Background Ribosomal DNA (rDNA) displays substantial inter-individual genetic variation in human and mouse. A systematic analysis of how this variation impacts epigenetic states and expression of the rDNA has thus far not been performed. Results Using a combination of long- and short-read sequencing, we establish that 45S rDNA units in the C57BL/6J mouse strain exist as distinct genetic haplotypes that influence the epigenetic state and transcriptional output of any given unit. DNA methylation dynamics at these haplotypes are dichotomous and life-stage specific: at one haplotype, the DNA methylation state is sensitive to the in utero environment, but refractory to post-weaning influences, whereas other haplotypes entropically gain DNA methylation during aging only. On the other hand, individual rDNA units in human show limited evidence of genetic haplotypes, and hence little discernible correlation between genetic and epigenetic states. However, in both species, adjacent units show similar epigenetic profiles, and the overall epigenetic state at rDNA is strongly positively correlated with the total rDNA copy number. Analysis of different mouse inbred strains reveals that in some strains, such as 129S1/SvImJ, the rDNA copy number is only approximately 150 copies per diploid genome and DNA methylation levels are < 5%. Conclusions Our work demonstrates that rDNA-associated genetic variation has a considerable influence on rDNA epigenetic state and consequently rRNA expression outcomes. In the future, it will be important to consider the impact of inter-individual rDNA (epi)genetic variation on mammalian phenotypes and diseases.

Epidemiological research suggests that paternal obesity may increase the risk of fathering small for gestational age offspring. Studies in non-human mammals indicate that such associations could be mediated by DNA methylation changes in spermatozoa that influence offspring development in utero. Human obesity is associated with differential DNA methylation in peripheral blood. It is unclear, however, whether this differential DNA methylation is reflected in spermatozoa. We profiled genome-wide DNA methylation using the Illumina MethylationEPIC array in a cross-sectional study of matched human blood and sperm from lean (discovery n = 47; replication n = 21) and obese (n = 22) males to analyse tissue covariation of DNA methylation, and identify obesity-associated methylomic signatures. We found that DNA methylation signatures of human blood and spermatozoa are highly discordant, and methylation levels are correlated at only a minority of CpG sites (~1%). At the majority of these sites, DNA methylation appears to be influenced by genetic variation. Obesity-associated DNA methylation in blood was not generally reflected in spermatozoa, and obesity was not associated with altered covariation patterns or accelerated epigenetic ageing in the two tissues. However, one cross-tissue obesity-specific hypermethylated site (cg19357369; chr4:2429884; P = 8.95 × 10 −8 ; 2% DNA methylation difference) was identified, warranting replication and further investigation. When compared to a wide range of human somatic tissue samples (n = 5,917), spermatozoa displayed differential DNA methylation across pathways enriched in transcriptional regulation. Overall, human sperm displays a unique DNA methylation profile that is highly discordant to, and practically uncorrelated with, that of matched peripheral blood. We observed that obesity was only nominally associated with differential DNA methylation in PLOS GENETICS

Ribosomal DNA (rDNA) displays substantial inter-individual genetic variation in human and mouse. Here we report that 45S rDNA units in the C57BL/6J mouse strain are epiallelic, existing as distinct genetic haplotypes that influence the epigenetic state and transcriptional output of any given unit. Epigenetic dynamics at these haplotypes are dichotomous and lifestage specific: at one haplotype, the DNA methylation state is sensitive to the in utero environment, but refractory to post-weaning influences, whereas other haplotypes entropically gain DNA methylation during ageing only. rDNA epiallelism is influenced by total rDNA copy number, and also found in other inbred mouse strains and humans. In the future, it will be important to consider the impact of inter-individual rDNA (epi)genetic variation on mammalian phenotypes and diseases.

BackgroundEpidemiological studies suggest that paternal obesity may increase the risk of fathering small for gestational age offspring. Studies in non-human mammals suggest that such associations could be mediated by DNA methylation changes in spermatozoa that influence offspring development in utero.Human obesity is associated with differential DNA methylation in peripheral blood. It is unclear, however, whether this differential DNA methylation is reflected in spermatozoa. We profiled genomewide DNA methylation using the Illumina MethylationEPIC array in matched human blood and sperm from lean (discovery n=47; replication n=21) and obese (n=22) males to analyse tissue covariation of DNA methylation, and identify whether this covariation is influenced by obesity. ResultsDNA methylation signatures of human blood and spermatozoa are highly discordant, and methylation levels are correlated at only a minority of CpG sites (~1%). While at the majority of these sites, DNA methylation appears to be influenced by genetic variation, obesity-associated DNA methylation in blood was not generally reflected in spermatozoa, and obesity did not influence covariation patterns. However, one cross-tissue obesity-specific hypermethylated site (cg19357369; chr4:2429884; P=8.95 ´ 10 -8 ; beta=0.02) was identified, warranting replication and further investigation. When compared to a wide range of human somatic tissue samples (n=5,917), spermatozoa displayed differential DNA methylation in pathways enriched in transcriptional regulation. ConclusionsHuman sperm displays a unique DNA methylation profile that is highly discordant to, and practically uncorrelated with, that of matched peripheral blood. Obesity only nominally influences sperm DNA methylation, making it an unlikely mediator of intergenerational effects of metabolic traits. BackgroundMultiple large-scale epigenome-wide association studies in humans have shown that environmental and acquired phenotypes, including smoking, ageing and obesity, are associated with altered DNA methylation in peripheral blood [1][2][3][4]. Whether such phenotypes also have the potential to induce epigenetic changes in gametes has generated considerable interest in recent years. Studies in nonhuman mammals suggest that the spermatozoal DNA methylome can be influenced by factors such as dietary alterations, toxicants and even psychological stress [5-10], although the majority of these results have yet to be replicated independently. A small number of studies also suggest that acquired traits in male mice induce epigenetic changes in sperm, which in turn influence the physiology of offspring [7,11,12].There is little evidence for such inter-and transgenerational effects of acquired phenotypes via epigenetic inheritance in humans. This is partly due to the fact that human sperm is rarely analysed outside of a reproductive medicine setting and is less accessible than, for example, peripheral blood.Further, it is ethically and practically impossible to perform a study of transgenerational effects in hum...