Genome-wide cytosine-5 DNA methylation increases sensitivity to oxidative stress and alkylating agents in E. coli

Centrala begrepp

While cytosine-5 DNA methylation (5mC) is an ancient epigenetic mark, its presence increases sensitivity to oxidative stress due to the formation of modified cytosines like 5fC, which are recognized as DNA damage, potentially explaining the loss of 5mC in some species.

Sammanfattning

• Bibliographic Information: Not applicable - information not provided in the content.
• Research Objective: This study investigates the fitness consequences of DNA methyltransferase-induced alkylation damage by introducing a eukaryotic-like 5mC system into E. coli. The researchers aimed to understand the impact of genome-wide 5mC on sensitivity to genotoxic stress, particularly oxidative stress.
• Methodology: The researchers expressed the CG methyltransferases M.SssI and M.MpeI in an E. coli strain lacking the McrABC restriction modification system. They then exposed these bacteria to various DNA-damaging agents, including MMS and H2O2, and assessed their sensitivity. ROS production was measured using a fluorescent sensor. Mass spectrometry was used to analyze DNA epigenetic modifications. Finally, transcriptomics was used to analyze gene expression changes in response to DNMT expression and H2O2 treatment.
• Key Findings: Introducing 5mC genome-wide in E. coli led to increased sensitivity to alkylating agents like MMS, especially in the absence of the repair enzyme AlkB. Unexpectedly, 5mC introduction also increased sensitivity to oxidative stress due to increased reactive oxygen species (ROS) formation. This heightened sensitivity is attributed to the non-enzymatic oxidation of 5mC to modified cytosines like 5hmC and 5fC, which E. coli recognizes as DNA damage.
• Main Conclusions: The study identifies increased sensitivity to oxidative stress and alkylating agents as negative consequences of genome-wide 5mC. This sensitivity, particularly to oxidative stress, offers a plausible explanation for the complete loss of 5mC in some species.
• Significance: This research provides a new perspective on the evolutionary implications of DNA methylation. It suggests that the trade-off between the potential benefits of 5mC and its susceptibility to oxidative damage may have shaped the evolution of DNA methylation pathways.
• Limitations and Future Research: The study was conducted in E. coli, and further research is needed to confirm if these findings hold true in eukaryotic systems. Further investigation is also needed to fully elucidate the mechanism by which DNMT expression leads to increased ROS production.

Statistik

Cells expressing either M.SssI or M.MpeI showed increased sensitivity to MMS treatment compared to WT C2523.
DNMT expression combined with deletion of AlkB had a greater effect on MMS sensitivity than would be expected from the effect of each individually (DNMT*alkB interaction p=0.0044 three-way Anova).
Induction of active DNMT expression through M.SssI led to a strongly exacerbated sensitivity to H2O2 treatment (p<1e-16 Two way anova).
The combined effect of expression of DNMT and mutation of Fpg was greater than the sum of the individual effects (Fpg*DNMT interaction p=0.032).
H2O2-induced ROS accumulation was markedly enhanced by addition of DNMT.
DNMT addition alone led to increased ROS accumulation, even without exposure to H2O2.
The effect of H2O2 and ROS was greater than the linear combination of the two effects separately (linear model interaction P-value<0.05).
Exposure of DNMT-expressing cells to H2O2 led to production of the 5mC oxidation products 5hmC and 5fC as measured by mass spectroscopy.
When combined with DNMT expression there was a strong increase in sensitivity beyond what would be expected from either DNMT expression or NgTET expression alone (Interaction TetDNMTIPTG p=1e-10 Anova).

Citat

"Our work identifies increased sensitivity to oxidative stress, as well as alkylating agents, as a negative consequence of genome-wide 5mC."
"Oxidative stress is frequently encountered by organisms in their environment, thus offering a plausible reason for total loss of 5mC in some species."

Viktiga insikter från

Introduction of cytosine-5 DNA methylation sensitizes cells to oxidative damage

by Krwawicz,J.,... på www.biorxiv.org 09-13-2024

https://www.biorxiv.org/content/10.1101/2024.09.13.612259v1

Djupare frågor

How do environmental factors, other than oxidative stress, influence the evolution and maintenance of DNA methylation in different organisms?

Beyond oxidative stress, a variety of environmental factors exert selective pressures that shape the evolution and maintenance of DNA methylation across organisms. These factors often interact with DNA methylation to influence gene expression, ultimately impacting an organism's fitness in its specific environment. Here are some key examples:

