Carrie L. Partch, PhD - US grants

DESCRIPTION (provided by applicant): Circadian rhythms exist to coordinate an organism's physiological and behavioral processes with the solar light/dark cycle. Dysfunctional circadian rhythms have been implicated in several psychiatric and sleep disorders and current light-based therapies have only limited success. Photoreceptors are required to convey light information to the molecular clock in the brain in order to synchronize its oscillation with the solar cycle. Both opsins and UV/blue light-absorbing cryptochromes have been identified as circadian photoreceptors in mammals. Recently, a UV/blue light photoreceptor was shown to induce clock gene expression in response to light in the zebrafish Z3 cell line. The main goal of this proposal is to determine if cryptochromes act as circadian photoreceptors in zebrafish and determine their mechanism of signal transduction using the Z3 cell line. This will be accomplished by: 1) determining the role of zebrafish cryptochromes in light-dependent gene induction using RNA interference; 2) characterizing the spectrometric properties of the zebrafish cryptochromes; and 3) identifying proteins that interact with zebrafish cryptochromes in a light-modulated manner. The results from these experiments will further our understanding of the role of cryptochromes in circadian phototransduction and will provide the groundwork for future studies in mammalian systems that will benefit the design of new circadian-based therapeutics for psychiatric and sleep disorders.

DESCRIPTION (provided by applicant): Aberrant cellular growth creates oxygen-poor microenvironments that serve to inhibit proliferation;tumors circumvent this homeostatic regulation through constitutive activation of the hypoxia response pathway, which up-regulates genes for anaerobic metabolism and angiogenesis to facilitate cellular growth that would otherwise be limited by low oxygen. The hypoxia response pathway is transcriptionally regulated by an oxygen-sensitive HIF-a (hypoxia-inducible factor-a) subunit and ARNT (aryl hydrocarbon nuclear translocator), which form a heterodimeric bHLH-PAS transcriptional activator that binds target sequences in DNA and recruits coactivators to initiate transcription. The objective of this proposal is to investigate the role of ARNT in transcriptional activation by examining the structural and functional basis of coactivator recruitment by ARNT PAS domains. We hypothesize that ARNT PAS domains simultaneously mediate interaction with HIF-a and coactivators by utilizing opposing interfaces on this small, modular domain. In Aim 1, we will identify minimal motifs for ARNT:coactivator interactions and define the molecular basis of coactivator specificity for ARNT. In Aim 2, we will determine the structural basis for the ARNT:coactivator interaction and characterize functional consequences of complex disruption in vivo. In Aim 3, we will screen ARNT PAS domains for artificial ligands to identify small molecule antagonists that disrupt the ARNT:coactivator interaction. This work will examine how ARNT contributes to transcriptional regulation in response to hypoxia and identify compounds that block ARNT function with potential therapeutic value in preventing tumor growth.

DESCRIPTION (provided by applicant): Mammals have an internal molecular clock that coordinates physiology into rhythms that coincide with the external solar day, providing enhanced evolutionary fitness by timing the peak activity of integrated biochemical processes. Loss of this internal 24-hour circadian clock leads to diabetes, metabolic syndrome, cancer, and premature aging by disrupting the temporal coordination of physiology with our behavior and the external environment. The long-term goal is to develop a deeper mechanistic understanding of how the molecular clock generates 24-hour timing in humans, in order to capitalize on this temporal regulation to develop new and innovative strategies to treat a broad spectrum of human diseases. The objective in this proposal is to identify the structural basis for transcriptional regulation by the primary circadian transcription factor, CLOCK:BMAL1, which controls expression of nearly 15% of the genome on a daily basis to drive circadian rhythms of physiology. Despite its critical importance in human physiology, very little is known about what governs the temporal switch from active to repressive CLOCK:BMAL1 complexes that create the intrinsic 24-hour period of the molecular clock. The central hypothesis is that CLOCK and BMAL1 transcriptional activation domains use intrinsic flexibility to regulate binding of activator and repressors to contribute to 24-hour timing of the molecular clock. Using nuclear magnetic resonance spectroscopy, quantitative biochemistry and cell-based studies, we will pursue two specific aims investigating 1) how a dynamic conformational switch in the BMAL1 activation domain regulates CLOCK:BMAL1 activity and 2) how competition for binding to the CLOCK activation domain regulates CLOCK:BMAL1 in normal clock function and in human cancer. Our innovative approach integrates diverse techniques from cell biology to solution NMR spectroscopy to generate biomedically relevant, atomic-level insight into clock function. The proposed research is significant, because it is expected to provide fundamentally new conceptual advances that address how CLOCK:BMAL1 works to generate 24-hour molecular rhythms and control global homeostasis. Ultimately, such knowledge has the potential to inform new strategies for basic and translational research into circadian control of human physiology.

