Introduction
Fish have evolved circadian system to synchronize to different environmental cyclic factors like light or food availability. These entraining factors can be referred as zeitgebers and they boosted the evolution of a self-sustainable molecular clock formed by positive and negative feedback loops. Clock and bmal represent the starting point and the positive loop; they work as transcriptional factors activating, among many genes, the negative loop formed primarily by per and cry, which in turn will work to repress clock and bmal transcription. This self-sustainable molecular system is the core of the vertebrate molecular clock, and its rhythmicity is transmitted to many physiological processes (Pando & Sassone-Corsi 2002). Even if this system is self-sustainable, several levels of regulation can be present. For instance, epigenetic mechanisms are involved not only as regulators of this system, but also as a target. Mechanisms of deacetylation and methylation seems to be crucial in this bidirectional system. In the deacetylation process, sirt1 plays an important role (Nakahata et al. 2009) counteracting the intrinsic acetylation activity of clock, which is responsible for chromatin remodeling that is associated to circadian control of gene expression. DNA methylation is another process that could be involved in the regulation of the circadian system since it has been proposed that dnmt3 might be responsible for the methylation of bmal’s promoter (Satou et al. 2013). At the same time, the same genes involved in methylation and de-methylation have been described as rhythmic in zebrafish gonads (Paredes et al. 2018), suggesting again the double nature of the interaction between the two molecular patterns. Moreover, sirtuins and DNA methyltransferases are linked to nutrient availability (Su et al. 2016), indicating that feeding time can act on the clock mechanism not only as a zeitgeber, but also passing through the epigenetic way. Therefore, the aim of this study was to investigate how feeding time affects the rhythms of the circadian clock and genes of the epigenetic system in the liver of the European sea bass (Dicentrarchus labrax). We also focused on the hypothalamus to understand if feeding time has the potential to affect the central pacemaker.
Materials and method
Fish were kept in an open system in a 14:10 LD cycle and fed with 1% of the fish body weight, once a day. Fish activity was monitored by mean of photocells. Two groups were made based on the feeding time: mid-light (ML) and mid-night (MD) feeding. After 30 days under these conditions, fish were sacrificed in a 24-h cycle at seven sampling points after light onset: ZT 0.5, 4, 7.5, 12, 16, 20 and 24.5 h to collect liver and hypothalamus. In the liver, clock genes and genes of the epigenetic system were analyzed, while in the hypothalamus clock genes were considered. Liver was also used to investigate the presence of a rhythm in the SAM/SAH ratio, which is an index of methylation potential.
Results
In the liver, the epigenetic genes analyzed (sirt1, dnmt1, dnmt3a, gadd45aa, mbd4 and tet2), presented rhythm in the ML group, but only dnmt3a conserved the rhythm also in the MD group. Additionally, all the acrophases were nocturnal. The analysis of the clock genes revealed that all of them (clock, bmal1, per1, per2, cry1 and cry2) presented rhythms in the ML group, while only for per2 the rhythm was maintained in the MD group. Finally, rhythms were found in the hypothalamus for the clock genes both for ML and MD groups, except cry2, where no rhythm was found. Concerning fish activity, both the groups displayed a diurnal activity.
Discussion and Conclusions
In the liver, the feeding time had a strong impact on the clock genes, since when the fish were fed in MD the rhythm disappeared with only one exception (per2). Moreover, when comparing ML with MD mean expression, in most of the cases they displayed a significant difference in most of the ZT points, demonstrating that also transcriptional levels are affected by feeding time. Sirt1 was rhythmic in ML group, but not in MD group and the differences between the two groups were significant in most time points. The explanation could be related to the availability of its cofactor, NAD+, that could have been affected by feeding time as well. Dnmt1 seems to be more affected by the feeding time than dnmt3a, since it presents rhythm only in ML group. This can suggest that the maintenance of the pattern of methylation in the cells could be affected as well by feeding. Also, the genes involved in de-methylation process present the same pattern observed for dnmt1, since the rhythm was only present in ML group. Moreover, comparing ML with MD group, is clear that feeding time has affected the transcriptional levels as well. In the hypothalamus, most of the clock genes analyzed displayed rhythm both in the ML and MD groups, and this is consistent with other data in fish that confirm that light is the main synchronizer in central tissues like the brain. However, when the two groups are compared, significant differences are present between the same ZT points. For this reason, it’s not possible to exclude an important effect of feeding time in the central pacemaker. Since both groups were diurnal, and most of the genes displayed a nocturnal achrophase, we can speculate that during the resting phase fish prepare the factors that are involved in the connection between clock system and epigenetic machinery. To better understand the connection between clock system and epigenetic mechanism, our research is currently focusing on the analysis of the methylation potential, to understand if methionine metabolism (1-C cycle) can be connected as well with the clock system.
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