Diurnal expression of Dnmt3b mRNA in mouse liver is regulated by feeding and hepatic clockwork

Abstract

DNA methyltransferase 3B (DNMT3B) is critically involved in de novo DNA methylation and genomic stability, while the regulatory mechanism in liver is largely unknown. We previously reported that diurnal variation occurs in the mRNA expression of Dnmt3b in adult mouse liver. The aim of this study was to determine the mechanism underlying the diurnal expression pattern. The highest level and the lowest level of Dnmt3b mRNA expression were confirmed to occur at dawn and in the afternoon, respectively, and the expression pattern of Dnmt3b closely coincided with that of Bmal1. Since the diurnal pattern of Dnmt3b mRNA expression developed at weaning and scheduled feeding to separate the feeding cycle from the light/dark cycle led to a phase-shift in the expression, it could be assumed that feeding plays a critical role as an entrainment signal. In liver-specific Bmal1 knockout (L-Bmal1 KO) mice, L-Bmal1 deficiency resulted in significantly higher levels of Dnmt3b at all measured time points, and the time when the expression was the lowest in wild-type mice was shifted to earlier. Investigation of global DNA methylation revealed a temporal decrease of 5-methyl-cytosine percentage in the genome of wild-type mice in late afternoon. By contrast, no such decrease in 5-methyl-cytosine percentage was detected in L-Bmal1 KO mice, suggesting that altered Dnmt3b expression affects the DNA methylation state. Taken together, the results suggest that the feeding and hepatic clockwork generated by the clock genes, including Bmal1, regulate the diurnal variation in Dnmt3b mRNA expression and the consequent dynamic changes in global DNA methylation.

Figure 1. Diurnal mRNA expression of Dnmts and circadian clock-related genes in the liver. The mRNA expressions of Dnmt1 (A), 3a (B), 3b (C) and circadian clock-related genes such as Bmal1 (D), Clock (E), Per1 (F), Per2 (G) and Cry1 (H) were examined in livers of male and female mice sacrificed at ZT 0, 4, 8, 12 and 16. n = 3 at each time point. Highest mRNA level in each figure was set as 1 and relative concentrations are shown.

Figure 2.Dnmts and Bmal1 mRNA expression at ZT 0 and 8 in the liver during postnatal development. There were no significant differences between ZT 0 and 8 through postnatal development in mRNA expressions of Dnmt1 (A) and Dnmt3a (B). Significant decrease of Dnmt3b mRNA in postnatal 3, 5, and 9 weeks at ZT 8 vs. ZT 0, although significant increase was found in postnatal 1 week (C). Significant increase of Bmal1 mRNA in postnatal 3, 5, and 9 weeks at ZT 8 vs. ZT 0 (D). The expression level in ZT 0 at postnatal 9 weeks was set as 1 and relative concentrations are shown. Numbers used in each group is shown in parentheses. *p < 0.05, **p < 0.01 (Student’s t-test).

Figure 3. Effect of scheduled feeding on Dnmt3b and Bmal1 mRNA expression patterns in the liver. Significant differences at all time points were found between groups in both Dnmt3b and Bmal1 mRNA expressions (**p < 0.01 vs. ad lib fed group, Student’s t-test). The expression level in ZT 16 for Dnmt3b and ZT 0 for Bmal1 of ad lib fed group was set as 1 and relative concentrations are shown. Numbers used in each group is shown in parentheses.

Figure 4. Effect of L-Bmal1 KO on diurnal expressions of Dnmts and Bmal1 mRNA in the liver. Slight but significant increase of Dnmt1 mRNA expression was demonstrated in L-Bmal1 knockout at ZT 10 (A, *p < 0.05, Student’s t-test). There was no significant difference between groups in Dnmt3a mRNA expressions (B). Significant increases of Dnmt3b mRNA expressions at ZT 4, 10, 16 and 22 by L-Bmal1 knockout was revealed [(C) **p < 0.01 at ZT 22, 10, 16 and *p < 0.05 at ZT4, Student’s t-test]. Significant reductions of Bmal1 mRNA expressions at ZT 4, 16 and 22 by L-Bmal1 knockout were confirmed [(D) **p < 0.01, Student’s t-test]. The expression level in ZT 22 of wild-type was set as 1 and relative concentrations are shown. Numbers used in each group is shown in parentheses.

Figure 5. Diurnal change in global 5-mC in liver genomic DNA from L-Bmal1 KO and wild-type mice. Comparison of 5-mC percentage at ZT0, 4, 8, 12, 16 and 20. ZT12 showed the lowest 5-mC percent value, being significantly different from that at ZT0 [(A) *p < 0.05, Student’s t-test]. In addition, 5-mC percent at ZT12 showed decreased tendency when compared with ZT4 and 8 (p = 0.07 and 0.08, respectively, Student’s t-test). In (B) we compared 5-mC percentage in wild-type and L-Bmal1 KO mice at ZT4, 10, 16, 22. In wild-type mice, significantly lower 5-mC percent at ZT10 was found, compared with other time points (**p < 0.01 vs. ZT4, p < 0.05 vs. ZT16 and 22, Student’s t-test). In L-Bmal1 KO mice, the time point-specific decline of 5-mC percentage (found in wild-type mice at ZT10) was not observed; instead, a significant increase in 5-mC percentage was found (#p < 0.001 vs. wild-type, Student’s t-test).