Steve A. Kay, Ph.D., D.Sc. - US grants

The circadian clock plays a vital role in the health and fitness of organisms by regulating cellular activities to specific times of the day and night. The long-term goal of this proposal is to understand how circadian clocks function within eukaryotic cells. Forward genetic approaches have been instrumental to initially identify many molecular components of the circadian clock in plants and animals revealing a shared molecular architecture. Recent progress in the circadian field has been focused on the characterization of the precise molecular wiring that builds the clock circuitry which ultimately will uncover how the circadian clocks control daily rhythms in physiology, metabolism and behavior in different species. By developing a complete transcription factor (TF) collection for the model organism Arabidopsis thaliana we have recently implemented a reverse genomic strategy that allowed us refine the transcriptional circuits that build the circadian clock in plants. Based on our initial discoveries we will continue using our TF collection to determine direct regulators of clock genes and implement the discovery of new transcriptional mechanisms by TF-focused gain-of-function screens. In addition, we propose computational approaches to build a map that connects identified clock components with clock-output genes and the genome-wide identification of mechanisms for the non-transcriptional circadian regulation of mRNA and protein levels. In sum, we propose a suite of genomic approaches to expand our transcriptional discovery program and extend our discovery pipeline to the post-transcriptional and post-translational levels. Given the ubiquity and relevance of the circadian clock, the identification of common clock mechanisms will help us understand how alterations in the circadian pacemaker have such a tremendous impact on human well- being.

Kay 9724120 Circadian clocks are known to regulate many essential cellular processes in all organisms so far examined. In higher plants most major metabolic pathways are subject to circadian regulation, as well as developmental events such as the photoperiodic control of flowering. However, the molecular components of these biological clocks remain unknown. Using Arabidopsis thaliana as a model plant and the luciferase gene fusion and bioluminescence imaging system developed in this laboratory, work will be continued on the identification and characterization of the DNA-protein interactions involved in regulated chlorophyll A binding protein (CAB2) gene transcription, the genetic analysis of the interactions between circadian and phototransduction pathways and genome-wide identification of clock-controlled genes in Arabidopsis. The long term goal is to further our understanding of how circadian clocks are built in eukaryotic cells. Although they are known to regulate many essential cellular processes in all organisms thus far analyzed, the molecular components of these biological clocks remain unknown. Using Arabidopsis as a model plant, genetic and molecular approaches will be used to enhance our knowledge of the mechanistic basis of the regulated response of gene expression to circadian rhythm.

DESCRIPTION (Adapted from applicant's abstract): This proposal addresses the molecular control of biological clocks in individual organisms, tissues and single cells. Circadian rhythms have been demonstrated to regulate many processes in all organisms in which they have been examined. Recent progress in several model systems has identified the presence of "clock" genes and proteins that represent the molecular components of these ubiquitous biological clocks. This proposal is concerned with understanding how these clock genes and proteins are regulated and how they contribute to the circadian organization of the organism at the tissue, cellular and molecular level. We plan to employ model systems that are tractable both genetically and molecularly in order to gain novel insights into the generation and utilization of circadian clocks. Given the ubiquitous nature of these clocks, knowledge gained in one particular system will have a broad impact. We have generated strains of transgenic Drosophila that contain the period clock gene fused to the firefly luciferase gene. We have used these strains to create an assay where we can monitor per gene transcription for the first time in a living animal, and this assay has already revealed novel features of per transcription that were previously unappreciated. We propose to employ this powerful assay to investigate the pattern of per transcription in live animals and to identify different tissues that contain autonomous circadian clocks. Furthermore, we will generate single cells from circadian tissues and use these to analyze clock regulation at the single cell level. These experiments will provide substantial insight into the circadian organization throughout the animals, as well as how circadian information is generated and transduced. We will gain novel information on the interaction between phototransduction and circadian-regulatory pathways, which in turn will broaden our understanding of how circadian clocks are integrated into cellular regulatory networks. Given the ubiquity of circadian-regulated physiology, the identification of common clock components and pathways will have a significant impact on understanding the pacemaker mechanisms and malfunctions associated with known features of human well-being.

