UC Berkeley

In the 40 years since Nüsslein-Volhard and Wieschaus published their Heidelberg mutants, detailed genetic, biochemical, and in situ experiments have filled in the identities of the sequence-specific transcription factors and gene gene regulatory network interactions that govern tissue patterning during the embryonic development of Drosophila melanogaster. In parallel, in vitro, in situ and genomic approaches have revealed the identities of much of the general transcriptional machinery and their roles in driving the transcription cycle in eukaryotes. Yet, the precise, in vivo timing of the recruitment of each of these general factors to the promoter by the sequence-specific transcription factors uncovered by the Heidelberg screen—let alone how this recruitment determines output transcriptional dynamics, such as bursting—remains elusive. In the work described in this thesis, I sought to bridge the gap between our detailed, in vitro understanding of the eukaryotic transcription cycle and our extensive knowledge of the key transcription factors that regulate Drosophila embryonic development by developing experimental and computational tools to enable us to correlate the microscopic and meso-scopic behavior of transcription factors with the transcriptional bursting that they regulate.

The work outlined in the first part of this dissertation describes our initial set of experiments designed to uncover correlations between the sub-nuclear dynamics of a transcriptional activator and transcriptional activity. I generated and characterized an extended palette of novel fluorophore CRISPR fusions to visualize and quantify the single-molecule and sub-nuclear dynamics of a key Drosophila developmental activator, Dorsal (Chapter 2). Using these new fusions, we simultaneously quantified Dorsal levels at its target loci and the resulting output transcriptional activity of these loci in single living cells, leading to the discovery that Dorsal is significantly enriched at actively transcribing target loci and forms stable clusters that traverse the nucleoplasm and preferentially interact with active sites of Dorsal-modulated transcription (Chapter 3). To expand our investigation of locus-specific transcription factor interactions, I adapted the ParB/intB genomic locus labeling system for simultaneous use with the MS2 nascent RNA labeling system, which will enable measurements of local increases of transcription factors both prior to transcriptional activation and during transcriptional repression (Chapter 4). Additionally, we developed a strategy to simultaneously and independently measure the transcriptional activity on sister chromatids, opening the door to determining whether the degree of coupling in gene regulation between tightly-associated promoters is distance-dependent due to a shared pool of transcriptional resources (Chapter 5).

In the second part of this thesis, I describe the development of two new fluorescence imaging tools that will more broadly improve the way we conduct quantitative experiments in the early Drosophila embryo. In the first project, we developed a series of modular cis-regulatory cassettes, consisting of multimerized, minimal enhancer regions from the vasa and nanos gene regulatory regions, that drive maternal protein expression levels spanning three orders of magnitude (Chapter 6). These have proven immediately useful for multiple projects described in this thesis, increasing the precision and efficiency of our development of novel in vivo fluorescence imaging tools for quantitative imaging in the early Drosophila embryo. In the second project, we adapted a suite of self-assembling protein nanocages for use as ratiometric fluorescence “standard candles” in the nuclei of the early Drosophila embryo, which will enable us to determine the absolute protein numbers present at transcriptional loci (Chapter 7).

This dissertation represents significant progress towards our goal of correlating the in vivo single molecule and sub-nuclear dynamics of transcription factors with the transcriptional bursts they regulate. The experimental and computational tools described here make it possible to conduct the types of live, quantitative microscopy studies that we believe are key to building a detailed, mechanistic understanding of the control of transcriptional bursting in the developing Drosophila embryo and other multicellular organisms.