Modeling the consequences of age-linked rDNA hypermethylation with dCas9-directed DNA methylation in human cells

  1. Yana Blokhina
  2. Abigail Buchwalter

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Abstract

Ribosomal DNA (rDNA) genes encode the structural RNAs of the ribosome and are present in hundreds of copies in mammalian genomes. Age-linked DNA hypermethylation throughout the rDNA constitutes a robust “methylation clock” that accurately reports age, yet the consequences of hypermethylation on rDNA function are unknown. We confirmed that pervasive hypermethylation of rDNA occurs during mammalian aging and senescence while rDNA copy number remains stable. We found that DNA methylation is exclusively found on the promoters and gene bodies of inactive rDNA. To model the effects of age-linked methylation on rDNA function, we directed de novo DNA methylation to the rDNA promoter or gene body with a nuclease-dead Cas9 (dCas9) – DNA methyltransferase fusion enzyme in human cells. Hypermethylation at each target site had no detectable effect on rRNA transcription, nucleolar morphology, or cellular growth rate. Instead, human UBF and Pol I remain bound to rDNA promoters in the presence of increased DNA methylation. These data suggest that promoter methylation is not sufficient to impair transcription of the human rDNA and imply that the human rDNA transcription machinery may be resilient to age-linked rDNA hypermethylation.

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    Reply to the reviewers

    We would like to thank the reviewers for their thoughtful evaluation of our work. Our point-by-point responses to reviewer critiques follow below. Please note that any referenced changes to the manuscript are highlighted in yellow in the revised manuscript text.

    Response to Common Critiques

    1. Reviewers 1 and 2 state that some elements of this study confirm previously published results (many in murine systems). However, the reviewers also acknowledge that the mouse and human rDNA repeats may be subject to quite distinct regulation because of the much denser CG content of the human rDNA promoter (26 CpGs) vs. the mouse rDNA promoter (only 2 CpGs); these potential differences in regulation motivated this study in human cells. We evaluate the functions of rDNA methylation in human cells, which is directly relevant to understanding the regulation of rDNA function in human aging, and to understanding the functional implications of DNA methylation "aging clocks" more generally. We also apply a recently developed technology (dCas9-mediated epigenome editing) to directly test the function of rDNA methylation. Novel findings reported in this study include:
    2. Pol I - engaged rDNA repeats are hypomethylated at sites both in the promoter and the gene body; this contrasts with Pol II transcription, which is coincident with gene body methylation.
    3. rDNA copy number remains stable with age in mammals, in striking contrast to findings in other eukaryotes. rDNA copy number instability has been proposed to be a universal feature of the aging genome, and this finding refutes that possibility.
    4. Induction of DNA methylation by an average of ~20% along 7-11 of the 26 CpGs in the human rDNA repeat does not measurably inhibit rDNA transcription.
    5. Human Pol I and UBTF remain bound to rDNA promoters in the presence of elevated CpG methylation, in contrast to the murine Pol I machinery.

    Reviewers 1 and 2 questioned our strategy of mapping sequencing data to the consensus ribosomal DNA (rDNA) repeat alone. We followed the approach of Wang & Lemos Genome Research 2019, who initially described the rDNA methylation clock. Wang & Lemos also mapped genomic data to rDNA consensus sequences alone due to the computational efficiency of this approach, and describe a head-to-head comparison of mapping performance outcomes in their Methods section. Importantly, their analysis indicated that the vast majority (>98%) of sequencing reads can be mapped uniquely to the consensus human rDNA repeat (U13369.1). When we launched our study, we also initially compared the performance of mapping to the rDNA repeat consensus sequence alone versus to the whole human genome. We noted very similar performance in both cases, with the possible exception of a modest increase in simple repeat sequences being erroneously mapped to the intergenic spacer (IGS) region of the rDNA when we mapped to the rDNA repeat alone. As the reviewers pointed out, the IGS contains simple repeat sequences that are also found at numerous other non-rDNA sites in the genome. However, the minor mis-mapping of simple repeats to the IGS did not affect our analyses of non-IGS sequences, which were the focus of this study. We therefore proceeded with mapping to the rDNA consensus sequence only.

