Circadian Rhythms, Behavior, and Gene Expression
When we began our studies of Drosophila circadian rhythms more than 25 years ago, our goal was to define the machinery that underlies the almost ubiquitous process of circadian rhythmicity. Our entrée into this problem was the period gene (per) of Drosophila melanogaster, discovered more than 10 years earlier in pioneering behavioral genetic experiments by Ronald Konopka and Seymour Benzer. In 1990, we discovered that per mRNA as well as its encoded protein (PER) undergoes fluctuations in level during the circadian cycle. These observations and others indicated that PER is important for a negative-feedback loop of gene expression. As PER was shown to be nuclear, we proposed that it inhibits the transcription of its own mRNA. Temporally controlled negative feedback at the transcriptional level is now an accepted feature of circadian timekeeping in mammals, plants, Neurospora, and even cyanobacteria. Moreover, the many additional clock components defined by genetics in Drosophila over the past 10–15 years are largely conserved and perform similar functions in the mammalian clock. This indicates that the machinery, as well as the principles, of the Drosophila clock is widely conserved, at least in animals.
Our current circadian work has two major goals: (1) to understand in mechanistic detail how the Drosophila circadian clock functions, including how circadian transcriptional regulation takes place; (2) to understand the neural circuit(s) relevant to circadian timekeeping within the fruit fly brain and the functions of individual circadian neurons.
Although transcription factors and transcriptional regulation play key roles in circadian rhythmicity, post-transcriptional regulation has received recent, prominent attention. This is due to the potent effect of kinase mutants on the Drosophila clock, as well as some remarkable results from the cyanobacterial circadian system. Nonetheless, our results on the first goal mentioned above continue to reinforce the importance of transcriptional regulation and the heterodimeric transcription factors Clock (CLK) and Cycle (CYC). This key complex drives the transcription of per and tim, and a small number of additional direct target genes have been identified. One is clockwork orange, and its protein contributes to feedback repression and complements the role of PER-TIM in inhibition of CLK-CYC activity.
We are using a variety of biochemical and genetic approaches to further dissect the transcriptional and post-transcriptional regulatory mechanisms that have an impact on the clock. Many of these new strategies are designed to identify new clock genes and simultaneously to identify new regulatory mechanisms of interest. One current approach is that of ChIP-tiling arrays or ChIP-seq, i.e., chromatin immunoprecipitation followed by tiling or by high-throughput sequencing. We have carried out circadian time course experiments, 6 time points/day, with antibodies against CLK, PER and RNA Pol II. The results indicate cyclical binding of CLK and PER to an enormous number of potential direct target genes. Some of these encode proteins that may contribute to transcriptional regulation. Others have potential roles in the post-transcriptional regulation of circadian function, suggesting that the core transcriptional feedback loop may be upstream of some modes of post-transcriptional regulation. One attractive explanation for the large number of direct target genes is the heterogeneity of the head tissue used for these kinds of experiments in Drosophila. Conceivably, the circadian machinery accesses different direct target genes in different neurons.
We are also using high-throughput sequencing to address old as well as new circadian issues. For example, how extensive are mRNA circadian oscillations in fly heads? This question has been addressed in many labs with affymetrix arrays, and we are now using high-throughput sequencing. Another newer question is, are there microRNAs (miRNAs) that undergo circadian oscillations in fly heads? The answer is yes, and some of these have impressive cycling amplitudes. This indicates that they must have short half-lives. This has implications for the metabolism of miRNAs, which are usually very stable; it is not known how their degradation is regulated. The observation also suggests that translational regulation makes important contributions to rhythms. Indeed, recent results show that the abundant miRNA bantam regulates the translation of the important circadian transcription factor CLK.
High-throughput sequencing is also being used to address the issue of nascent splicing. We have been interested for many years in the extent to which splicing takes place prior to 3'-end formation. Although we have only studied this question in yeast, we are now addressing it in Drosophila tissue culture cells. To this end, we are trying to measure at a genomic level how much nascent splicing takes place. What are the general rules, and which are the exceptional genes? We are also addressing the circadian regulation of alternative splicing in flies: which splicing events appear to be under circadian regulation? We are currently in a descriptive phase, but we hope to move on to two more mechanistic issues: how does the regulation take place and do any of these splicing events make a contribution to core circadian timekeeping?
We have focused these past few years on various brain-anatomical aspects of Drosophila rhythms. There are only seven neuronal groups in the adult brain, comprising about 150 cells on each side; they express high levels of clock genes and usually undergo circadian oscillations of clock gene mRNA and protein in synchrony. One neuronal group, the small ventral-lateral neurons (s-LNvs), controls the characteristic morning activity peak of the insect activity pattern, and another controls the evening peak. In addition, the morning group is the master pacemaker in constant darkness. This nighttime oscillator sends a daily resetting signal to the evening or daytime oscillator under these standard light:dark conditions, which ensures that the two groups stay in sync. The relationship between these two oscillators can switch as a function of environmental conditions, as the daytime cells become the masters in constant light.
These different relationships to light are mirrored by more direct relationships to light: one set of cells appears directly light-sensitive to dawn, whereas another is probably light-sensitive to dusk or to lights-off. The role of light and its relationship to circadian cells also have an impact on fly arousal and sleep. Intriguingly, the dawn cells are arousal cells, whereas the lights-off cells appear to promote sleep—as one might expect for a diurnal animal. We are trying to identify additional groups of brain neurons with a major impact on sleep, and preliminary results focus attention outside of the circadian network. By combining these different neuronal groups with different in vivo approaches to manipulate neuronal function, we are trying to gain insight into the circuitry and rules that underlie sleep regulation.
The increasing awareness of neuronal specificity within the circadian network also offers opportunities for gene discovery, i.e., profiling mRNAs within circadian neuronal subtypes. Although core clock genes are highly enriched in all brain circadian neurons, many mRNAs appear to be enriched in a circadian neuron-specific manner, which may provide a link to the distinct functions of different neuronal subgroups. One specific mRNA, enriched in dorsal neurons, suggests that translational regulation may be important for the response to light signals. mRNAs differentially expressed within specific neuron subsets should also provide invaluable tools for manipulating specific circadian neurons without affecting others.