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Studies on the Bursting Kinetics of ​even-skipped​ Transcription


Enhancers drive the transcription of genes in defined spatio-temporal patterns of gene expression that orchestrate the development of animal body plans. The spatial aspects of enhancer function and gene expression pattern formation have been elucidated with elegant experiments in ​Drosophila melanogaster and other organisms. However, our knowledge regarding the temporal dynamics of pattern formation is still incomplete.

In this work, I coupled the MS2 reporter system, which enables real-time, single-locus analysis of transcription, with the regulatory sequences of the gene ​even-skipped (eve)​, a fine model of enhancer function active in seven antero-posterior stripes that ring the fruit fly embryo before gastrulation.

Our results show highly dynamic expression patterns that emerge from stochastic bursts of transcription in single loci. We found that the dynamic expression of ​eve stripes stems from shared transcriptional kinetics. Nuclei at the center of all stripes increase their frequency of transcriptional bursting. Nuclei in the inter-stripe boundaries remain quiescent and do not show any detectable bursting. All the while stripes shift anteriorly, propelled by expression flow. The bursting kinetics in the ectopic patterns of eve expression that result from lifting the repression of ​eve enhancers are similar to the kinetics in the endogenous stripes. These observations show that the various ​eve enhancers generate similar bursting kinetics regardless of the myriad of distinct inputs that shape their activity.

This dissertation consists of four chapters. Chapter 1 describes the construction of our MS2-based ​eve reporter and the generation of the transgenic fruit flies carrying it. Chapter 2 goes through our imaging methods and their optimization. It ends with an account of our data analysis to turn movies of transcription in embryonic development into meaningful and quantifiable data. Chapter 3 summarizes our findings regarding the conserved kinetics of ​even-skipped transcriptional bursting. Finally, Chapter 4 explores the activity of ​eve​ enhancers in body regions where they are not normally active.

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