Progress in cutting-edge biomedical studies has become increasingly dependent upon the technology required to measure biological process as they occur in living organisms, tissues, and individual cells. In recent years, increasing numbers of probes have been developed that can detect cellular process in live cells as have the technologies and tools to monitor these activities. Research groups at The Scripps Research Institute have initiated activities to image processed in live cells in several areas. Using green fluorescent protein (GFP) to track intra- and intercellular dynamics of many proteins, including movement of centromeric proteins during mitosis, targeting of proteins to the nuclear envelope, virus movement, neuronal cytoskeleton dynamics and monitoring expression of clock proteins. Others are developing and applying novel fluorescent analogs to indicators for studies in live cell function. Our goal is to establish at TSRI a unique facility with equipment and software that will enable us to study live cells in real-time with the capacity to capture light in optical sections, to deconvolve the capture light, carry out ratio image, and to reconstruct patterns of fluorescence through cells. The system will perform bright field and differential interference contrast as well as low-light fluorescence and luminescence imaging of animal and plant cells and tissues. We propose to acquire a Deltavision digital microscopy system that will emphasize the following features: optimized optical paths to reduce light loss, several types of stage chambers for the maintenance of living specimens during image acquisition, a high resolution/high readout rate cooled CCD camera, optical sectioning capabilities and proven deconvolution software for assembling 3D images, and silicon graphics workstations for rapid convenient image acquisition and processing. While each of the participants has existing fluorescence microscopy capabilities within their labs, this equipment will enable areas of research far beyond those currently available at this Institute. Furthermore, this Digital Microscopy Resource system for live cell processes will be included within the physical facility that also contains confocal and electron microscopes, and will serve a large diverse biology community at TSRI. It will also initiate opportunities for interdisciplinary collaboration with other well established TSRI programs in computational biology, imaging science, and biological structure through member participation on the Advisory board that will oversee the use of the instrument.

microarray technology; biomedical equipment purchase;

DESCRIPTION (provided by applicant) This research will be done primarily in Argentina as an extension of NIH grant # RO1 MH51573. An organism's clock serves it as a temporal filter to time gene expression, cell metabolism, physiology and behavior to the most critical moments in the day, thus contributing to the organism's adaptation to a changing environment. Substantial progress has been made in elucidating the molecular process that imparts this temporal control. It is based in a transcriptional feedback loop, a widely conserved strategy for generating circadian oscillations; the ubiquity of this mechanism ensures that progress in one model will be of use to all. However, less information is available as to how the clock controls locomotor behavior. Gaining understanding in a model system like Drosophila most likely will impact the current thinking of how the clock controls sleep/activity cycles in mammals.The aim of the present proposal is to investigate the role of a calcium-dependent voltage-gated potassium channel, slowpoke, in the consolidation of the activity cycles in the fruit fly. The Specific Aims cover the analysis of mRNA and protein temporal profiles of slo and slob, two candidate genes putatively involved in circadian control of behavior; locomotor behavior analysis of strains carrying mutations in either gene under entrained and free running conditions; the molecular characterization of core oscillator function in a slo null (loss of function) mutant background and the analysis of the resetting response of the circadian pacemaker in a hypomorphic mutation (incomplete loss of function).

