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

Abstract

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 Drosophila clock neurons. Using high-resolution imaging and DNA-fluorescence in situ hybridization techniques, we demonstrate that Drosophila 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, period and timeless, 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 per 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.

Keywords: circadian rhythms; live imaging; nuclear organization.

Fig. 1.

PER protein is organized into a few discrete nuclear foci during the circadian repression phase. (A) Schematic of the Drosophila circadian clock network, with the major classes of clock neurons labeled. (B) Schematic of the core molecular clock in the Drosophila clock neurons. During the activation phase, the CLK protein complex binds to the E-box sequence of the per gene and drives its transcription. During the repression phase, the PER protein enters the nucleus and inhibits CLK transcription factor activity, silencing its own expression. (C) Data from per-mNeonGreen;Clk-GAL4>UAS-CD4-tdTomato flies entrained to LD cycles (ZT0: lights on; ZT12: lights off). Representative images of PER foci (green) in sLNvs (cell membrane labeled with tdTomato and shown in red) over the circadian cycle. N denotes the nucleus and C denotes the cytoplasm. (D and E) Quantitation of PER foci intensity (D) and foci number per sLNv (E) at specific ZTs over the LD cycle. The '0' denotes that there is no PER protein in the sLNvs. (F) Representative time-lapse images of sLNvs showing PER foci undergoing fusion. (G) Representative images of PER foci at ZT0 in other groups of clock neurons (lLNv, DN1_p, and DN2). (H) Quantitation of PER foci intensity in all classes of clock neurons at ZT0. (Scale bars, 1 μm.) The statistical test used was a Kruskal–Wallis test (D, E, and H). *P < 0.05, **P < 0.005, ****P < 0.0001. Individual data points, mean, and SEM are shown. 'n' refers to the number of neurons. See Dataset S2 for detailed statistical analysis.

Fig. 2.

PER foci persist in constant darkness and are stereotypically positioned at the nuclear envelope. (A) per-mNeonGreen;Clk-GAL4>UAS-CD4-tdTomato flies are entrained to LD cycles for 5 d and released into DD. Representative images of PER foci in sLNvs at 2-h intervals on day 1 of DD. CT refers to circadian time. (B and C) Quantitation of PER foci intensity (B) and foci number per sLNv (C) at specific CTs during day 1 of DD. (D) Representative images of PER foci in sLNvs from per-EGFP;Clk-GAL4>UAS-unc84-tdTomato flies. PER foci are located close to the nuclear envelope (marked in red) during peak repression phase. N denotes the nucleus, and C denotes the cytoplasm. (E) Quantitation of the percentage of PER foci that are less than 0.5 μm away from the nuclear envelope at different ZTs. (F) Quantitation of the distance between PER foci and the nuclear envelope at different ZTs. (Scale bars, 1 μm.) The statistical test used was a Kruskal–Wallis test (B, C, E, and F). *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001. Individual data points, mean, and SEM are shown. 'n' refers to the number of neurons. See Dataset S2 for detailed statistical analysis.

Fig. 3.

PER foci dynamics and spatial organization are regulated by DBT kinase. (A and B) Representative images of sLNvs from per-EGFP;Clk-GAL4>UAS-dbt^L,UAS-CD4-tdTomato flies (A) and per-EGFP;Clk-GAL4>UAS-dbt^S,UAS-CD4-tdTomato flies (B) at different ZTs over the circadian cycle. (C and D) Quantitation of PER foci intensity in sLNvs from dbt^L (C) and dbt^S (D) mutants. (Scale bars, 1 μm.) The statistical test used was a Kruskal–Wallis test (C and D). **P < 0.005, ****P < 0.0001. Individual data points, mean, and SEM are shown. 'n' refers to the number of neurons. See Dataset S2 for detailed statistical analysis.

Fig. 4.

