Genetic variation at mouse and human ribosomal DNA influences associated epigenetic states.

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.

DNA methylation in human sperm: a systematic review.

BACKGROUND: Studies in non-human mammals suggest that environmental factors can influence spermatozoal DNA methylation, and some research suggests that spermatozoal DNA methylation is also implicated in conditions such as subfertility and imprinting disorders in the offspring. Together with an increased availability of cost-effective methods of interrogating DNA methylation, this premise has led to an increasing number of studies investigating the DNA methylation landscape of human spermatozoa. However, how the human spermatozoal DNA methylome is influenced by environmental factors is still unclear, as is the role of human spermatozoal DNA methylation in subfertility and in influencing offspring health. OBJECTIVE AND RATIONALE: The aim of this systematic review was to critically appraise the quality of the current body of literature on DNA methylation in human spermatozoa, summarize current knowledge and generate recommendations for future research. SEARCH METHODS: A comprehensive literature search of the PubMed, Web of Science and Cochrane Library databases was conducted using the search terms 'semen' OR 'sperm' AND 'DNA methylation'. Publications from 1 January 2003 to 2 March 2020 that studied human sperm and were written in English were included. Studies that used sperm DNA methylation to develop methodologies or forensically identify semen were excluded, as were reviews, commentaries, meta-analyses or editorial texts. The Grading of Recommendations, Assessment, Development and Evaluations (GRADE) criteria were used to objectively evaluate quality of evidence in each included publication. OUTCOMES: The search identified 446 records, of which 135 were included in the systematic review. These 135 studies were divided into three groups according to area of research; 56 studies investigated the influence of spermatozoal DNA methylation on male fertility and abnormal semen parameters, 20 studies investigated spermatozoal DNA methylation in pregnancy outcomes including offspring health and 59 studies assessed the influence of environmental factors on spermatozoal DNA methylation. Findings from studies that scored as 'high' and 'moderate' quality of evidence according to GRADE criteria were summarized. We found that male subfertility and abnormal semen parameters, in particular oligozoospermia, appear to be associated with abnormal spermatozoal DNA methylation of imprinted regions. However, no specific DNA methylation signature of either subfertility or abnormal semen parameters has been convincingly replicated in genome-scale, unbiased analyses. Furthermore, although findings require independent replication, current evidence suggests that the spermatozoal DNA methylome is influenced by cigarette smoking, advanced age and environmental pollutants. Importantly however, from a clinical point of view, there is no convincing evidence that changes in spermatozoal DNA methylation influence pregnancy outcomes or offspring health. WIDER IMPLICATIONS: Although it appears that the human sperm DNA methylome can be influenced by certain environmental and physiological traits, no findings have been robustly replicated between studies. We have generated a set of recommendations that would enhance the reliability and robustness of findings of future analyses of the human sperm methylome. Such studies will likely require multicentre collaborations to reach appropriate sample sizes, and should incorporate phenotype data in more complex statistical models.

DNA methylation clocks as a predictor for ageing and age estimation in naked mole-rats, Heterocephalus glaber.

The naked mole-rat, Heterocephalus glaber (NMR), the longest-lived rodent, is of significance and interest in the study of biomarkers for ageing. Recent breakthroughs in this field have revealed 'epigenetic clocks' that are based on the temporal accumulation of DNA methylation at specific genomic sites. Here, we validate the hypothesis of an epigenetic clock in NMRs based on changes in methylation of targeted CpG sites. We initially analysed 51 CpGs in NMR livers spanning an age range of 39-1,144 weeks and found 23 to be significantly associated with age (p<0.05). We then built a predictor of age using these sites. To test the accuracy of this model, we analysed an additional set of liver samples, and were successfully able to predict their age with a root mean squared error of 166 weeks. We also profiled skin samples with the same age range, finding a striking correlation between their predicted age versus their actual age (R=0.93), but which was lower when compared to the liver, suggesting that skin ages slower than the liver in NMRs. Our model will enable the prediction of age in wild-caught and captive NMRs of unknown age, and will be invaluable for further mechanistic studies of mammalian ageing.

Early life diet conditions the molecular response to post-weaning protein restriction in the mouse.

BACKGROUND: Environmental influences fluctuate throughout the life course of an organism. It is therefore important to understand how the timing of exposure impacts molecular responses. Herein, we examine the responses of two key molecular markers of dietary stress, namely variant-specific methylation at ribosomal DNA (rDNA) and small RNA distribution, including tRNA fragments, in a mouse model of protein restriction (PR) with exposure at pre- and/or post-weaning. RESULTS: We first confirm that pre-weaning PR exposure modulates the methylation state of rDNA in a genotype-dependent manner, whereas post-weaning PR exposure has no such effect. Conversely, post-weaning PR induces a shift in small RNA distribution, but there is no effect in the pre-weaning PR model. Intriguingly, mice exposed to PR throughout their lives show neither of these two dietary stress markers, similar to controls. CONCLUSIONS: The results show that the timing of the insult affects the nature of the molecular response but also, critically, that 'matching' diet exposure either side of weaning eliminates the stress response at the level of rDNA methylation and small RNA in sperm.

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

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.