Making it or breaking it: DNA methylation and genome integrity

Abstract

Cells encounter a multitude of external and internal stress-causing agents that can ultimately lead to DNA damage, mutations and disease. A cascade of signaling events counters these challenges to DNA, which is termed as the DNA damage response (DDR). The DDR preserves genome integrity by engaging appropriate repair pathways, while also coordinating cell cycle and/or apoptotic responses. Although many of the protein components in the DDR are identified, how chemical modifications to DNA impact the DDR is poorly understood. This review focuses on our current understanding of DNA methylation in maintaining genome integrity in mammalian cells. DNA methylation is a reversible epigenetic mark, which has been implicated in DNA damage signaling, repair and replication. Sites of DNA methylation can trigger mutations, which are drivers of human diseases including cancer. Indeed, alterations in DNA methylation are associated with increased susceptibility to tumorigenesis but whether this occurs through effects on the DDR, transcriptional responses or both is not entirely clear. Here, we also highlight epigenetic drugs currently in use as therapeutics that target DNA methylation pathways and discuss their effects in the context of the DDR. Finally, we pose unanswered questions regarding the interplay between DNA methylation, transcription and the DDR, positing the potential coordinated efforts of these pathways in genome integrity. While the impact of DNA methylation on gene regulation is widely understood, how this modification contributes to genome instability and mutations, either directly or indirectly, and the potential therapeutic opportunities in targeting DNA methylation pathways in cancer remain active areas of investigation.

Keywords: Cancer; DNA damage; DNA methylation; DNA synthesis and repair; epigenetics; genome integrity.

Figure 1.. Potential roles of DNA methylation in genome maintenance

A simplified model depicting the putative roles of DNA methylation in the maintenance of genome integrity. DNA is methylated commonly at CpG sites, transposable elements, sites of tissue-specific gene silencing, X-chromosome inactivation and genome imprinting. DNA can undergo spontaneous deamination causing mutations or encounter roadblocks during replication from secondary structures such as R-loops and G-quadruplexes (G4), which may confer aberrant methylation patterns across the genome affecting gene transcription or impacting DNA DSB repair in response to DNA damage. Collectively, DNA methylation has the potential to affect the DDR as illustrated. Likewise,alterations in these pathways could also alter DNA methylation, which warrants consideration.

Figure 2.. An overview of DNA methylases and demethylases

(A) Structural depiction of cytosine methylation to form 5mC. Upon TET activity, 5mC can give rise to 5-hydroxymethylcytosine (5hmC). TETs can iteratively oxidize 5hmC to 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). 5fC and 5caC can be recognized and excised by the action of TDG forming cytosine through BER. (B) DNA methylation in adenine. Adenine can be methylated by N6AMT1 in DNA. The m6A RNA methyltransferase MettL3–MettL14 has been suggested to also methylate ssDNA. Demethylation has been suggested to occur via ALKBH1 activity. DNMT - includes DNMT1, DNMT3A, DNMT3B; TETs - TET1, TET2, TET3. Abbreviations: BER, base excision repair; N6AMT1, N⁶-adenine-specific DNA methyltransferase 1; TDG, thymine DNA glycosylase.

Figure 3.. Readers of DNA methylation

DNA methylation is recognized by several proteins which include: MBD, UHRF, Zn finger domain proteins including ZBTB33 (Kaiso), ZBTB4, ZBTB38, CTCF, KLF4, WT1, EGR1, ZFP57, basic leucine zipper-containing TFs (bZIP) and homeodomain-containing TFs.

Figure 4.. DNA methylation and mutations

(A,B) Cytosine and 5mC undergo deamination either spontaneously or when exposed to agents that are hydrolytic or alkylating. Hydrolytic deamination (removal of -NH₂ group as ammonia) from cytosine leads to the formation of uracil. Since uracil is not present in DNA, it is readily recognized by UDG, resulting in the substitution of uracil to cytosine. However, when 5mC undergoes deamination, it leads to the formation of thymine, a base normally present within DNA. TDG recognizes and corrects these misincorporated bases but inappropriate repair causes C→T transition mutations within the genome. (C) Transposable elements present in the genome are generally silenced by hypermethylation. DNA demethylation leads to the activation of these transposable elements, with the potential to increase mutations including insertions.

Figure 5.. Roles of DNMT1 in genome integrity

DNMT1 plays several critical roles in maintaining genome stability. These include: altered activity of DNA methylation can result in mitotic catastrophe; deficiency of DNMT1 has been found to be genetically unstable; DNMT1 is recruited to sites of laser damage; DNMT1 interacts with the replisome clamp PCNA during DNA replication and repair processes.

Figure 6.. Future perspectives

When normal cells encounter DNA damage, readers, writers and erasers of DNA methylation may contribute to the cellular response to DNA damage via gene regulation, DDRs and repair processes to ensure the maintenance of genome and epigenome integrity. However, in cancer cells, the function of readers, writers and erasers of DNA methylation may be altered. The changes in the methylation landscape could result in genomic and epigenomic instability due to differential gene expression, mutations and endogenous DNA damage, resulting in genome instability, a hallmark of cancer. Increased understanding of the mechanisms surrounding DNA methylation upon DNA damage and maintenance of genome integrity is necessary to extend current therapeutic strategies. Combinatorial treatments of inhibitors of DNA methylation along with DNA damaging agents and drugs targeting the DDR (i.e. PARP inhibitors) could offer promising drug treatment options to target cancer cells with altered DNA methylation patterns.