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Scholarly Works (7 results)

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UC Irvine Electronic Theses and Dissertations (2021)

ABSTRACT OF THE DISSERTATIONMechanisms of robustness and competition in transcriptional control By Rachel Victoria Waymack Doctor of Philosophy in Developmental and Cell Biology University of California, Irvine, 2021 Assistant Professor Zeba Wunderlich, Chair

During normal development, organisms must establish and maintain precise patterns of gene expression that are also robust to internal and external perturbations. The key cell fate decisions made during early development that establish the organism’s body plan rely on these precise gene expression patterns and even relatively minor deviations can cause significant defects. This precision and robustness must be achieved in the context of the inherently noisy process of transcription, which is controlled by the stochastic molecular interactions between DNA, transcription factors (TFs), and the other pieces of the transcriptional machinery. While the question of how regulatory regions of DNA, such as enhancers, accurately interact with TFs to control gene expression has been the topic of many studies, there is still much we do not know about how enhancers and TFs combine to regulate gene expression. Here, I investigate novel mechanisms of gene expression regulation by enhancers and TFs. In Chapter 2, I show that shadow enhancers, groups of seemingly redundant enhancers, are able to buffer fluctuations in upstream regulators by separating TF inputs between individual enhancers in a case study of the shadow enhancers regulating Kruppel expression during early embryonic Drosophila development. In Chapter 3, I investigate competition observed between transcriptional reporters and find that transgenic reporters compete with one another and an endogenous gene for TF activation, likely due to the non-homogenous nature of the nucleus. Then, complementing my findings in Chapter 2, I analyze the TF regulation of a large set of Drosophila shadow enhancers and show that these groups of enhancers display a wide range of regulatory logic configurations in Chapter 4. In Chapter 5, I discuss a proposed project to investigate the relationship between TF dynamics and enhancer-controlled transcription during NF-kB signaling in the immune response. Taken together, these projects inform our understanding of how gene expression is regulated by enhancers and TFs in early development and novel regulatory mechanisms that likely apply more broadly throughout the genome and life stages.

