CLOCK and NPAS2 have overlapping roles in the suprachiasmatic circadian clock

Abstract

Heterodimers of CLOCK and BMAL1, bHLH-PAS transcription factors, are believed to be the major transcriptional regulators of the circadian clock mechanism in mammals. However, a recent study shows that CLOCK-deficient mice continue to exhibit robust behavioral and molecular rhythms. Here we report that the transcription factor NPAS2 (MOP4) is able to functionally substitute for CLOCK in the master brain clock in mice to regulate circadian rhythmicity.

Figure 1

Locomotor activity rhythms are abolished in double knockout mice. (a–d) Representative activity rhythms of (a) wild-type (left), CLOCK-deficient (Clock^−/−; middle) and NPAS2-deficient mice (Npas2^−/−; right), (b) CLOCK-deficient mice having one functional allele of Npas2 (Clock^−/−; Npas2^+/−), (c) double knockout mice (Clock^−/−; Npas2^−/−), and (d) NPAS2-deficient mice having one functional allele of Clock (Clock^+/−; Npas2^−/−). Each horizontal line represents 48 h; the second 24-h period is plotted both to the right and below the first. Vertical bars represent periods of wheel-running activity. Animals were initially housed in a 12-h light, 12-h dark cycle (LD) and were then transferred to constant darkness (DD). The timing of the LD cycle is indicated by the bar above the top records, and gray shading in the records indicates darkness. Numbers on the left indicate days of study. The yellow lines delineate the phase of activity onset in constant darkness. (e) Periodogram estimates of period for each genotype. Each bar is the mean ± s.e.m.; the number of animals is indicated in each bar. *P < 0.05, ANOVA compared with wild type (Tukey HSD for unequal n), AR indicates arrhythmicity. (f) Circadian amplitude (power from periodogram analyses) from days 6–15 in DD. The four rhythmic genotypes did not differ (ANOVA, P > 0.05).

Figure 2

Clock gene mRNAs are arrhythmic in the SCN of double knockout mice. (a) Brains from wild-type mice (black lines), NPAS2-deficient mice (Npas2^−/−, green lines) and double-knockout mice (Clock^−/−;Npas2^−/−, brown lines) were collected at 4-h intervals on the first day in constant darkness. mRNA levels for mPer1, mPer2, Bmal1 and Rev-erbα were determined by in situ hybridization. Each point represents the mean ± s.e.m. (n = 3–4 animals for each time point except for circadian time (CT) 2 in which n = 2 for mPer1 and Bmal1 in the double knockout mice). The horizontal bar at the bottom of each panel represents the light-dark cycle (gray = light, black = dark) the animals were housed in before placement in constant dark. For each gene, the mRNA levels were rhythmic in wild-type and NPAS2-deficient SCN (ANOVA, P < 0.05), whereas the mRNA levels were not rhythmic in the double knockout SCN (ANOVA, P > 0.1). (b) Representative in situ hybridization autoradiographs showing mPer1 and Bmal1 mRNA expression levels in wild-type and double-knockout (Clock^−/−;Npas2^−/−) brains collected at CT 6. Consecutive coronal sections, at the level of the SCN, are shown for each genotype.

Figure 3

SCN molecular rhythms persist in culture without CLOCK. Bioluminescence rhythms from cultures of the SCN of wild-type mice expressing mPeriod2::Luciferase (Clock^+/+;mPer2^Luc, top panel) and CLOCK-deficient mice expressing the fusion protein (Clock^−/−;mPer2^Luc, bottom panel). The data shown (n = 2, wild-type SCN; n = 2, CLOCK-deficient SCN) were normalized by the average luciferase activity over the duration of the experiment and 24-h trends were removed. The raw data from these and additional experiments are shown in Supplementary Figure 1.