Investigation of the interaction of A-MYB and TCFL5 on pachytene PIWI-interacting RNA genes in two sub-species of mice, mus musculus musculus and mus musculus castaneus

Abstract: Spermatogenesis is a complex process that involves the production of sperm cells from germ cells in order to transmit genetic information to progeny. The PIWI-interacting RNA (piRNA) is a non-coding small silencing RNA that guides the PIWI protein to silence target transposon transcripts to regulate the production of germ cells. In mammals, distinct sets of piRNAs function in gonocytes, spermatogonia, and developing spermatocytes. Coinciding with puberty, pachytene piRNAs, expressed at pachytene stage of meiosis I, silence mRNAs to regulate spermatogenesis. At the onset of meiosis I, the transcription factors A-MYB and TCFL5 initiate the transcription of pachytene piRNA genes via binding to their transcription start sites. This study aims to detect which pachytene piRNA genes are bound by these two transcription factors in two sub-species of mice, Mus Musculus musculus and Mus Musculus castaneus, and if there is a variation in binding regions between these sub-species. Here, chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed to map the binding regions of A-MYB and TCFL5 in the genomes of mouse sub-species. Three testis tissues from each sub-species were sonicated for ChIP-seq on A-MYB and three from each mouse for TCFL5. Sonication was optimized at high power for 40 cycles of 60 sec on, 30 sec off, for 1 h. After 20 cycles, lysates were transferred to a fresh tube, vortex, and sonicated for 20 more cycles. Library DNAs were average 333-360 bp, and qPCR showed enough DNA for Illumina sequencing. Analysis of sequencing data is ongoing. The transcription start sites of pachytene piRNAs could be identified after analysis.