Diet: Dietary components can directly impact DNA methylation patterns. For instance, folate and other methyl-donating nutrients play a crucial role in the one-carbon metabolism pathway, which is essential for providing methyl groups for DNA methylation. Diets deficient in these nutrients can lead to altered methylation patterns, potentially affecting gene expression and disease susceptibility.
Temperature: Temperature fluctuations, particularly extreme ones, can influence DNA methylation. Studies in plants have shown that temperature stress can induce changes in DNA methylation, affecting genes involved in stress response and development. This epigenetic plasticity likely allows for rapid adaptation to changing environmental conditions.
Salinity: In organisms inhabiting environments with fluctuating salinity, DNA methylation plays a crucial role in osmoregulation. Changes in salinity can trigger differential methylation patterns, modulating the expression of genes involved in ion transport and stress response, ultimately aiding in adaptation to saline environments.
Exposure to Toxins: Exposure to environmental toxins, such as heavy metals and pollutants, can disrupt DNA methylation patterns. These epigenetic alterations can have detrimental effects on gene expression, potentially leading to developmental defects, impaired immune function, and increased disease susceptibility.
Symbiotic Interactions: Interactions with symbiotic microorganisms can also influence DNA methylation patterns. For example, in plants, symbiotic fungi can induce changes in DNA methylation in the host plant's roots, promoting nutrient uptake and enhancing stress tolerance.
The interplay between these environmental factors and DNA methylation highlights the dynamic nature of epigenetic regulation. Organisms have evolved sophisticated mechanisms to sense and respond to environmental cues, with DNA methylation acting as a crucial interface between the environment and the genome. Understanding these interactions is essential for unraveling the complexities of phenotypic plasticity, adaptation, and the evolutionary implications of epigenetic modifications.

Could the increased sensitivity to oxidative stress induced by 5mC be leveraged for developing novel therapeutic strategies against pathogens or cancer cells?

The discovery that 5mC increases sensitivity to oxidative stress presents a promising avenue for developing novel therapeutic strategies against pathogens and cancer cells. By exploiting this vulnerability, we could potentially target these cells more effectively while minimizing damage to healthy cells. Here are some potential strategies:

Enhancing ROS Production: Developing drugs that specifically increase ROS production in cancer cells or pathogens with high 5mC levels could selectively induce oxidative stress and cell death. This approach could exploit the existing vulnerability to ROS conferred by 5mC, leading to targeted therapeutic intervention.
Inhibiting ROS Scavenging Systems: Cancer cells and some pathogens often upregulate ROS scavenging systems to counteract increased oxidative stress. Inhibiting these systems, such as superoxide dismutase (SOD) or catalase, could further sensitize these cells to ROS-mediated damage, particularly in the presence of high 5mC levels.
Combining with Existing Therapies: Combining ROS-inducing agents or inhibitors of ROS scavenging systems with conventional chemotherapy or antimicrobial drugs could enhance their efficacy. This synergistic approach could potentially overcome drug resistance and improve treatment outcomes by exploiting the heightened sensitivity to oxidative stress in target cells.
Targeting 5mC Oxidation Pathways: Developing drugs that specifically target the oxidation of 5mC to 5hmC and 5fC could enhance the accumulation of these potentially cytotoxic DNA lesions. This approach could further sensitize cells with high 5mC levels to oxidative damage, leading to targeted cell death.
While promising, translating these strategies into effective therapies requires careful consideration of potential challenges. These include ensuring specificity to target cells, minimizing off-target effects on healthy cells, and overcoming potential resistance mechanisms. Further research is crucial to explore these avenues and develop safe and effective therapeutic interventions that exploit the link between 5mC and oxidative stress sensitivity.

If biological systems are constantly evolving to mitigate risks and enhance survival, how might the presence and role of 5mC evolve in the future?

Predicting the future of 5mC is a fascinating exercise in evolutionary speculation. Given the dynamic interplay between 5mC, environmental pressures, and genomic stability, several evolutionary trajectories are conceivable:

Enhanced Regulation and Protection: Organisms might evolve more refined mechanisms to regulate DNMT activity and minimize off-target alkylation damage. This could involve tighter control of DNMT expression, increased fidelity of DNMT enzymes, or enhanced repair pathways to counteract 3mC lesions. Such adaptations would allow for the maintenance of beneficial 5mC functions while mitigating its mutagenic potential.
Specialization of Function: The role of 5mC might become increasingly specialized in different lineages. Some organisms might retain 5mC for its role in gene regulation, while others might evolve alternative mechanisms for epigenetic control, rendering 5mC dispensable. This specialization could reflect the diverse ecological niches and selective pressures experienced by different organisms.
Emergence of Novel Functions: Evolution often repurposes existing molecular machinery for new functions. It's conceivable that 5mC, or its oxidized derivatives like 5hmC and 5fC, could acquire novel roles beyond gene regulation and DNA damage. These could include mediating interactions with symbiotic organisms, sensing environmental cues, or even participating in cellular signaling pathways.
Loss in Specific Lineages: The ongoing loss of DNMTs and 5mC in certain lineages might continue. This could be driven by the inherent mutagenic potential of 5mC, particularly in environments with high oxidative stress or exposure to alkylating agents. In these lineages, alternative mechanisms for epigenetic regulation would likely evolve to compensate for the loss of 5mC-dependent processes.
Ultimately, the evolutionary fate of 5mC will depend on the complex interplay between its benefits, risks, and the ever-changing selective pressures imposed by the environment. Understanding the evolutionary dynamics of 5mC will provide valuable insights into the plasticity of epigenetic systems and their role in shaping the diversity of life on Earth.