[unreadable] DESCRIPTION (provided by applicant): The long term goal of this proposal is to understand how circadian clock networks are constructed in eukaryotic cells. Circadian clocks are known to regulate many essential cellular processes widely distributed across biological systems, including humans. In higher plants, the clock network regulates diverse processes ranging from photosynthesis to cell elongation to the control of flowering time. We have chosen Arabidopsis as a model organism and have identified several clock genes from genetic screens. The reciprocal regulation amongst these clock components defined an initial molecular feedback loop which forms the basis for elaborated models of multiple loop clock networks in higher plants. We have made substantial progress on all of the previous specific aims, including the identification and characterization of several new clock genes. The experiments in this proposal aim to build on the current clock models by continuing to identify clock factors involved in regulating the transcription of known key clock components such as CCA1 and TOC1. To this end, we have created a unique functional genomics resource consisting of a library of more than 200 cycling transcription factors that can be used to detect DNA binding as well as protein-protein interactions on a spectrum of targets. This has led to the discovery of a novel transcription factor TCP21 that binds to the CCA1 promoter. This proposal aims to characterize TCP21 and identify additional transcriptional regulators within the core network. We have also identified a novel clock transcription factor LUX that will be characterized in terms of its DNA target genes and interaction partners. We propose to extensively characterize the time dependent protein-protein interactions and post- translational mechanisms that add critical additional layers of control to the transcriptional components of the clock. Finally, we plan to use a systems biology approach to characterize the logic underlying the output networks of the clock. We wish to explore the similarities in system architecture between plant and animal clock networks. Given the ubiquity of circadian-regulated physiology, characterization of circadian systems in model organisms will impact our understanding of the pacemaker mechanisms and malfunctions associated with many known features of human well-being. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Circadian rhythms exist in almost all organisms to anticipate the daily changes in the environment and to temporally coordinate biological processes. Although much progress has been made to identify the components of the mammalian clock and the mechanisms by which it controls physiology, the knowledge base is by no means complete. We propose to further elucidate the architecture of the liver clock and its relationship with metabolism by generating and integrating divergent types of large-scale data, followed by validation of these data in vivo. In order to isolate and study the cell-autonomous clock, we have identified a unique cell line, Met Murine Hepatocyte-Day 3 (MMH-D3) that retains many metabolic functions and exhibits robust circadian rhythms. Thus we will be able to interrogate both the clock and metabolic pathways and study their interactions. We have already obtained transcriptomic and metabolomic data for MMH-D3 time-courses (every 2-hours for 48-hours) to identify cycling entities. Furthermore, we will perform chromatin immunoprecipitation followed by sequencing (ChIP-seq) for REV-ERB1, a nuclear hormone receptor that is part of the clock and a regulator of lipid metabolism, to explore one of the mechanisms of how the clock controls metabolism. To identify novel proteins that act as systemic cues for the liver clock, we will perform high- throughput screening of an invaluable library of over 6,000 secreted proteins. To our knowledge, this is one of the broadest and diverse data sets collected for the analysis of the circadian clock. To generate the most comprehensive model of the clock, these data sets will not only be evaluated individually, but will be integrated to reconstruct biological networks using methods based on vector auto-regression. Such networks have the ability to predict regulation and interaction within and between the different layers, as well as their associations with metabolic pathways. The hypotheses generated by the network model will be verified using adenovirus techniques in vivo, which will also enable us to differentiate between cell-autonomous and systemic signals governing the liver clock. The clock clearly has an influence on proper physiological function, thus a better understanding of circadian clockwork will improve our understanding of the mechanisms that underlie human disease; this will refine diagnostic and therapeutic strategies.

