ABSTRACTCircadian clocks regulate ∼24 hour oscillations in gene expression, behavior, and physiology. While the molecular and neural mechanisms of circadian rhythms are well characterized, how cellular organization of clock components controls circadian clock regulation remains poorly understood. Here, we elucidate how clock proteins regulate circadian rhythms by controlling the spatiotemporal organization of clock genes. Using high-resolution live imaging techniques we demonstrate that Drosophila clock proteins are concentrated in a few discrete foci and are organized at the nuclear envelope; these results are in contrast to longstanding expectations that clock proteins are diffusely distributed in the nucleus. We also show that clock protein foci are highly dynamic and change in number, size, and localization over the circadian cycle. Further, we demonstrate that clock genes are positioned at the nuclear periphery by the clock proteins precisely during the circadian repression phase, suggesting that subnuclear localization of clock genes plays an important role in the control of rhythmic gene expression. Finally, we show that Lamin B receptor, a nuclear envelope protein, is required for peripheral localization of clock protein foci and clock genes and for normal circadian rhythms. These results reveal that clock proteins form dynamic nuclear foci and play a hitherto unexpected role in the subnuclear reorganization of clock genes to control circadian rhythms, identifying a novel mechanism of circadian regulation. Our results further suggest a new role for clock protein foci in the clustering of clock-regulated genes during the repression phase to control gene co-regulation and circadian rhythms.SIGNIFICANCEAlmost all living organisms have evolved circadian clocks to tell time. Circadian clocks regulate ∼24-hour oscillations in gene expression, behavior and physiology. Here, we reveal the surprisingly sophisticated spatiotemporal organization of clock proteins and clock genes and its critical role in circadian clock function. We show, in contrast to current expectations, that clock proteins are concentrated in a few discrete, dynamic nuclear foci at the nuclear envelope during the repression phase. Further, we uncovered several unexpected features of clock protein foci, including their role in positioning the clock genes at the nuclear envelope precisely during the repression phase to enable circadian rhythms. These studies provide fundamental new insights into the cellular mechanisms of circadian rhythms and establish direct links between nuclear organization and circadian clocks.