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Open Access Publications from the University of California

Scholarly Works (6 results)

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

The survival and fitness of an organism rely on faithful transmission of the genome. In sexually reproducing organisms, genome transmission requires a specialized cell division named meiosis, in which the parental genome is shuffled and halved. The kinetochore, a conserved protein complex that mediates chromosome segregation, is a key player in meiosis. Here we investigated how the budding yeast kinetochore undergoes dynamic changes in this specialized cell division. The microtubule-binding part of the kinetochore (outer kinetochore) dissociates from the chromosomes in meiotic prophase and reassembles before the meiotic divisions. This dynamic behavior is crucial to establish the meiosis-specific chromosome segregation. We have discovered a multi-level network that regulates the dynamic kinetochore behavior by controlling the abundance of one single kinetochore subunit, Ndc80. Ndc80 is downregulated in meiotic prophase and later re-synthesized during the meiotic divisions. This fluctuation in the Ndc80 level requires regulation on Ndc80 synthesis and turnover. Central to the Ndc80 synthesis regulation is the toggling of two NDC80 mRNAs: (1) a coding mRNA that is translated into Ndc80 protein, and (2) a 5’-extended mRNA termed LUTI. Rather than coding for Ndc80, LUTI expression impedes the expression of the coding mRNA via chromatin modifications, leading to Ndc80 synthesis repression. Two meiotic master transcription factors, Ime1 and Ndt80, regulate the expression of LUTI and the coding mRNA, respectively, and integrate Ndc80 regulation into the larger meiotic program. Besides synthesis, Ndc80 degradation is temporally controlled: Ndc80 is degraded in meiotic prophase and not in metaphase I. Ndc80 degradation requires active proteasomes, a degron sequence at Ndc80’s N-terminus, and Aurora B phosphorylation on Ndc80, which is known to correct erroneous microtubule-kinetochore attachments and is rewired to degrade Ndc80 in meiotic prophase. Crucially, mis-regulation of Ndc80 abundance disrupts chromosome segregation, gamete formation, and progeny fitness, highlighting the importance of Ndc80 modulation. Altogether, this work reveals a beautiful case in evolution in which the gene expression, turnover, and activity of the kinetochore are tuned to accommodate the specialized chromosome segregation program meiosis.

    Cover page: Meiotic regulation of kinetochore composition and function
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2018)
    Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene NDC80 during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in other yeast strains and growth conditions.
      Cover page: Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2017)
      Cell differentiation programs require dynamic regulation of gene expression. During meiotic prophase in Saccharomyces cerevisiae, expression of the kinetochore complex subunit Ndc80 is downregulated by a 5' extended long undecoded NDC80 transcript isoform. Here we demonstrate a transcriptional interference mechanism that is responsible for inhibiting expression of the coding NDC80 mRNA isoform. Transcription from a distal NDC80 promoter directs Set1-dependent histone H3K4 dimethylation and Set2-dependent H3K36 trimethylation to establish a repressive chromatin state in the downstream canonical NDC80 promoter. As a consequence, NDC80 expression is repressed during meiotic prophase. The transcriptional mechanism described here is rapidly reversible, adaptable to fine-tune gene expression, and relies on Set2 and the Set3 histone deacetylase complex. Thus, expression of a 5' extended mRNA isoform causes transcriptional interference at the downstream promoter. We demonstrate that this is an effective mechanism to promote dynamic changes in gene expression during cell differentiation.
        Cover page: Transcription of a 5’ extended mRNA isoform directs dynamic chromatin changes and interference of a downstream promoter
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2020)
        The kinetochore complex is a conserved machinery that connects chromosomes to spindle microtubules. During meiosis, the kinetochore is restructured to accommodate a specialized chromosome segregation pattern. In budding yeast, meiotic kinetochore remodeling is mediated by the temporal changes in the abundance of a single subunit called Ndc80. We previously described the regulatory events that control the timely synthesis of Ndc80. Here, we report that Ndc80 turnover is also tightly regulated in meiosis: Ndc80 degradation is active in meiotic prophase, but not in metaphase I. Ndc80 degradation depends on the ubiquitin ligase APCAma1 and is mediated by the proteasome. Importantly, Aurora B-dependent Ndc80 phosphorylation, a mark that has been previously implicated in correcting erroneous microtubule-kinetochore attachments, is essential for Ndc80 degradation in a microtubule-independent manner. The N terminus of Ndc80, including a 27-residue sequence and Aurora B phosphorylation sites, is both necessary and sufficient for kinetochore protein degradation. Finally, defects in Ndc80 turnover predispose meiotic cells to chromosome mis-segregation. Our study elucidates the mechanism by which meiotic cells modulate their kinetochore composition through regulated Ndc80 degradation, and demonstrates that Aurora B-dependent regulation of kinetochores extends beyond altering microtubule attachments.
          Cover page: Aurora B-dependent Ndc80 degradation regulates kinetochore composition in meiosis.
          • 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 (2022)
            The African turquoise killifish is an exciting new vertebrate model for aging studies. A significant challenge for any model organism is the control over its diet in space and time. To address this challenge, we created an automated and networked fish feeding system. Our automated feeder is designed to be open-source, easily transferable, and built from widely available components. Compared to manual feeding, our automated system is highly precise and flexible. As a proof of concept for the feeding flexibility of these automated feeders, we define a favorable regimen for growth and fertility for the African killifish and a dietary restriction regimen where both feeding time and quantity are reduced. We show that this dietary restriction regimen extends lifespan in males (but not in females) and impacts the transcriptomes of killifish livers in a sex-specific manner. Moreover, combining our automated feeding system with a video camera, we establish a quantitative associative learning assay to provide an integrative measure of cognitive performance for the killifish. The ability to precisely control food delivery in the killifish opens new areas to assess lifespan and cognitive behavior dynamics and to screen for dietary interventions and drugs in a scalable manner previously impossible with traditional vertebrate model organisms.
              Cover page: An automated feeding system for the African killifish reveals the impact of diet on lifespan and allows scalable assessment of associative learning.
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