Project Summary The ability to encode time at the molecular level is universally used to synchronize biological processes into ~24-hour rhythms that coincide with the solar day for health and homeostasis. How do we measure time at the molecular level and use it as the basis for biological regulation? The goal of the current project is to use the simplest model system for circadian cycling to explore the structural basis for generating a molecular clock that keeps 24-hour time. Cyanobacteria have a biochemically tractable clock composed of three Kai (cycle) proteins that keep ~24-hour time in vitro, requiring only the addition of ATP as an energy source. KaiC is the central pacemaker of this clock, with two tandem ATPase domains (C1 and C2) that each assemble into hexameric rings connected by a flexible linker. Information about the time of day is transmitted between the rings to influence interactions with KaiA and KaiB, as well as with proteins that transmit information about the environment to the clock and control biological timing in vivo. Despite its relatively simple composition, the lack of structures for intermediate states formed by Kai protein complexes has limited our understanding of this molecular clock. The proposed research will provide structural, biochemical, and in vivo functional data in support of a new model for Kai protein interactions, leading to a major shift in our understanding of the cyanobacterial circadian oscillator. We are pursuing a hypothesis that competition for the KaiC C1 domain, a central hub for clock protein interactions, is essential for circadian timekeeping. Our approach is built on the discovery that KaiB undergoes a metamorphic transition to a new protein fold that is needed to regulate assembly of clock protein complexes with KaiC. Using a version of KaiB locked into its rare, active conformation, we present three new structures of clock protein complexes that demonstrate the vastly underappreciated role that the KaiC C1 domain plays in generating the molecular clock. In Aim 1, we will determine how the hexameric KaiC ATPase controls assembly of Kai protein complexes. In Aim 2, we will identify the structural basis for communication between the two ATPase rings of KaiC. In Aim 3, we will determine how competitive interactions at the KaiC C1 domain control clock signaling throughout the day and night. The proposed studies will demonstrate the structural basis by which KaiC integrates interactions with clock proteins to keep 24-hour time and transform our understanding of the cyanobacterial circadian clock.

We request partial support for the 22nd Chronobiology Gordon Research Conference (GRC) and the accompanying 5th Gordon Research Seminar (GRS), which will be held Bates College in Lewiston, Maine from June 20-25th, 2021. Any funds that we receive from the NIH will be used to support registration fees and/or travel costs for half the speakers/session chairs at GRC and GRS with a particular focus on supporting the younger researchers, postdoctoral fellows and graduate students. The GRC/GRS meetings on Chronobiology have a rich history of attracting a wide-range of researchers working at many different levels with diverse methodologies and model organisms. The GRS will focus on training and mentorship, while the GRC will present the latest unpublished work from both senior and emerging stars of the field. The sub-theme for the 2021 Chronobiology meeting is 'Inputs, circadian oscillators and outputs: From single cells to human health' to emphasize the study of diverse organisms and the application of these insights in the clinic and workplace, achieved with molecular, cellular, physiological and behavioral approaches. In metazoans, the master circadian clock is in the brain, and chronic circadian disruption by modern lifestyles has important implications for human behavior, metabolism and healthy aging, so the conference fits well within the mission of the NINDS. Speakers and discussion leaders represent senior established figures and younger emerging stars of the field and have been selected by consulting with an informal committee consisting of mid-career and junior figures in the field. We are enriched for female speakers/session chairs with several underrepresented minorities as well as representation from a range of nationalities, from the Americas, Europe and Far East to promote cultural, racial and gender diversity. Apart from mentorship activities, the GRS will feature 10 trainee speakers, one of whom will be promoted to give a short talk at the GRC along with 9 other younger scientists selected from abstracts, providing a useful learning experience for the trainees and early career scientists.

Project Summary Circadian rhythms arise from genetically encoded clocks that are intimately linked to external cues like light to synchronize physiology and behavior with the 24-hour solar cycle. Although the genetic networks that give rise to circadian rhythms are now relatively well established, we still don?t understand many of the fundamental, molecular steps that determine the ~24-hour basis of these clocks and how they respond to external time-setting cues. By integrating structural biology and solution biophysical methods with biochemistry and cell biology, we aim to determine the underlying biochemical principles that lead to the day-long timescale of circadian signaling and uncover the mechanisms that allow biological clocks to faithfully maintain intrinsic timing and respond robustly to external cues. With prior NIGMS funding, we studied clock systems from mammals and cyanobacteria to discover how different clock proteins assemble into regulatory complexes and identified how protein dynamics, enzyme activity and/or post-translational modifications impact clock timing. Our comparative biochemical approach highlighted surprising commonalities, such as the competition for mutually exclusive binding sites, between these clocks despite their different molecular architectures. Here, we will continue to pursue the structural basis of protein assemblies from diverse biological clocks, determine the consequences of post- translational modifications on clock protein function, study the molecular basis for entrainment of clocks to external cues, and seek out new inroads for pharmacological intervention. Funding from the MIRA program would provide us with the resources and flexibility to explore commonalities in mechanisms of biological timekeeping across a diverse array of species from cyanobacteria to humans.