on chromatin where MLE binds during 0-2 Hr pre-MZTand 2-4 Hr post-MZT embryonic stages in males. In 0-2 Hr and 2-4 Hr old control embryos 28,279 and 24,910 MLE peaks were detected. After depletion of maternal CLAMP, 41.97% of 0-2 Hr peaks (22183 remaining, 6096 lost) and 59.1% of 2-4 Hr peaks (15735 remaining, 9175 lost) are lost. We also found loss of maternal CLAMP resulted in 26% and 35.25% new MLE peaks at 0-2 hr pre-MZT and 2-4 Hr post-MZT embryonic stages. Our motif search shows presence of putative motifs for MLE binding on chromatin which needs further validation. Conclusion: This is the first study showing genome-wide MLE occupancy in sexed embryos during 0-2 hr pre-MZT and 2-4 hr post-MZT stages. We show that the maternally deposited transcription factor CLAMP affects MLE occupancy on chromatin in early embryos. Thus, we conclude that maternal transcription factors can affect spliceosome function by regulating occupancy of their components on chromatin. Male embryos with maternal CLAMP (control, MTD-GAL4>GFPRNAi mothers) and depleted maternal CLAMP (experimental, MTD-GAL4>CLAMPRNAi mothers) at two time points-1)0-2 Hr pre-MZT and 2) 2-4 Hr post-MZT collected. Using anti-MLE antibody the DNA fragments bound to MLE protein captured (Cut and Run protocol) and DNA libraries made. Pair-end sequencing of DNA libraries in a Hi-seq illumina platform with average 7-10 million reads per sample. Multiple batches of crosses were set and embryos collected from multiple timed egg-layings used to make DNA libraries, 2 replicates for each genotype and time point.