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Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2019)

It has long been appreciated that organization of macromolecules within the cell is paramount to proper cellular function. As the number of tools in the molecular and cell biologist’s toolbox grows, the list of ways cells achieve organization also grows. This is especially true for the process of transcription and its regulation, where the fundamental questions remain a challenge to address, but where a convergence of genetic, genomic, structural and imaging techniques offer the possibility of answers. The proper loading of an RNA polymerase on a gene requires the coordinated action of hundreds of proteins and other macro molecules, as well as cellular genome to be maintained in a topologically permissive state; yet as a system, gene regulation requires interactions to be dynamic enough to respond to the changing needs of the cell in response to internal and external ques. How a cell constructs a system that is, on the one hand robust, and on the other hand flexible remains a challenge to answer.

In Chapter 1 of this thesis, we will introduce the study of transcription regulation as a general topic, and discuss a number of fluorescence imaging techniques that have fundamentally changed the our understanding of transcription regulation. Chapter 2 will focus on one particular aspect of cellular organization which has recently come into vogue—that of membraneless compartment formation through liquid-liquid phase separation. A thorough investigation of the literature, particularly in light of the experiments which we foreshadow and introduce in more depth in Chapter 3, suggests that much more work is needed before phase separation as a means of biological organization can become a general paradigm. Chapter 3 presents a particularly poignant example of the issues raised in Chapter 2. Using Herpes Simplex Virus as a model system because of its ability to form compartments in the nucleus, we demonstrate that recruitment of Pol II and other proteins can be explained through the availability of nonspecific binding sites rather than phase separation. Lastly, Chapter 4 will present technical findings on the optimal fluorescent dyes to use for in vivo labeling of proteins for imaging applications.

In summary, this dissertation underscores the way in which new imaging techniques reveal new biological principles and challenge old models. It will be of great interest to watch these ideas and techniques mature beyond the work presented in this dissertation.

    Cover page: Advanced imaging methods unveil new mechanisms transcriptional regulation in the context of nuclear organization
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2019)
    The idea that liquid-liquid phase separation (LLPS) may be a general mechanism by which molecules in the complex cellular milieu may self-organize has generated much excitement and fervor in the cell biology community. While this concept is not new, its rise to preeminence has resulted in renewed interest in the mechanisms that shape and drive diverse cellular self-assembly processes from gene expression to cell division to stress responses. In vitro biochemical data have been instrumental in deriving some of the fundamental principles and molecular grammar by which biological molecules may phase separate, and the molecular basis of these interactions. Definitive evidence is lacking as to whether the same principles apply in the physiological environment inside living cells. In this Perspective, we analyze the evidence supporting phase separation in vivo across multiple cellular processes. We find that the evidence for in vivo LLPS is often phenomenological and inadequate to discriminate between phase separation and other possible mechanisms. Moreover, the causal relationship and functional consequences of LLPS in vivo are even more elusive. We underscore the importance of performing quantitative measurements on proteins in their endogenous state and physiological abundance, as well as make recommendations for experiments that may yield more conclusive results.
      Cover page: Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2017)
      Differentiation programs such as meiosis depend on extensive gene regulation to mediate cellular morphogenesis. Meiosis requires transient removal of the outer kinetochore, the complex that connects microtubules to chromosomes. How the meiotic gene expression program temporally restricts kinetochore function is unknown. We discovered that in budding yeast, kinetochore inactivation occurs by reducing the abundance of a limiting subunit, Ndc80. Furthermore, we uncovered an integrated mechanism that acts at the transcriptional and translational level to repress NDC80 expression. Central to this mechanism is the developmentally controlled transcription of an alternate NDC80 mRNA isoform, which itself cannot produce protein due to regulatory upstream ORFs in its extended 5' leader. Instead, transcription of this isoform represses the canonical NDC80 mRNA expression in cis, thereby inhibiting Ndc80 protein synthesis. This model of gene regulation raises the intriguing notion that transcription of an mRNA, despite carrying a canonical coding sequence, can directly cause gene repression.
        Cover page: Kinetochore inactivation by expression of a repressive mRNA
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2017)
        TERT promoter mutations (TPMs) are the most common noncoding mutations in cancer. The timing and consequences of TPMs have not been fully established. Here, we show that TPMs acquired at the transition from benign nevus to malignant melanoma do not support telomere maintenance. In vitro experiments revealed that TPMs do not prevent telomere attrition, resulting in cells with critically short and unprotected telomeres. Immortalization by TPMs requires a gradual up-regulation of telomerase, coinciding with telomere fusions. These data suggest that TPMs contribute to tumorigenesis by promoting immortalization and genomic instability in two phases. In an initial phase, TPMs do not prevent bulk telomere shortening but extend cellular life span by healing the shortest telomeres. In the second phase, the critically short telomeres lead to genome instability and telomerase is further up-regulated to sustain cell proliferation.
          Cover page: Mutations in the promoter of the telomerase gene TERT contribute to tumorigenesis by a two-step mechanism
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2020)
          Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Unbiased detection through proximity labeling of transient protein interactions in cell-free Xenopus laevis egg extract identified the dimeric histone chaperone facilitates chromatin transcription (FACT) as an interactor of substrate-bound Cas9. FACT is both necessary and sufficient to displace dCas9, and FACT immunodepletion converts Cas9's activity from multi-turnover to single turnover. In human cells, FACT depletion extends dCas9 residence times, delays genome editing, and alters the balance between indel formation and homology-directed repair. FACT knockdown also increases epigenetic marking by dCas9-based transcriptional effectors with a concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.
            Cover page: The Histone Chaperone FACT Induces Cas9 Multi-turnover Behavior and Modifies Genome Manipulation in Human Cells
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