Figure 1Immunostaining, quantification, and genomic mapping of 5-hmC across three ages and two brain regions in mouse
(a–f) 5-hmC–specific immunostaining in P7 (a,c) and 6 week (b,d) cerebellum, P7 (e) and 6 week (f) hippocampus. Dentate gyrus granular neurons in P7 are indicated by the dashed lines. (g,h) Quantification of 5-hmC–specific immunoblot in cerebellum and hippocampus at P7, 6 weeks and 1 year (n = 3, mean ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001, ANOVA). (i) Heat map of genome-wide Pearson correlations between input normalized 5-hmC binned data (10-kb bins) in hippocampus and cerebellum. In all heat maps, red indicates cerebellum and blue indicates hippocampus. (j,k) Example of tissue-specific 5-hmC loci in cerebellum and hippocampus based on genome-wide binned data (10-kb bins) identified by hierarchical clustering (uncentered complete linkage). (l) Example of developmentally programmed 5-hmC loci in cerebellum based on genome-wide binned data (10-kb bins) identified by hierarchical clustering (uncentered complete linkage). (m) Heat map of normalized 5-hmC binned data (10-kb bins) on the X chromosome (hierarchical clustering, uncentered complete linkage). For all heat maps, increasingly darker color represents increased age. In j–m, the chromosome number is indicated on the right. (n) Chromosomal read densities in hippocampus and cerebellum at each age for 5-hmC–enriched DNA, input DNA and values expected by chance. (o) Depletion of 5-hmC on the X chromosome relative to autosomes in male and female mouse brain. For the female sample, normalized 5-hmC densities were divided by input densities from the same female (GSE25398). Dashed line corresponds to 1, or no enrichment.