Browsing by Subject "Circadian Clocks"
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Item Architecture of the Mammalian Circadian Repressive Complex(2017-07-27) Rosensweig, Clark Jeffrey; Hibbs, Ryan E.; Green, Carla B.; Takahashi, Joseph; Konopka, GenevieveIntricate timing systems have evolved to help organisms in all walks of life organize their physiology to the solar day. Mammalian circadian clocks are driven by a transcription/translation feedback loop composed of positive regulators (CLOCK/BMAL1) and repressors (CRY1/2 and PER1/2). To understand what drives periodicity within this clock, I took structural approaches with the hope of identifying atomic-level details that inform behavioral outputs. Despite high sequence identity, null mutations of Cry1 or Cry2 have divergent effects on periodicity, accelerating and decelerating the clock speed, respectively. To understand the unique roles of CRY1 and CRY2, we used statistical coupling analysis to identify co-evolving residues within the CRY protein family. We identified an evolutionary hotspot, an ancestral secondary cofactor-binding pocket, which has been repurposed for direct interaction with CLOCK and BMAL1. Mutations weakening binding between CLOCK/BMAL1 and CRY1 lead to acceleration of the clock, revealing a novel mode of period regulation in the mammalian clock. Subtle divergence between CRY1 and CRY2 at the secondary pocket underlies differences in affinity for CLOCK/BMAL1. The lower affinity interaction with CRY2 is strengthened by co-expression of PER2, suggesting that PER expression limits the length of the repressive phase in CRY2-driven rhythms. In order to better understand PER's role, we collaborated with another lab to solve and validate a structure of CRY2 bound to a fragment of PER. In so doing, we discovered that interaction between PER and CRY is necessary for rhythmic derepression, providing insight into the role of a key interaction in the molecular clockwork.Item Cellular Basis of Behavioral Circadian Rhythms in Mammals: The Role of Neuromedin S (Nms)-Producing Cells in the Suprachiasmatic Nucleus(2013-08-07) Lee, Ivan T.; Elmquist, Joel; Russell, David W.; Greene, Robert W.; Yanagisawa, Masashi; Takahashi, JosephBehavioral circadian rhythms in mammals are controlled by highly heterogeneous populations of neurons located in the suprachiasmatic nucleus (SCN). Lesion and transplantation studies have established that the SCN is both necessary and sufficient for the generation of daily rhythms in locomotion. It remains uncertain, however, whether this pacemaking property of the SCN is limited to certain subsets of cells or intrinsic to all neurons within the SCN. To dissect out the cellular properties of circadian rhythms, we utilized a BAC transgenic mouse line in which Cre recombinase (iCre) is driven by the promoter of neuromedin S (Nms), a neuropeptide that has restricted expression in ~40% of cells within the SCN. Using this cell-type specific driver, we genetically altered the molecular oscillation of Nms-positive cells by overexpressing the ClockΔ19 or the Period2 transgene. ClockΔ19 is a semi-dominant mutation that leads to lengthened behavioral circadian periods when expressed in the majority of SCN cells. Likewise, Period2, when overexpressed in all or almost all of the SCN neurons, lead to the loss of behavioral circadian rhythms. We found that, intriguingly, the transgenic expression of ClockΔ19 only in Nms-positive neurons leads to a lengthened period in circadian rhythms while the overexpression of Per2 in Nms-expressing neurons causes the loss of daily rhythms altogether, suggesting that behavioral rhythms can be controlled by the molecular oscillation of Nms-positive cells. Next, to ascertain whether Nms-expressing neurons are required for normal behavioral circadian rhythms, we utilized a tetanus toxin-based technology that permits the inducible and reversible inhibition of neurotransmission. Surprisingly, this genetic manipulation revealed that synaptic neurotransmission from Nms neurons is essential for the generation of behavioral circadian rhythms. Taken together, these results indicate that Nms marks a specialized subgroup of neurons that is both necessary and sufficient for the production of circadian rhythms in behavior.Item Regulation of Adaptive and Innate Immunity by the Circadian Transcription Factor NFIL3(2014-06-13) Yu, Xiaofei; Yarovinsky, Felix; Hooper, Lora V.; Alto, Neal; Green, Carla B.; van Oers, Nicolai S. C.The day-night cycle has a profound impact on animal physiology, which has been shown to be mediated by an intracellular timing system called the circadian clock. However, little is known about whether and how the day-night cycle and the circadian clock influence host immunity. Here, I show that the circadian transcription factor NFIL3 is critical for both adaptive and innate immunity by regulating TH17 cells and innate lymphoid cells (ILCs), respectively. First, NFIL3 transcriptionally represses RORgt, the master regulator of TH17 cells, by directly binding to the Rorgt promoter and thereby suppresses TH17 cell development from naïve T helper cells. Consistent with oscillation of Nfil3 expression during the circadian cycle, Rorgt expression also oscillates in mice, with higher expression at noon and lower expression at midnight. Accordingly, naïve T helper cells show greater potential to develop into TH17 cells at noon than at midnight. Furthermore, artificially disturbing the circadian cycles of mice by manipulating their light exposure results in circadian clock-mediated disruption of TH17 homeostasis and increased susceptibility to experimentally-induced colitis. Therefore, NFIL3 regulates TH17 cell development in a circadian manner. Second, NFIL3 is essential for the development of all major types of innate lymphoid cells (ILCs) by regulating the generation of ILC progenitors in the bone marrow. One of the NFIL3-dependent progenitor populations, aLP, can differentiate into all major types of ILCs in vivo. NFIL3 controls progenitor development by activating a High Mobility Group (HMG) transcription factor Tox directly in common lymphoid progenitors (CLPs). Accordingly, restoring Tox expression in Nfil3-deficient progenitors rescues ILC development in mice. So NFIL3 regulates ILC development by activating Tox expression in bone marrow precursors. Taken together, my work demonstrates that NFIL3 is a critical regulator of host immunity and that it modulates immune functions along the circadian cycle. This study is among the first to reveal the influence of the circadian clock on host immunity and provides novel insights into the regulatory mechanisms underlying variations of immune cell development and function during the circadian cycle.Item Regulation of Body Composition by the Microbiota and the Circadian Clock(2017-11-21) Wang, Yuhao; Green, Carla B.; Liu, Yi; Wan, Yihong; Hooper, Lora V.The intestinal microbiota has been identified as an environmental factor that markedly impacts energy storage and body fat accumulation, yet the underlying mechanisms remain unclear. In this dissertation, I show that the microbiota regulates body composition through the circadian transcription factor NFIL3 in intestinal epithelial cells. First, epithelial NFIL3 promotes lipid absorption and export in the intestine by regulating a circadian expression program of epithelial lipid metabolic genes. Second, Nfil3 transcription oscillates diurnally in intestinal epithelial cells and the amplitude of the circadian oscillation is controlled by the microbiota through group 3 innate lymphoid cells (ILC3), STAT3, and the epithelial cell circadian clock. These findings provide mechanistic insight into how the intestinal microbiota regulates body composition and establish NFIL3 as an essential molecular link among the microbiota, the circadian clock, and host metabolism.Item [Southwestern News](2002-11-21) Maier, Scott; Haduch, Bill