The molecular clock machinery in mammals

Already in the 1930s, Erwin Bünning reported the inheritance of circadian period in plants [13]. However, it was not until almost 50 years later, that the basic components of the molecular clock machinery started to be discovered in Drosophila. Despite cyanobacteria and erythrocytes, which do not required a transcriptional machinery to generate circadian oscillations [50], [138], circadian clocks of most living organism follow the same molecular design principle: transcriptional-translational feedback loops (TTFL) generate circadian oscillations by self-sustained, temporally regulated cycles of clock gene expression in combination with time delayed repression or activation of these genes by their own protein products (Figure 1-4).

Circadian TTFLs are generated by a defined set of genes, the so-called clock genes, which contribute to different interlocked feedback loops generating circadian

oscillations in a tissue-specific fashion [81]. Components of the “core feedback loop”

are Clock, Bmal1 (also called Arntl), Period1/2/3, and Cry1/2. Following translation CLOCK and BMAL1 form basic helix-loop-helix (bHLH) transcription factor complexes that induces the expression of their target genes Period1/2/3 and Cry1/2 by binding to E-box enhancer elements in their promoter regions [139]. After a defined time delay, necessary to establish ~24 hour oscillations, PER and CRY protein products relocate to the nucleus where they suppress CLOCK/BMAL1 transcriptional activity either by direct or by indirect interaction [140], [141]. During the first half of a circadian cycle CLOCK/BMAL1 transcriptional activity is increasing until the accumulation of PER and CRY proteins results in the formation of repressive complexes. Depending on the half-lives of PER/CRY complex components, transcriptional repression is progressively relieved throughout the second half of a circadian cycle and until CLOCK/BMAL1 activity is restored, leading to the initiation of a new cycle [142], [143]. Post-translational modifications, most importantly phosphorylation and ubiquitination, have been described to regulate the activity and degradation of PER and CRY proteins [142]. CLOCK/BMAL1 heterodimers also regulate the E-box driven expression of Nr1d1/2 (also called Rev-erba/b), Rora/b, and Dbp [10], [142], [144], which serve to fine-tune oscillations generated by the core loop. The nuclear receptors REV-ERBa/b and RORa/b regulate Bmal1 transcription by competitive binding to its RevDR2 and RORE enhancer elements and ensure nearly anti-phasic expression of Bmal1 and Per1/2/3 [145], [146]. Nr1d1/2 and Rora/b driven feedback on Bmal1 transcription is often referred to as “stabilizing feedback loop”. While the core loop (Bmal1, Clock, Per, Cry) is essential for the generation of circadian oscillations per se, the stabilizing loop seems to be important for the temporally organized expression of core loop components and with that for the regulation of circadian phase and period. Indeed, it has been shown that REV-ERBa knock-out mice, with regard to their locomotor activity, display shortened rhythms, larger period diversity, as well as increased and accelerated phase responses to light pulses during the second half of the night [145]–

[147]. Expression of NFIL3 (also called E4BP4) is regulated by competitive action of REV-ERBs and RORs on their respective enhancer elements in the Nfil3 promoter [148]. The proline and acidic amino acid-rich basic leucine zipper (PAR bZip) transcription factor DBP induces, while its anti-phasic bZip transcription factor NFIL3 suppresses D-box dependent gene transcription [148]. Just like REV-ERBa/b and RORa/b nuclear receptors, DBP and NFIL3 transcription factors compete for their

D-box binding sites [148], thereby regulating the expression of D-D-box target genes Nr1d1/2, Rora/b, and Per1/2/3 [149]. Thus, based on their interconnection with core and stabilizing loops, DBP and NFIL3 transcriptional activity forms a third and so-called

“accessory feedback loop”. Due to their antagonist transcriptional activity and anti-phasic expression, it has been suggested that the accessory loop is important for the generation of high amplitude circadian oscillations [148], [150].

Figure 1-4: The mammalian core clock network

The transcription factors CLOCK and BMAL1 drive the E-box dependent expression of target proteins, including Pers, Crys, Rev-erbs (Nr1d1/2), Rors, Dbp (E-box sites not shown for all genes, but see [142], [144]). In the core feedback loop PER and CRY protein products form complexes and suppress their

proteins is regulated by casein kinase 1 and E3 ubiquitin ligase pathways. In a stabilizing feedback loop, the nuclear receptors REV-ERBa/b (encoded by Nr1d1/2) and the retinoic acid-related orphan receptors RORa/b (encoded by Rora/b), competitively suppress and activate Bmal1 transcription, by binding to its RevDR2 and RORE promoter elements. A third, accessory, feedback loop is generated by Nifl3, regulated by REV-ERBa/b and RORa/b, and Dbp, which competitively regulate the expression of a number of clock genes via binding to their D-box promoter elements. These three interlocked TTFLs constitute the mammalian circadian oscillator, with clock genes being expressed in specific relation to each other (see graph at the top right). Expression of rhythmic output genes, so-called clock-controlled genes (CCG), is regulated by the three TTFLs via binding to their respective enhancer elements in promoters of the CCGs. (AMPK = 5’AMP-activated protein kinase, CK1 = casein kinase 1, CRE = cAMP response element, FBX = F-box protein, SCF = SKP1-cullin-F-box protein, SRE = serum response element, Ub = ubiquitin). (adapted from [143])

Activation and repression of distinct clock-controlled promoter elements has been shown to be very important for the regulation of amplitude and transcriptional delay times required for the generation of ~24 hour oscillations [150]. Moreover, timing and order of regulation of these clock-controlled elements seems to be critical for the phase of circadian oscillations: E-boxes, RevDR2, and RORE sites follow a repressor-precedes-activator pattern, while D-boxes follow a repressor-antiphasic-to-activator pattern [151]. The temporal coordination of cis-regulatory DNA elements by interlocked TTFLs, appears to be an inherent design principle of mammalian circadian clocks and ensures both, robustness of circadian rhythms despite variations in gene expression levels, as well as plasticity with regard to the phases of gene expression. Moreover, TTFL dependent transcriptional regulation is not restricted to clock genes, but also appears at promoter sites of so-called clock-controlled genes (CCG). Thereby, circadian oscillations of 3-10% of mammalian transcripts are generated in a tissue-specific manner [41]–[46], [81].