DNA methylation and demethylation

Maintenance of DNA methylation patterns

It is essential for plants to maintain the global DNA methylation patterns to keep transposons silenced, preserve cell type identity, or establish an epigenetic memory against environmental stresses (Law & Jacobsen, 2010). According to the sequence context of the cytosine sites (CG, CHG, and CHH), DNA methyltransferases control and regulate their methylation due to cooperative or competing interactions by different mechanisms (Meyer, 2011; H. Zhang et al., 2018). The efficiency of the different DNA methyltransferases is reflected in the methylation level at their preferred target sites. Symmetric methylation (CG and CHG) is most efficient, with most sites being 80–100% methylated, while methylation levels at nonsymmetric (CHH) target sites vary between 10% to 20% (Martínez-García et al., 2010; P. R. V. Satyaki & Gehring, 2017a).

The sequence context further influences DNA methylation in plants even beyond the mentioned dinucleotide and trinucleotide sites. For example, CG sites are undermethylated when the exact four-base context is ACGT. Also, CHG and CHH sites are less efficiently methylated when another cytosine follows the C, as in CCG instead of CAG or CTG. These more specific sequence contexts seem to reduce the efficiency of the methyltransferases. However, the reason is yet unknown. For example, in Arabidopsis thaliana, some methyltransferases share similar sequence specificities to probably provide a methylation backup system and avoid harmful alterations in the plant genome (Law & Jacobsen, 2010; Li et al., 2018; Meyer, 2011).

Maintenance of CG methylation

In plants, genetic analyses have demonstrated that CG methylation is regulated like in mammals through homolog proteins. CG cytosine methylation is maintained by the DNA METHYLTRANSFERASE 1 (MET1), which recognizes hemimethylated CG dinucleotides following DNA replication and methylates the unmodified cytosine in the new complementary strand (Fig. 2). To do this, MET1 probably is recruited to the replication complex by the VARIANT IN METHYLATION (VIM) protein family of SRA (SET- and RING-associated) domain proteins (Law & Jacobsen, 2010; Zhang et al., 2018). This interaction with VIM was proposed because VIM1 loss of function mutants of Arabidopsis thaliana lose the DNA methylation of their centromeres (J. Kim, Kim, Richards, Chung, & Woo, 2014).

Prevalence of non-CG methylation in plants

Non-CG sites refer to symmetrical trinucleotide CHG sites and to asymmetrical CHH trinucleotide sites. Non-CG methylation is maintained by plant-specific enzymes such as the CHROMOMETHYLASE (CMT) family (Kenchanmane Raju, Ritter, & Niederhuth, 2019). CHG methylation is maintained via a reinforcing loop, which involves histone and DNA methylation (Fig. 3) (Law & Jacobsen, 2010). The DNA methyltransferases CHROMOMETHYLASE 3 (CMT3) and, to less extent, CMT2 catalyze the maintenance of CHG methylation in A. thaliana (Fig. 3A) (Zhang et al., 2018). However, they require a whole complex of other proteins. For example, the histone methyltransferases SUPPRESSOR OF VARIEGATION 3-9 HOMOLOGs: SUVH4 (also known as KRYPTONITE (KYP)), SUVH5, and SUVH6 bind non-CG methylated regions and methylate H3K9 (Fig. 3B)(Kenchanmane Raju et al., 2019). SUVH4, primarily responsible for H3K9 demethylation, is an essential partner, as suggested by a dramatic decrease of DNA CHG methylation in loss of function mutants (Law & Jacobsen, 2010). Law & Jacobsen (2010) suggested that the reason for the interdependence of CMT3 and SUVH4 could be found in their multidomain structure. Because CMT3 can bind to the methylated histone H3 with a chromodomain, SUVH4 can bind to CHG methylated DNA with its SRA domain (Law & Jacobsen, 2010; Zhang et al., 2018). Interestingly, CMT3 appears to have a role also in gene-body methylation (gbM) in several plant species (Wendte et al., 2019). In Eutrema salsugineum, a plant that naturally lacks gbM, the gain of CMT3 triggered a new establishment of gbM in genes homolog to naturally methylated genes in A. thaliana. The gained gbM was maintained even in following generations (Wendte et al., 2019). However, this observation is still a very recent one.