    Reviewers 1 and 2 pointed out that our dCas9-DNMT strategy induced only a 15-20% increase in rDNA methylation and questioned whether we could expect to detect downstream effects in rDNA transcription. While Reviewer 2 suggested that multiple sgRNAs could enhance methylation efficiency, it turns out that this has already been tested for other target genes and shown that multiple sgRNAs cannot increase efficiency of CpG methylation by dCas9-DNMTs (Stepper et al., Nucleic Acids Research 2017). Separately, the goal of this study was to model the effects of age-linked rDNA hypermethylation, which increases by 15-20% over mammalian lifespan (Wang & Lemos 2019; see also our Figure 1). Importantly for interpreting these data, induction of promoter methylation to a similar extent on the mouse rDNA repeat was able to direct detectable repression of rDNA transcription (Santoro et al., 2011). Further, dCas9-DNMT has been previously shown to induce a ~20% increase in CpG methylation of the Pol II target gene EpCAM and cause measurable transcriptional repression that was detectable by qPCR (Stepper et al., 2017). In contrast, we were able to induce rDNA methylation to a similar extent and observed no change in the levels of either pre-rRNA or mature rRNA. Because we see that UBF and Pol I remain bound to rDNA in spite of higher CpG methylation (Fig. 7 and Fig. S4), we interpret these data together to indicate that the human Pol I machinery can continue to engage with rDNA in the presence of intermediate levels of CpG methylation.

    Reviewer 1

    inactivation of rDNA transcription per se does not affect chromatin accessibility, to date only depletion or deletion of UBTF has been found to do this and even this does not enhance CpG methylation, these published findings should be referenced.

    Our analyses in Figure 2 focus on defining the relationships between chromatin accessibility, transcriptional activity, and CpG methylation throughout the human rDNA repeat. We cannot determine causation from this analysis - meaning whether chromatin accessibility influences CpG methylation or vice versa - and this point is beyond the scope of our study. Our major goal was to test whether induced CpG methylation affects transcription output.

    The authors overstate their results by writing "actively transcribed rDNA repeats are hypomethylated at their promoter" despite only one SmaI site but many CpG sites exist in the human promoter, the latter having not been assayed.

    We analyzed several pieces of data to come to this conclusion. First, ATAC-Me indicates that ATAC-accessible rDNA repeats are completely devoid of methylation both in their promoter and throughout the gene body; as UBTF binding controls rDNA accessibility (Sanij et al., JCB 2008; Hamdane et al., PLoS Genet 2014), we infer that ATAC-accessible repeats are engaged with the Pol I transcription machinery and hypomethylated. To more directly probe this question, we evaluated the methylation status of Pol I-bound rDNA repeats at five separate sites by ChIP-chop: two sites in the 5' regulatory region (5' ETS and core promoter, pooled together as "promoter" in Figure 2F) and three sites within the gene body (18S, 5.8S, and 28S, pooled together as "gene body" in Figure 2F). These data clearly indicate that Pol I preferentially binds to these regions when they are hypomethylated, as the extent of CpG methylation at these same sites is higher in input DNA and lower in Pol I-ChIPped DNA. While we do not comprehensively profile CpG methylation status of Pol I-bound DNA, these ChIP-chop analyses are consistent with our interpretation that "actively transcribed (that is, Pol I-engaged) rDNA repeats are hypomethylated at their promoter".

    Pol I's preference for binding hypomethylated promoters has been previously described in mouse cells (Santoro & Grummt 2001) and human cells (Brown & Szyf Mol Cell Biol 2007). We confirm this and also report the novel finding that rDNA gene bodies bound by Pol I are hypomethylated. This contrasts with known relationships between Pol II and CpG methylation, where genes actively transcribed by Pol II often have dense gene body CpG methylation.

    While we think it is reasonable to infer from ATAC-Me data and ChIP-chop data together that accessible and hypomethylated rDNA repeats reflect transcriptionally active repeats, we appreciate the reviewer's point that we analyzed only a select few CpG sites by Pol I ChIP-chop. We have adjusted the text to make our interpretation more parsimonious (see highlights).

