Principles of Enhancer Biology in the Developing Drosophila Embryo
The regulation of transcription underlies most major cellular processes. In order for a cell to have the appropriate set of proteins to perform biological functions, it must transcribe the genes that code for those proteins in a temporally coordinated manner. In eukaryotic organisms, this coordinati...
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| Format: | Dissertation |
| Language: | English |
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ProQuest Dissertations & Theses
01-01-2022
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| Online Access: | Get full text |
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| Summary: | The regulation of transcription underlies most major cellular processes. In order for a cell to have the appropriate set of proteins to perform biological functions, it must transcribe the genes that code for those proteins in a temporally coordinated manner. In eukaryotic organisms, this coordination is achieved through the activity of non-coding sequences of DNA called enhancers, which are capable of selectively activating nearby genes in response to signals transduced by transcription factors. While the broad outline of this process has been known for decades, the physical mechanisms that govern how this occurs is relatively poorly understood. In chapter 1 of this thesis, I identify the unique challenges of this problem, and highlight how recent advancements in imaging techniques may allow us to overcome them.. In chapter 2, I utilized these techniques to understand how pioneer transcription factors facilitate the activation of enhancers over a gradient of a transcriptional activator. I used two reporter transgenes for the Drosophila gene short gastrulation (sog), which contained 24xMS2 stem loops allowing for real-time visualization of transcription in living embryos. The first was driven by the wildtype distal enhancer of sog, while the second contained a modified version of the enhancer lacking any binding sites for pioneer transcription factor Zelda. By comparing the kinetic parameters of transcription between the two over the Dorsal morphogen gradient, I was able to see how the pioneer factor influenced the effectiveness of Dorsal mediated transcriptional activation at various concentrations of Dorsal. I found that Zelda increased Dorsal’s ability to activate the reporter particularly at low concentrations of Dorsal. The number of nuclei that activated transcription, speed of activation, and intensity of activation were all affected in a concentration dependent manner. By imaging Dorsal protein in fixed embryos, I found that this was likely driven by Zelda’s ability to increase the local concentration of Dorsal at the site of transcription, effectively flattening the Dorsal gradient. In chapter 3, I investigated how the endogenous sog utilized two enhancers in a coordinated manner. I created enhancer deletion lines for both the distal and proximal enhancer of sog, and inserted the same MS2 sequence into the first intron of sog. I found that the enhancers were tuned to different portions of the Dorsal gradient, and they summed these transcription domains in the wildtype version of sog. However, the two enhancers appeared to integrate the signals of activation and repression in different manners, and the timing and robustness of activation was altered in unique ways in each enhancer mutant. These differences caused the downstream signaling cascade sog is responsible for refining to be altered in idiosyncratic ways. Finally, in chapter 4 I review the implications of this research for future study, and put forth a model for how enhancers physically interact during the process of transcriptional activation. |
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| ISBN: | 9798837544453 |