Writing about a biological subject based on one species can limit the scope of the discussion, but in the case of DNA methylation and Arabidopsis, the restriction is entirely appropriate. Unlike many other popular model organisms, Arabidopsis has retained and embellished a multi-layered methylation system that contributes to gene and transposon silencing, imprinting, and genome stability. Many of the findings from Arabidopsis are applicable to other eukaryotes. The field has relied in large part on forward and reverse genetic screens and the study of methylation and its consequences at one or a few loci. These sorts of experiments are still providing novel insights, especially when combined with the natural genetic variation present within Arabidopsis thaliana. Additionally, the field is increasingly moving towards genomics to understand the interplay between methylation, demethylation, chromatin state, and gene expression. Cytosine can be methylated at the carbon five position, and in plants this can occur on any cytosine regardless of the sequence context (Figure 1). In general, 5-methylcytosine is associated with transcriptional silencing. How this is achieved is still not well understood. Methylation can block transcription factor binding and prevent transcription or it can recruit chromatin-modifying complexes that mediate silencing through changes to the underlying histones. In this chapter we provide an introduction to the mechanisms of DNA methylation and demethylation and consider the biological relevance of these activities at different types of sequences. First, we provide a brief background on how methylation status is empirically determined, then address in more detail the enzymes and systems that add and remove it, and the interaction of DNA methylation with chromatin. Finally, we consider possible regulatory roles of DNA methylation in Arabidopsis. Figure 1 Chemical structure of cytosine and 5-methylcytosine. Measuring Methylation If you're interested in methylation at a certain sequence, how do you go about measuring it? Several methods of varying utility have been devised to determine the methylation status of specific sequences and entire genomes; most rely on chemical differences between 5-methylcytosine and cytosine or on the ability of restriction enzymes and antibodies to discriminate between the two. Several restriction enzymes are inhibited by methylation in CG, CHG, or CHH (H = A, T, or C) sequence contexts. Genomic DNA can be digested with a methylation sensitive restriction enzyme and the sequence of interest probed by Southern blot, with the amount of methylation determined by the completeness of digestion. The major disadvantage to this method is that the methylation status of only a few nucleotides can be queried at once. Methylation is often found in clusters in Arabidopsis. Although clusters of methylation are highly heritable, differences do arise at individual nucleotides within those regions even in closely related lines (Tran et al., 2005a). Given the potential for site-to-site variability in DNA methylation, drawing any conclusions about methylation based on one or a few enzyme sites can be tenuous. The McrBC enzyme is also of use; it imprecisely cleaves methylated DNA if there are methylated sites containing a methyl-cytosine preceded by a purine within 40 bp to 3 kb of one another (Sutherland et al., 1992). After genomic DNA is digested with McrBC, regions of interest can be amplified by PCR—the more highly methylated a sequence is the less it will be amplified (Vaughn et al., 2007). An advantage of both of the methods that employ methylation-sensitive enzymes is that all molecules can be assayed simultaneously. Affinity purification is an attractive route for assaying methylation. A commercially available 5-methylcytosine antibody can be used to pull down the methylated fraction of the genome. Particular sequences can then be analyzed by comparing PCR amplification between input and pull-down fractions or between genotypes. The methyl-binding domain (MBD) from mammals has been used in a similar manner, but it only binds methylated CG sites. Bisulfite sequencing provides the most detailed data about methylation of particular cytosines in a sequence. Treatment of restriction digested or sheared single-stranded DNA with sodium bisulfite followed by desulfonation converts cytosine to uracil but leaves 5-methylcytosine intact (Figure 1) (Frommer et al., 1992; Clark et al., 1994). A region of interest, usually less than 700 base pairs, is then amplified in a strand-specific manner by PCR; after cloning and sequencing of the PCR product the original cytosines will be read as Ts, and 5-methylcytosines as Cs. Comparison to the reference sequence allows determination of the methylation status of each cytosine. Although bisulfite sequencing is the gold standard for methylation analysis, biases can be introduced at several points in the procedure, including PCR amplification and cloning of the PCR products (Warnecke et al., 2002). Sequencing the PCR product directly using pyrosequencing can eliminate one aspect of this bias, as long as information about individual molecules is not required (Tost and Gut, 2007). Because bisulfite treatment produces DNA strands that are no longer complementary, two separate PCR reactions must be performed if methylation information for each strand is desired. This information might be particularly relevant in plants since asymmetric cytosines are methylated and thus the number of potentially methylated sites differs on each strand. Hairpin bisulfite PCR cleverly solves this problem by joining the complementary strands of DNA using a hairpin linker, allowing them to be assessed simultaneously (Laird et al., 2004), but has so far not been applied in plant studies. Many of the above methods can be, or have already been, extended to whole genome methylation analysis if combined with microarrays or high-throughput sequencing (Yazaki et al., 2007; Zilberman and Henikoff, 2007). As the technology has improved, the definition of what constitutes “genome-wide” and “high-resolution” methylation mapping has also changed. Initial efforts to globally map methylation in Arabidopsis relied on methylation-sensitive enzymes and microarrays of limited genomic coverage (Tompa et al., 2002; Lippman et al., 2004; Tran et al., 2005a; Tran et al., 2005b), but provided important information about methylation location at a gross level. Methylation has also been mapped by immunoprecipitating the methylated fraction of the genome with a 5-methylcytosine antibody or MBD and hybridizing the bound or unbound DNA to tiling microarrays. This has allowed detailed examination of methylation patterns across the genome and within specific regions (Zhang et al., 2006; Penterman et al., 2007c; Zilberman et al., 2007). Methylation mapping in methyltransferase, demethylase, and RNA interference mutants is giving us a broader understanding of how methylation patterns are established, maintained, and potentially changed. Recently, methylation has been mapped genome-wide at single base resolution by bisulfite-treating DNA, sequencing it using Solexa high-throughput sequencing technology, and aligning the sequences back to the genome (Cokus et al., 2008). This has provided a level of detail unprecedented for any organism. Another new method, which requires only small amounts of DNA, involves hybridizing bisulfite-treated DNA to tiling microarrays (Reinders et al., 2008). It is expected that the use of high-throughput sequencing or microarrays in combination with affinity purification or bisulfite treatment will continue to further refine the Arabidopsis methylation map in different genotypes, tissues, or conditions. Once a methylation pattern is determined, deriving meaning from it can be challenging. Often differences are compared between wild type and mutant plants or between different conditions. Although differences might be statistically significant, biological significance is harder to assess. As will become clear in the following sections, large questions still remain as to how methylation exerts the influence that it does, or if methylation at particular sequences is relevant to biological function.