Abstract
Numerous signal pathways are epigenetically controlled during brain development and ageing. Thereby, both 5-methylcytosine (5mC) and the newly described 5-hydroxymethylcytosine (5hmC) are highly exhibited in the brain. As there is an uneven distribution of 5hmC in the brain depending on age and region, there is the need to investigate the underlying mechanisms being responsible for 5hmC generation and decline. The aim of this study was to quantify expression levels of genes that are associated with DNA methylation/demethylation in different brain regions and at different ages. Therefore, we investigated frontal cortex and cerebellum of 40 mice (strain C57BL/6), each eight mice sacrificed at day 0, 7, 15, 30 and 120 after birth. We performed expression profiling of methylation/demethylation genes depending on age and brain region. Interestingly, we see significant expression differences of genes being responsible for methylation/demethylation with a significant reduction of expression levels during ageing. Validating selected expression data on protein level using immunohistochemistry verified the expression data. In conclusion, our findings demonstrate that the regulation of methylation/demethylation pathways is highly controlled depending on brain region and age. Thus our data will help to better understand the complexity and plasticity of the brain epigenome.
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Acknowledgments
This work was supported by the Förderprogramm für Foschung und Lehre (FöFoLe) of the Ludwig-Maximilians-University (# 28/2011 to TFJK, HAK and SK) and by the Friedrich-Baur Foundation (# 12/11 to TFJK).
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All procedures performed in this study were in accordance with the ethical standards of the institute at which the experiments were conducted.
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Supplementary material 1. Figure 1: Determining the influence of sex on the global brain epigenome. The influence of sex on the global brain epigenome was determined by integrating a previously published dataset of 22 brain samples (Kraus et al. 2012). We found that there was no significant influence of sex on the global brain epigenome in both cortex (a) and subcortical white matter (b) (TIFF 381 kb)
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Supplementary material 2. Figure 2: Correlation analysis of 5mC amount and Dnmt3a, Tdg and Ki67 positive cells. In the frontal cortex (a-c) there are no significant correlation of 5mC amount and the number of Dnmt3a (a), Tdg (b) and Ki67 (c) positive cells. In the external granular cell layer (EGCL) of the cerebellar cortex (d-f) there are no significant correlations of 5mC amount and Dnmt3a (d), Tdg (e) and Ki67 (f) positive cells. In the internal granular cell layer (IGCL) (g-i) there are no significant correlation of 5mC amount and Dnmt3a (g), Tdg (h) and Ki67 (i). In the molecular cell layer (MCL) (j-l) there is no significant correlation of 5hmC amount and Dnmt3a (j), Tdg (k) and Ki67 (l). EGCL: external granular cell layer, MCL: molecular cell layer, IGCL: internal granular cell layer. p > 0.05 in all performed analysis (TIFF 1355 kb)
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Supplementary material 3. Figure 3: Correlation analysis of 5hmC and 5mC amounts. In the frontal cortex there is no significant correlation of 5hmC and 5mC amounts (a). In the external granular cell layer (EGCL) of the cerebellar cortex there is no significant correlation of 5hmC and 5mC amounts (b). In the internal granular cell layer (IGCL) there no significant correlation of 5hmC and 5mC amounts (c). In the molecular cell layer (MCL) there is no significant correlation of 5hmC and 5mC amounts (d). EGCL: external granular cell layer, MCL: molecular cell layer, IGCL: internal granular cell layer. p > 0.05 in all performed analysis (TIFF 528 kb)
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Supplementary material 4. Table 1: Primer sequences used for quantification of expression levels using qPCR. For: forward primer sequence, rev: reverse primer sequence (XLSX 9 kb)
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Supplementary material 5. Table 2: Immunohistochemically determined absolute and relative counts of 5hmC positive cells in all investigated cases. These data were integrated from a previous publication (Kraus et al. 2014) into this analysis to get a complete picture of methylation/demethylation during brain development and ageing. EGCL external granular cell layer, IGCL internal granular cell layer MCL molecular cell layer. M: identification number of mouse (XLSX 12 kb)
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Supplementary material 6. Table 3: Immunohistochemically determined absolute and relative counts of 5mC positive cells in all investigated cases. EGCL external granular cell layer, IGCL internal granular cell layer MCL molecular cell layer. M: identification number of mouse (XLSX 11 kb)
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Supplementary material 7. Table 4: Expression levels for Dnmt1, Dnmt3a, Dnmt3b, Tet1, Tet2, Tet3, Apobec1, Apobec2, Apobec3, Mbd4, Smug1 and Tdg in the frontal and cerebellar cortex. M: identification number of mouse (XLSX 19 kb)
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Supplementary material 8. Table 5: Inter-region comparison of gene expression levels. A comparison of the expression levels of Dnmt1, Dnmt3a, Dnmt3b, Tet1, Tet2, Tet3, Apobec1, Apobec2, Apobec3, Mbd4, Smug1 and Tdg in frontal cortex with cerebellar cortex at the age of 0, 7, 15, 30 and 120 days of age showed that gene expression levels are also region specific. Indicated are p-values of inter-region comparisons using t-test. Significant p-values are indicated in grey boxes (XLSX 9 kb)
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Supplementary material 9. Table 6: Region specific absolute and relative counts of Dnmt3a, Tdg and Ki67 positive nuclei in all investigated cases. EGCL external granular cell layer, IGCL internal granular cell layer MCL molecular cell layer. M: identification number of mouse (XLSX 15 kb)
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Kraus, T.F.J., Kilinc, S., Steinmaurer, M. et al. Profiling of methylation and demethylation pathways during brain development and ageing. J Neural Transm 123, 189–203 (2016). https://doi.org/10.1007/s00702-015-1469-2
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DOI: https://doi.org/10.1007/s00702-015-1469-2