**6. Studies on light-dependent regulation of zebrafish circadian clock have revealed links of circadian clock with DNA repair and cellular DDR**

To guarantee that an organism's behavior remains tied to the rhythms of its environment, the circadian clocks must respond to environmental stimuli to be reset [2, 10]. The main cue for animals is light, which is provided by the day-night cycle. The mammalian route for circadian entrainment by light uses the retinohypothalamic tract, which connects directly to the central clock located in the suprachiasmatic nucleus of the brain [42]. This makes it difficult to analyze the light entrainment mechanisms of the circadian clocks, especially at cellular levels. Zebrafish peripheral cellular clocks display a striking feature as they are directly light responsive [43]. Notably, in the zebrafish-cultured cell lines, oscillations of clock gene expression can be entrained to new light-dark cycle, and expression of clock genes, such as zebrafish *Cryptochrome1a* (*zCry1a*) and *Period2* (*zPer2*), is transactivated by an acute light pulse [44–46]. These observations show that zebrafish cultured cells have the clock components required for a light-induced reset of circadian clock, therefore, providing a valuable tool for the study of general light-dependent regulation of cellular clocks.

Studies using zebrafish-cultured cells have contributed to identification of cellular signaling cascades involved in the light-dependent regulation of cellular clocks [47]. In several organisms, external stimuli are connected to a cell's nucleus via MAPK signaling pathways, such as p38 and extracellular signal-regulated kinase (ERK) [48]. Light has been reported to activate these signaling cascades in zebrafish cells (**Figure 2**) [49]. By a pharmacological approach, it has also been reported that the light-induced ERK activation triggers expression of *zPer2* and *zCry1a* genes, whereas the light-induced p38 activation suppresses it, highlighting a MAPKmediated cross-regulatory mechanism of the expression of circadian clock genes [49, 50]. Importantly, an increased understanding of the light-dependent cellular clock regulation in zebrafish has suggested intriguing associations of the circadian clock with DNA repair and cellular DDR as described below.

*Oncogenes and Carcinogenesis*

the mammalian circadian clock [21].

**4. Roles of circadian clocks in regulation of cell cycle**

Circadian clock proteins appear to play roles in cell cycle control, acting as tumor suppressors. They control the timing of cell proliferation by transcriptional control of key cell cycle genes such as *Wee1*, *c-Myc*, and cyclin-dependent kinase inhibitor 1d (20 kDa protein, *p20*) [27–29]. In mammals, PER proteins directly interact with ATM and Chk2 proteins, inducing cell growth inhibition, cell cycle arrest, and apoptosis [17]. In addition, it has been also reported that PER1 and PER2 interact with the androgen receptor (AR) or estrogen receptor (ER), respectively, in that PER1 inhibits AR-dependent transcription and PER2 induces ER degradation [19, 30]. These findings support the idea that clock proteins act as key players in the cell cycle by interacting directly with and regulating the functions of the cell cycle

In zebrafish, the cell cycle is directly regulated by light [31, 32]. Light determines the timing of mitosis (M phase) and DNA synthesis (S phase), establishing a circadian rhythm for cell cycle progression. At the molecular level, cellular clocks establish the circadian expression of the cell cycle genes, zebrafish *Wee1* and *p20* [29, 32]. The Wee-l kinase controls the timing of the G2/M transition by directly phosphorylating and inhibiting cell division cycle2 (Cdc2)/cyclin B, leading to the suppression of mitotic cell division. In contrast, p20 regulates the G1/S transition of the cell cycle. Thus, the circadian control of these cell cycle regulators could be a mechanism establishing the circadian rhythm of cell cycle. Both cell cycle and circadian clock are endogenous pacemakers, and these mechanisms coexist in most eukaryotic cells and share several conceptual characteristics. The abovementioned findings point to functional links between the cell cycle and circadian clock in

(**Figure 1B**).

regulators.

in tumorigenesis [8, 9]. Importantly, mice with mutations in the *Bmal1* gene show premature aging phenotype [15]. In addition, human CLOCK has been suggested to be involved in metastasis of colorectal cancer [16]. These findings implicate the core circadian machinery in the regulation of DDR and the cell cycle. Indeed, the circadian regulators have been demonstrated to interact with crucial components of cellular stress response pathways including the ATM, the checkpoint kinase 2 (Chk2) kinase [17], sirtuin1 (SIRT1) deacetylase [18], and nuclear receptors

19, 20], whereas it has been reported that DNA damage can act as a resetting cue for

Histone acetyltransferases (HATs) such as CBP/p300 are known to acetylate nonhistone targets and have also been recognized as tumor suppressors [22, 23]. Translocation, amplification, overexpression, or mutation of HAT genes are known to occur in several forms of cancer, and several key cell cycle proteins (including p53 and c-MYC) are known targets of HATs. These observations suggest that HATs can also affect cell proliferation and differentiation in multiple ways, in addition to chromatin remodeling. It was previously reported that a core circadian regulator, CLOCK, has intrinsic HAT activity [24] and further that it acetylates a nonhistone target, the heterodimeric CLOCK-binding partner BMAL (**Figure 1B**) [25]. CLOCK also acetylates the glucocorticoid receptor and the argininosuccinate synthase, negatively regulating the transactivation capacity and the enzymatic activity, respectively [20, 26]. It is conceivable that CLOCK would directly interact with and regulate key DDR regulators, leading to the acetylation of these proteins and thereby modulating their activities

**16**

different organisms.

### **Figure 2.**

*A proposal model of light signaling pathways involved in shared control of the circadian clock and DNA repair in zebrafish. In a variety of organisms, light induces ROS production. In zebrafish cells, the light-induced ROS stimulate intracellular MAPK/ERK signaling pathway, which transduces photic signal to zCry1a expression. The light-induced zCRY1a interacts directly with the zCLOCK:zBMAL complex and modifies its transcriptional capacity. Notably, the zCLOCK:zBMAL complex regulates the transcription of a variety of genes involved in cellular stress responses and DDR. UV component of sunlight induces DNA damage. Light-induced ROS and activation of MAPK/ERK pathway also induce expression of a DNA repair enzyme, zPHR. The induced zPHR repairs UV-damaged DNA in a light-dependent manner.*
