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Open Access Publications from the University of California

Scholarly Works (10 results)

  • Article
  • Peer Reviewed
UC Santa Cruz Previously Published Works (2018)
Abstract Controlling protein activity and localization is a key tool in modern biology. Mammalian steroid receptor ligand-binding domains (LBDs) fusions have been used in a range of organisms and cell-types to inactivate proteins of interest until the cognate steroid ligand is applied. Here, we demonstrate that the glucocorticoid receptor LBD confers ligand-gated control of a heterologous gene expression system (Q system) and the DAF-16 transcription factor in C. elegans . These experiments demonstrate provide a powerful tool for temporal control of protein activity, and will bolster existing tools used to modulate gene expression and protein activity in this animal.
    • Article
    • Peer Reviewed
    UC Santa Cruz Previously Published Works (2019)
    Controlling protein activity and localization is a key tool in modern biology. Mammalian steroid receptor ligand-binding domain (LBD) fusions have been used in a range of organisms and cell types to inactivate proteins of interest until the cognate steroid ligand is applied. Here, we demonstrate that the glucocorticoid receptor LBD confers ligand-gated control of a heterologous gene expression system (Q system) and the DAF-16 transcription factor in Caenorhabditis elegans These experiments provide a powerful tool for temporal control of protein activity, and will bolster existing tools used to modulate gene expression and protein activity in this animal.
      Cover page: A New Tool for Inducible Gene Expression in Caenorhabditis elegans.
      • Thesis
      • Peer Reviewed
      UCLA Electronic Theses and Dissertations (2013)

      Chronobiologists focus on understanding at least two ways of regulating the time of life: biological clocks that anticipate environmental conditions, and temporal switches that regulate sequential events. Of the former, the best-characterized biological clocks regulate circadian rhythms in metazoans. These self sustaining oscillators involve transcriptional-translational feedback loops that affect the physiology of the organism. In contrast, progression through development requires the proper temporal coordination of factors that control sequential events. However, the timing mechanisms that coordinate both cyclical and successive events in development are not generally well understood.

      The molting cycle of C. elegans is an example of both a periodic and sequential developmental program. Molting of the exoskeleton involves the coordinated synthesis, secretion, and assembly of a new skeleton, as well as degradation of the old one. Quiescent behaviors also accompany the molting cycle. Worms molt four times, approximately once every eight hours when cultivated at room temperature. The molts are coordinated with successive transitions in the temporal fates of epidermal stem cells, which are programmed by genes in the heterochronic regulatory network. Every molt is a critical developmental transition and mis-coordination of the either the sequential or reiterative events can lead to larval arrest and death.

      This thesis describes how the C. elegans heterochronic gene lin-42a, homologous to the mammalian circadian clock protein PERIOD, is required for cyclical and sequential progression of development. The oscillatory expression of lin-42a in the epidermis peaks during the molts. Inactivation of lin-42a results in arrhythmic molts and continuously abnormal epidermal stem cell dynamics. In contrast, forced expression of lin-42a leads to anachronistic larval molts and lethargy in adults. These results suggest that rising and falling levels of LIN-42A allow the start and completion, respectively, of larval molts. Ancillary factors of the molting timer likely include the conserved nuclear hormone receptors NHR-23/RORα and NHR-25/SF1, and the family of let-7 microRNAs. Thus, interconnected positive and negative regulatory interactions among LIN-42A, NHR-23 and -25, and let-7 may drive transitions through each molt. I propose that these factors regulate molting cycles in much the same way that PERIOD-based oscillators drive rhythmic behaviors and metabolic processes in mature mammals.

      The LIN-42-based timer may synchronize the periodic molts with fluctuations in external or internal conditions, in much the same way that circadian clocks synchronize rhythmic biological processes of higher metazoans with recurrent fluctuations in zeitgebers. To identify novel factors that regulate these molting cues, we designed a forward genetic screen to identify novel factors that regulate the timing of the molt. I identified that the LRP-2/Megalin receptor is required for

      the cessation of larval molting cycles. The lrp-2 transcripts are detected predominately in the anterior head neurons, suggesting that that major site of action of LRP-2 is in the nervous system. LRP-2 may function to sequester neuroendocrine cues from other tissues to control the terminal fate of larval molts.

      By analyzing the genetic and molecular mechanisms of that regulate the molting cycle, we anticipate the discovery of the broader mechanisms that couple biological periodicity and rapid development in higher organisms.

