1
Cytosolic and Transcriptional Cycles Underlying Circadian Oscillations

Michael H. Hastings1 and John S. O'Neill2

1 Division of Neurobiology, Medical Research Council Laboratory of Molecular Biology, Cambridge, UK

2 Division of Cell Biology, Medical Research Council Laboratory of Molecular Biology, Cambridge, UK

1.1 Introduction

Circadian (circa- approximately, -dian a day) clocks are the internal pacemakers that drive the daily rhythms in our physiology and behavior that adapt us to the 24-hour world (Duffy et al., 2011). They thereby maintain temporal coherence in our core metabolism, even when individuals are held in isolation, experimentally deprived of external timing cues such as light-dark (LD) cycles. As a result of this ability of our endogenous circadian system to define internal time and use it to drive daily rhythms, our brains and bodies can be viewed as 24-hour machines, alternating between states of wakefulness and sleep, catabolism and anabolism, growth/repair and physical activity. It is now widely recognized that disturbance of this daily program can carry significant costs for morbidity and even mortality (Hastings et al., 2003). Some personal insights into this can come from the subjective experiences of jet lag. More insidiously, however, the disturbance of nocturnal sleep, and consequent disaffected mood, loss of mental capacity and social disruption, is a common element of neurodegenerative and psychiatric conditions (Hatfield et al., 2004; Wulff et al., 2010; Bliwise et al., 2011) (This volume, several chapters). Moreover, epidemiological evidence now associates increased risk of cancer as well as cardiovascular and metabolic diseases with extensive experience of rotational shift-work (Knutsson, 1989; Viswanathan et al., 2007; Huang et al., 2011) (This volume, Chapter 13), a life-style that will inevitably compromise circadian coherence, and which represents a major and growing hazard to public health. Evolution has programmed us to live by a 24-hour day and where genetic, pathological, environmental or social factors drive us against this program, we pay a heavy price. Conversely, the recognition that our body is a 24-hour machine, with different metabolic and physiological states across day and night, provides a route into enhancing therapeutic efficacy by administering medicines on a schedule that maximizes their bioavailability and by targeting disease states at their most critical and vulnerable phases of the day (Levi and Schibler, 2007).

Key to appreciating the role of the circadian clock in both health and illness, and thereby identifying novel therapeutic strategies, is the unravelling of its molecular and cellular bases. Whilst the formal properties of circadian clocks have been understood for over 60 years, and the identification in 1972 of the?suprachiasmatic nucleus (SCN) as the brain's principal pacemaker provided a neuroanatomical focus to circadian biology (Weaver, 1998; Chapter 3) (Fig. 1.1a-e), proper mechanistic understanding of the timing process proved to be elusive. This changed dramatically from the late 1970s onwards, when "circadian clock genes" and their mechanisms of action were identified: firstly in Drosophila, then in Neurospora, and more recently in mouse (Takahashi et al., 2008). The outcome of these studies was to reveal that an autoregulatory negative feedback oscillator, based on sequential transcriptional and posttranslational processes, lies at the heart of the circadian timepieces of these divergent groups. Even though the molecular components may differ, the "logic" of the mechanism is conserved. But things move on, and there is growing realization that these transcriptionally based clocks do not operate in isolation; rather, they are mutually dependent upon intrinsically rhythmic cytosolic signals (cAMP, Ca2+, kinases), such that the cell as a whole has a resonant structure, tuned to 24-hour operation (Hastings et al., 2008). Finally, the most recent development has been to show that even in cells lacking transcriptional apparatus (most notably mammalian erythrocytes), circadian cycles of metabolic state can be sustained (O'Neill and Reddy, 2011). The purpose of this chapter is to review the development of this molecular and cellular model of the circadian clockwork of mammals.

