PMC

Light-induced phase shifts of PER2::LUC rhythms in Per2^Luc;mOpn4 cells. (A) Per2^Luc;mOpn4 cells exhibit rhythmic bioluminescence that can be reset by light. Representative examples of bioluminescence rhythms [relative light units/min (RLU)] and phase shifts in response to 10-min light pulses are shown. Shaded area is enlarged in the Inset. (B) Magnitude of phase shift [mean ± SD (n = 3)] increases with increasing duration of light pulse (3 × 10¹⁵ photons cm⁻² s⁻¹, 480 nm) at CT3 and CT22 but not at CT12 (CT12 = peak PER2::LUC level).

Light-induced CREB phosphorylation and Per gene transcription in Per2^Luc;mOpn4 cells. (A) Immunoblot analysis shows light-induced rise in pCREB levels after light stimulation at CT22. (B and C) Chromatin immunoprecipitation with antiacetylated H3K9 (B) and anti-pSer-133 CREB antibody (C) indicate enrichment of immunoprecipitated Per2 promoter DNA sequences relative to the antibody control in response to light pulses at CT12, CT22, or CT3 for acetylated H3K9 and at CT22 for pCREB. (D–F) q-PCR analysis shows that light exposure at CT12 (D), CT22 (E), or CT3 (F) increased Per2 mRNA levels compared with dark-treated samples. Samples were collected before light pulse (prepulse, or PP) as well as at indicated times (x axis) after 10-min light stimulation. Values in B–F are mean ± SEM (B and C) or mean ± SD (D–F); n = 3; *, P < 0.01 compared with the prepulse; Student's t test.

Circadian modulation of PER2::LUC induction correlates with pCREB binding to the Per2 promoter. (A) Median normalized bioluminescence counts (thin colored lines) from 14 different cultures in 12-h windows starting immediately after a 10-min light pulse or a control culture (thick green line) are shown. Color-matched arrows along the x axis mark the times of light pulses. (B) Immunoblot analysis of CREB and pSer-133-CREB levels in whole-cell lysates from cells harvested 15 min after a 10-min light (+) or dark (−) treatment at various circadian times. (C) ChIP assay for pCREB at the Per2 promoter CRE site shows both circadian and light modulation of its binding. Chromatin-immunoprecipitated DNA was quantified by qPCR and normalized to the signal from protein-A agarose beads. Average normalized signals (± SD; n = 3 qPCR runs) from unstimulated cells or from cells collected 30 min after 10-min light pulses are shown. ChIP signal from stimulated cells was significantly higher from control cells at all time points (*, P < 0.05 Student's t test). Data are representative of three independent experiments.

Circadian modulation of phase shifts and amplitude changes in Per2^Luc;mOpn4 fibroblasts. (A) Double-plotted PRC from 56 different light-treated samples. Each point represents the magnitude of phase shift (y axis) upon light treatment at a given time (x axis). (B) The average relative amplitude (filled square, ± SEM; n = 5–11) of the same 56 samples receiving light pulses in 4-h bins shows clear circadian modulation. Each point represents relative amplitude of individual samples (y axis) after a light pulse, expressed as percentage of amplitude of the untreated cultures during a comparable interval. Average amplitude of the untreated samples (solid horizontal line) and SEM (two parallel dashed lines, n = 5) are also shown. Statistically significant changes in amplitude (Student's t test, P < 0.05) compared with the controls are indicated by asterisks. Samples where no reliable rhythm or phase could be determined are shown in red (A and B). (C) Normalized bioluminescence traces from three representative plates that received a 10-min light pulse near CT12 (green) or CT24 (red) or received no light (black), showing a phase-dependent effect on transient induction of Per2, phase shift, and amplitude after the light pulse. For clarity, data from 3.5 to 5 days is magnified in the Inset.

Phase shifts and amplitude changes in individual oscillators. (A) Bioluminescence heatmap of 166 fibroblasts from five different experiments in which cells received light stimulation (cyan line). (B) Overall bioluminescence from two separate cultures that received light (arrow) at the peak (orange) or the trough (blue) of the PER2::LUC rhythm show changes of amplitude in the subsequent days. (C) The respective color-coded phases (triangles) of individual fibroblasts before (Left) and after (Right) light stimulation are shown. (D and E) Sample traces of four individual cells that received light (arrow) either in the CT8–16 interval (D) or in the CT20–4 interval (E). y axis, photons/min. (F) Phase transition plot shows the phases of 166 cells (filled circles) before and after a light pulse. Light pulse at approximately CT12 resulted in small and relatively homogeneous phase shifts, whereas light at CT24 produced extremely heterogeneous phase shifts. (G) Based on the time of light pulse, cells in F were binned into six different 4-h bins. The old (Upper) and new phases (Lower) of these six groups of cells are shown in phase plots. (H) Relative average amplitudes (± SEM; n = 16–43) expressed as percentage of amplitude before light stimulation (y axis) for each of the six 4-h binned groups show circadian modulation (*, P < 0.05; Student's t test). Average amplitude before light pulse (solid horizontal line) and SEM (two parallel dashed lines, n = 166) are shown. In C and G, circadian times from 0 to 24 are plotted around the circumference of the phase plots. Arrow direction indicates average phase of the cells, and its length indicates phase coherence among cells. Values are in SI Tables 1 and 2.