Circadian expression of DNA methylation and demethylation genes in zebrafish gonads

ABSTRACT

This research aimed at investigating the light synchronization and endogenous origin of daily expression rhythms of eight key genes involved in epigenetic mechanisms (DNA methylation and demethylation) in zebrafish gonads. To this end, 84 zebrafish were distributed into six tanks, each one containing 14 fish (7 males and 7 females). Animals were subjected to 12 h light:12 h dark cycles (LD, lights on at ZT0 h) and fed randomly three times a day during the light phase. Locomotor activity rhythms were recorded in each tank for 20 days to test their synchronization to light. Then, zebrafish were fasted for one day and gonad samples were collected every 4 h during a 24 h cycle (ZT2, 6, 10, 14, 18, and 22 h). The results revealed that most of the epigenetic genes investigated exhibited a significant daily rhythm. DNA methylation genes (dnmt4, dnmt5, dnmt7) exhibited a daily rhythm of expression with a nocturnal acrophase (ZT14:01~ZT22:17 h), except for dnmt7 in males (ZT2:25 h). Similarly, all DNA demethylation genes (tet2, tdg, mb4, gadd45aa, and apobec2b) revealed the existence of statistically significant daily rhythms, except for gadd45aa in females. In females, tdg, mb4, and apobec2b presented a nocturnal peak (ZT14:20 ~ ZT22:04 h), whereas the tet2 acrophase was diurnal (ZT4:02 h). In males, tet2, tdg, and gadd45aa had nocturnal acrophases (ZT18:26~ZT21:31 h), whereas mb4 and apobec2b displayed diurnal acrophases (ZT5:28 and ZT4:02 h, respectively). To determine the endogenous nature of gene expression rhythms, another experiment was performed: 12 groups of 14 fish (7 males and 7 females) were kept in complete darkness (DD) and sampled every 4 h during a 48 h cycle (CT2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, and 46 h). Under DD, most of the genes (7 out of 8) presented circadian rhythmicity with different endogenous periodicities (tau), suggesting that the epigenetic mechanisms of DNA methylation and demethylation in the gonads follow an internal control, functioning as part of the translation network linking the environment into somatic signals in fish reproduction.

KEYWORDS:

• Gene expression
• dnmts
• tet2
• tdg
• mb4
• gadd45aa
• apobec2b
• Danio rerio

Acknowledgment

The authors would like to thank José Antonio Oliver for his help in daily fish management and sampling; the personnel of the Molecular Biology Section from the Research Support Service (SAI) of the University of Murcia for their help with the qPCR assays.

Declaration of Interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

Additional information

Funding

This research was funded by the project “SOLEMBRYO” (AGL2013-49027-C3-1-R) and “BLUESOLE” (AGL2017-82582-C3-3-R) granted by the Spanish Ministry of Economic Affairs and Competitiveness (MINECO) co-funded with FEDER, and “CHRONOHEALTH” granted by Fundación Seneca (19899/GERM/15) to FJSV. J.F.LO was funded through a research fellowship granted by MINECO (Juan de la Cierva Program).