Fig. 1.1 The suprachiasmatic nucleus (SCN) as circadian pacemaker. (a) Frontal MRI view of human brain to identify location of SCN (boxed) in anterior hypothalamus at junction of third ventricle and optic chiasm (courtesy of Dr Adrian Owen, MRC CBU, Cambridge UK). (b) Comparable view of mouse brain labelled auto-radiographically to reveal SCN in ventral hypothalamus. (c) Recording of wheel-running activity of mouse (double-plotted on 48-h time base) free-running in continuous dim red light, with a sustained circadian period of slightly less than 24 h (King et al., 2003). (d) Behavior of same mouse following ablation of SCN - note total loss of circadian organization in absence of SCN, but no change in overall activity level. (e) Behavior of same SCN-lesioned mouse following intracerebral graft of SCN from a Clockdelta19 mouse. Note modest restoration of circadian patterning to behavior, but with a period longer than 24 h as determined by graft genotype. This genetic specification of circadian period proves that the rhythm is controlled by the grafted SCN and, thus, the SCN is the definitive pacemaker to circadian behavior. (f) Schematic representation of conventional TTFL at the heart of the SCN circadian pacemaker. (See text for details.)

1.2 Assembling the transcriptional feedback loop

1.2.1 Discovering clock genes and their actions in lower species

The idea that a complex behavioral trait such as the circadian cycle of rest and activity could be understood from the viewpoint of single gene actions was, for some time, contentious in both the circadian field and also more widely. Nevertheless, the creation by Ron Konopka and Seymour Benzer of mutant Drosophila with atypically short or long periods to their circadian behavior, and the subsequent cloning of the Period gene as the molecular target of these mutations, initiated a revolution in clock biology (Konopka, 1987 ). Alongside the Frq (Frq) gene of Neurospora, cloned by Jay Dunlap and colleagues (Loros et al., 1989), Period (Per) provided an entry point into the molecular mechanisms of clocks: changes in the encoded proteins could make the clock run faster, or slower or not at all. They therefore MUST be an intrinsic part of the clockwork. Moreover, it became apparent that the key action of the encoded proteins was to inhibit the expression of their cognate genes. Given that there is an inevitable time lag between transcriptional activation and nuclear entry of the fully formed protein, an oscillation is bound to ensue, as in any other delayed negative feedback system (Hardin et al., 1990; Aronson et al. 1994). Indeed, autoregulation of this type is well recognized in molecular biology, with oscillations commonly occurring over a couple of hours. The critical property here, however, is that the dynamics of the contributory stages (gene activation, protein synthesis, intracellular transport, protein degradation) are extended such that the cycle runs for approximately 24 hours. Subsequent mutational and biochemical studies revealed that Per and Frq are components of dynamic, multiprotein complexes, the assembly of which is facilitated in part by their protein interaction domains (Hardin, 2005; Crosthwaite et al., 1997). Of particular note were the so-called?PAS (Per-Arnt-Sim) interaction domains of Per. The positive drives to the feedback loops that stimulate expression of Per and Frq, comes from additional PAS-containing proteins: CLOCK and CYCLE in flies (Allada et al., 1998; Rutila et al., 1998), and WHITE COLLAR 1 and 2 in Neurospora (Crosthwaite et al., 1997; de Paula et al., 2007). After forming heteromers, these positive factors activate transcription via specific regulatory sequences in the enhancer regions of Per and Frq, respectively. Thus, positive factors drive the expression of negative factors, which in turn oppose the positive drive leading to a decline in negative factor abundance, which allows the cycle to start again approximately 24 hours after the previous point of initiation.

Although both systems are light sensitive - a prerequisite for synchronization with solar cycles and, thereby, environmental time, their molecular basis to entrainment is different. In flies, the stability of PER is dependent on association with another circadian protein, TIMELESS (Myers et al., 1996; Koh et al., 2006), which in turn is subject to degradation by CRY, a light-dependent factor with similarity to photolyase DNA repair proteins (Cashmore, 2003). Consequently, PER protein can only accumulate in the night, thereby stably entraining the entire molecular cycle to solar time. In contrast, the light-sensitive component in the Neurospora loop is the positive factor White Collar-1, which binds?FAD (flavin adenine dinucleotide) as a chromophore (Crosthwaite et al., 1997; de Paula et al., 2007). Thus,...