one-step-crispr-based-strategy-for-endogenous-gene-tagging-drosophila

One-step CRISPR-based Strategy for Endogenous Gene Tagging in <em>Drosophila melanogaster</em>

The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent of techniques that ...

University of Michigan

Circadian clocks regulate ∼24-h oscillations in gene expression, behavior, and physiology. While the genetic and molecular mechanisms of circadian rhythms are well characterized, what remains poorly understood are the intracellular dynamics of circadian clock components and how they affect circadian rhythms. Here, we elucidate how spatiotemporal organization and dynamics of core clock proteins and genes affect circadian rhythms in  clock neurons. Using high-resolution imaging and DNA-fluorescence in situ hybridization techniques, we demonstrate that  clock proteins (PERIOD and CLOCK) are organized into a few discrete foci at the nuclear envelope during the circadian repression phase and play an important role in the subnuclear localization of core clock genes to control circadian rhythms. Specifically, we show that core clock genes,  and , are positioned close to the nuclear periphery by the PERIOD protein specifically during the repression phase, suggesting that subnuclear localization of core clock genes might play a key role in their rhythmic gene expression. Finally, we show that loss of Lamin B receptor, a nuclear envelope protein, leads to disruption of PER foci and  gene peripheral localization and results in circadian rhythm defects. These results demonstrate that clock proteins play a hitherto unexpected role in the subnuclear reorganization of core clock genes to control circadian rhythms, revealing how clocks function at the subcellular level. Our results further suggest that clock protein foci might regulate dynamic clustering and spatial reorganization of clock-regulated genes over the repression phase to control circadian rhythms in behavior and physiology.

Clock proteins regulate spatiotemporal organization of clock genes to control circadian rhythms.

Circadian clocks are cell autonomous timekeepers that regulate ∼24-h oscillations in the expression of many genes and control rhythms in nearly all our behavior and physiology. Almost every cell in the human body has a molecular clock and networks of cells containing clock proteins orchestrate daily rhythms in many physiological processes, from sleep-wake cycles to metabolism to immunity. All eukaryotic circadian clocks are based on transcription-translation delayed negative feedback loops in which activation of core clock genes is negatively regulated by their cognate protein products. Our current understanding of circadian clocks has been accumulated from decades of genetic and biochemical experiments, however, what remains poorly understood is how clock proteins, genes, and mRNAs are spatiotemporally organized within live clock cells and how such subcellular organization affects circadian rhythms at the single cell level. Here, we review recent progress in understanding how clock proteins and genes are spatially organized within clock cells over the circadian cycle and the role of such organization in generating circadian rhythms and highlight open questions for future studies.

Yangbo Xiao

Department of Cell and Developmental Biology

University of Michigan

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

Yangbo Xiao

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cell and Developmental Biology

University of Michigan

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

Department of Cell and Developmental Biology