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

Transcription factor CLAMP associates with RNA helicase, Maleless MLE as part of Male-specific MSL complex in males, which regulates dosage compensation in males. Both CLAMP and MLE are maternally deposited proteins (protein expression detectd in 0- Hr embryo, before zygotic genome activation) and MLE is also a conserved component of spliceosome. Moreover, CLAMP too associates with many other RNA binding protein compoments of spliceosomes. Both these proteins are well expressed in females, but apart from that might influence splicing in females, not much is known if they have any female-specific function. Furthermore, whether they regulate each other's function in females is not known as well, especially before zygotic genome activation starts, in a pre-MZT embryo (0-2 Hr Embryo). Therefore, we performed CutnRun assay to detrmine MLE binding sites on chromatin in female embryos before (0-2 Hr) and after MZT (2-4 Hr). And then identify, in absence of maternal CLAMP, whether there is change is distribution of control MLE female peaks. We identified regions on chromatin where MLE binds during 0-2 Hr pre-MZT and 2-4 Hr post-MZT embryonic stages in females. CLAMP unlike to males do not affect MLE distribution on chromatin in females. Overall design: Female embryos with maternal CLAMP (control, MTD-GAL4>GFPRNAi mothers) at two time points-1)0-2 Hr pre-MZT and 2) 2-4 Hr post-MZT and depleted maternal CLAMP (experimental, MTD-GAL4>CLAMPRNAi mothers) at one time points-1)0-2 Hr pre-MZT collected. Embryos were sexed using the meiotic drive system that produces sperm with only X chromosomes (Reider et al 2017), resulting in progeny of only female genotypes. Using anti-MLE antibody the DNA fragments bound to MLE protein captured using Cut and Run protocol (Uyehara and McKay 2019) and DNA libraries made. Pair-end sequencing of DNA libraries in a Hi-seq illumina platform. Multiple batches of crosses were set and embryos collected from multiple timed egg-layings used to make DNA libraries.