Fig. 3

Widespread changes in DNA methylation and gene expression point to disruptions in melanocortin and GABAergic neural signaling. a Illustration of experimental strategy to limit cellular heterogeneity by microdissection of ARH at P12 followed by NeuN immunolabelling to isolate neuronal nuclei by FACS. b Representative FACS plot showing gating strategy used to isolate neural (NeuN+) nuclei (see also Supplementary Fig. 3A). c Violin plot showing genomic localization and methylation levels of hypomethylated and hypermethylated DMRs in ARH neurons as determined by whole-genome bisulfite sequencing. Hypermethylated DMRs predominate in F/F mice except in CpG island, 5′-UTR and enhancer regions. d Proportional genomic distribution of hypo- and hypermethylated DMRs. e Gene ontology (GO) function analysis of GREAT-defined²² cis-regulatory region-associated DMRs shows significant enrichment of genes associated with melanocortin signaling, GABAergic neurotransmission, and TGF-β signaling among hypomethylated DMRs (left). Genes belonging to the melanocortin signaling GO term tend to be downregulated in F/F mice, while genes associated with GABAergic neurotransmission tend to be upregulated in F/F mice (right). f Correlogram showing Pearson correlation coefficients (R) between DMR methylation level in each genomic region relative to other regions in the same gene, and expression of the associated gene. g Significant changes in the expression of GABA synthesis genes (Gad1), receptor subunits (Gabra2, 4, 5), and transporters (Slc6a1, Slc6a11). h Differential expression of genes specific to defined ARH neural cell types²⁵. Cell types are ranked according to ratio of down- to upregulated genes characteristic of that cell type. Numbers of significantly down- and upregulated genes associated with each cell type are provided. Inset: representative density plot showing log₂ fold change values for differentially expressed genes enriched in Agrp_Sst neurons relative to a background list of all genes with significant differential expression. p-Values represent results of χ² test against background list of significantly up- and downregulated genes. Values reflect mean ± SEM; n = 3–5, *p < 0.05, ***p < 0.001. Source data for g provided as a Source Data file.