    Cover page: Mechanisms of robustness and competition in transcriptional controlCreative Commons 'BY-NC' version 4.0 license
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2021)
    Transgenic reporters allow the measurement of regulatory DNA activity in vivo and consequently have long been useful tools for studying enhancers. Despite their utility, few studies have investigated the effects these reporters may have on the expression of other genes. Understanding these effects is required to accurately interpret reporter data and characterize gene regulatory mechanisms. By measuring the expression of Kruppel (Kr) enhancer reporters in live Drosophila embryos, we find reporters inhibit one another's expression and that of a nearby endogenous gene. Using synthetic transcription factor (TF) binding site arrays, we present evidence that competition for TFs is partially responsible for the observed transcriptional inhibition. We develop a simple thermodynamic model that predicts competition of the measured magnitude specifically when TF binding is restricted to distinct nuclear subregions. Our findings underline an unexpected role of the non-homogenous nature of the nucleus in regulating gene expression.
      Cover page: Molecular competition can shape enhancer activity in the Drosophila embryo
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2021)
      Shadow enhancers are seemingly redundant transcriptional cis-regulatory elements that regulate the same gene and drive overlapping expression patterns. Recent studies have shown that shadow enhancers are remarkably abundant and control most developmental gene expression in both invertebrates and vertebrates, including mammals. Shadow enhancers might provide an important mechanism for buffering gene expression against mutations in non-coding regulatory regions of genes implicated in human disease. Technological advances in genome editing and live imaging have shed light on how shadow enhancers establish precise gene expression patterns and confer phenotypic robustness. Shadow enhancers can interact in complex ways and may also help to drive the formation of transcriptional hubs within the nucleus. Despite their apparent redundancy, the prevalence and evolutionary conservation of shadow enhancers underscore their key role in emerging metazoan gene regulatory networks.
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2021)
        Proper development depends on precise spatiotemporal gene expression patterns. Most genes are regulated by multiple enhancers and often by multiple core promoters that generate similar transcripts. We hypothesize that these multiple promoters may be required either because enhancers prefer a specific promoter or because multiple promoters serve as a redundancy mechanism. To test these hypotheses, we studied the expression of the knirps locus in the early Drosophila melanogaster embryo, which is mediated by multiple enhancers and core promoters. We found that one of these promoters resembles a typical “sharp” developmental promoter, while the other resembles a “broad” promoter usually associated with housekeeping genes. Using synthetic reporter constructs, we found that some, but not all, enhancers in the locus show a preference for one promoter. By analyzing the dynamics of these reporters, we identified specific burst properties during the transcription process, namely burst size and frequency, that are most strongly tuned by the specific combination of promoter and enhancer. Using locus-sized reporters, we discovered that even enhancers that show no promoter preference in a synthetic setting have a preference in the locus context. Our results suggest that the presence of multiple promoters in a locus is both due to enhancer preference and a need for redundancy and that “broad” promoters with dispersed transcription start sites are common among developmental genes. Our results also imply that it can be difficult to extrapolate expression measurements from synthetic reporters to the locus context, where many variables shape a gene’s overall expression pattern.
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2019)
          Shadow enhancers, groups of seemingly redundant enhancers, are found in a wide range of organisms and are critical for robust developmental patterning. However, their mechanism of action is unknown. We hypothesized that shadow enhancers drive consistent expression levels by buffering upstream noise through a separation of transcription factor (TF) inputs at the individual enhancers. By measuring transcriptional dynamics of several Kruppel shadow enhancer configurations in live Drosophila embryos, we showed individual member enhancers act largely independently. We found that TF fluctuations are an appreciable source of noise that the shadow enhancer pair can better buffer than duplicated enhancers. The shadow enhancer pair is uniquely able to maintain low levels of expression noise across a wide range of temperatures. A stochastic model demonstrated the separation of TF inputs is sufficient to explain these findings. Our results suggest the widespread use of shadow enhancers is partially due to their noise suppressing ability.
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2020)
            Shadow enhancers, groups of seemingly redundant enhancers, are found in a wide range of organisms and are critical for robust developmental patterning. However, their mechanism of action is unknown. We hypothesized that shadow enhancers drive consistent expression levels by buffering upstream noise through a separation of transcription factor (TF) inputs at the individual enhancers. By measuring the transcriptional dynamics of several Kruppel shadow enhancer configurations in live Drosophila embryos, we showed that individual member enhancers act largely independently. We found that TF fluctuations are an appreciable source of noise that the shadow enhancer pair can better buffer than duplicated enhancers. The shadow enhancer pair is also uniquely able to maintain low levels of expression noise across a wide range of temperatures. A stochastic model demonstrated the separation of TF inputs is sufficient to explain these findings. Our results suggest the widespread use of shadow enhancers is partially due to their noise suppressing ability.
              • Article
              • Peer Reviewed
              UC Irvine Previously Published Works (2021)
              Directed evolution has been used for decades to engineer biological systems at or below the organismal level. Above the organismal level, a small number of studies have attempted to artificially select microbial ecosystems, with uneven and generally modest success. Our theoretical understanding of artificial ecosystem selection is limited, particularly for large assemblages of asexual organisms, and we know little about designing efficient methods to direct their evolution. Here, we have developed a flexible modelling framework that allows us to systematically probe any arbitrary selection strategy on any arbitrary set of communities and selected functions. By artificially selecting hundreds of in silico microbial metacommunities under identical conditions, we first show that the main breeding methods used to date, which do not necessarily let communities reach their ecological equilibrium, are outperformed by a simple screen of sufficiently mature communities. We then identify a range of alternative directed evolution strategies that, particularly when applied in combination, are well suited for the top-down engineering of large, diverse and stable microbial consortia. Our results emphasize that directed evolution allows an ecological structure-function landscape to be navigated in search of dynamically stable and ecologically resilient communities with desired quantitative attributes.
                Cover page: Engineering complex communities by directed evolution.
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