Modeling the Effects of Age-Linked Ribosomal DNA Hypermethylation in Human Cells

Core Concepts

Age-linked hypermethylation of ribosomal DNA (rDNA) does not impair rRNA transcription or ribosome biogenesis in human cells, as the human rDNA transcription machinery remains resilient to increased DNA methylation.

Abstract

The authors investigated the consequences of age-linked rDNA hypermethylation in human cells. They found that actively transcribed rDNA repeats are completely hypomethylated, while silent repeats are densely methylated. Contrary to expectations, inducing rDNA methylation using a dCas9-DNMT3A3L fusion did not impair rRNA transcription, nucleolar morphology, or cellular growth rate.
Key highlights:

Mammalian rDNA repeats undergo extensive hypermethylation during aging and senescence, but rDNA copy number remains stable.
Active, Pol I-engaged rDNA repeats are hypomethylated across the promoter and gene body, while silent repeats are densely methylated.
Directing de novo methylation to the rDNA promoter or gene body using dCas9-DNMT3A3L had no detectable effect on rRNA transcription, nucleolar morphology, or cellular growth.
The human rDNA transcription factor UBF remains bound to methylated rDNA promoters, unlike in mouse cells where promoter methylation displaces UBF.
These findings suggest that the human rDNA transcription machinery is resilient to age-linked rDNA hypermethylation, in contrast to the mouse system. The authors propose that additional epigenetic mechanisms beyond DNA methylation may be required to impair rDNA transcription in human cells.

Stats

Mammalian rDNA repeats undergo 15-20% increase in CpG methylation during aging.
Active, Pol I-bound rDNA repeats are 95% hypomethylated compared to the total rDNA pool.
Inducing 15-20% increase in rDNA promoter or gene body methylation using dCas9-DNMT3A3L had no effect on rRNA transcription, nucleolar morphology, or cellular growth rate.

Quotes

"Surprisingly, however, we saw no evidence for impaired rRNA transcription after inducing methylation within either the promoter or the gene body, as the levels of both nascent pre-rRNA and mature 28S rRNA remained unchanged."
"These data indicate that increased CpG methylation is not sufficient to impair rRNA transcription in human cells."
"UBF remained bound to rDNA promoters in the presence of methylation; we confirmed that this binding was specific and significantly enriched above background signal."

Key Insights Distilled From

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

by Blokhina,Y. ... at www.biorxiv.org 10-19-2023

https://www.biorxiv.org/content/10.1101/2023.10.18.562830v2

Deeper Inquiries

What other epigenetic mechanisms, beyond DNA methylation, may be involved in regulating rDNA transcription in human cells during aging?

In addition to DNA methylation, several other epigenetic mechanisms may play a role in regulating rDNA transcription in human cells during aging. One key mechanism is histone modification, particularly histone acetylation and methylation. Histone acetylation is associated with active transcription, while histone methylation can either activate or repress gene expression depending on the specific histone residue modified and the extent of methylation. Changes in histone modifications at the rDNA locus could impact the accessibility of the DNA to transcription factors and RNA polymerase, thereby influencing rDNA transcription during aging.
Another important epigenetic mechanism is nucleosome positioning. The positioning of nucleosomes along the rDNA repeats can affect the binding of transcription factors and RNA polymerase, ultimately influencing rDNA transcription. Changes in nucleosome positioning, which can be regulated by ATP-dependent chromatin remodelers, may occur during aging and impact rDNA transcription efficiency.
Furthermore, non-coding RNAs, such as small nucleolar RNAs (snoRNAs) and long non-coding RNAs (lncRNAs), can also regulate rDNA transcription. SnoRNAs are involved in rRNA processing and modification, while lncRNAs can act as scaffolds for chromatin-modifying complexes or as regulators of transcription factor activity. Alterations in the expression or function of these non-coding RNAs could impact rDNA transcription dynamics during aging.

How do the differences in rDNA regulation between mouse and human cells contribute to their distinct responses to age-linked rDNA hypermethylation?

The differences in rDNA regulation between mouse and human cells can contribute to their distinct responses to age-linked rDNA hypermethylation. One key difference is the sensitivity of the transcription machinery to DNA methylation. In mice, even a single CpG methylation event in the rDNA promoter can displace the essential transcription factor UBF and inhibit transcription. This high sensitivity to methylation may lead to more pronounced effects of age-linked hypermethylation on rDNA transcription in mouse cells compared to human cells, where UBF remains bound to methylated rDNA promoters.
Additionally, the genomic organization and sequence of the rDNA locus may differ between mouse and human cells, leading to variations in the distribution of CpG sites and regulatory elements. These differences could result in distinct responses to age-linked hypermethylation, with human cells potentially having evolved mechanisms to maintain rDNA transcription even in the presence of increased methylation.
Moreover, the chromatin structure and epigenetic landscape surrounding the rDNA repeats may vary between mouse and human cells. Differences in histone modifications, nucleosome positioning, and the expression of non-coding RNAs at the rDNA locus could influence the impact of age-linked hypermethylation on rDNA transcription in each species.

How might the resilience of the human rDNA transcription machinery to methylation impact cellular and organismal aging processes?

The resilience of the human rDNA transcription machinery to methylation could have significant implications for cellular and organismal aging processes. One key impact is on protein synthesis and cellular function. The maintenance of rDNA transcription despite age-linked hypermethylation ensures the continuous production of ribosomal RNAs, which are essential for ribosome biogenesis and protein synthesis. This resilience may help to preserve cellular homeostasis and function in aging cells, preventing the decline in protein synthesis efficiency that is often associated with aging.
Furthermore, the ability of the human rDNA transcription machinery to tolerate methylation may contribute to the overall stability of the genome during aging. By ensuring the continued transcription of rDNA repeats, even in the presence of increased methylation, human cells can maintain the integrity of the ribosome biogenesis pathway and prevent disruptions in protein production that could lead to cellular dysfunction and senescence.
On an organismal level, the resilience of the human rDNA transcription machinery to methylation may contribute to healthy aging and longevity. By preserving the capacity for efficient ribosome biogenesis and protein synthesis, cells can maintain essential cellular functions and support overall organismal health. This resilience may help to mitigate the impact of age-related changes in rDNA methylation and contribute to the maintenance of tissue homeostasis and function throughout the aging process.