Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2010)
The RNA polymerase II core promoter is a diverse and complex region of DNA that contains the transcription start site. Core promoter elements are conserved DNA sequences within the core promoter that direct transcription to the start site and function in the regulation of gene expression. Here I describe the TCT motif, a newly discovered core promoter element that encompasses the transcription start site and is conserved among the ribosomal protein genes. Mutational analysis of three ribosomal protein gene promoters, RpLP1, RpS12, and RpS15, demonstrates the importance of this DNA element. A severe down-regulation of transcription is seen when the wild-type sequence is mutated, with positions 3-5 (+3 to + 5 relative to the +1 transcription start site) being the most important. I also show that the TCT does not work synergistically with other previously characterized core promoter elements, and the RpLP1 promoter is not bound by Transcription Factor IID (TFIID). The TCT is a necessary core promoter element for the transcription of the ribosomal protein genes. Its inability to work synergistically with other known promoter elements, along with its inability to bind TFIID, may suggest a parallel mechanism of RNA polymerase II transcription for the ribosomal protein genes that is independent of the mechanism used for all previously studied genes
    Cover page: The TCT motif, a core promoter element for the translational machinery
    • Thesis
    • Peer Reviewed
    UC San Francisco Electronic Theses and Dissertations (2015)

    Ubiquitin and ubiquitin-like proteins impart regulatory information during innate immune responses to infection, but the scope of their role in coordinating the responses of immune cells to infection is unknown. Recent advances in immmunoaffinity purification of ubiquitylated peptides, paired with high accuracy mass-spectrometry, have provided a new platform to measure global cellular ubiquitylation with extraordinary depth. Here we report the application of this methodology to quantitatively measure changes in ubiquitylation of the macrophage proteome during infection with three intracellular bacterial pathogens: Mycobacterium tuberculosis, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes. This has revealed thousands of ubiquitylation events that occur early after infection, most of which were previously unknown. Unlike transcriptional responses to bacterial infection, which are largely monotonic, our results reveal a surprising diversity in ubiquitin-mediated responses to each pathogen. Likewise, using well-defined attenuated bacterial mutants revealed a remarkable ability of macrophages to discriminate virulent from non-virulent bacteria, suggesting macrophages can integrate information about the nature of the engulfed bacteria and elicit unique responses. Many of these changes likely play functional roles in host resistance, as we have identified a novel ubiquitin-modified pathway, the antiviral OASL1-IRF7 pathway, that is critical for Mycobacterium tuberculosis growth in vitro and in vivo.

      Cover page: Identifying functional ubiquitylation during intracellular bacterial pathogen infection
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2020)
      Macrophages are highly plastic cells with critical roles in immunity, cancer, and tissue homeostasis, but how these distinct cellular fates are triggered by environmental cues is poorly understood. To uncover how primary murine macrophages respond to bacterial pathogens, we globally assessed changes in post-translational modifications of proteins during infection with Mycobacterium tuberculosis, a notorious intracellular pathogen. We identified hundreds of dynamically regulated phosphorylation and ubiquitylation sites, indicating that dramatic remodeling of multiple host pathways, both expected and unexpected, occurred during infection. Most of these cellular changes were not captured by mRNA profiling, and included activation of ubiquitin-mediated autophagy, an evolutionarily ancient cellular antimicrobial system. This analysis also revealed that a particular autophagy receptor, TAX1BP1, mediates clearance of ubiquitylated Mtb and targets bacteria to LC3-positive phagophores. These studies provide a new resource for understanding how macrophages shape their proteome to meet the challenge of infection.
        Cover page: Dynamic post-translational modification profiling of M. tuberculosis-infected primary macrophages
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2018)
        Although macrophages are armed with potent antibacterial functions, Mycobacterium tuberculosis (Mtb) replicates inside these innate immune cells. Determinants of macrophage intrinsic bacterial control, and the Mtb strategies to overcome them, are poorly understood. To further study these processes, we used an affinity tag purification mass spectrometry (AP-MS) approach to identify 187 Mtb-human protein-protein interactions (PPIs) involving 34 secreted Mtb proteins. This interaction map revealed two factors involved in Mtb pathogenesis-the secreted Mtb protein, LpqN, and its binding partner, the human ubiquitin ligase CBL. We discovered that an lpqN Mtb mutant is attenuated in macrophages, but growth is restored when CBL is removed. Conversely, Cbl-/- macrophages are resistant to viral infection, indicating that CBL regulates cell-intrinsic polarization between antibacterial and antiviral immunity. Collectively, these findings illustrate the utility of this Mtb-human PPI map for developing a deeper understanding of the intricate interactions between Mtb and its host.
          Cover page: An Mtb-Human Protein-Protein Interaction Map Identifies a Switch between Host Antiviral and Antibacterial Responses
          Top