        Cover page: Unifying Mechanisms of Developmental Timing: Genetic and Molecular Analysis of the Molting Cycle Timer of Caenorhabditis elegans
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2012)
        Animal development requires temporal coordination between recurrent processes and sequential events, but the underlying timing mechanisms are not yet understood. The molting cycle of C. elegans provides an ideal system to study this basic problem. We recently characterized LIN-42, which is related to the circadian clock protein PERIOD, as a key component of the developmental timer underlying rhythmic molting cycles. In this context, LIN-42 coordinates epithelial stem cell dynamics with progression of the molting cycle. Repeated actions of LIN-42 may enable the reprogramming of seam cell temporal fates, while stage-specific actions of LIN-42 and other heterochronic genes select fates appropriate for upcoming, rather than passing, life stages. Here, we discuss the possible configuration of the molting timer, which may include interconnected positive and negative regulatory loops among lin-42, conserved nuclear hormone receptors such as NHR-23 and -25, and the let-7 family of microRNAs. Physiological and environmental conditions may modulate the activities of particular components of this molting timer. Finding that LIN-42 regulates both a sleep-like behavioral state and epidermal stem cell dynamics further supports the model of functional conservation between LIN-42 and mammalian PERIOD proteins. The molting timer may therefore represent a primitive form of a central biological clock and provide a general paradigm for the integration of rhythmic and developmental processes.
          Cover page: Toward a unified model of developmental timing
          • Article
          • Peer Reviewed
          UC Santa Cruz Previously Published Works (2019)
          Controlling protein activity and localization is a key tool in modern biology. Mammalian steroid receptor ligand-binding domain (LBD) fusions have been used in a range of organisms and cell types to inactivate proteins of interest until the cognate steroid ligand is applied. Here, we demonstrate that the glucocorticoid receptor LBD confers ligand-gated control of a heterologous gene expression system (Q system) and the DAF-16 transcription factor in Caenorhabditis elegans These experiments provide a powerful tool for temporal control of protein activity, and will bolster existing tools used to modulate gene expression and protein activity in this animal.
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2012)
            Abstract Background Studies with the nematode model Caenorhabditis elegans have identified conserved biochemical pathways that act to modulate life span. Life span can also be influenced by the composition of the intestinal microbiome, and C. elegans life span can be dramatically influenced by its diet of Escherichia coli. Although C. elegans is typically fed the standard OP50 strain of E. coli, nematodes fed E. coli strains rendered respiratory deficient, either due to a lack coenzyme Q or the absence of ATP synthase, show significant life span extension. Here we explore the mechanisms accounting for the enhanced nematode life span in response to these diets. Results The intestinal load of E. coli was monitored by determination of worm-associated colony forming units (cfu/worm or coliform counts) as a function of age. The presence of GFP-expressing E. coli in the worm intestine was also monitored by fluorescence microscopy. Worms fed the standard OP50 E. coli strain have high cfu and GFP-labeled bacteria in their guts at the L4 larval stage, and show saturated coliform counts by day five of adulthood. In contrast, nematodes fed diets of respiratory deficient E. coli lacking coenzyme Q lived significantly longer and failed to accumulate bacteria within the lumen at early ages. Animals fed bacteria deficient in complex V showed intermediate coliform numbers and were not quite as long-lived. The results indicate that respiratory deficient Q-less E. coli are effectively degraded in the early adult worm, either at the pharynx or within the intestine, and do not accumulate in the intestinal tract until day ten of adulthood. Conclusions The findings of this study suggest that the nematodes fed the respiratory deficient E. coli diet live longer because the delay in bacterial colonization of the gut subjects the worms to less stress compared to worms fed the OP50 E. coli diet. This work suggests that bacterial respiration can act as a virulence factor, influencing the ability of bacteria to colonize and subsequently harm the animal host. Respiratory deficient bacteria may pose a useful model for probing probiotic relationships within the gut microbiome in higher organisms.
              Cover page: Delayed accumulation of intestinal coliform bacteria enhances life span and stress resistance in Caenorhabditis elegans fed respiratory deficient E. coli
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2020)
              Despite decades of clinical use, mechanisms of glucocorticoid resistance are poorly understood. We treated primary murine T lineage acute lymphoblastic leukemias (T-ALLs) with the glucocorticoid dexamethasone (DEX) alone and in combination with the pan-PI3 kinase inhibitor GDC-0941 and observed a robust response to DEX that was modestly enhanced by GDC-0941. Continuous in vivo treatment invariably resulted in outgrowth of drug-resistant clones, ~30% of which showed markedly reduced glucocorticoid receptor (GR) protein expression. A similar proportion of relapsed human T-ALLs also exhibited low GR protein levels. De novo or preexisting mutations in the gene encoding GR (Nr3c1) occurred in relapsed clones derived from multiple independent parental leukemias. CRISPR/Cas9 gene editing confirmed that loss of GR expression confers DEX resistance. Exposing drug-sensitive T-ALLs to DEX in vivo altered transcript levels of multiple genes, and this response was attenuated in relapsed T-ALLs. These data implicate reduced GR protein expression as a frequent cause of glucocorticoid resistance in T-ALL.
                Cover page: Loss of glucocorticoid receptor expression mediates in vivo dexamethasone resistance in T-cell acute lymphoblastic leukemia
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2020)
                Despite decades of clinical use, mechanisms of glucocorticoid resistance are poorly understood. We treated primary murine T lineage acute lymphoblastic leukemias (T-ALLs) with the glucocorticoid dexamethasone (DEX) alone and in combination with the pan-PI3 kinase inhibitor GDC-0941 and observed a robust response to DEX that was modestly enhanced by GDC-0941. Continuous in vivo treatment invariably resulted in outgrowth of drug-resistant clones, ~30% of which showed markedly reduced glucocorticoid receptor (GR) protein expression. A similar proportion of relapsed human T-ALLs also exhibited low GR protein levels. De novo or preexisting mutations in the gene encoding GR (Nr3c1) occurred in relapsed clones derived from multiple independent parental leukemias. CRISPR/Cas9 gene editing confirmed that loss of GR expression confers DEX resistance. Exposing drug-sensitive T-ALLs to DEX in vivo altered transcript levels of multiple genes, and this response was attenuated in relapsed T-ALLs. These data implicate reduced GR protein expression as a frequent cause of glucocorticoid resistance in T-ALL.
                  • Article
                  • Peer Reviewed
                  UCLA Previously Published Works (2014)
                  Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit β is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit β and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.
                    Cover page: The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.
                    • Article
                    • Peer Reviewed
                    UCLA Previously Published Works (2014)
                    Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit β is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit β and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.
                      Cover page: The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR
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