1. INTRODUCTION

Circadian rhythm is 24-hour cycle rhythmicity in organisms, which is endogenous and can be seen externally as changes in behaviour or may be internal in form of gene expression. These rhythms are synchronized or entrained to environmental signals called zeitgebers [1]. There are three important features of circadian rhythm—a 24-hour endogenous free running period, entrainment (adjustment of the period to the surrounding environmental signals, such as light and temperature, in time-dependent manner), and temperature compensation (the periodicity of the circadian rhythm is maintained even when there is a variation in the temperature within a physiological range) [1–4].

Circadian clock, that drives circadian rhythm [5], is present in all the cells and tissues of the body [6]. The circadian clock is mainly of two types—primary or central and peripheral. The central clock in Drosophila is a group of 5–6 bilaterally symmetric small ventral lateral neurons located in the lateral brain [5]. The circadian clock in mammals was first discovered in central nervous system [3]. The central clock is located in the suprachiasmatic nucleus (SCN) of ventral anterior hypothalamus [5]. The other oscillators, besides the central clock, present in the body are the peripheral oscillators. In Drosophila, peripheral clocks can be directly synchronized by the environmental signals [1]. In mammals, the peripheral clock is entrained by SCN through some hormonal signals, such as glucocorticoids [7]. It is slow in response to light because it takes a feeding time of 4–12 hours to receive the signal from the SCN [1] and its rhythmicity is lost just after 4–5 days [8].

Three components are involved in the maintenance of circadian timing system—entrainment/input pathway which connects the pacemaker to the environment through retinohypothalamic tract (RHT); pacemaker, which generates the circadian signal [2] and output pathway which activates behavioural and physiological processes [9] and involves both neuronal and hormonal signals [2].

The circadian system constitutes non-visual photoreceptor cells of retina, SCN, pineal gland, and many peripheral oscillators [10]. Retina contains non-visual photoreceptor cells, called intrinsically photosensitive retinal ganglion cells (ipRGCs) [6]. These cells express a photopigment called melanopsin which makes ipRGCs photosensitive. SCN, the central clock of mammals, acts as a co-ordinator between the external environmental cues and the body of an organism by receiving the environmental cues like light and by sending the signals to the circadian time-keeping system. The entrainment of the central clock is rapid in response to light [1] and the rhythm in response to entrainment is maintained for more than 15 days [8]. The SCN receives information from the ipRGCs through RHT which is helpful in entrainment [1,6]. Pineal gland is an endocrine gland which synthesizes a hormone, called melatonin (N-acetyl-5-methoxytryptamine). This hormone is secreted in a circadian manner. It plays a role as a hormone of darkness because it is secreted maximally at night in the absence of light [11].

2. CLOCK GENES

Studies in many organisms, such as photosynthetic bacteria, Arabidopsis thaliana, Drosophila, Chlamydomonas, Neurospora, hamster, and mice have shown that oscillations in transcription of some clock genes generate and maintain circadian rhythm [12]. Clock genes identified in Drosophila are period (PER), timeless (TIM), clock (CLK), cycle (CYC), and doubletime (DBT) [13]. In mammals, some of the genes are circadian locomoter output cycles kaput protein (CLOCK or CLK) gene and its paralogs, such as neuronal PAS domain protein2 (NPAS2), period1 (PER1), period2 (PER2), period3 (PER3), brain and muscle arnt-like1 (BMAL1 or ARNTL, or MOP3), cryptochrome1 (CRY1), and cryptochrome2 (CRY2) [1,2,6].

3. PERIOD GENES

In Drosophila, PER gene is located on the X chromosome. It encodes PER protein which is essential for the circadian rhythms in eclosion (emergence of the adult fly from the pupa) and locomoter activity [4].

