Affiliation: School of Biosciences, Cardiff University, Cardiff, UK
Keywords: a. spermatogenesis; h. translational regulation
Spermatogenesis provides an excellent model system to study many biological processes, including co- and post-transcriptional gene regulation. The adult Drosophila male germline is highly transcriptionally active during the primary spermatocyte stage. The transcriptional complexity in Drosophila spermatogenesis is well known, however, the underpinning molecular mechanism regulating the expression of more than 1000 genes, specifically in testes in Drosophila remains largely undetermined. Using predominantly D. melanogaster as a model, we are employing various state-of-art techniques in the field to understand the cellular and molecular basis of testes specific gene regulation. Testis Meiotic Arrest Complex (tMAC) is required for the activation of transcription of a large number of genes in spermatocytes. This complex comprises several proteins with DNA binding activity and several additional non-DNA binding proteins. We are dissecting how this complex interacts with target promoters by identifying in vitro binding motif for each of the DNA-binding domain-containing proteins within tMAC using high throughput SELEX sequencing. For in vivo identification of binding sites of each tMAC subunit, we are generating GFP-tagged lines and using high-resolution Chip-Exo, RNA-Seq, and Chip-qPCR in Drosophila testes. Testis-specific transcription typically depends on short DNA regions immediately flanking the transcription start sites (TSS). We have generated a set of promoter-reporter constructs and are investigating how these short promoter regions interact with tMAC, and other transcriptional regulatory complexes, to enable gene expression. For some genes, a small number of mutations are sufficient to convert a non-functional promoter into a functional promoter. By integrating the in vivo data with the in vitro binding site motif information, we will determine which DNA-binding proteins contribute to the binding of tMAC to any specific promoter sequence. This study will further deepen our understanding of how testes specific gene regulation achieves at co- and post-transcriptional levels in Drosophila.