The Intertwined Story of DNA Methylation and Epigenetic Aging

April 28, 2025 By Stuart P. Atkinson

Cartoons of dCas9–DNMT3A and CRISPRoff constructs. — Schematic of dCas9–DNMT3A fusion versus CRISPRoff systems targeting CpG methylation, showing components and linker distances. From Liesenfelder et al.

The Intertwined Story of DNA Methylation and Epigenetic Aging

Epigenetic aging describes the genome-wide alterations to DNA methylation (DNAm) at specific CpGs that occur during normal human aging (Koch and Wagner). Our ever-improving appreciation of this mechanism has supported the development of epigenetic clocks that underpin aging-based studies (Bell et al. and Horvath and Raj) and help to identify factors that accelerate epigenetic aging, which has been linked to higher all-cause mortality (Lin et al., Zhang et al., Marioni et al., and Levine et al.). Importantly, whether altered DNAm levels directly affect the aging process remains an unanswered question (Horvath and Raj, Lpez-Otin et al., and Kabacik et al.). Recent studies have provided some clarity by suggesting the existence of crosstalk between epigenetic modifications within a network. Bozic et al. reported how inter-allelic epigenetic crosstalk may underlie the symmetric nature of acute myeloid leukemia patient-specific aberrant DNAm patterns on both alleles, Hernando-Herraez et al. described the coherent evolution of age-associated DNAm alterations in single-cell analysis, and Tarkhov et al. noted that DNAm changes can originate from CpG clusters coherently modified with age.

In their recent Nature Aging paper (Liesenfelder et al.), researchers from the laboratory of Wolfgang Wagner (RWTH Aachen University) sought to define the impact of epigenetic editing of DNAm at aging-associated CpGs. In Part 1 of this series, the Epigenome Technologies blog reports how CRISPR-mediated DNAm editing at individual aging-related CpGs prompted highly reproducible genome-wide bystander effects that impacted other age-associated sites. Part 2 will report on the consequences of multiplexed The range of products and services that Epigenome Technologies provides can empower your research aims with flexible, high-resolution technologies that turn hidden regulatory layers into actionable discoveries ready to transform our understanding of health and disease.

Scatter of DNAm change on Chr19 and DNAm time-course. — Scatter of DNA methylation change across chromosome 19 for dCas9–DNMT3A and CRISPRoff (target CpG in PDE4C highlighted), and time-course of DNAm levels in edited versus control cells. From Liesenfelder et al.

The Site-specific and Stable Effects of CRISPR-guided Epigenetic Editing

Modifying DNAm levels at the PDE4C locus in HEK293T cells employed dCAS9-DNMT3A (Tarjan et al.) or CRISPRoff (Nunez et al.) with specific guide (g)RNAs

The PDE4C locus undergoes increased DNAm in various tissues during aging (Koch and Wagner) and has a high correlation with chronological age in blood (Weidner et al.)
Pyrosequencing/Illumina BeadChip (EPIC) analysis detected increased DNAm at all CpGs at the target site
The increased DNAm reflects the age-associated DNAm dynamics of the PDE4C locus well
Detailed analyses via bisulfite amplicon sequencing revealed a greater level of epigenetic editing efficiency (due to DNAm heterogeneity at neighboring CpGs detected by the previous methods)
Cells proliferated well and lost CRISPR construct plasmid expression after a few days
however, DNA hypermethylation remained stable for up to 100 days

Bisulfite barcoded amplicon sequencing revealed that neighboring CpGs at the targeted loci did not display coherent modification

However, regions with lower DNAm increased to match neighboring CpGs with higher DNAm over time, suggesting that homogeneous local DNAm levels take months to occur after initial CRISPR activity

Scatter of DNAm change correlation and bar chart of enrichment. — Correlation of DNA methylation changes between CRISPRoff and dCas9–DNMT3A (R² indicated), and enrichment of bystander methylation effects across genomic regions. From Liesenfelder et al.

CRISPR-guided Epigenetic Editing Evokes Genome-wide Bystander Effects

While the target site underwent the highest gain in DNAm, EPIC BeadChip data revealed significantly increased DNAm at 4,864 (dCAS9-DNMT3A) and 3,326 (CRISPRoff) sites and decreased DNAm at 7 (dCAS9-DNMT3A) and 14 (CRISPRoff) sites, which agrees with previous observations (Lin et al.)

