The Center for Research on Biological Clocks

Program Project

Many CRBC faculty members participate in a Program Project funded by the National Institute of Neurological Disorders and Stroke entitled "Coordination of Circadian Physiology of Diverse Species (P01 NS39546-01A1). The overall goal of the program is to infer fundamental properties of circadian mechanisms through comparative genomics, physiology, and biochemistry among the following diverse instructive model systems: cyanobacteria, fungi, mammalian cell cultures, and rodents, and birds.

The group has recently published a review that summarizes the outcomes of the first four years of P01 activities:

Bell-Pedersen, D., Cassone, V.M., Earnest, D.J., Golden, S.S., Hardin, P.E., Thomas, T.L., and Zoran, M.J. 2005. Regulation of circadian rhythms by multiple oscillators: lessons from diverse organisms. Nature Reviews Genetics, in press. 6:544-56

Overall Summary, Program Project
Coordination of Circadian Physiology in Diverse Species
Vincent M. Cassone

Circadian rhythms are endogenous oscillations of physiological functions that occur in diverse organisms. These rhythms share many formal and biochemical properties, raising the possibility that common mechanisms underlie all circadian clocks from cyanobacteria to humans. With this theoretical framework, the original P01 grant proposed that, by using common technologies in technical cores and shared data, a common process could be studied in 4 instructive model systems: the cyanobacterium Synechococcus , the fungus Neurospora , the chick pineal gland, and the rodent suprachiasmatic nucleus (SCN). The research instead revealed that all circadian systems, from cyanobacteria to mammals and birds: (1) comprise multiple circadian oscillations that behave independently under some experimental conditions and are integrated by complex processes; (2) regulate intermediary metabolism and redox state as both downstream processes of the clock and perhaps as part of the clock mechanism itself; (3) regulate protein dynamics in terms of both protein synthesis and catabolism; and (4) incorporate post-translational regulation as the rule rather than the exception, such that transcription/translation loop models are not sufficient to explain the clock. Further, the data told us that homology of known oscillator components is not a common element: (1) The sequences of known clock genes are very different if one discounts common protein-protein interaction motifs; (2) The temporal dynamics of clock gene transcription and translation are disparate enough that a deep evolutionary relationship among these diverse model systems is unlikely. The present proposal seeks to extend these findings by placing the circadian clock in the context of these organisms' cellular machinery, metabolism, and protein dynamics. We will ask: (1) How are multiple circadian oscillatory mechanisms coordinated within and among cells?; (2) How do metabolic processes interact with clock machinery in each of these systems?; (3) How does the regulation of protein dynamics fit into the overall scheme of circadian organization?; and (4) What are the fundamentally conserved properties of the clocks among these diverse organisms?
Lay summary: This research will lead the way to a comprehensive systems biology approach to biological clocks that will elucidate chronobiological disorders associated with sleep and wakefulness, many of which are attended by metabolic, endocrine, and cell-cycle disorders.

Project 1
The Circadian Clock in the Context of the Cyanobacterial Cell
Susan S. Golden

