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Can epigenetics decode a cell’s history? Linking somatic DNA methylation, DNA repair, and gene expression

Can epigenetics decode a cell's history?  Linking somatic DNA methylation, DNA repair, and gene expressionMammalian Genomic DNA (mainly cytosines, in the doublet CpG) can be covalently modified by methylation, which is layered on the primary genetic information and alters gene expression. There are two patterns of DNA methylation. The first is stable methylation, called imprinting. Imprinting is inherited in a sex-specific fashion and is invariant among individuals and cell types (X inactivation, for example). The second pattern, metastable methylation, is unstable and variable among individuals and cell types and is associated with cancer and aging.1 Both types of these methylation patterns are essential for the cellular storage of large DNA molecules, such as mammalian genomes. In fact, DNA strands 2 meters in length can be compacted into a single 10 microns nucleus of a eukaryotic cell – that’s 3 billion bases in each human cell! Additionally, the compaction of large segments of methylated DNA provides a structural platform that allows selective activation of genes during development and somatic life. Loss of the enzymes responsible for DNA methylation is detrimental to embryo development and generates genome instability.2

Despite the large amount of information available today, we do not fully understand the mechanisms leading to methylation during embryogenesis, development, and somatic life of an individual cell. The methylation of a specific CpG is neither sequence, or site specific, and it is not known why, when, or where a DNA seqment undergoes de novo methylation. We do, however, know the mechanisms through which DNA methylation marks are propagated to the replicating DNA to the next generation: The structure of the main methylating enzyme, DNMT1 – which is associated to the replicating complex (replisome) – shows that it recognizes the hemi-methylated substrate (the template strand, for example) with a much higher affinity than the un-methylated substrate. This system simply permits the inheritance of methylation marks through selective methylation of only hemi-methylated DNA template.3 However, heterogeneous methylation of the same DNA segment within a cell population is a common hallmark of cancerous and aged cells. What remains unclear, is why – despite this methylation-replicating system – variations in methylation patterning occurs.

An explanation for this may lie in the cell’s history: the variation found in somatic DNA methylation may be a consequence from previous DNA damage. This theory, which has been gaining popularity, suggests that segments of DNA that have been damaged and faithfully repaired by homologous recombination can be marked by methylation – acting as a scar in the damaged tissue. Evidence supporting the association of DNA methylation and damage & homologous repair include observations that germline DNA methylation positively correlates with regional levels of meiotic recombination4,5, and that double strand breaks (DSBs) repaired by homologous recombination generate cells with methylated DNA around the break.6,7 Additionally, it has been shown that DNMT1 is recruited to the DSB just minutes after damage.8

The direct link between DNA damage and DNA methylation has been tested in a precisely controlled system in which recombination between partial duplications of a chromosomal Green Fluorescent Protein (GFP) gene is initiated by a DSB in one copy of the gene. The unique DSB is generated by cleavage with the meganuclease I-SceI, which does not cleave the eukaryotic genome. The DSB is repeatedly formed and repaired, until the I-SceI site is lost by homologous, (HR) or non-homologous error-prone (NHEJ) repair, or depletion of the I-SceI enzyme. Resulting recombination products can be detected by direct analysis of the DNA flanking the DSB, or by the appearance of functional GFP.  Two cell types are generated after recombination: clones expressing high levels of GFP and clones expressing low levels of GFP, termed H and L clones respectively. Relative to the parental gene, the repaired GFP is hypomethylated in H clones and hypermethylated in L clones. The altered methylation pattern is largely restricted to a single segment, just 3 to the DSB along the direction of transcription. Hypermethylation of this tract modifies the chromatin and significantly reduces transcription, although it is 2000 bp distant from the strong cytomegalovirus promoter that drives GFP expression. This series of events has even been documented by a time lapse movie that records the first appearance of the repaired and methylated GFP gene, which progressively leads to silencing of the GFP protein expression!7

Surprisingly, the researchers found that during a limited period after repair (2-3 weeks after the initial damage) the methylation and expression of the repaired gene was not consistent from cell to cell! During transcription some methyl groups were removed by a process of “active” demethylation in some of the cells – generating heterogeneous methylation patterns and levels of expression of the repaired gene in the cell population. However, four weeks after the damage and homologous repair, methylation and expression of the repaired gene stabilized and did not change hereafter (even after 3 years). The authors conclude that HR first and transcription later were able to modify the methylation status of the repaired gene and generate a great variety of methylated epialleles (alleles differing only by their methylation) which eventually become stable and govern the stochastic expression of the genes in complex cell populations (Figure 1).