In mammals, three period genes were discovered—PER1 [14], PER2 [14,15], and PER3 [15,16]. A fourth human period gene, PER4 [17] has been identified as a pseudogene, which is descended by retrotransposition of PER3 gene [18]. The four PER paralogues have been evolved via two genome duplications from a single ancestral gene. The duplicated gene, which is of functional importance are retained, such as PER3 and the others, are lost from the genome such as PER4 [19].

4. TRANSCRIPTION/TRANSLATION FEEDBACK LOOPS (TTFLS)

Approximately, 43% of all transcriptomes of the mammalian body shows circadian rhythmicity in organ-specific manner. Organ-specificity means the clock genes are active in the whole body but the output is different in each organ. The rhythmic genes are clustered together in the genome and are longer than non-rhythmic genes. Liver has the largest number of circadian genes and the brain regions, such as hypothalamus, have the minimum number [30].

A nearly 24-hour period oscillation in levels of PER mRNA and PER proteins occurs which are expressed rhythmically [31]. There is a rapid transcription of PER gene just after sunset/lights off (zeitgeber time, ZT12), accumulation of PER protein in the cytoplasm 2–6 hours after sunset (ZT10-17) and at ZT18, PER reaches its maximum level. These proteins are translocated to the nucleus 5–8 hours after lights off, where they repress their own transcription. PER protein levels become the lowest during the sunrise/lights on (ZT0/24) [32] (Fig. 1). The delay between PER expression and its repressor activity in the nucleus acts as a checkpoint for a stable circadian oscillation generation [33]. Moreover, a recent study suggests that transcription delay of PER2 and CRY1 and degradation rates of CRY1 and REV-ERBα are the major factors which influence phases of the genes involved in circadian timing network [34].

There are two feedback loops—positive and negative, which interact with each other to drive the circadian rhythm correctly. These loops are discussed in the following section for Drosophila and mammals.

4.1. TTFLs in Drosophila

4.1.1 Transcriptional activators

In Drosophila, the bHLH-PAS (basic helix–loop–helix, Period–Arnt–Single-minded) transcription factors, CLOCK (dCLK) and CYCLE (CYC) are the activators of positive feedback loop. dCLK is rhythmically expressed which means that its RNA and protein levels oscillate over 24-hour period but CYC is constitutively expressed [5,31,35].

4.1.2. Transcriptional inhibitors

PER and TIM are the transcriptional inhibitors of this feedback loop. The levels of RNAs and proteins of both PER and TIM oscillate rhythmically with same period and phase [31,36]. Though PER is required for the expression of TIM in the nucleus [36], it is primary repressor of the transcription of the positive elements of the loop [31]. Degradation of TIM is rapid through a proteasome-mediated pathway and is due to the formation of heterodimer with dCRY in response to light [5,36]. In Drosophila, dCRY plays a role in circadian photoreception [37].

Figure 1: Circadian rhythm pattern. [Click here to view]

TIM lacks a PAS domain, so it associates with PER by a heterotypic protein interaction [31]. The interaction between TIM and PER occurs at two sites as follows:

1)Cytoplasmic localization domains (CLDs) of both proteins; which not only helps in formation of TIM-PER heterodimer but also allows the nuclear entry of the complex

2)between PAS of PER protein with nuclear localization signal (NLS) of TIM [33].

Nuclear entry of each protein is prevented by the CLD either by binding the monomeric PER and TIM to the cytoplasmic anchor or by inhibiting the NLSs of both proteins [31]. Thus, TIM is essential for PER stabilization and its transport to the nucleus [32,33] possibly due to the physical association of both PER and TIM that may suppresses the activity of CLD [33].

4.1.3. Positive feedback loop

The positive feedback loop involves dCLK transcription regulation. Transcripts of dCLK and those of PER and TIM oscillate out of phase. Thus, when the level of dCLK mRNA peaks (in the late night and in early morning), the levels of PER and TIM transcripts are the lowest [5,31]. dCLK-CYC heterodimer enhances the transcription of negative elements, PER and TIM genes. The complex constitutively binds specifically to CACGTG nucleotide sequence of E-box enhancer elements of the target gene promoters [31,38]. The RNA levels of PER and TIM increase, and thus PER and TIM proteins accumulate in the cytoplasm as heterodimer [31].

