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Non-homologous end joining induced alterations in DNA methylation: A source of permanent epigenetic change
Brittany Allen1, Antonio Pezone2, Antonio Porcellini3
1College of Medicine, Burnett School of Biomedical Sciences, University of Central Florida, Orlando, FL, USA.
Abstract:
In addition to genetic mutations, epigenetic revision plays a major role in the development and progression of cancer; specifically, inappropriate DNA methylation or demethylation of CpG residues may alter the expression of genes that promote tumorigenesis. We hypothesize that DNA repair, specifically the repair of DNA double strand breaks (DSB) by Non-Homologous End Joining (NHEJ) may play a role in this process. Using a GFP reporter system inserted into the genome of HeLa cells, we are able to induce targeted DNA damage that enables the cells, after successfully undergoing NHEJ repair, to express WT GFP. These GFP+ cells were segregated into two expression classes, one with robust expression (Bright) and the other with reduced expression (Dim). Using a DNA hypomethylating drug (AzadC) we demonstrated that the different GFP expression levels was due to differential methylation statuses of CpGs in regions on either side of the break site. Deep sequencing analysis of this area in sorted Bright and Dim populations revealed a collection of different epi-alleles that display patterns of DNA methylation following repair by NHEJ. These patterns differ between Bright and Dim cells which are hypo- and hypermethylated, respectively, and between the post-repair populations and the original, uncut cells. These data suggest that NHEJ repair facilitates a rewrite of the methylation landscape in repaired genes, elucidating a potential source for the altered methylation patterns seen in cancer cells, and understanding the mechanism by which this occurs could provide new therapeutic targets for preventing this process from contributing to tumorigenesis.
Insights
DNA repair through Non-Homologous End Joining (NHEJ) can alter DNA methylation patterns. This epigenetic rewriting following double-strand break repair may contribute to cancer development and offers potential therapeutic targets.
Area of Science:
- Molecular Biology
- Epigenetics
- Cancer Research
Background:
- Epigenetic alterations, including aberrant DNA methylation, are hallmarks of cancer.
- DNA methylation changes can dysregulate gene expression, promoting tumorigenesis.
- The role of DNA repair mechanisms in shaping the epigenome is not fully understood.
Purpose of the Study:
- To investigate the potential role of Non-Homologous End Joining (NHEJ) in altering DNA methylation patterns after double-strand break (DSB) repair.
- To determine if NHEJ-mediated repair influences the methylation status of CpG residues near the repair site.
- To explore the implications of these findings for cancer development and potential therapeutic strategies.
Main Methods:
- Utilized a GFP reporter system in HeLa cells to induce and track targeted DNA damage and subsequent NHEJ repair.
- Segregated cells based on GFP expression levels (Bright vs. Dim) post-repair.
- Employed a DNA hypomethylating agent (AzadC) to assess the impact on GFP expression.
- Performed deep sequencing analysis to characterize methylation patterns in sorted cell populations.
Main Results:
- Differential GFP expression (Bright vs. Dim) correlated with distinct DNA methylation statuses of CpGs flanking the DSB site.
- NHEJ repair resulted in unique DNA methylation patterns (epi-alleles) compared to original, uncut cells.
- Bright and Dim populations exhibited hypo- and hypermethylation, respectively, indicating repair-associated epigenetic modifications.
- These findings suggest NHEJ actively reshapes the local methylation landscape.
Conclusions:
- NHEJ-mediated DNA repair can induce significant epigenetic alterations, rewriting the local DNA methylation landscape.
- This repair-associated epigenetic modification represents a potential source of the altered methylation patterns observed in cancer.
- Understanding this mechanism could unveil novel therapeutic targets for cancer prevention and treatment.
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