CLK protein is concentrated in nuclear foci in the clock neurons during the repression phase. (A) Schematic of CRISPR/Cas9 genome editing to generate Clk-mScarlet-I flies. (B) Clk-mScarlet-I flies were entrained to LD cycles (ZT0: lights on; ZT12: lights off) for 5 d and released into DD for 7 d. Averaged population locomotor-activity profiles of Clk-mScarlet-I flies (n = 62) in LD and DD with rest–activity shown for two consecutive days in the same line. These flies display rhythmic behaviors with a period of 24.39 ± 0.10 h, with activity peaks around the time of lights on and lights off (see SI Appendix, Fig. S10B for details). (C) Representative images of CLK (red) in DN1_ps (cell membrane labeled with GFP and shown in green) from Clk-GAL4>UAS-CD8-GFP;Clk-mScarlet-I flies over the circadian cycle. N denotes the nucleus, and C denotes the cytoplasm. (D and E) Quantitation of CLK foci intensity (D) and foci number per DN1_p (E) at specific ZTs over the 24-h LD cycle. 'D' denotes that CLK protein is diffusely distributed in the nucleus and is not concentrated in foci at those time points. (F) Representative images of CLK and PER foci colocalization at ZT0 in DN1_ps from per-mNeonGreen;Clk-mScarlet-I flies. A total of 86% of CLK foci colocalize with PER foci (from 65 CLK foci from 10 brains). (G) Representative images of CLK foci in DN1_ps at ZT0 from control (+;Clk-GAL4>UAS-CD8-GFP;Clk-mScarlet-I) and per⁰¹;Clk-GAL4>UAS-CD8-GFP;Clk-mScarlet-I mutant flies. (H) Quantitation of the percentage of DN1_ps with CLK foci at ZT0 in control and per⁰¹ mutant flies. Each of the data points corresponds to measurements from an individual brain. (Scale bars, 1 μm.) Statistical tests used were a Kruskal–Wallis test (D and E) and a Mann–Whitney U test (H). *P < 0.05, **P < 0.005, ****P < 0.0001. Individual data points, mean, and SEM are shown. 'n' refers to the number of neurons. See Dataset S2 for detailed statistical analysis.

Fig. 5.

Core clock genes are positioned close to the nuclear periphery by PER protein during the repression phase. (A) Representative images of DNA-FISH results using per-gene probes (green dots) during repression (ZT0) and activation (ZT12) phases in lLNvs from WT and per⁰¹ null mutant flies. Anti-PDF antibody (red) was used to identify lLNv clock neurons, and DAPI (blue) was used to mark the nucleus boundary. N denotes the nucleus, and C denotes the cytoplasm. White arrowheads denote the location of per gene in the nucleus. (B) Representative images of DNA-FISH results using tim-gene probes (white dots) at ZT0 and ZT12 in lLNvs from WT and per⁰¹ null mutant flies. White arrowheads denote the location of tim gene in the nucleus. (C and D) Quantitation of the distance between per gene and the nucleus boundary (C) and tim gene and the nucleus boundary (D) at ZT0 and ZT12 in lLNvs from WT and per⁰¹ mutant flies. (Scale bars, 1 μm.) The statistical test used was a Kruskal–Wallis test (C and D). ****P < 0.0001. Individual data points, mean, and SEM are shown. 'n' refers to the number of neurons. See Dataset S2 for detailed statistical analysis.

Fig. 6.

LBR is required for tethering per gene to the nuclear envelope and for circadian rhythms. (A) Representative images of DNA-FISH results using per-gene probes (green dots) at ZT0 and ZT12 in lLNvs from control (+>UAS-LBR-RNAi) and Clk-GAL4>UAS-LBR-RNAi flies. (B) Quantitation of distance between per gene and the nucleus boundary at ZT0 and ZT12 in lLNvs from control (+>UAS-LBR-RNAi) and Clk-GAL4>UAS-LBR-RNAi flies. (C) Representative images of PER foci in sLNvs in control (perEGFP;Clk-GAL4>UAS-CD4-tdTomato,+) and experimental (per-EGFP;Clk-GAL4>UAS-CD4-tdTomato,LBR-RNAi) flies at CT48 on the third day of DD3. (D) Quantitation of percentage of sLNvs with PER foci from control and experimental flies at CT48. Each of the data points correspond to measurements from an individual brain. (E) A model for spatiotemporal localization of clock protein–chromatin complexes in clock neurons over the circadian cycle. (Scale bars, 1 μm.) The statistical test used was a Kruskal–Wallis test (B and D). ****P < 0.0001. Individual data points, mean, and SEM are shown. 'n' refers to the number of neurons. See Dataset S2 for detailed statistical analysis.