    The human rDNA promoter contains many CpGs which may not affect transcription when methylated. RRBS and WGBS data can't tell us much if we don't understand which sites, when methylated, affect transcription*. *

    We agree, and this ambiguity is what motivated us to induce methylation and evaluate the consequences. In plasmid reporter experiments where the human rDNA promoter was fused to a luciferase reporter, it was shown that in vitro methylation of the plasmid potently inhibited transcription in human cells (Ghoshal et al., J Biol Chem 2004). In this study, methylation of 7/26 CpGs was sufficient to induce >75% inhibition of reporter plasmid transcription, while methylation at single sites could induce ~50% inhibition. We neglected to site this relevant study and have included a reference to it in the revised manuscript. Importantly, this plasmid reporter assay does not assess the effects of CpG methylation on the full rDNA repeat in its endogenous genomic context. We were able to induce significant CpG hypermethylation on 11/26 promoter CpGs with one guide (P+G) and on 7/26 CpGs with a second guide (P+A) (Figure 3D). This level of methylation did not induce detectable silencing of rRNA transcription. Instead, we found that both UBF (Fig. 7) and Pol I (Fig. S4) remained bound to rDNA in the presence of CpG hypermethylation.

    The argument that the mouse rDNA Pol I machinery is "exquisitely sensitive" to CpG methylation is a little misleading as there are only two CpGs in the mouse rDNA promoter. Which of the 26 human CpGs are the critical ones?

    Immediately following this statement in the Discussion, we state that "the human rDNA promoter is significantly more CG-rich than the mouse rDNA promoter". We have revised this section to emphasize the difference (26 CpGs in human vs. only 2 in the mouse) and discuss this point raised by the reviewer: which are the critical CpGs in the human rDNA? Here again it is relevant to cite the human rDNA promoter reporter assays performed by Ghoshal et al., J Biol Chem 2004. These data indicate that CpG methylation of 7/26 promoter CpGs interferes with transcription from an rDNA reporter plasmid. Notably, it is unclear how generalizable findings from reporter assays are to the genomic context of the endogenous full length rDNA sequence. Our data indicate that partial methylation of 7-11 CpGs in the human rDNA promoter causes no detectable rDNA inhibition, and indeed does not displace UBF or Pol I (Fig. 7; Fig. S4).

    Antibody SC13125 used for UBF ChIP sees nearly exclusively the shorter transcriptionally inactive UBF2 variant. These data need to be repeated with an antibody that detects both UBF forms.

    We thank the reviewer for raising the important issue of UBTF splice isoforms. Relevant citations demonstrating that the SC13125 antibody recognizes only UBF2 would have been very helpful. The human UBTF gene is alternatively spliced into full-length UBF1 (exon 8 retained) and UBF2 (exon 8 spliced out). The deletion of exon 8 results in a 37 amino acid deletion in UBF2 corresponding to residues 221-268 in HMG box 2 of UBF1 (see Ensembl entry ENSG00000108312.16). The truncation of HMG box 2 makes UBF2 a far less potent transcriptional activator than UBF1. Because of the small molecular weight difference between these two isoforms, preference of an antibody for one vs. another isoform is not readily apparent by Western blotting. However, according to the manufacturer of the UBTF antibody used in this study, the immunogen corresponds to residues 1-220 of UBTF1, which is immediately N-terminal to the residues deleted in UBF2 (AAs 221-268, encoded by exon 8). The antibody's immunogen is thus entirely sequence that is shared between UBF1 and UBF2. Further, a previous study performed immunoprecipitation followed by mass spectrometry using this antibody and reported detection of UBF1-specific peptides (Drakas et al., PNAS 2004). Therefore, absent our knowledge of any evidence to the contrary, we conclude that this antibody recognizes UBF1 and possibly also UBF2.

    We thank the reviewer for raising this point and have adjusted the text to avoid the misleading implication that we are unambiguously detecting only the UBF1 isoform; all mentions of "UBF1" in the revised text have been replaced with "UBTF".

