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  1. Evaluation Summary:

    This manuscript extends the evidence that ribosomal DNA has substantial interindividual variation, and presents evidence that variants are associated with differences in DNA methylation. The authors show that some rDNA types respond to environmental signals during in utero development, whereas others are changed during the aging process - thus broadening the known communication between development/nutrition/aging and the cellular protein synthesis machinery. These findings have relevance for the influence of such epialleles on gene expression and disease risk.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  2. Reviewer #1 (Public Review):

    Work by the authors and others previously revealed that there are different sequence variants of the DNA encoding for ribosomal RNA in mouse, that these variants have different DNA methylation state and are affected differently by "the environment" (by diet). Previous work was focused on very specific single nucleotide variants but a global picture of all the different rDNA copies/haplotypes and their DNA methylation state was missing. The aim of the current paper was to fill this gap and it achieved this.

    Because the sequence of the different rDNA sequence variants (that vary even between animals of the same inbred mouse strain) are not fully annotated and included in the mouse genome assemblies, the authors first cataloged them using their own short read and ultra-long read whole genome sequencing from DNA from four mice of the most widely used inbred mouse strain (C57BL/6J). This revealed the presence of four different haplotypes, that form two pairs based on genetic similarity. One of the genetic differences that discriminates the two pairs of haplotypes is the A/C variant at position -104 previously analysed by this group in publications in 2016 and 2018. One of the four haplotypes (the ATA haplotype) was found to be highly methylated in all samples. The authors then showed that DNA methylation of this specific haplotype is affected by dietary interventions (this way refining the observations from their 2016 and 2018 papers) and also that (extreme) loss of DNA methylation leads to its transcriptional activation. This data provides indirect evidence that "the environment" can potentially affect the expression of different rDNA haplotypes. This is an exciting result showing a potential interconnection between the environment, the genotype (already previously reported by this group) AND a molecular phenotype. However the link with the phenotype requires more work to become convincing and to also show that - even if this interconnection is indeed true - that it affects a physiological phenotype.

    The paper then attempts to test whether different rDNA haplotypes with distinct DNA methylation profiles also exist in other mouse strains and also in humans. Through DNA sequencing, DNA methylation sequencing (WGBS) and rRNA sequencing they show that indeed different sequence variants with different DNA methylation level exist in other mouse strains and one also is identified in human. An interesting additional observation is that both human and mouse data support a link between rDNA copy number and silencing, as previously reported by other groups in yeast.

    Strengths:
    * The paper presents the most extensive annotation of the different rDNA haplotypes in the C57BL/6J mouse genome. This annotation and the generated data will be very valuable to anyone interested in rDNA in this species.
    * DNA methylation data complement the generated rDNA sequence data and provide a picture (from a few tissues/cell types) of the DNA methylation level of these haplotypes and their variation. One of the four main haplotypes appears to be highly DNA methylated. This will also be useful to scientists interested in rDNA regulation and also to those interested in how the environment affects the regulation of specific haplotypes.
    * Similar data is also generated for five additional mouse strains, although the analysis and interpretation of this data is not as extensive as for the C57BL/6J strain.
    * Evidence is shown that supports the hypothesis that rDNA copy number affects rDNA silencing in mammals, as previously observed in yeast.

    Weaknesses:
    * The manuscript follows up from previous work by the same group (Holland et al Science 2016 and Danson et al BMC Biology 2018). The work presented in this manuscript is clearly the most complete, but the conclusions from the analysis presented here are consistent with the conclusions from analyses from data already in previous papers and therefore the conceptual advances are - in relative terms - small. This does not undermine the importance of performing an extensive analysis of the mouse rDNA variants and making the new data and results public.
    * A lot of data is generated for five mouse strains in addition to C57BL/6J and rDNA variants and DNA methylation variant positions are also identified. However, the data from these strains is not as extensively analysed as for C57BL/6J. So, for example, there is no annotation of haplotypes, presumably because of the lack of nanopore sequencing data. A more detailed comparison of the variants found in these strains would be helpful to the reader.
    * The analysis of human data is mainly used to provide some evidence that rDNA alleles with different DNA methylation also exist in human. A single significant nucleotide variant is found with allele-dependent DNA methylation (site 7980). At this site the authors also report the correlation between copy number and DNA methylation of rDNA in human and that there is anti correlation between DNA methylation at this site and its relative abundance in rRNA - as found in mice. Although this observation is exciting, it is based on a single nucleotide variant and even for this variant I found the evidence weak.