4.1.4. Negative feedback loop

The negative feedback loop involves repression of the positive element dCLK-CYC. This repression is achieved when PER and TIM form a heterodimer due to increase in the amount of PER and TIM proteins. This heterodimer moves into the nucleus and causes a conformational change in dCLK-CYC or decreases its DNA-binding activity without affecting the association between dCLK and CYC which causes fall in the levels of PER and TIM proteins [38]. According to Bae et al. [35], CYC is present in abundant amount, approximately 200 times more than the amount of dCLK. PER and TIM bind with dCLK or dCLK-CYC complex rather than with the free CYC. Thus, dCLK acts as a limiting agent for the PER-TIM and dCLK feedback loops [35]. Figure 2 illustrates that dCLK-CYC heterodimer enhances the transcription of negative elements, PER and TIM genes. The RNA levels of PER and TIM increase, and thus PER and TIM proteins accumulate in the cytoplasm as heterodimer. This heterodimer moves into the nucleus and inhibits dCLK-CYC heterodimer which causes fall in the levels of PER and TIM proteins. DBT is a kinase which degrades monomeric PER but does not show its activity on PER-TIM heterodimer. Shaggy, another protein kinase, is constitutively expressed. It phosphorylates TIM protein and also helps in nuclear translocation of PER/TIM.

4.2. TTFLs in mammals

Mammalian core clock acts via enhancer elements in their promoters, such as E-boxes, D-boxes, and ROR-elements [39] and includes mainly five regulators, such as activators BMAL1 and DBP (D-box regulator) and the inhibitors PER2, CRY1, and REV-ERBα. There are three feedback loops, which are essential for circadian rhythm generation. These are autoinhibitions of PER and CRY, BMAL1/REV-ERBα loops [40] and repressilator motifs which contains PER2, CRY1, and REV-ERBα genes [41]. These loops are tissue-specific. The primary feedback loop PER-CRY autoinhibition is particularly found in SCN, whereas BMAL1/REV-ERBα loops are found in the heart. BMAL1/REV-ERBα loop and repressilators form the largest group of oscillators in liver. The co-existence of these feedback loops provides redundancy and enhances robustness and flexibility of the circadian core clock [40].

Figure 2: Feedback loop in Drosophila. [Click here to view]

4.2.1. Transcriptional activators

CLK (or NPAS2) and BMAL1 are the bHLH-PAS transcription factors constituting the positive elements of the loop. Unlike dCLK, mammalian CLK is expressed constitutively [5] in SCN but its oscillations are cyclic in peripheral tissues [42], whereas BMAL1 RNA and protein levels have a cycle of over 24-hour period [5,31].

4.2.2. Transcriptional inhibitors

mCRY and mPER are the transcriptional inhibitors of the loop. mCRY, a member of the blue light-sensitive family of photoreceptor proteins [43] and the primary repressor in mammals [44] has two types—CRY1 and CRY2 which are rhythmically expressed [45]. Although CRY1 is a stronger transcriptional repressor than CRY2 and can maintain the rhythm alone [46], CRY1 and CRY2 bind to CLOCK and BMAL1 with the same affinity when PER2 is co-expressed [47].

PER1 and PER2 respond differentially toward light [14]. This is because the regulatory region of PER1 mainly responds to light but that of PER2 is affected by hormonal and other signals [14]. Though mPERs (mPER1 and mPER2) are expressed rhythmically [48], there is a delay of 4 hours between their expressions. PER1 transcripts are formed prior to PER2 transcripts may be due to the fact that a minimum amount of PER1 is required to initiate PER2 expression [14].