Circadian rhythms in the cyanobacterium Synechococcus elongatus share the properties of those driven by eukaryotic clocks: an endogenous free-running period of about 24 h, resetting of the phasing of the rhythms by environmental cues, and temperature compensation of circadian period. However, the cyanobacterial circadian oscillator proteins, KaiA, KaiB, and KaiC, are not homologous to those of eukaryotes, and the fundamental clock mechanism is different. A circadian oscillation in phosphorylation of KaiC can be reconstituted in vitro with only the three Kai proteins and ATP. Thus, the mechanism of circadian oscillation has become tractable to biophysical analyses. The current proposal will complement in vitro data by revealing mechanisms that integrate circadian oscillation with fundamental cellular processes. Molecular structures of the oscillator proteins, and the S. elongatus genome sequence, are known. Prior results showed that sigma factors of RNA polymerase affect circadian gene expression, and inactivation of one, SigC, revealed two different periods of oscillation in cells. We created mutations in 68% of all S. elongatus genes and identified new loci that affect circadian period and tie the clock to protein metabolism and redox regulation. A temporally-controlled modification of KaiA was detected in association with a redox sensor, LdpA. An effective fluorescent reporter for real-time intracellular localization of clock proteins was developed. Project 1 will capitalize on these data to provide a comprehensive understanding of a circadian clock at a systems level. Our new Specific Aims are to: (1) determine the connection between redox sensing and clock components by identifying the modification on the subpopulation of KaiA that co-purifies with LdpA, and testing the hypothesis that redox state affects circadian period by changing the ratios of Kai proteins; (2) define unknown components of the periodosome (clock complex) that tie the oscillator to input and output pathways by using ion trap electrospray mass spectrometry of peptides from affinity-tagged complexes; and (3) track the intracellular localization of clock proteins during the circadian and cell cycles and determine whether clock proteins associate with the bacterial nucleoid.
Lay summary: This project will explain how circadian oscillations are tied to fundamental processes in living cells, and how the clock can be manipulated. Lessons can be extrapolated to human health and disease, such as metabolic syndrome.

Project 2
Interaction of Circadian Oscillators within the Neurospora Cell
Deborah Bell-Pedersen

Organisms from bacteria to humans use a circadian clock to control daily biochemical, physiological, and behavioral rhythms. This clock affects human physiology, and disruptions of normal clock function can cause health problems, including manic depression, and sleep disorders. We now have a firm understanding of the molecular oscillators that form the core of the circadian timing system. However, new data reveal that the circadian system is universally more complex than a single molecular feedback loop oscillator regulating all overt rhythmicity. Evidence now indicates that multiple circadian oscillators exist within single cells of microbial organisms and among the cells and tissues of multi-cellular organisms. We hypothesize that distinct multiple oscillators comprise the Neurospora crassa clock, and that these communicate with each other to generate a coordinated rhythmic program of cellular activities. We have identified two Neurospora mutant strains (Light Mutant 1 [LM1] and LM2) that display circadian rhythms in the absence of the FRQ/WC oscillator (FWO), previously considered to be the core of the fungal clock. Thes e mutant strains uncover a novel circadian oscillator, called the LMO, which can function in cells that lack the FWO, but that is coupled to the FWO when the system is intact. A critical question that is relevant to the organization of all clocks, including the human clock, is: how do multiple oscillators communicate with each other to coordinately regulate circadian rhythms? To address this question, we will first determine the role of the LM1 and LM2 genes in the function of the LMO. Second, we will use genetic and physical methods to identify central components of the LMO. To test our hypothesis, we will determine if the LMO components physically interact with constituents of the FWO. In addition, LMO components will be inactivated in wild-type cells to determine if loss of the LMO affects the expression of key elements of the FWO and/or overt rhythmicity. To characterize LMO-specific outputs, we will use transcriptional profiling to identify genes that are rhythmically expressed in cells that have a functional LMO, but that lack the FWO.
Lay Summary: These studies will provide the first molecular description of a second cellular oscillator, and will uncover the mechanisms by which circadian oscillators communicate with each other to coordinately control rhythmic processes. This in turn will facilitate new approaches for therapies for human conditions that result from circadian dysfunction.

