A mechanistic analysis of gene regulation and its evolution in a Drosophila model

The body plans and adorning characteristics of animals have evolved impressively diverse morphologies. These body plans and characteristics are the products of networks of genes whose expression are controlled by cis-regulatory elements (CREs) that can be positioned at great distances from the gene(s) whose expression is being regulated. The pattern of expression a CRE imparts on a gene stems from its collection of binding sites for transcription factor proteins, what is referred to as a regulatory logic. The overarching goal for my thesis was to understand how animal traits develop and evolve at the levels of gene regulatory networks, CREs, and CRE regulatory logics. Chapter 2 presents research into the male-pattern of abdominal pigmentation that seemingly originated, diversified, and was lost in fruit fly species of the Sophophora sub-genus. This research focused on CREs for the terminal differentiation genes yellow and tan that independently evolved a male-specific pattern of gene regulation. Through the use of reporter transgenes we found evidence that these CREs activities evolved during the trait\u27s origin, but diversity and trait loss were more impacted by changes elsewhere in the genome (in trans) than to these CREs. Moreover, we revealed that similar activities of the two CREs regulating co-expressed genes stems from unique regulatory logics, of which key activators and repressors remain unknown. CREs, such as the tan gene CRE, are often located at a distance from the promoter of the gene they regulate. Thus, CREs and promoters must find each other in the nucleus and interact to facilitate transcription. Key studies suggest that CREs and promoters encode information to make these interactions take place by the binding of regulatory proteins. However, conventional reporter transgenes study CREs when they are directly adjacent to a promoter. In Chapter 3, research is presented for a novel reporter transgene system we developed that can test the activity of a CRE when it is both proximal and distal to reporter genes. We optimized CRE spacing and fluorescent reporter parameters for this system, tested it with various CREs and promoters, and describe an experimental scheme that can be used to dissect the sequences involved in long distance gene expression regulation. This new reporter system offers the promise to find sequences involved in CRE-promoter interactions, but the critical proteins binding these sequences will then need to be found by another method. In a similar vein, the outcomes for Chapter 2 ran into an all too frequent difficulty with the molecular dissection of CREs; finding functional CRE sequences but being unable to identify the interacting transcription factors. The work presented in Chapter 4 was my attempt to utilize a high-throughput yeast one-hybrid assay to test a library of fruit fly transcription factors for interactions with various functional CRE sequences. This approach identified many interactions available for further investigations. In the future this yeast approach seems well suited to be paired with more traditional methods of CRE study to expedite an understanding of gene regulation and its evolution