Chan, S. W. L., Zhang, X., Bernatavichute, Y. V. & Jacobsen, S. E. Two-step recruitment of RNA-directed DNA methylation to tandem repeats. PLoS Biol. 4, 1923–1933 (2006). Quadrana, L. & Colot, V. Plant transgenerational epigenetics. Annu. Rev. Genet. 50, 467–491 (2016). Xie Z, Johansen LK, Gustafson AM, Kasschau KD, Lellis AD, et al. (2004) Genetic and functional diversification of small RNA pathways in plants. PLoS Biol 2: E104. He XJ, Hsu YF, Pontes O, Zhu J, Lu J, et al. (2009) NRPD4, a protein related to the RPB4 subunit of RNA polymerase II, is a component of RNA polymerases IV and V and is required for RNA–directed DNA methylation. Genes Dev 23: 318–330.

Funding: Support was provided by a Leukemia and Lymphoma Special Fellowship (IRH), National Cancer Institute award F32CA137995 (GAH), and National Institutes of Health grant GM60398 (SEJ). SEJ is an investigator of the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Zhang X, Yazaki J, Sundaresan A, Cokus S, Chan SW, et al. (2006) Genome-wide high-resolution mapping and functional analysis of DNA methylation in arabidopsis. Cell 126: 1189–1201.Pandey, N. & Pandey-Rai, S. Deciphering UV-B-induced variation in DNA methylation pattern and its influence on regulation of DBR2 expression in Artemisia annua L. Planta 242, 869–879 (2015). Sign in to the AWS Management Console and choose the region you would like to use for the DR environment. For crystallization, the DRM2-DNA complexes were each mixed with 1 mM SAH. Crystals for all the DRM2-DNA complexes were generated using sitting-drop vapor-diffusion method at 4°C. Each drop was prepared by mixing 0.5 μl of DRM2-DNA complex sample with 0.5 μl of precipitant solution [for DRM2-CTT, DRM2-CCG, and DRM2 C397R-CCG complexes: 2% v/v Tacsimate (pH 6.0), 0.1 M bis-tris (pH 6.5), and 20% w/v polyethylene glycol 3350; for DRM2-CAT complex: 0.1 M sodium acetate trihydrate (pH 7.0) and 12% w/v polyethylene glycol 3350; for DRM2-CCT complex: 0.1 M sodium formate (pH 7.0) and 12% w/v polyethylene glycol 3350; and for DRM2-CTG: 0.2 M potassium iodide and 20% w/v polyethylene glycol 3350 (pH 7.0)]. The crystal quality was further improved using the microseeding method. To harvest crystals, the crystals were soaked in cryoprotectants made of mother liquor supplemented with 30% glycerol before being flash-frozen in liquid nitrogen.

Zhu, X. et al. Effects of ultraviolet B exposure on DNA methylation in patients with systemic lupus erythematosus. Exp. Ther. Med. 5, 1219–1225 (2013). Dominguez-Salas, P. et al. Maternal nutrition at conception modulates DNA methylation of human metastable epialleles. Nat. Commun. 5, 3746 (2014). Bergman, Y. & Cedar, H. DNA methylation dynamics in health and disease. Nat. Struct. Mol. Biol. 20, 274–281 (2013). Zilberman D, Cao X, Johansen LK, Xie Z, Carrington JC, et al. (2004) Role of Arabidopsis ARGONAUTE4 in RNA–directed DNA methylation triggered by inverted repeats. Curr Biol 14: 1214–1220.Henderson, I. R. & Jacobsen, S. E. Tandem repeats upstream of the Arabidopsis endogene SDC recruit non-CG DNA methylation and initiate siRNA spreading. Genes Dev. 22, 1597–1606 (2008). A synthetic DNA fragment encoding the methyltransferase domain of A. thaliana DRM2 (residues 270 to 626) was cloned into pRSFDuet-1 vector (Novagen), preceded by an N-terminal His 6-SUMO tag. The expression plasmid was transformed into Escherichia coli BL21 DE3 (RIL) cells, and the cells were grown at 37°C. After the cell density reached an optical density at 600 nm of 0.8, the temperature was lowered to 16°C. Subsequently, the cells were induced by 100 μM isopropyl-β-D-thiogalactopyranoside and continued to grow overnight. The cells were collected and resuspended in lysis buffer [50 mM tris-HCl (pH 8.0), 1 M NaCl, 25 mM imidazole, and 1 mM phenylmethylsulfonyl fluoride] and lysed using an Avestin Emulsiflex C3 homogenizer. After centrifugation, the supernatant was applied to a Ni 2+–nitrilotriacetic acid affinity column and the His 6-SUMO-DRM2 fusion protein was eluted with elution buffer [20 mM tris-HCl (pH 8.0), 300 mM NaCl, and 300 mM imidazole]. The His 6-SUMO tag was then removed by ubiquitin-like protease 1–mediated cleavage. The tag-free protein was further purified through ion-exchange chromatography on a Heparin HP column (GE Healthcare) and size exclusion chromatography on a 16/600 Superdex 200 pg column (GE Healthcare). The final protein sample was concentrated and stored in −80°C freezer for future use. The license received by the "Appstore SDK" (or potentially a fake masquerading as such) has been tampered with during transit. Henderson IR, Zhang X, Lu C, Johnson L, Meyers BC, et al. (2006) Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nat Genet 38: 721–725.

Law JA, Jacobsen SE (2010) Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet 11: 204–220. Kawakatsu, T. et al. Unique cell-type-specific patterns of DNA methylation in the root meristem. Nat. Plants 2, 16058 (2016). Eun, C. et al. AGO6 functions in RNA-mediated transcriptional gene silencing in shoot and root meristems in Arabidopsis thaliana. PLoS ONE 6, e25730 (2011).Freitag M, Lee DW, Kothe GO, Pratt RJ, Aramayo R, et al. (2004) DNA methylation is independent of RNA interference in Neurospora. Science 304: 1939. Pontier D, Yahubyan G, Vega D, Bulski A, Saez-Vasquez J, et al. (2005) Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis. Genes Dev 19: 2030–2040. All necessary IAM Roles and Policies needed to make the solution work, following least privilege principles. Jeddeloh JA, Bender J, Richards EJ (1998) The DNA methylation locus DDM1 is required for maintenance of gene silencing in Arabidopsis. Genes Dev 12: 1714–1725.