    Setting aside the question about the UBTF antibody reagent used, we observe consistent results by evaluating both UBTF (Figure 7) and Pol I (Figure S4) binding to rDNA in spite of CpG methylation; therefore, we conclude that the human Pol I machinery is not displaced from the human rDNA promoter by intermediate levels of CpG methylation.

    Reviewer 2

    There is very little discussion concerning the methylation status of the IGS...the Kobayashi lab has convincingly demonstrated that rDNA repeats fall into 2 classes. Those in which the supposedly active repeats lack methylation on promoters and coding regions and those in which both promoters and coding regions are heavily methylated. In both cases the IGS is fully methylated.

    We cite this study in the Discussion (reference 18 in bibliography) and agree that this work is relevant to ours; we have adjusted the text to emphasize this point. Notably, this previous analysis of CpG methylation patterns by long-read sequencing implied that active repeats may be entirely hypomethylated along their coding sequence; our data more directly demonstrate this both by ATAC-Me and by Pol I ChIP-chop (Fig. 2).

    There is no description of how rRNA levels were assessed. I suggest this could be further complemented by in vivo incorporation studies such as EU labeling.

    We apologize for this lack of clarity. rRNA levels were assessed by qPCR of the 45S pre-rRNA (Fig. 3A) and of mature 28S rRNA (Fig. 3B), and these data are presented as a fold change in each rDNA-targeting sgRNA compared to a non-targeting control sgRNA. The primersets used are listed in Supplementary Table 1.

    While we agree that EU labeling could be useful for detecting nucleolar transcription, qPCR detection of the 45S rRNA also sensitively reports nascent transcription and we think is sufficient to address this question.

    Reviewer 3

    The study points to differences between mouse and human rDNA and the effect of DNA methylation on transcriptional output. Did the mouse rDNA dataset also measure transcription output to correlate with DNA methylation age differences?

    The original study that defined the rDNA methylation clock (Wang & Lemos Genome Research 2019) did not evaluate rDNA transcription in parallel. More generally, the relationship of age-linked "clock" CpG methylation sites to expression / function of CpG methylated loci is very unclear, and testing the potential relationship between age-linked rDNA methylation and function was the major goal of this study.

    Did the spacer promoter also get methylated and did that affect UBF and Pol I binding?

    While the existence and function of a spacer promoter has been more clearly defined in the mouse rDNA repeat, recent evidence indicates that the Pol I transcription machinery also binds a second location about 800 bp upstream of the core promoter in the human rDNA repeat (Mars et al G3 2018). The guides that we used to direct CpG methylation recognize single unique sites in the core rDNA promoter and do not recognize sequences in this putative spacer promoter, and we did not analyze methylation at the spacer promoter. Analysis of the spacer promoter is generally beyond the scope of this study, as it is unknown whether there is any relationship between spacer promoter methylation and aging progression.

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript Modeling the consequences of age-linked rDNA hypermethylation with dCas9-directed DNA methylation in human cells studies the DNA methylation during aging at the rDNA. The study is well performed and provides several new insights into rDNA transcriptional regulation. The main finding is that in human cells, rRNA methylation does not affect transcription output, UBF and RNA pol I binding, even though the bound gene copies are less methylated than the silent ones. The experimental approach is excellent; the data mining and experiments are appropriate and shows essential points. The results are very interesting and provides new aspects to the state of rDNA that will further the understanding of ribosomal transcription.

    Minor concerns

    The study points to differences between mouse and human rDNA, and the effect of DNA methylation on transcriptional output. Did the in the mouse rDNA data-set also measure transcription output to correlate with DNA methylation age-differences.

    Some rRNA genes, including the human gene repeat, has a second promoter 7-800 base pairs upstream of the promoter. This site also contains a CTCF binding site, upstream of which nucleosomal chromatin state is found. Downstream of the spacer promoter, a UBF associated chromatin state assembles, presumable on active copies. Did the spacer promoter also get methylated and did that affect UBF binding and pol I binding?

