Profiling Doublesex-DNA interactions with targeted DamID in Drosophila melanogaster

Thesis

The transcriptional regulators doublesex (dsx) and fruitless (fru) function together to specify sexual physiology and behaviour in Drosophila melanogaster. Whilst fru has only been located in insect lineages, dsx is structurally and functionally conserved throughout the animal kingdom. The role of FRU proteins as transcriptional regulators has been studied extensively with DamID – a method that uses DNA adenine methylation to create ‘fingerprints’ in the genome that correspond to where Dam-tagged proteins have interacted with DNA. Comparatively little research has focused on DSX, and there are few direct dsx target genes in the CNS. In this study, we implement the novel targeted DamID (TaDa) approach for profiling DsxM and DsxF expressing neurons in the Drosophila adult CNS, marking the first time these neurons will be profiled in a cell-specific manner. TaDa harnesses the phenomenon of low frequency ribosome re-initiation, and the introduction of a primary open reading frame (ORF) encoded before the Dam-fusion, to enable low level expression of the Dam-fusion in a cell-type specific manner when driven using Gal4/UAS. We generate a rich list of putative dsx targets in the CNS. Comparisons with a published DSX-fat body DamID dataset, an organ in which dsx is known to be widely expressed, reveal a tendency towards tissue-specificity. Our Gene Ontology analyses reveal DSX targets are involved in nervous system development, confirming the known role attributed to dsx, and motif analyses identify novel putative GATA cofactors. Alongside independent protein-DNA interaction screens (ChIP-seq and yeast-one hybrid), we identify the neuropeptides Diuretic Hormone 31 (Dh31), Tachykinin 1 (Tk1) and Neuropeptide-like precursor 1 (Nplp1) for further analysis. dsx/ Nplp1 expression analyses reveal a single cluster of neurons in the female brain, likely pMN1, which we speculate is involved in post-mating behaviour. The wealth of putative dsx target genes generated here pave the way for further characterisation of the dsx machinery and thus a better understanding of its role in specifying sexual physiology and behaviour.

Authors: D'Souza, LA

• DOI: 10.5287/ora-jnoqxkr5r
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: doublesex; CNS target genes; DamID; Drosophila melanogaster; protein-DNA interactions; targeted DamID
• Subjects: Molecular Biology; Neuroscience; Physiology, Anatomy and Genetics