Our laboratories and research are directed towards understanding the mechanism by which eukaryotic organisms keep time on a daily basis, and how this capacity to keep time is used to regulate metabolism and development. Circadian clocks with fundamentally identical characteristics are found in all groups of eukaryotic organisms, but the uses to which these clock are put reflects the diversity of evolution. Phylogenetically this ranges from the control of cell division and enzyme activities in unicells, to a firmly established involvement in plant and animal photoperiodism and in avian and insect celestial navigation, to multiplicity of human systems including endocrine function, work-rest cycles and sleep, and drug tolerances and effectiveness. Cell division in many tissues and organs within the human body is regulated on a daily basis by the clock, providing the theoretical basis for chronotherapy of cancer, and manipulation of internal circadian rhythms provides treatments for several kinds of sleep and affective disorders. The general layout of the feedback loop that makes up the clock and the identity molecular identity of some of the components, particularly the heterodimeric positive elements WC-1 and WC-2 that serve as positive elements in the loop, appear to be conserved among the Crown Eukaryotes (Dunlap Science 280, 1548-49, 1998; Cell 96, 271-290, 1999).
We are pursuing the analysis of the clock chiefly by using as a model system the fungus Neurospora crassa, although similarities between this basic model system and the mammalian circadian oscillator have led us to examine the molecular biology of mammalian clocks also. As a lower eukaryote, Neurospora has a biological clock that is functionally equivalent to those seen in higher organisms, but at the same time Neurospora is a simple organism upon which can be brought to bear all of the current techniques of classical and molecular genetics and biochemistry.
Our research is presently proceeding along four lines (see figure). One of these is aimed at identifying the components of the Neurospora oscillator and understanding how they work together to generate time information. Classical genetic analysis has revealed the existence of seven different genetic loci which, when mutated, result in an organism with an altered biological clock; these are putative "clock genes". In this work, supported by NIGMS, we are engaged in the molecular cloning of these clock genes as a first step towards elucidating their organization, products, and ultimately their function as a part of the circadian oscillator (e.g. Science, 263:1578-84, March 18, 1994; Science 276:763-69, May 2, 1997). This work was the first to identify positive elements in the circadian cycle, and the first to show widespread conservation of molecular hallmarks of rhythm genes, the PAS domains.
Currently our work on Neurospora is most closely focused on the frq and white collar gene complexes. The circadian system in Neurospora comprises an autoregulatory feedback cycle, wherein the White Collar (WC1 and WC2) proteins promote the expression of the frequency (frq) gene which encodes two forms of the FRQ protein, each of which can feed back via interactions with the WCs to depress the level of transcript arising from the frq gene. As predicted, both frq RNA and protein cycle in abundance showing peaks in the subjective day with frq mRNA peaking about 4 hrs after subjective dawn (CT4) and FRQ peaking about 4 hrs later at CT 8-10 (Aronson et al, Science 263, 1578, 1994; Garceau et al Cell 89, 469, 1997; Merrow et al, PNAS 94, 3877, 1997). The two forms of FRQ arise from alternative translation initiation sites; ambient temperature influences the clock by determining both the absolute amount of FRQ and the ratio between the two forms (Garceau et al Cell 89, 469, 1997; Liu et al, Cell 89,477, 1997). FRQ is phosphorylated as soon as it is made and is processively re-phosphorylated over the course of the day (Garceau et al Cell 89, 469, 1997), a modification that appears to play a role in regulating turnover. FRQ spends the early part of each day immediately following its synthesis in the nucleus, and the timing of its localization there appears to be regulated (Luo et al, EMBO J. 17, 1228, 1998). Nuclear localization is required for FRQ function. Within a few hours after FRQ acts to depress the level of its transcript, FRQ levels in the nucleus begin to fall although they continue to rise in the cytoplasm for a few hours before beginning to fall there also. Some FRQ persists in the cytoplasm till the mid-subjective night when positive acting factors, encoded by the wc-1 and wc-2 genes, turn on synthesis of frq and begin the cycle again (Crosthwaite et al Science 276, 763, 1997). Interestingly, the transcription of neither wc gene appears to be strongly rhythmic although WC1 levels appear to cycle and mutations in wc-2 can lead to period length effects. The WC-1 and WC-2 proteins have PAS domains similar to clock-associated proteins from the mouse (mPER1,2,3, CLOCK, BMAL) and the fly (PER), and also similar to light-response associated proteins from a number of systems, suggesting that clock molecules may have arisen from ancient proteins involved in light responsivity (Crosthwaite et al Science 276, 763, 1997).
