Fig. 2

DNA methylation and mRNA expression of the Dao gene. a Dao promoter region structure showing the position of the analyzed CpGs. The numbers of the CpG sites refer to the putative transcriptional start site (TSS), indicated with + 1. White box indicates the putative upstream regulatory region; black box indicates the first exon; and gray box indicates the first intron. Black arrows at the top of the map specify the position of the primers used for bisulfite amplification. PR1 denotes the promoter region downstream of the TSS analyzed in all brain areas. PR2 indicates the promoter region upstream of the TSS explored in the cerebellum area. The Dao sequence was retrieved by Ensembl with the following accession number: ENSMUSG00000042096. b Average Dao methylation (%) is shown for HIPP, CX, and CB at the P1, P15, P30, and P60 developmental stages (n = 3 mice/stage). Comparisons between developmental stages in each area were performed using one-way ANOVA followed by Tukey’s multiple comparison post-hoc test. *p ≤ 0.001. c mRNA expression levels are reported for each analyzed brain area during brain development. Dao mRNA expression was normalized to the mean of two housekeeping genes and is expressed as 2^–∆Ct values, * p ≤ 0.001 (one-way ANOVA followed by Tukey’s multiple comparison post-hoc test). d Graphs represent the average methylation at single CpG sites in promoter region 1 (PR1), downstream of the TSS, in HIPP, CX, and CB during ontogenesis. e Methylation levels at CpG sites located in promoter region 2 (PR2, see a) are shown. The methylation trend of the seven CpG sites is indicated at each developmental stage. f A comprehensive view of single CpG methylation levels upstream and downstream of the Dao promoter region in the cerebellum during the transition from P1 to P15. Nine CpG sites are indicated, and their position is referred to as the TSS. CpGs with average methylation < 30% are shown in green, while CpG sites with average methylation > 50% are indicated in red