    Significance

    This is a very interesting and novel study which just needs to be extended to other feature of the rDNA to provide a full picture. The results presented in the manuscript are novel and contributes to the understanding of ribosomal transcription, in particular the outstanding question about the impact of DNA methylation on the transcriptional output and chromatin states. It provides important insight into how to think about rRNA transcription in different cell lines, states and diseases, such as cancer. The general aspects of the study suggest a broad broad.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    Mammalian genomes typically contain between 150 and 250 copies of a ribosomal gene repeat (rDNA) that are transcribed by RNA polymerase I to yield pre-rRNAs that encode rRNAs. It is generally accepted, that in most cells as many as 50% of repeats are transcriptionally silent. It is now appreciated that the regulatory elements and transcribed regions of these "silent" repeats are heavily methylated. Thus rDNA hypermethylation correlates with silence. However, whether this is a driver of silencing or a consequence of silencing is open to debate. This manuscript weighs into this debate. Initial experiments remap existing bisulfite sequencing data from both the mouse and humans. These results confirm previous data that rDNA hypermethylation correlates with aging. Next, to strengthen links between hypermethylation and silencing, they remap methylation-resolved ATAC sequencing data. This confirms that hypomethylated rDNA is in a more open chromatin conformation, presumably the "active repeats". In mammals there have been competing claims regarding changes in rDNA copy number during aging. Notably it has been claimed previously that rDNA copy number drops during human aging. A potential flaw in that study is that it studies of rDNA copy number utilised genetically diverse human populations. Here, using digital PCR, they survey rDNA copy number in various tissues of an inbred mouse strain. Analysing young mice and old mice, they find no evidence for age related rDNA loss. While the above experiments are well performed the results are largely confirmatory in nature. The next set of experiments attempt to address a critical question, namely, is rDNA hyper methylation a 'cause' of a 'consequence' of silencing. They generated an inducible nuclease dead CAS9 fused with de novo methyltransferase function (dCas9-3A3L) and gRNAs targeting either the promoter of the 28S coding region. Experiments performed in transformed and non-transformed human cell lines demonstrated a 15-20% methylated rDNA. Analysis of pre and mature rRNAs as well as cell staining reveal that transcript levels and nucleolar morphology are unaltered. Furthermore, the finding that UBF 'chipped' rDNA is more heavily methylated argues that directed methylation of the human rDNA promoter does not displace UBF. These experiments suggest that rDNA hypermethylation is more of a consequence of silencing than a cause of silencing.

    Major comments:

    It is not clear from the methods how previous rDNA was mapped onto rDNA repeats. Did they generate a customised reference genome with rDNA added, or simply map reads to rDNA in isolation. This is of critical importance as only reads that uniquely map to rDNA should be considered. Mammalian genomes typically contain many rDNA pseudo genes. Furthermore, the rDNA intergenic spacer (IGS) contain many retro/repeated elements that are distributed throughout the genome.

    There is very little discussion concerning the methylation status of the IGS. Using nanopore sequencing the Kobayashi lab has convincingly demonstrated that rDNA repeats fall into 2 classes. Those in which the supposedly active repeats lack methylation on promoters and coding regions and those in which both promoters and coding regions are heavily methylated. In both cases the IGS is fully methylated.

    In the targeted methylation experiments the increase in rDNA methylation remains both local and modest 15-20% increase. Would it be possible to increase the number of gRNA so as to achieve a higher level and more distributed change in rDNA methylation.

    Minor comments:

    The older U13369 rDNA reference has many sequence errors and should be avoided.

    There is no description of how rRNA levels are assessed. I suggest this could be further complemented by in vivo incorporation studies such as EU/click-chemistry.

    Significance

    Around 50% of data presented in this manuscript (Figs 1-3) is confirmatory rather than novel. While the data regarding targeted methylation of "active rDNA repeats is interesting, and I think pointing us in the right direction, it is not comprehensive enough to overturn the pervasive notion that methylation causes silencing.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The manuscript attempts to provide an answer to why methylation of the human rDNA correlates with aging. They conclude that this correlation is not connected with changes in rDNA activity of copy numbers.