DESCRIPTION (provided by applicant): Transcription factors (TF) have a crucial role in controlling gene expression, and exploring their molecular targets, binding partners and mode of regulation is essential to understand any plant biological process. Arabidopsis offers some unique advantages for the development of large-scale genomic approaches for the study of TFs function such as the ease and low cost to generate large transgenic collections, or the propensity of most gene-regulatory regions to be circumscribed to a short region upstream the transcription start site. The potential of these types of strategies is greatly exemplified in a recent report from our laboratory where, by using a fraction of the full TF collection, a novel clock component was identified. For these reasons, we started gathering all available Arabidopsis TFs from the different ORFeome resources (Salk, Pekin-Yale, REGIA, TIGR and RIKEN) to generate a complete TF collection. However, our findings from resequencing the different clones revealed a large overlap between the collections and several hundred mislabeled or missing ones, resulting in a final coverage close to 75% of all the Arabidopsis transcription factors and regulators. Here, we propose to generate an homogenous gold standard GATEWAYTM compatible collection containing every Arabidopsis TF. The corresponding coding sequences will be cloned in the same vector using the available ORFeomes as the template resource when possible. The remaining 25% missing ones will be generated by following different complementary amplification protocols and subsequently cloned in the same vector backbone. In addition, we propose to create and distribute to the community, nine application-ready genomic collections containing each TF in fusion with different tags for a multitude of applications. These nine collections will allow (1) overexpression screens in plants of wild type as well as EAR or VP64 translational fusions, (2) protein-protein interaction screens, (3) protein-DNA interaction screens, (4) subcellular localization and, (5) bacterial recombinant protein expression. In addition, in collaboration with Dr. Joe Ecker at the Salk Institute, we propose to test and compare the efficiency of different protein epitope-tags suitable to perform ChIP-seq experiment. A collection of TF tagged with the selected epitope will be generated. Finally, we propose to devise a simple protocol to perform yeast one-hybrid screens with full TF collections at a reasonable cost. We strongly believe these resources have the potential to greatly enhance research in Arabidopsis and other crop species. As the knowledge gap is being filled, the study of transcriptional networks in Arabidopsis will ultimately help us understand the biochemical complexity of multicellular organisms and positively impact the biomedical research community.

DESCRIPTION (provided by applicant): The circadian clock serves as a temporal filter to time gene expression, cell metabolism, physiology and behavior to the most critical moments in the day, thus contributing to the organism's adaptation to a changing environment. While the molecular mechanisms that generate and sustain rhythmicity at the cellular level are well understood, it is less clear how this information is further structured to control specific behavioral outputs. Rhythmic release of pigment dispersing factor (PDF) has been proposed to propagate the (time of day) information from core pacemaker (PDF reactive) cells to downstream targets underlying rhythmic locomotor activity. Indeed, such circadian changes in PDF intensity represent the only known mechanism through which the PDF circuit could communicate with its output. Recently, we reported a novel circadian phenomenon involving extensive remodeling in the axonal terminals of the PDF circuit which display higher complexity during the day and significantly lower complexity at nighttime. Thus, clock-controlled structural plasticity could be a candidate mechanism contributing to the transmission of the information downstream of pacemaker cells. The aim of the present proposal is to extend this initial observation and characterize the structure of the PDF circuit by time- lapse imaging in cultured brains. This approach, coupled with immunohistochemistry, will shed light on whether synaptogenesis and synapse elimination is concomitantly taking place. In addition, the molecular mechanisms responsible for such substantial remodeling will be explored employing novel genetic tools that allow spatial and temporal control of gene expression. Gaining understanding in a model system like Drosophila most likely will impact the current thinking of how the clock controls sleep/activity cycles in mammals. PUBLIC HEALTH RELEVANCE: Circadian systems evolved as a mechanism that allows organisms to adapt to the environmental changes in light and dark which occur as a consequence of the rotation of the Earth. Because of its unique repertoire of genetic tools, Drosophila is a well established model for the study of the circadian clock. Although the biochemical components underlying the molecular oscillations have been characterized in detail, the mechanisms used by clock neurons to convey information to the downstream pathways remain elusive. We have recently discovered a novel form of network plasticity whereby a circuit that is central to rhythmic rest-activity cycles undergoes substantial circadian remodeling and might be critical for clock control of behavior. The aim of this proposal is to extend the initial observations and characterize the molecular mechanisms underlying this form of structural plasticity. Thus, this project will shed light into some novel mechanisms by which neuronal circadian clocks regulate physiology and behavior.