CRY repressor activity depends upon its synthesis, post-translational modifications, nuclear shuttling, and its degradation. These activities are regulated by the interaction of CRY to different proteins, such as PER1/2 [49], E3 ligases (FBXL3 and FBXL21), and CLOCK. Conserved core structure called photolyase homology region of CRY binds with all these proteins. PER competes for the binding of CRY to CLOCK/BMAL1 complex [50]. Binding site of PER to CRY overlaps with the binding site of FBXL3 and CLOCK/BMAL1 which regulates the degradation and repression activities of CRY. Another site, Ser71 of CRY1 is phosphorylated by nutrient-responsive AMP-activated kinase (AMPK) which enhances the binding of FBXL3 and reduces the stability of CRY1. CRY1 also entrain peripheral clocks metabolically by phosphorylation of AMPK through nutrient signals [51]. Cys412 of CRY1 forms an intramolecular disulfide bond with its Cys363 [52], which weakens mCRY1-mPER2 interactions and an intermolecular disulfide bond with FBXL3. Moreover, zinc enhances the formation of the reduced state of mCRY1 and stabilizes the mCRY1/mPER2 complex [53].

Constitutively nuclear mammalian homolog of Drosophila TIM shows no change in its RNA and protein levels and is not degraded by light exposure [48]. mTIM does not affect PER1 nuclear translocation but when PER1 enters the nucleus, mTIM interferes in CLK-BMAL1-mediated transcription [48].

4.2.3. Positive feedback loop

It involves the BMAL1 transcription regulation. There is an interval of 12 hours between the peak RNA levels of BMAL1 and PER & CRY. CLK (or NPAS2) and BMAL1 heterodimer remains bound specifically to CACGTG nucleotide sequence of E-box enhancer elements of the target gene promoters constitutively. During the day, when co-activators such as p300, which occupy the binding site of CRY1 [54], are bound to this heterodimer, it enhances the transcription of negative elements, PER and CRY genes and also activates transcription of retinoic acid-related orphan nuclear receptor gene—REV-ERBα through E-box enhancers. REV-ERBα protein then represses the transcription of BMAL1 gene. Thus, the RNA levels of BMAL1 fall and those of PER and CRY increase [55]. RORα, another orphan nuclear receptor protein, competes with REV-ERBα to bind retinoic acid-related orphan receptor response elements present in the BMAL1 promoter. It enhances the transcription of BMAL1 gene [56]. An E3 ubiquitin ligase, TNF receptor-associated factor 2, decreases the stability of BMAL1, thus decreases PER1 mRNA expression [57]. CLK-BMAL1 heterodimer also regulates the expression of clock-controlled genes (CCG) such as genes related to cell growth, apoptosis and DNA repair [58].

4.2.4. Negative feedback loop

It involves the repression of the positive element CLK-BMAL1 by the negative regulators. As the amount of PER and CRY proteins increases, these proteins stabilize by forming heterodimer involving PAS domain. The heterodimer may be between any one of the three PER proteins with one of the two cryptochrome proteins (CRY1 and CRY2). PER2 has a stabilization sequence that stabilizes the complex. As PER-CRY heterodimer moves into the nucleus, CRY proteins repress the transcription of PER and CRY and also that of REV-ERBα by inhibiting the positive regulators of the loop. As a result, the levels of PER and CRY fall and those of BMAL1 rise during night [55]. Recently, it is shown in mouse liver cell extracts that the three PER proteins form a mature cytoplasmic multi-globular complex with the two CRYs and casein kinase 1 delta which migrates to the nucleus and inhibits CLOCK/BMAL1 [59]. Figure 3 illustrates feedback loops in mammals. ipRGCs of retina receive light signal and convey the signal to the SCN core through RHT which releases glutamate and pituitary adenylate cyclase activating polypeptide (PACAP) onto cells in the SCN core. It results in phosphorylation of cAMP-response-element-binding protein which binds to the cAMP-response element present in the promoter of PER1 and PER2, inducing their transcription. Different neurotransmitters, such as VIP, GRP and GABA, help in communication between the SCN core and SCN shell. CLOCK-BMAL1 heterodimer binds to E-boxes in the promoter region of PER, CRY, REV-ERBα, RORα, and other CCG and induces their transcription. High levels of PER and CRY proteins in the cytoplasm cause them to dimerize which then binds with casein kinase 1 and translocate to the nucleus where they inhibit the activity of CLOCK–BMAL, thus inhibiting their own transcription. At the same time, REV-ERBα inhibits the transcription of BMAL1.