Light and temperature act to synchronize the internal clock with external environmental cycle in Neurospora as they do in all circadian systems, and in a second effort in the lab the mechanisms of this synchronization have first been worked out in Neurospora. Light delivered at any point within the cycle acts rapidly through the WC proteins to increase the level of frq transcript thereby resetting the clock (Crosthwaite et al. Cell 81, 1003 - 1012, 1995); mammalian circadian rhythms are reset in part in a similar manner (Shigeyoshi et al Cell 91, 1043 - 1053, 1997). Temperature acts posttranscriptionally to determine the absolute level of FRQ in the cell and the site of initiation of translation within the frq transcript, thereby dictating the ratio of long FRQ versus short FRQ (Liu et al, Cell 89, 477 - 486, 1997). Resetting of the clock by changes in temperature can be understood in terms of changes in these set points (Liu et al, Science 281, 825 - 29, 1998).
In a third approach (see figure), we seek to understand the molecular nature of time information and to describe the pathways and mechanisms whereby clocks act to regulate the metabolism and behavior of cells. A first step in this process has been to focus on clock control of gene expression. In work supported by NSF and by NIMH, we have isolated through the use of subtractive and differential hybridization, libraries of genes whose activities are controlled on a daily basis by the clock (Loros et al, Science 243: 385 - 388, 1989; Bell Pedersen et al, PNAS 93: 13096 - 13101, 1996). We are thus in a unique position to study the mechanisms by which the clock can act to bring about transcriptional regulation of genes, and thus effect genetic and metabolic control. Nuclear runon analysis has established that these clock-controlled genes (ccg's) are regulated at the level of transcription (Loros and Dunlap, MCB, 11: 558 - 563, 1991). Thus, there must exist cis-acting regulatory sequences that serve to mediate regulation by the clock. The identification of these sequences (e.g. Bell Pedersen et al, MCB 16: 513 - 521, 1996) and the trans-acting factors that interact with them is now an obvious goal. More than a dozen circadianly expressed genes are known to act downstream of the clock. These clock-controlled genes include a hydrophobin (eas=ccg-2), trehalose synthase (ccg-9), and glyceraldehyde 3-P dehydrogenase (ccg-7=gpd) and play roles in clock regulation of development, stress responses, and intermediary metabolism (Loros, Curr. Opin. Microbiol. 1, 698 - 706).
Finally, in work supported y the NIMH we have recently begun to use genomics approaches as a tool for understanding circadian regulation in both Neurospora and in the mouse. We have, in collaboration with Bruce Roe at the University of Oklahoma, carried out bulk sequencing of cDNAs from morning and evening libraries. The subsequent assembly of these ESTs into sequence contigs (http://www.genome.ou.edu/fungal.html) allows the counting of "hits" of an individual gene in either morning or evening RNAs, and an estimate of the number and identify of ccgs which now appear to number more than 100 . In collaboration with William Schwartz at UMass Worcester and Jeffrey Trent at the National Human Genome Research Institute we have also begun to use DNA microarrays to pursue the global circadian regulation of gene expression in the murine SCN. We have generated time-of-day specific cDNA libraries from the SCN and have isolated timed RNAs that are being used in transcriptional profiling and cluster analysis.
Overall then we are interested in understanding how the circadian system in organized in Neurospora and in mammals, and why evolution has done it in this way. For each of our approaches, our aim is to understand the organization of cells as a function of time, and ultimately to achieve an appreciation of life as a four-dimensional process. There is every reason to believe that the circadian system of Neurospora will be the first circadian system to be completely described at the molecular level, and that what we learn from both fungi and mice will lead us to a better appreciation and understanding of circadian timing in people.
|Organization:||Dartmouth-Hitchcock Medical Center|