Project Summary / Abstract Circadian rhythms are pervasive among organisms, allowing them to anticipate and adapt to the predictable 24-hour day-night cycle. Their function is to temporally coordinate physiological processes, such as metabolism, within the organism. Consequently, the disruption of circadian rhythms leads to desynchronized internal clocks and complex metabolic disorders, such as diabetes. The mechanism of how the clock controls downstream metabolic pathways is not well-established. One of the central players in this relationship is the core clock gene Cryptochrome (Cry). CRY is necessary to maintain rhythmicity and determine period length, but it has also been implicated in diabetes and glucose tolerance. Until recently, CRY was thought to function only in the nucleus; our recent unanticipated findings indicate it also inhibits gluconeogenesis in the cytoplasm through its interaction with Gs?. In parallel, nuclear CRY also regulates gluconeogenesis, albeit through a completely different pathway. Together, this two-pronged approach allows CRY to fine-tune its regulation of glucose homeostasis; however, its presence in two subcellular locales has made it difficult to study its compartment-specific mechanisms. To overcome this challenge, we created two unique reagents that localize CRY to each region. Cytosolic CRY will be studied at the atomic and cellular level to identify its binding partners and how their interactions determine their biochemical functions. Nuclear CRY will be investigated on the genomic scale to uncover its interactions with other transcription factors in the enhancers of gluconeogenesis genes. Chromosome conformation capture techniques will enable us to model nucleus-wide hepatocyte-specific enhancer architecture. On the therapeutic front, we will characterize the mechanism of action of novel clock-modifying chemical compounds identified from our screens. These compounds have the potential to identify novel clock genes and to regulate metabolism, paving the way towards clinical translation. The use of these techniques to study how CRY controls gluconeogenesis will be a proof-of-concept for how the clock achieves precision in modulating a tissue-specific metabolic pathway. The success of these studies will significantly improve the understanding of the crosstalk between the biological clock and physiology or disease states, as well as provide a proof-of-concept model for applying cell-based findings in improving human health.

Glioblastomas rank among the most lethal of all human cancers. Current therapy includes maximal surgical resection, followed by combined radiotherapy and oral chemotherapy (temozolomide), and adjuvant temozolomide. Maximal current therapy offers only palliation. Median survival for glioblastoma patients has been reported to be 15-21 months, but these data are derived from patients with favorable age and performance status. Recurrent glioblastoma therapy is limited with little evidence for effective therapy. Treatment failure is derived from numerous causes, including the presence of stem-like tumor cells, called glioblastoma stem cells (GSCs). GSCs contribute to radioresistance, chemoresistance, invasion, immune escape, and angiogenesis. GSCs display dependencies on specific signal transduction pathways and epigenetic regulation, associated with metabolic reprogramming. Almost all living organisms on earth are exposed to a regular 24-hour day-night cycles generated by planet?s rotation around its own axis, which in return leads to the evolution of intrinsic, entrainable circadian rhythm driven by cell autonomous biological clocks. Molecular oscillation of transcriptional circuitry to regulate circadian rhythms include positive regulation by the BMAL1 and CLOCK transcription factors, with two negative regulatory loops that either transcriptionally downregulate BMAL1 or bind and inhibit BMAL1:CLOCK transcriptional complexes. In our proposed studies, we leverage preliminary findings that the circadian rhythm machinery serves distinct cellular and molecular roles in maintenance of GSCs. We will determine the necessity for circadian rhythm regulation in GSCs mediate through metabolic reprogramming and selective activation of oncogenic pathways. To translate these efforts into novel clinical paradigms, we are using a novel class of agents that target circadian clock function. These small molecule inhibitors are brain penetrant and can be combined with other therapies to create synergistic targeting of GSCs. To generate the most effective therapeutic paradigm, we will interrogate the preclinical utility of novel targeted therapies that disrupt the circadian rhythm oscillatory loop that could accentuate the efficacy of conventional therapy. Collectively, the proposed studies will lay the foundation for improved understanding of circadian rhythm regulation in cancer stem cell biology with possible application to improved oncologic care.