    Major comments:

    The authors reanalyze public data from RRBS and WGBS that suggests a correlation between aging and rDNA methylation. They then use public ATAC-Me sequence data and show a good correlation between chromatin accessibility and lack of CpG methylation. This correlation has been known for some time, but the ATAC-Me approach is a nice confirmation that it extends through the coding region and probably the promoter and enhancer sequences. In referring to the correlation between open chromatin and hypomethylation the authors state that "these data imply that methylation of the rDNA promoter and gene body both occur exclusively on non-transcribed, silent repeats" However, it is known that inactivation of rDNA transcription per se does not affect chromatin accessibility, to date only depletion or deletion of UBTF has been found to do this and even this does not enhance CpG methylation, these published findings should be referenced. The authors do recognize this and use the so-called ChIP-chop (not ChIP-ChOP) method to analyze methylation of PolI ChIPped DNA at a single SmaI site in the 47S promoter and a site within the 28S (Table S1 showing primers was not available to me to define the exact regions, the ref to Santoro for the technique should be 2014 not 2013). The ChIP-chop assay repeats previous work but here is done on HEK293T, the cell line they use for later study. The authors also overstate their results by writing "actively transcribed rDNA repeats are hypomethylated at their promoter" despite only one SmaI site but many CpG sites exist in the human promoter, the latter having not been assayed.

    The authors do go on to convincingly show rDNA copy numbers are constant with age by assaying various mouse tissues from young and old mice, hence excluding this as an affector of aging. They then attempt to use targeted de novo methylation to ask if this has any effect on rDNA transcription. Such effects have been extensively claimed as a source of rDNA regulation, though there is little evidence that this occurs in vivo. The authors use dCas9 targeted DNMT to locally enhance methylation using two promoter and one 28S guide RNAs and are able to show mean increases of 15 to 20% by ChIP-chop (but 40 to 50% at other CpGs by WGB-seq (BSAS), not discussed). Measurement of pre-rRNA and 28S abundance (relative to what control is not stated), cell proliferation, PolI nucleolar distribution and UBF (incorrectly referred to UBF1, see comment below) occupancy at the promoter are all suggested to show no effects of this targeted methylation. Hence the authors conclude that "These data suggest that promoter methylation is not sufficient to impair transcription of the human rDNA and imply that the human rDNA transcription machinery may be resilient to age-linked rDNA hypermethylation" But in fact no more than a 20% change due to the targeted methylation should be expected in any of the parameters measured. It is not at all evident that such a small effect would be detected by the authors.

    Specific points:

    Mapping was to the rDNA repeat unit in the absence of the human genome. This may bias the mapping data since the human genome contains rDNA pseudogenes and intermediate repetitive elements that are also present in the rDNA unit. These will be present in all the RRBS and WGS datasets, may or may not change methylation levels with age and will be mapped onto the single copy of the rDNA used in the data alignment. These factors need to be controlled.

    The human rDNA promoters contain many 26 CpGs, most of which may have no effect on transcription when methylated. Thus, very little of significance can be gleaned from RRBS data and this goes for WBS data without understanding which sites when methylated affect transcription.

    The argument that the mouse rDNA is "mouse Pol I machinery is exquisitely sensitive to a single CpG methylation event in the UCE, which blocks UBF binding and prevents transcription". Here the reference is to one of only two CpGs in the mouse promoter and, in this reviewer's opinion, the effect of its methylation has never been convincingly shown in vivo on the endogenous genes. However, if true, it also opens the question of which of the 26 CpGs in the human promoter are critical ones.

    Antibody SC13125 used for UBF ChIP sees near exclusively the shorter transcriptionally inactive UBF2 variant. These data need to be repeated with an antibody that detects both UBF forms.

    Significance

    We believe that the authors are correct in their conclusion that rDNA activity is not significantly affected by the level of CpG methylation. This said, the data presented in the manuscript does not provide strong support for this notion and hence, does not significantly advance our understanding of the role of rRNA in aging.

  5. Version published to 10.1101/2023.10.18.562830v2 on bioRxiv
  6. Version published to 10.1101/2023.10.18.562830v1 on bioRxiv