Early-life nutrition modulates the epigenetic state of specific rDNA genetic variants in mice

Abstract

A suboptimal early-life environment, due to poor nutrition or stress during pregnancy, can influence lifelong phenotypes in the progeny. Epigenetic factors are thought to be key mediators of these effects. We show that protein restriction in mice from conception until weaning induces a linear correlation between growth restriction and DNA methylation at ribosomal DNA (rDNA). This epigenetic response remains into adulthood and is restricted to rDNA copies associated with a specific genetic variant within the promoter. Related effects are also found in models of maternal high-fat or obesogenic diets. Our work identifies environmentally induced epigenetic dynamics that are dependent on underlying genetic variation and establishes rDNA as a genomic target of nutritional insults.

Figure 1. Maternal protein restriction (PR) induces a correlation between rDNA methylation and weaning weight

A. Statistical tests were performed using litter means (large symbols (n_L)), individuals are represented with small symbols (n). Control = black, PR = red. Litters of 5-10 pups included. Weaning weight of PR males (n = 83, n_L = 17) were reduced compared to Control (n = 135, n_L = 20) (left; t-test, P = 2 x10^-6). B. Methylation across the rDNA promoter and coding region was determined in sperm of using RRBS. PR sperm (red, n = 8) is relatively hypermethylated compared to Control (black, n = 8). Mean methylation % (line) and individual mice (points) are shown. The relative position of the transcriptional start site (TSS) and rRNA subunits are shown (top). The region of the rDNA 98% homologous to the differentially methylated region on chromosome 17 (mm10) is shaded blue. C. The correlation coefficient (τ) between weaning weight and DNA methylation for each CpG site covered (> 100X) with RRBS is shown for the rDNA promoter and coding region in sperm. Control (black, n = 8), PR sperm (red, n = 8). Illustrations for the derivation of the correlation coefficient are provided when positive (green point), close to zero (purple point) and negative (orange point) on the left hand side. The relative position of the transcriptional start site (TSS) and rRNA subunits are shown (top). CpG-133 is circled in blue. Non coding regions include the promoter, ETS = external transcribed spacer, ITS = internal transcribed spacer.

Figure 2. Diet induced dynamics are restricted to a specific genetic variant of rDNA and reflect the relative copy number of that variant

A. BisPCR-seq amplicons were generated spanning both CpG-133 (methylation is indicated by black circle) and the genetic variant (A or C) at position -104, relative to the TSS (left panel). The percentage of CpG-133 methylation in sperm for each genetic variant is shown for Control sperm (black, n=15), and PR sperm (red, n=17). B. Methylation of CpG-133 in A-variant rDNA copies (CpG-133^A, x-axis) does not correlate with weaning weight (y-axis) in Control sperm (black, n=15; τ = 0.20, P = 0.30). Methylation of CpG-133 in A-variant rDNA copies (CpG-133A, x-axis) correlates negatively with weaning weight (y-axis) in PR sperm (red, n=17; τ = -0.43, P = 0.017). C. Methylation of CpG-133 in A-variant rDNA copies (CpG-133^A, x-axis) does not correlate with weaning weight (y-axis) in Control liver (black, n=26; τ = -0.14, P = 0.32). Methylation of CpG-133 in A-variant rDNA copies (CpG-133^A, x-axis) correlates negatively with weaning weight (y-axis) in PR liver (red, n=24; τ = -0.46, P =0.0016). D. Methylation of CpG-133 in A-variant rDNA copies (CpG-133^A, x-axis) does not correlate with the percentage of total rDNA copies with an A-variant (%A, y-axis) in Control sperm (black, n=15; τ = -0.07, P = 0.77). Methylation of CpG-133 in A-variant rDNA copies (CpG-133^A, x-axis) correlates positively with the percentage of total rDNA copies with an A-variant (%A, y-axis) in PR sperm (red, n=17; τ = 0.71, P = 1.9 x 10^-5).

Figure 3. Functional consequences of altered rDNA dynamics

A. pRNA is transcribed from early replicating rDNA copies (assumed to be unmethylated at CpG-133). If this assumption is true, the percentage of pRNA reads that encode an A at position -104 (pRNA(%A); indicated in blue; right) is a reflection of not only the relative abundance of A-variant rDNA copies (%A; indicated by A in black; left), but also the methylation of the A-variant (CpG-133^A, methylation is indicated by black circles). B. The percentage of pRNA reads that encode an A at position -104 (pRNA(%A); y-axis) positively correlates with the percentage of unmethylated A-variant reads as a total of all rDNA copies (%A^UN) in both Control (black) and PR (red) liver (total, n=23, τ = 0.61, P = 1.4 x10^-5). C. The percentage of unmethylated A-variant reads as a total of all rDNA copies (%AUN, x-axis) is not correlated with the abundance of unprocessed 45S-rRNA (y-axis) in liver of Control (black; n=14, τ = 0.03, P = 0.91), but is positively correlated in liver of PR (red; n=12, τ = 0.52, P = 0.021).

Figure 4. High fat or Obesogenic diet perturbs rDNA and a model for PR induced rDNA interactions.

A. RRBS raw sequencing reads (obtained from Cannon et al., 2014) were mapped to the rDNA consensus. Dams were fed either a low (M^L) or high fat (M^H) diet prior to conception and up until the pups were weaned. At weaning pups were placed onto either a low (A^L) or high fat (A^H) diet, to give rise to four dietary categories as indicated (x-axis). Data is from liver of 9 week old male offspring. A-variant CpG-133 methylation (CpG-133^A, y-axis) was significantly higher in the liver of 9 week old male offspring fed M^HA^L (t-test, P = 0.0098) and M^HA^H (t-test, P = 0.0079), compared to M^LA^L. n=10 for each dietary group. B. Dams were fed either a Control (C) or Obesogenic (O) diet 6 weeks prior to conception and up until the pups were weaned. At weaning pups were placed onto either a Control (C) or Obesogenic (O) diet, to give rise to four dietary categories as indicated (x-axis). Data is from liver of 6 month old male offspring. A-variant CpG-133 methylation (CpG-133^A, y-axis) was significantly higher in the liver of 6 month old male offspring exposed in early development to an obesogenic diet, but weaned onto control diet (OC, t-test, P = 0.017), compared to CC. (CC: n=7, CO: n=8, OC: n=8, OO: n=7). C. There is natural variation in the relative copy number of rDNA with an A-variant at position -104 (%A, indicated left, grey). Methylation is established independently of copy number shortly after fertilisation and this is maintained throughout development in Control fed animals. However, in animals fed a PR diet, animals with higher %A silence more A-variants through methylation at CpG-133 and this correlates with reduced weaning weight. Once established, this methylation pattern is maintained into adulthood, in the absence of further exposure.