    Overall, I consider that the most significant part of this paper is the generation of mouse rDNA haplotypes for the C47BL/6J mouse strain. This will be very useful to the scientific community interested in rDNA in mouse and in further testing the effect of environmental exposures on rDNA activity. Other than that, it strengthened previous reports that there are different rDNA alleles in mouse with different DNA methylation and that the environment affects these alleles differently (as reported in Holland et al). An additional interesting result - consistent with findings in other organisms - is that there is some evidence of correlation between epigenetic silencing and rDNA copy number in mammals. However, in my opinion, these latter results appear rather preliminary at this stage.

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  3. Reviewer #2 (Public Review):

    In this manuscript, Rodriguez-Algarra and co-authors show that certain ribosomal DNA (rDNA) variants respond to insults of in utero development by accumulating DNA methylation, whereas others are altered only due to long term aging. They undertake this work using multiple methods, but probably the most compelling and novel relies upon long read nanopore sequencing data that can simultaneously give a read out for DNA sequence and cytosine methylation. They characterise 4 major haplotypes of rDNA which they term ATA, ATG, CCA and CTA. The ATA haplotype has significant levels of methylation (~60% and greater) and appears to be sensitive to perturbations in-utero. In contrast, the other haplotypes have low methylation levels, but gain methylation during aging.

    In doing this work, Rodriguez-Algarra et al. show that rDNA has an epiallelic quality that is intrinsically linked to multiple environmental signals (i.e. early developmental nutrition and aging), in a clearly dichotomous nature. To confirm this new insight has broader relevance, they examined additional strains of mice finding rDNA methylation was correlated with its copy number (as with the original strain), an observation further supported by analysis in Human.

    Nevertheless, the manuscript could be improved by a broader outlook, incorporating understanding from non-mammalian systems. In addition, more could be done to understand the linkage of rDNA haplotypes and their genomic location.

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  4. Reviewer #3 (Public Review):

    This manuscript characterizes inter-individual variants in rDNA at the 45S rDNA unit; these variants are associated with variable DNA methylation and histone modification and sensitivity to environmental stimuli (thus, epialleles). Moreover, rDNA copy number was associated with variant frequency and DNA methylation.

    Abundant data in the primary and supplementary figures. provide support for the conclusions in the paper. The data consistently demonstrate an association between the ATA variant and increased rDNA methylation, increased H3K9me3, and relative depletion of H3K27me3, and reduced binding of UBTF (which binds unmethylated sites). Moreover, the ATA variant appears to be more sensitive than other variants to prenatal and postnatal dietary factors. Moreover, the authors demonstrate that allele frequencies can be detected whether or not ribosomal depletion steps are performed prior to RNA seq.

    Several questions remain in order to more fully integrate these data into concepts of regulation of rDNA and rRNA and responsiveness to environmental stimuli:

    1. What is the relationship between allelic variants and physiology? Are allelic variants associated with differential expression of rRNA or ribosome subunits /composition at a protein level? The current manuscript provides only preliminary data from a small number of samples (Figure S16) to support a relationship between variant frequency in RNA and in polysome-associated RNA. Expansion of these data to assess ribosomal function will be required in future studies to provide more functional relevance for the findings.

    2. rDNA promoter methylation varies across mouse strains, in proportion to rDNA copy number. It remains uncertain whether differences in allelic variant distribution are driving differences in copy number across strains, or between animals of the same strain, or vice versa. Expansion of sample number within a strain would help to address this question and allow examination of additional determinants of rDNA copy number within individual animals.

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