Project 3-
Intercellular Integration of SCN Output Signals
David Earnest

During the course of the previous project, our understanding of the organization of the mammalian circadian system changed considerably as a result of evidence indicating that many peripheral tissues in vivo and fibroblast cell lines in vitro also express oscillations in molecular components of the canonical clockworks. Yet, despite these oscillatory properties, peripheral tissues and fibroblast cell lines cannot function as pacemakers by regulating circadian rhythms in other cells or downstream processes. Only oscillators derived from the suprachiasmatic nucleus (SCN) possess the capability to restore behavioral rhythmicity when transplanted into SCN-lesioned hosts, and to coordinate molecular and physiological oscillations in co-cultured cells. The pacemaker function of SCN oscillators is presumably derived from the distinctive nature of their outputs that mediate cellular communication between cell-autonomous clocks within the SCN and from the SCN to downstream oscillators. Using immortalized rat SCN cells (SCN2.2), we have shown that pacemaker regulation of circadian rhythms in other co-cultured cells is mediated by SCN-specific diffusible factors. Therefore, the long-term objective of this project is to identify the diffusible outputs that communicate rhythmicity to co-cultures of other cell types in vitro and restore circadian wheel-running behavior when transplanted into arrhythmic hosts in vivo . Experiments will use multi-faceted approaches to determine whether: 1) antisense/siRNA and/or pharmacological inhibition of candidates for circadian diffusible signals in SCN2.2 cells or their receptor/response elements in co-cultured cells disrupts rhythmicity in these downstream cells; 2) clonal lines of immortalized SCN cells that express a specific output signal are capable of generating endogenous oscillations and coordinating rhythms in co-cultured cells; and 3) clonal SCN cell lines with pacemaker properties in vitro also confer behavioral rhythmicity to SCN-lesioned rats and transgenic mice with mutant circadian phenotypes. These studies will yield novel information onhow SCN circadian outputs coordinate oscillations in different downstream tissues and cells, and what cellular processes distinguish the function of SCN oscillators as a circadian pacemaker.
Lay Summary: Such information will lead to new developments in the understanding, diagnosis and treatment of disorders in human health and performance that may result from internal desynchronization of body processessuch as depression, sleep disturbances, diabetes, obesity and carcinogenesis.

Project 4
Pacemaker Properties of the Avian Pineal Gland
Vincent M. Cassone

The avian pineal gland is an important model system for the study of cellular and molecular bases of circadian clocks and sleep. However, although the mechanisms of the biosynthesis of the major output of this gland, melatonin, are largely understood, the molecular bases for the rhythm generating mechanism has been elusive, and the cellular/molecular basis for its pacemaker properties is completely unknown. Transcriptional profiling and the development of new cell culture methods in the original P01 grant identified several candidate genes that were likely to be important in rhythm generation and showed that melatonin broadly affected metabolic and ionic homeostasis without affecting clock gene rhythms. The present proposal seeks to test the hypothesis that pineal pacemaker activity regulates CNS and peripheral metabolic rhythms via mechanisms that are independent of or at least differentially regulated from output regulation through clock gene rhythms in chicks. (1) We will determine whether surgical disruption of circadian organization by pinealectomy and retinectomy differentially affects glucose metabolism and clock gene rhythms (mRNA and protein) in CNS and peripheral tissues in vivo. We will also ask whether administration of exogenous melatonin differentially affects glucose metabolism and clock genes (mRNA and protein) in CNS and peripheral tissues in vivo by employing both quantitative PCR and DNA microarrays. (2) We have developed a new experimental system in which primary cultured pinealocytes are co-cultured with CNS and peripheral target cells. We will determine the dynamics of pacemaker properties by monitoring melatonin rhythms in the media, and both metabolic activity and clock gene rhythms in pacemaker and target cells simultaneously. Finally, (4) we will determine the roles played by clock genes and other genes identified in our transcriptional profiling by up-regegulating and knocking down expression of these and other genes in pacemaker and target cells by lentiviral transfection.
Lay Summary: The results of these studies will provide the first molecular analysis of pineal pacemaker activity. It will also build a bridge between metabolic and clock gene regulation in sleep-wake cycles in a species whose metabolic demands are more similar to diurnal humans than are those of nocturnal rodents.