Analysis suggested that DNAm alterations represent consistent epigenetic bystandereffects and did not occur due to the random footprints of the constructs

Regions predicted as gRNA off-target sites did not undergo significant alterations in DNAm levels

Instead, hypermethylated bystander CpGs displayed enrichment in CpG islands and shores (but not at shelf and open sea areas) and occurred at upstream promotor regions
CpG context may relate to sequence preferences of DNA-methyltransferase 3A (DNMT3A) (Gao et al.)

Violin plots and odds ratio curves for CpG ATAC signal and overlap. — Violin plots of ATAC signal at PDE4C bystander (a) and aging (b) CpGs, and odds ratio versus interaction rpm for PDE4C bystander sites (d) and array CpGs (e). From Liesenfelder et al.

Age-associated CpGs Suffer from Bystander Effects

Exploring links between epigenetic bystander effects and aging-associated sites employed data from an epigenome-wide association study of 18,413 individuals that identified the top 10,000 CpGs linearly associated with age (5,328 hyper- and 4,389 hypo-methylated CpGs passed the filter criteria) (Bernabeu et al.)
Analysis of whether bystander alterations associated with PDE4C locus modification displayed enrichment for age-associated hyper-/hypo-methylated CpGs revealed that the 5,328 CpGs with age-associated hypermethylation displayed a significant methylation gain compared to remaining CpGs
Targeting an alternative gene known to undergo aging-associated DNAm alterations (FHL1; Han et al.) again revealed bystander effects

Pyrosequencing/EPIC BeadChip analysis revealed significant DNAm gains at age-hypermethylated sites
Comparisons revealed some correlation of genome-wide methylation effects between FHL2 or PDE4C

Composite panels of CRISPR sgRNA screens, DNAm and gene expression. — CRISPR sgRNA library screens in HEK293T cells: panels a–d show methylation and expression changes at PDE4C and candidate loci by day 7; panels e–h display DNAm profiling of additional targets at days 3 and 15. From Liesenfelder et al.

Do Bystander Effects Occur Through Chromatin Interactions?

Analysis of ATAC-seq data (Calviello et al.) revealed that PDE4C/FHL2-associated reproducible bystander effects represented sites of open chromatin

Age-hypomethylated CpGs associated with closed chromatin, while age-hypermethylated CpGs tended to display higher chromatin accessibility
These data suggest that bystanders occurred due to alterations in chromatin accessibility; however, logistic regression analysis indicated that the chromatin state alone does not explain the bystander effect

4C-sequencing suggested that altered chromatin interactions may also prompt bystander effects

Overall, the interaction of epigenetic bystander modifications and age-associated CpGs suggested an interacting epigenetic network

Density and cumulative DNAm change at day 3 and day 15 — Density (i, k) and cumulative distribution (j, l) of DNA methylation changes for all, hypo-, and hyper-CpGs at day 3 (i, j) and day 15 (k, l). From Liesenfelder et al.

Epigenetic Editing at Individual Age-related CpGs Has a Wide-reaching Impact

These findings also suggest the greater stability of epigenetic editing at regions that gain methylation with age; however, hypermethylated and hypomethylated epigenetic clock sites that display greater susceptibility to the accumulation or loss of DNAm, respectively, may explain their consistent age-related alterations. Finally, these data suggest that chromatin interactions between genomic sites may support coherent bystander modifications but do not fully explain their extent.

As with any good study, these findings evoke yet more unanswered questions; can Epigenome Technologies offer the means to answer them? The profiling of multiple histone modifications combined with simultaneous RNA sequencing at the single-cell level may provide an understanding of the complementary role of another level of epigenetic regulation. Paired-Tag from Epigenome Technologies generates joint epigenetic and gene expression profiles at the single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with an efficiency comparable to single-nucleus RNA-seq/ChIP-seq assays. As such, the application of Paired-Tag technology may enable giant leaps forward in understanding gene regulation and, in this case, epigenetic aging.

For more on the consequences of altering DNA methylation at individual age-linked CpGs, see Nature Aging, March 2025.