Oscillating perceptions: the ups and downs of the CLOCK protein in the mouse circadian system

Abstract

A functional mouse CLOCK protein has long been thought to be essential for mammalian circadian clockwork function, based mainly on studies of mice bearing a dominant negative, antimorphic mutation in the Clock gene. However, new discoveries using recently developed Clock-null mutant mice have shaken up this view. In this review, I discuss how this recent work impacts and alters the previous view of the role of CLOCK in the mouse circadian clockwork.

Figure 1

Model of the mammalian circadian clockwork. The bHLH-PAS transcription factors CLOCK and BMAL1 heterodimerize, and bind E-box elements to drive expression of the mPer1, mPer2 and mCry1 and mCry2 clock genes. The PER (P) and CRY (Cr) proteins form a complex along with CKI δ/ε (CK) and translocate into the nucleus where they bind CLOCK:BMAL1 to inhibit transcription (−), completing the essential negative feedback loop. Posttranslational modification (black lollipops) of several of these components appears to help maintain ~24 h rhythmicity. CLOCK:BMAL1 heterodimers also drive expression of Ror’s (Ro) and Rev-erb α/β (Re), which are transcriptional activators (+) or repressors (−), respectively, that drive cyclical Bmal1 expression via Ror elements within the Bmal1 promoter, in a secondary feedback loop that appears to stabilize rhythms. Clock controlled genes (CCG’s) are output genes directly regulated by this central clockwork (adapted from Reppert and Weaver (2002), and Emery and Reppert (2004)).

Figure 2

Behavioural rhythms persist in Clock^−/− mice, but not Clock^−/−; Npas2^−/− mice. (A) Representative double-plotted actograms depicting behavioural rhythms obtained from mice of the genotypes indicated. Each horizontal line represents two days of recording, and data from each day are plotted twice: on the upper line from 24 to 48 h and the subsequent lower line from 0–24 h. Activity levels are depicted by black marks above each horizontal line. Alternating white and black bars at the top of each plot represents the light cycle, the animals were maintained on, and the white and gray within the records indicates time when the animals were in the light or dark, respectively (data shown were adapted from DeBruyne et al. (2007a) with permission from Nature Neuroscience). (B) Representative mRNA abundance profiles in the SCN of wild-type and Clock^−/− mice (the data shown were adapted from DeBruyne et al. (2006), with permission from Neuron).

Figure 3

SCN mPER2::LUC rhythms are abolished in Clock^−/−; Npas2^−/− double-knockout mice. Representative normalized bioluminescence rhythms obtained from SCN isolated from the indicated genotypes. Two individual records are shown in each graph (data are replotted from DeBruyne et al. (2007b), with permission from Current Biology).

Figure 4

Circadian oscillator function in isolated liver and lung explants is abolished in CLOCK-deficient animals. Representative normalized bioluminescence profiles obtained from isolated liver (left side) and lung (right side) explants taken from the indicated genotypes. Each panel contains data from two independent animals (data are replotted from DeBruyne et al. (2007b), with permission from Current Biology).

Figure 5

Schematic depiction of the spatial relationship between CLOCK and NPAS2 dependent oscillators. Two possible spatial relationships of CLOCK and NPAS2 within the SCN are depicted in the SCN diagram, labelled A and B, and correspond to the simplified clockwork models labelled A and B in the bottom left. The simplified clockwork models illustrate either CLOCK (C) and NPAS2 (N) heterodimerizing with BMAL1 (B) and binding E-box elements to drive circadian gene expression and ultimately oscillator function (oscillator symbol). In A, different SCN neurons contain either CLOCK-dependent (yellow) or NPAS2 dependent (red) oscillators, with no overlap. In B, most cells contain CLOCK-dependent oscillators, but some cells also have oscillators that can use CLOCK or NPAS2 more or less interchangeably (orange). The nonSCN and peripheral oscillators shown are drawn assuming the model shown in A extends to these tissues. The liver and lung contain CLOCK dependent circadian oscillators (yellow), whereas there may be some nonSCN and peripheral tissues that contain NPAS2-dependent oscillators (red). The forebrain may contain a strictly NPAS2-dependent oscillator (Dudley et al. 2003), but it is not known if rhythms in the forebrain are driven by cell-autonomous oscillators. The model shown in B is also a possibility with non-SCN and peripheral tissues (not shown).