Core A- the Genomics, Proteomics and Bioinformatics Core
Terry L. Thomas

The overall goal of Core A is to provide genomics, proteomics and bioinformatics support for P01 investigators. Core A will maintain whole genome and custom DNA microarray platforms to facilitate the gene expression experiments of P01 investigators. Core A will also provide quantitative real-time PCR (qPCR) support for P01 projects. Proteomics activities will be supported within Core A. This will include facilitating yeast two-hybrid screens and preparation of protein samples for Ion Trap Electrospray Mass Spectrometry (IE-MS) to be carried out in the Texas A&M Protein Chemistry Laboratory. Core A will provide robust bioinformatics platform(s) for P01 investigators. Local and distributed servers will be provided for data analysis, storage and archiving. Additional data mining tools will be maintained locally for further gene expression, protein structure, and comparative data analysis. Core A will acquire or develop new technologies to facilitate P01 investigator progress. As new genomics, proteomics and bioinformatics platforms emerge, they will be evaluated by Core A and implemented where appropriate.

The research supported by the Genomics, Proteomics and Bioinformatics Core will lead to an understanding of the molecular and biochemical basis of circadian timing and of the systems-level consequences of circadian rhythmicity. This in turn will facilitate the development of new therapies for diseases and conditions related to circadian pathologies.

Core B, Real-Time Imaging of Circadian Processes Core
Mark J. Zoran

Core B, the Real-Time Imaging of Circadian Processes Core, will provide P01 investigators with optical and imaging equipment for quantitative analysis of cellular physiology and gene expression in living cells. The specific aims of the Real-Time Imaging Core are to provide facilities for the visualization and digital image capture of cellular events, provide facilities for the detection and monitoring of gene expression reporters of living cell in real-time, to support the analysis and archival of real-time imaging data files, to facilitate the acquisition of new optical technologies and real-time approaches to the analysis of clock-components and/or clock-controlled cellular events, and to provide Core users with consultation regarding experimental design of imaging studies and advice on interpretation of imaging data.

The Director of Core B is Dr. Mark Zoran. He will oversee all technical work done by the Core and provide technical advice to the Core users. Mr. Jeff Burkeen manages the day-to-day operation of the facility. Core facilities are equipped with a Real-Time Microscopy System, a Cryo-Cooled CCD Camera System, a 2-Detector, 40-Plate Stacker Topcount, UVP BioChemiluminescent Imaging System, a NanoDrop ND-1000 Spectrophotometer, and a networked data analysis workstation. Research conducted and published by Core B users includes studies of vertebrate brain cell function, modulation of glial cells by clock-controlled signals, pacemaker properties of brain cells, and clock-controlled gene expression in fungal and bacteria cells. Other Core B users have published studies of nervous system regeneration, neuronal synapse formation, and microbial development and genetics.

Our long-tern goal is the discovery of mechanisms used by diverse biological systems, from single cells to animals brains, to coordinate intra- and intercellular processes that govern biological timing and neural integration. Specifically, Core B provides researchers with tools for imaging these cellular functions. Disorders and degeneration of neural function often derive from, or result in, compromise of cell signaling. Discovery of these basic cellular mechanisms will benefit medical science and, ultimately, the public health.

Core C, Administrative Core
Susan S. Golden and Vincent M. Cassone

The overall goal of Administrative Core C is to facilitate the scientific interactions among the Projects and Cores A and B, and to foster additional connections among the P01 researchers, Texas A&M, and external scientific communities. Core C will achieve this goal by providing an administrative structure and Staff Assistant to organize activities of the P01 participants and integrate these with related educational and research activities. The Specific Aims of Core C for the next budget period are to: (1) organize meetings of the Executive Committee (P01 Co-PIs) and Internal and External Advisory Boards at appropriate intervals; (2) assist with preparation of manuscripts and grant proposals; (3) facilitate communication among participating laboratories and cores; and (4) provide information about the P01 to the greater scientific community through an updated website. The Administrative Core will provide administrative support for the research projects within the Program Project, will help integrate research and training initiatives for the group, and will provide a format for future research initiatives. Through these efforts, Core C will strengthen the ties among participants and support a culture of continuous improvement. Core C will facilitate the goals of the Program Project to integrate multidisciplinary approaches to biological clocks in diverse species, yielding insights that will ultimately elucidate chronobiological disorders associated with sleep and wakefulness, many of which are attended by metabolic, endocrine, and cell-cycle disorders.