Age-related epigenome-wide DNA methylation and hydroxymethylation in longitudinal mouse blood

Figures & data

Table 1. Differential 5mC and 5hmC in mouse blood by age.

Δ5mC/5hmC	Age
	DMCs*	DHMRs‡
Hyper-	17,460	160
Hypo-	10,736	8446
Both	0	7
Total	28,196	8613

The DSS and csaw R packages were used to examine the effect of age on 5mC and 5hmC levels across the epigenome. For all models, age was included as an ordinal variable (2, 4, and 10 months). Therefore, the effect of age was modeled as a trend – changes that occur from 2 months (baseline) to 4 months to 10 months old. Models also included a paired mouse ID variable to account for within-individual effects, as well as a sex variable. Directionality of DMCs and DHMRs are indicated in separate rows; direction is relative to 2-month-old samples. DMC and DHMR calling thresholds: *DMCs = Δ > 0.05 and FDR < 0.05; ‡DHMRs = FDR < 0.10.

Figure 1. Sequencing data collection and analysis workflow. Genomic DNA was isolated from matched offspring blood samples at 2 months (n = 6), 4 months (n = 6), and 10 months of age (n = 6). DNA samples were then split for separate processing steps specific to the ERRBS and HMeDIP-seq methods. All processed samples were amplified and sequenced on an Illumina HiSeq 4000 sequencer using single-end, 50 nt read length. Separate bioinformatics pipelines were used to analyze ERRBS and HMeDIP-seq data. Differentially methylated CpGs (DMCs) were compared to differentially hydroxymethylated regions (DHMRs) for regions of chromosomal overlap. Regions of overlap were then annotated and visualized in the genome browser. The Nfic gene was validated using RT-qPCR on available RNA from the blood samples.

Figure 2. Venn diagrams of intersecting age-related DMCs and DHMRs by chromosomal location. The bedtools intercept function was used to test for overlap between DMCs and DHMRs by exact chromosomal location. Comparing age-related DMCs and DHMRs, we identified 65 regions that overlapped by chromosomal position. The age-related DMC and DHMR overlap sites annotated to 40 unique gene IDs. Of these 40 genes, three had multiple DMCs that overlapped a DHMR – Fbln1, Ldlrad3I, and Nfic.

Figure 3. ERRBS, HMeDIP-seq, and RT-qPCR data for the Nfic gene DMC and DHMR overlap region. ERRBS and HMeDIP-seq data at the Nfic DMC/DHMR overlap region (chr10: 81433551–81433,800) were visualized using the Gviz, csaw, and RnBeads R packages. Of the 8,446 hypo-hydroxymethylated DHMRs, the Nfic DHMR was the 172nd most significant region (FDR = 0.0002). Y-axes show mouse ID across the three blood sample ages; sample ages are separated by horizontal lines. (a) The EERBS data show significant age-related decreases in 5mC at five CpG sites annotated to the Nfic gene, as indicated by the black box with an asterisk. Percentage DNA methylation is represented by a heatmap, with low % methylation represented by light red, and high % methylation represented by dark red; white indicates a lack of data coverage for ERRBS. A line plot comparison of 5mC across the region for all three age groups (2 months = orange, 4 months = purple, 10 months = green) is included at the bottom of the output. (b) The HMeDIP-seq data at this gene region also show a significant decrease in 5hmC peaks with age in longitudinal mouse blood samples. Blue and red peaks represent forward and reverse strand 5hmC enrichment, respectively. (c) RT-qPCR was used to measure Nfic expression levels using the 2^−ΔΔCt method. There was a significant decrease in mean Nfic expression between 2 and 10 month blood samples (P = 0.04).

Table 2. Age-related DMCs annotated to the Esr1 locus.

Chr	Start	End	Strand	Location	Δ % Methylation*
chr10	4666894	4666894	+	1 to 5 kb	−25.7%
chr10	4709922	4709922	+	Promoter	10.0%
chr10	4712221	4712221	+	Intron	41.2%
chr10	4712716	4712716	+	Exon	−25.4%
chr10	4932943	4932943	+	Intron	30.4%
chr10	4971465	4971465	+	Intron	36.2%
chr10	4972470	4972470	+	Intron	−14.9%

List of significant DMCs annotated to the Esr1 locus. Location of CpG sites is based on the uc056yfz.1 transcript variant. *Change in % methylation is defined as 10-month-old level relative to 2-month-old level.