Expression differences of Tet genes generating hydroxymethylation during brain aging beginning at embryonic day 16
In order to generate a complete picture of regulation and expression levels of enzymes that are responsible for demethylation during brain development and aging we integrated our previously published Tet expression data [11], attached starting with embryonic day 16 (E16). Investigating the expression levels of the Tet genes in murine frontal cortex and cerebellum from embryonic day 16 (E16) till the age of 120 days (P120) we found distinct differences of gene expression (Fig. 1a-1c, 2d-2f). In frontal cortex the expression levels of Tet 2 significantly decreased starting with E16 and ending with P120 (p<0.05, p<0.01, p<0.001, p<0.0001, ANOVA with Newman-Keuls post hoc) (Fig. 1b).
The expression levels of Tet1 in frontal cortex (Fig. 1a) at E16 have significant lower Tet 1 expression levels than at P0 (p<0.001), P7 (p<0.01) and P15 (p<0.001). Comparing P30 and P120 with P0 (p<0.0001), P7 (p<0.001) and P15 (p<0.0001) shows significant decreased expression (ANOVA with Newman-Keuls post hoc). The same applies for Tet3 expression (ANOVA with Newman-Keuls post hoc) in frontal cortex (Fig. 1c) despite of differences in significance.
The significantly increased expression levels of Tet 2 in cerebellum (Fig. 2e) are starting with E16 and are lasting till P0. At both days the Tet 2 expression in cerebellum are significantly higher compared with P7, P15, P30 and P120 (p varies between <0,05 and <0,001, ANOVA with Newman-Keuls post hoc).
Comparing adult cerebellum of mice at the age of day 30 (p<0.01, ANOVA with Newman-Keuls post hoc) and 120 (p<0,05, ANOVA with Newman-Keuls post hoc) with embryonic day 16 it confirms a decreased expression level of Tet1 (Fig. 2d). Tet 1 expression at P0 (p<0,05) is significantly higher than at P30 (ANOVA with Newman-Keuls post hoc) (Fig. 2d). At day 7 of adult cerebellum there is a significant increased expression level of Tet 1 compared with the other expressions of adult age (p varies between <0,05 and <0,001, ANOVA with Newman-Keuls post hoc) (Fig. 2d).
Tet 3 expression in cerebellar tissue at day 30 is lower than adult day 7 and embryonic day 16 (each p<0,01, ANOVA with Newman-Keuls post hoc) (Fig. 2f).
Tet1 regulation at H3K4me3 and H3K27me3
In order to determine the regulation level of Tet genes at transcriptional activated and inactivated chromatin status in adult murine cortex ChIP-qPCR was carried out on H3K4me3 and H3K27me3 marks. ChIP-qPCR data reveal a significant gain of Tet levels at both marks. Analysis confirm in murine cerebrum of fetal (E16), adolescent, adult and elderly (P0, P7, P15, P30) as well as of aged brains (P120) a correlation between H3K4me3 or H3K27me3 and the activation or inactivation status of Tet1. As a mark of transcriptional inactivation H3K27me3 correlates with inactivation status of Tet1. There is increasing inactivation after E16, P7 and P30. In status of transcriptional activation (H3K4me3) there is nearly steady level of Tet 1 activation regardless of the age. These results do not show any significant changes (Fig. 3g).
Tet2 regulation at H3K4me3 and H3K27me3
A further evidence for the role of H3K4me3 as a transcriptional activator is shown by the result of the regulation status of Tet2. Assessments of the activation and inactivation levels reveal only a slight change in the age groups. But concurrently, a significant increase of inactivation status in Tet2 in murine brain of P120 is detected (p between <0,01 and <0,001, ANOVA with Newman-Keuls post hoc) (Fig. 3h). In status of transcriptional activation (H3K4me3) there is nearly steady level of Tet 2 activation regardless of the age and without any significant changes (Fig. 3h).
Tet3 regulation at H3K4me3 and H3K27me3
Next, we examine the correlation between aging and activation of Tet3. H3K4me3 leads to no significant levels over the life span (Fig. 3i). In contrast, an inverse alteration is observed at H3K27me3 mark: Tet3 in brain of embryonic day E16 and of P120 old mice has a significant higher inactivation compared with the remaining age groups (Fig. 3i). A significant increase of inactivation status in Tet3 is detected in murine brain as well of E16 compared with P0 (p<0,01), P7 (p<0,01), P15 (p<0,05) and P30 (p<0,01) as of P120 compared with P0 (p<0,05), P7 (p<0,05) and P30 (p<0,05) (ANOVA with Newman-Keuls post hoc) (Fig. 3i).