These experiments offer insight into the development of the heterogeneous phenotypes observed in cancer and aging. DNA methylation may be able to be read as a damage-repair code that modifies the expression of genes in cell populations and drives adaptation to environmental challenges. Selection of specific epialleles in each cell may soon prove to be relevant for the rapid evolution of cancer cell phenotypes.

Sequence of events leading to silencing or expression of HR DNA segments

Figure 1 illustrates the sequence of events leading to silencing or expression of HR DNA segments. Red circles represent de novo methylated CpGs induced by HR. Black circles represent methylated CpGs before HR. Since silencing depends on the location of de novo methylated CpGs and DNA damage is random, HR-induced methylation is also random. If the expression of the repaired gene is harmful, only cells inheriting the silenced copy will survive. Conversely, if the function of the repaired gene is beneficial, cells inheriting the under methylated copy will have a selective advantage. doi:10.1371/journal.pgen.0030110.g012

 

 

References

1. Smith ZD, & Meissner A (2013). DNA methylation: roles in mammalian development. Nature reviews. Genetics, 14 (3), 204-20 PMID: 23400093

2. Rizwana R, & Hahn PJ (1999). CpG methylation reduces genomic instability. Journal of cell science, 112 ( Pt 24), 4513-9 PMID: 10574701

3. Song J, Teplova M, Ishibe-Murakami S, & Patel DJ (2012). Structure-based mechanistic insights into DNMT1-mediated maintenance DNA methylation. Science (New York, N.Y.), 335 (6069), 709-12 PMID: 22323818

4. Sandovici I, Kassovska-Bratinova S, Vaughan JE, Stewart R, Leppert M, & Sapienza C (2006). Human imprinted chromosomal regions are historical hot-spots of recombination. PLoS genetics, 2 (7) PMID: 16839189

5. Sigurdsson MI, Smith AV, Bjornsson HT, & Jonsson JJ (2009). HapMap methylation-associated SNPs, markers of germline DNA methylation, positively correlate with regional levels of human meiotic recombination. Genome research, 19 (4), 581-9 PMID: 19158364

6.  Cuozzo C, Porcellini A, Angrisano T, Morano A, Lee B, Di Pardo A, Messina S, Iuliano R, Fusco A, Santillo MR, Muller MT, Chiariotti L, Gottesman ME, & Avvedimento EV (2007). DNA damage, homology-directed repair, and DNA methylation. PLoS genetics, 3 (7) PMID: 17616978

7. Morano A, Angrisano T, Russo G, Landi R, Pezone A, Bartollino S, Zuchegna C, Babbio F, Bonapace IM, Allen B, Muller MT, Chiariotti L, Gottesman ME, Porcellini A, & Avvedimento EV (2014). Targeted DNA methylation by homology-directed repair in mammalian cells. Transcription reshapes methylation on the repaired gene. Nucleic acids research, 42 (2), 804-21 PMID: 24137009

Supplementary Data – mov file

Supplementary Data – m4v file

8. Ha K, Lee GE, Palii SS, Brown KD, Takeda Y, Liu K, Bhalla KN, & Robertson KD (2011). Rapid and transient recruitment of DNMT1 to DNA double-strand breaks is mediated by its interaction with multiple components of the DNA damage response machinery. Human molecular genetics, 20 (1), 126-40 PMID: 20940144

9. Horvath S (2013). DNA methylation age of human tissues and cell types. Genome biology, 14 (10) PMID: 24138928

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Enrico Avvedimento

Enrico Avvedimento