Asymmethric DNA methylation

CHH methylation cannot be copied from the old strand, and therefore is maintained by constant de novo methylation. De novo DNA methylation is a process that involves plant-specific pathways and enzymes. For instance, the methyltransferases DRM2 and CMT2 catalyze this reaction, depending on the broader sequence context (Henderson et al., 2010). DRM2 is part of the RNA-directed DNA methylation (RdDM) pathway (described in more detail in Chapter 3) that guides DRM2 to small-RNA target regions rich in transposons or other repeat sequences primarily located in the Euchromatin. On the contrary, CTM2 targets H1-containing Heterochromatin sites (Zhang et al., 2018). While the Euchromatine is the region containing most of the protein-coding genes, Heterochromatin contains most of the transposons and other repeat sequences. Other enzymes may affect the maintenance of asymmetric CHH methylation, like MET1, CMT3, SuvH2, and SuvH9, so there is also some cross-talk with H3K9 methylation. However, only CTM2 and DRM2 can catalyze this reaction. But in turn, these two enzymes can also de novo methylate cytosines in other sequence contexts (Zhang et al. 2018).

Notably, CMT2 maintained DNA methylation is reduced by mutations in DECRESED DNA METHYLATION 1 (DDM1), a chromatin-remodeling protein (Zhang et al., 2018). This association was used in a pivotal study to produce a population of Arabidopsis thaliana recombinant inbred lines with a nearly identical genomic sequence but different cytosine methylation patterns, called EpiRiLs (Johannes et al., 2009). The study demonstrated for the first time that differences only in DNA methylation produced phenotypic variation.

DNA demethylation

It is known that DNA methylation is a stable epigenetic mark across species; however, during different plant lifetime processes, a decrease in global methylation has been observed (Law & Jacobsen, 2010). A reduction of methyltransferases activity or low levels of methyl donors present may result in failure to conserve methylation during DNA replication , which is known as passive DNA demethylation. On the contrary, removing the methyl group by an enzymatic process is described as active DNA demethylation (Zhang et al., 2018).

Active demethylation

Active demethylation implies a family of DNA demethylases, 5-mC DNA glycosylases–apurinic/apyrimidinic lyases, which drive the base excision repair (BER) pathway (Law & Jacobsen, 2010; Zhang et al., 2018). In A. thaliana, the group of glycosylases involves REPRESSOR OF SILENCING 1 (ROS1), TRANSCRIPTIONAL ACTIVATOR DEMETER (DME), DEMETER-LIKE PROTEIN 2 and 3 (DML2 and DML3), which are able to identify and extract 5-mC from all cytosines in a double-stranded DNA sequence (Law & Jacobsen, 2010; Zhang et al., 2018).

During DNA demethylation, DME/ROS1 behave as glycosylases hydrolyzing the glycosylic bond between the cytosine and the deoxyribose molecule, then apurinic or apyrimidinic lyases cut the DNA backbone and produce an excision of the methyl-cytosine base, which will be filled through a DNA polymerase and ligase enzymes with a non-methylated cytosine base (Figure 4). However, the exact process, how the reposition of the unmethylated cytosine occurs is still unidentified (Parrilla-Doblas, Roldán-Arjona, Ariza, & Córdoba-Cañero, 2019; H. Zhang et al., 2018).

The glycosylases ROS1, DML2, and DML3 are expressed preferably in vegetative tissues, possibly to counteract robust DNA methylation by the RdDM pathway. Law & Jacobsen (2010) suggest that the shared target sequences of these glycosylases with the RdDM pathway regulate gene expression activity by removing methylation cytosines in genes and preventing their silencing (Fig.5A). However, these enzymes also maintain an adaptable inactive state to keep silenced transposons. In addition, it has been shown that the activity of genes nearby transposons is negatively affected by the RdDM pathway, which maintains the methylation in Euchromatic regions (Fig. 5B) (M. Y. Kim & Zilberman, 2014; Law & Jacobsen, 2010; H. Zhang et al., 2018).

Unlike ROS1 and DML, DME has been observed preferentially expressed in plant gamete cells and being involved in imprinting, as will be further outlined in the section DNA methylation and imprinting (Wöhrmann et al., 2012).

Passive demethylation in plants

Passive demethylation has been characterized in plant endosperm where MET1 activity decreases during female gametogenesis and results in a drop of the global methylation (Jullien et al., 2008). The reduction of MET1 expression levels activates demethylation pathways directed by DME. Together this activates genes that are expressed in a parent-of-origin-specific manner (Law & Jacobsen, 2010).