DNA methylation confers a cerebellum-specific identity in non-human primates

Abstract

Selective regulation of gene expression across distinct brain regions is crucial for establishing and maintaining subdivision identities. DNA methylation, a key regulator of gene transcription, modulates transcriptional activity through the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). While DNA methylation is hypothesized to play an essential role in shaping brain identity by influencing gene expression patterns, its direct contribution, especially in primates, remains largely unexplored. This study examined DNA methylation landscapes and transcriptional profiles across four brain regions, including the cortex, cerebellum, striatum, and hippocampus, using samples from 12 rhesus monkeys. The cerebellum exhibited distinct epigenetic and transcriptional signatures, with differentially methylated regions (DMRs) significantly enriched in metabolic pathways. Notably, genes harboring clustered differentially hydroxymethylated regions (DhMRs) overlapped with those implicated in schizophrenia. Moreover, 5mC located 1 kb upstream of the ATG start codon was correlated with gene expression and exhibited region-specific associations with 5hmC. These findings provide insights into the coordinated regulation of cerebellum-specific 5mC and 5hmC , highlighting their potential roles in defining cerebellar identity and contributing to neuropsychiatric diseases.

Keywords: 5-Hydroxymethylcytosine; 5-Methylcytosine; Cerebellum; Gene expression; Non-human primates; Tissue identity.

Figure 1

Cerebellum-specific 5mC patterns and DMRs in rhesus monkey brains A: PCA of 5mC in all samples and in 2-, 8-, and 17-year-old groups. B: Cerebellum-specific DMRs in 2-, 8-, and 17-year-old groups. DMRs for the three groups were 8 497, 26 446, and 40 354, respectively. C: Volcano plots and GO functional analysis of genes with cerebellum-specific DMRs in gene body regions. D: Volcano plots and GO functional analysis of genes with cerebellum-specific DMRs in promoter regions. Cere: Cerebellum; Ctx: Cortex; Hipp: Hippocampus; Stri: Striatum.

Figure 2

Unique gene expression patterns in the cerebellum A: PCA of cerebellum-specific gene expression patterns in rhesus monkey brains. B: PCA of three age groups. C: DEGs were enriched in environmental information processing and organismal systems. Most DEGs were enriched in pathways related to signaling transduction and neuronal functions. Y-axis shows GO terms, x-axis shows significance of P-values and groups. Cere: Cerebellum; Ctx: Cortex; Hipp: Hippocampus; Stri: Striatum.

Figure 3

Comparison of 5mC and 5hmC distribution in genomic components and cerebellum-specific DhMR enrichment A: 5mC read density in promoter, CpG island, gene body, and repeat regions in four brain regions. B: 5hmC read density in promoter, CpG island, gene body, and repeat regions in four brain regions. C: Hypermethylated-DhMR and hypomethylated-DhMR genes were enriched in schizophrenia-related pathways. Boxes represent genes related to diseases and cerebellum functions. Hyper-DhMR genes (red) and hypo-DhMR genes (green) are shown. Cere: Cerebellum; Ctx: Cortex; Hipp: Hippocampus; Stri: Striatum.

Figure 4

Schizophrenia-related gene expression in the cerebellum A: Transcriptional levels of GRM4, GRIN2C, ALS2CL, PAX6, GNB3, GFAP, PLLP, and CHRM4. qPCR was applied to quantify the relative expression of cerebellum-specific genes (n=3). Cere: Cerebellum, Ctx: Cortex, Stri: Striatum, Hipp: Hippocampus. B: Protein levels of GRIN2C, PAX6, ALS2CL, GFAP, and CHRM4 in different brain regions (n=4) based on western blot analysis. C: Immunofluorescence staining of cortex and cerebellum of a 9-year-old rhesus monkey with antibodies against GRIN2C and PAX6. Purkinje cells were immunopositive for CD-28K, granule cells were positive for NeuN. D: 5mC and 5hmC levels in promoter regions of PAX6 and GRIN2C in the cerebellum and other brain regions (referred to here as the brain). Cere: Cerebellum. Data were analyzed by one-way ANOVA, with results presented as mean±SEM.***: P<0.001.

Figure 5

Coordinated regulation of 5mC and 5hmC on gene expression A, B: Cerebellum-specific 5mC (A) and 5hmC (B) in promoter or gene body regions that overlapped with gene expression changes. Pos: Positive correlation between DNA methylation level and gene expression level. Neg: Negative correlation between DNA methylation and gene expression level. C: GO functional analysis of 5mC-negatively correlated genes. D: GO functional analysis of 5hmC-positively correlated genes. Y-axis shows GO terms, x-axis shows significance test.

Figure 6

DNA methylation in promoter regions of cerebellum-specific genes A: Reduced DNA methylation 1 000 bp upstream of promoter was associated with higher expression of cerebellum-specific genes. B: Increased DNA methylation 1 000 bp upstream of promoter was associated with lower expression of cerebellum-specific genes. X-axis represents promoter position. TSS site was 0. Y-axis indicates DNA methylation level. qPCR results confirmed cerebellum-specific gene expression. Data were analyzed by one-way ANOVA, with results presented as mean±SEM. ^*: P<0.05; ^**: P<0.01; ^***: P<0.001; ^****: P<0.0001. Cere: Cerebellum, Brain: other brain regions, Ctx: Cortex, Stri: Striatum, Hipp: Hippocampus.

Figure 7

Two modes of coordinated regulation of gene expression by 5mC and 5hmC A: 5mC and 5hmC regulated genes in opposite directions, accompanied by lower gene expression. B: 5mC and 5hmC regulated genes in the same direction, accompanied by higher gene expression. X-axis represents promoter position. TSS site was 0. Y-axis indicates 5mC or 5hmC levels. qPCR results confirmed cerebellum-specific gene expression. Data were analyzed by one-way ANOVA, with results presented as mean±SEM. ^*: P<0.05; ^**: P<0.01; ^***: P<0.001; ^****: P<0.0001. Cere: Cerebellum, Brain: other brain regions, Ctx: Cortex, Stri: Striatum, Hipp: Hippocampus.