DNA methylation of the endogenous PAI genes in Arabidopsis.

histone proteins to form chromatin allows packaging of DNA into the nucleus. However, the degree of chromatin compaction varies along the length of each chromosome, with some regions present in relatively open transcriptionally active euchromatin, and some regions present in more compact transcriptionally silent heterochromatin. In particular, repetitive arrays associated with centromeres and ribosomal RNA-encoding genes (rDNA), as well as transposable element sequences (both DNA transposons and retrotransposons), are assembled into heterochromatin. In the case of repeat arrays, heterochromatin is likely to serve as a means of stabilizing these structures against rearrangement, whereas in the case of transposable elements heterochromatin serves as a means of genome defense against deleterious effects of their transcription and movement. Thus, the integrity of chromatin organization is critical for correct patterns of gene expression and genome stability. Key questions are how chromatin organization patterns are established, and how these patterns are maintained through cell divisions. In mammals and plants, as well as some species of fungi such as Neurospora crassa, heterochromatic regions of the genome are marked by cytosine methylation. Because this covalent DNA modification can be inherited as hemimethylated DNA after each round of replication, it provides an epigenetic memory of heterochromatin patterning in the previous generation. In mammals, cytosine methylation occurs almost exclusively in CG contexts, whereas in plants and Neurospora both CG and non-CG cytosines can be methylated. These differences reflect the different substrate specificities of DNA methyltransferase (DMTase) enzymes present in each organism. Heterochromatin is also associated with particular patterns of posttranslational modifications on the amino-terminal “tails” of histone proteins, which extend outward from the globular core of the histone octamer. For example, in many eukaryotes heterochromatin-associated modification patterns include methylation of histone H3 at lysine 9 (H3 mK9) and lack of acetylation (deacetylation) of lysines on H3 and H4. Conversely, euchromatin is associated with methylation of H3 at lysine 4 (H3 mK4) and hyperacetylation of lysines on H3 and H4. These and other posttranslational histone modifications are thought to constitute a “histone code” that guides the formation of heterochromatin, euchromatin, or specialized chromatin structures, most likely through recruitment of appropriate chromatin remodeling factors to histones carrying particular combinations of modifications (Jenuwein and Allis 2001). A striking recent discovery in Neurospora, Arabidopsis, and mammals is that cytosine methylation patterns can be guided by one of the heterochromatin-associated histone modifications, methylation of H3 at lysine 9. Neurospora presents the simplest example of this relationship: Loss of H3 mK9, either by mutation of the H3 K9 histone methyltransferase (HMTase) DIM-5 or by mutation of the K9 residue of the single H3-encoding gene, abolishes cytosine methylation, similar to mutation of the DMTase DIM-2 (Kouzminova and Selker 2001; Tamaru and Selker 2001). In Arabidopsis, mutation of the H3 K9 HMTase SUVH4/KYP (hereafter called SUVH4) reduces non-CG but not CG patterns of cytosine methylation, similar to mutation of the DMTase CMT3 (Jackson et al. 2002; Malagnac et al. 2002). In the mouse, double mutation of the hSUVH391 and hSUV392 H3 K9 HMTases causes demethylation of major satellite repeat sequences (Lehnertz et al. 2003). Similarly, mutation of the mouse G9a H3 K9 HMTase causes demethylation of an imprinted locus (Xin et al. 2003). Taken together, these findings suggest that DMTases can be recruited to appropriate target sites by recognizing the H3 mK9 mark. Another emerging area in our understanding of how cytosine methylation and heterochromatin are targeted is the finding that RNA molecules can guide these epigenetic modifications. For example, in plant systems that express double-stranded RNA (dsRNA) and “diced” small interfering RNAs (siRNAs) associated with RNA interference (RNAi), DNA sequences with identity to the aberrant RNAs can acquire cytosine methylation (Bender 2004). This RNA-directed DNA methylation (RdDM) is typically very specifically targeted, and results in dense CG and non-CG methylation. Interestingly, an examination of transgene and transposon targets of RdDM in Arabidopsis suggests that different loci are differentially sensitive to methylation mutations. Some silenced loci are demethylated and reactivated by mutation of the MET1 CG-specific DMTase and the HDA6 histone deacetylase, whereas other silenced loci are demethylated and reactivated by mutations in CMT3 and SUVH4, which control CNG and other patterns of non-CG methylation (Lindroth et al. 2001; Murfett et al. 2001; Aufsatz et al. 2002; Cao and Jacobsen 2002a; Jackson et al. 2002; Cao et al. 2003; DNA Methylation of the Endogenous PAI Genes in Arabidopsis