The interaction between these positive and negative loops is thus essential for the proper functioning of circadian rhythm [9,55]. The entire cycle takes approximately 24 hours to complete. The yield of PER and CRY proteins is regulated by E3 ubiquitin ligase complexes [6]. The nuclear entry and repressor activity of PER-CRY complex occur through the interaction of PER with KPNB1, an importin-β component, without the involvement of importin-α [60]. CLOCK/BMAL1 recruits Ddb1 (DNA damage binding protein 1)–Cullin-4 (Cul4) E3 ubiquitin ligase to E-boxes of the target gene which enhances mono-ubiquitination of H2B histone protein at Lys-120. This enzymatic action helps in the stable interaction between the PER complex and CLOCK/BMAL1 complex which causes the modification in the chromatin and thus repression of transcription [61]. CRY binds to BMAL1’s C-terminal transactivation domain with its C-terminal α-helix tail and competes with other coactivators for binding to BMAL1 [62]. The secondary pocket of CRY binds with the PAS-B domain of CLOCK protein [63].

Figure 3: Feedback loop in mammals [Modified from figure 1 (Antle & Silver, 2005)]. [Click here to view]

Since PER has a rate-limiting role in the formation of a negative-feedback complex with CRY, its rhythmic expression is critical in the circadian oscillations. PER2 has a great binding affinity with CLOCK and BMAL1 which connects the negative complex (PER:CRY) to the positive complex (CLOCK:BMAL1), a key step to regulate the circadian rhythm [64]. PER2 LCCLL motif between the two PAS domains triggers the interaction of PER2 with CLOCK or BMAL1. This motif may not be flexible enough or may not be accessible to interact with CLOCK/ BMAL1 in PER1. PER2 also binds to the nuclear receptors, peroxisome proliferator-activated receptor-α (PPARα, present in the liver) and REV-ERBα which regulates the transcription of BMAL1 gene [65].

5. CONCLUSION

The feedback loops generate the molecular mechanism of circadian clock. The feedback loops are tissue-specific. PER-CRY autoinhibition are characteristic for SCN clocks, while BMAL1/REV-ERBα loops are found in the heart and repressilators motifs are found in liver. Tissue-specific use of a network of co-existing synergistic feedback loops could account for functional differences between organs. These co-existing feedback loops enhances robustness and flexibility of the circadian core clock [40]. Further work on the details of molecular clocks is needed to know their roles in peripheral tissues and to know how these are associated with the behavioural and physiological systems of the body. Some clock-related disorders can also be cured by knowing these associations.

ACKNOWLEDGMENT

The author would like to express his special thanks of gratitude to UGC, New Delhi, for providing fellowship throughout his work period.

ABBREVIATIONS

SCN suprachiasmatic nucleus

RHT retinohypothalamic tract

ipRGC intrinsically photosensitive retinal ganglion cell

PER period

TIM timeless

CLK clock

CYC cycle

DBT doubletime

NPAS2 neuronal PAS domain protein2

BMAL1 brain and muscle arnt-like1

CRY cryptochrome

PAS Period–Arnt–Single-minded

bHLH basic helix–loop–helix

CLD cytoplasmic localization domain

NLS nuclear localization signal

AMPK AMP-activated kinase

CCG clock-controlled genes

Ddb1 DNA damage binding protein 1

Cul4 cullin-4

PPARα peroxisome proliferator-activated receptor-α

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