Genome-Wide H3K9m2 Profiling Figure 6. H3K9m2 methylation of transposable elements. The majority of transposons in pericentromeric regions have high levels of H3K9m2 (blue bars), while a large number of transposons in the arms are unmethylated (red line). X axis represents the average H3K9m2 level (Z-score) for the element and Y axis represents the percentage of elements falling into each category. doi:10.1371/journal.pone.0003156.g006 underscore the differences between H3K9m2 patterns in pericen- tromeric heterochromatin relative to euchromatic arms. Conclusion We have profiled H3K9m2 methylation across the Arabidopsis genome and combined the analysis with recently published high- resolution BS-seq data to understand the relationship between H3K9m2 and DNA methylation. The H3K9m2 methylation data, along with repeat annotations and other epigenomic datasets are available to the community at our local UCSC genome browser site: http://epigenomics.mcdb.ucla.edu/H3K9m2/ Our bioinformatics analyses, as well as manual inspection of the data in genome browsers, shows that H3K9m2 and CHG DNA methylation are very tightly correlated throughout the genome, showing that the relationship between these two marks may be universal. These results are consistent with the proposed self- reinforcing loop mechanism operating between the CHG methyltransferase (CMT3) and the primary H3K9m2 methyl- transferase KYP [30]. Our data also show that the CG only methylation associated with roughly one third of expressed genes is not correlated with H3K9m2, showing that this type of methylation is maintained by a different mechanism. Finally, our data suggest that different mechanisms are operating to maintain high levels of H3K9m2 in large blocks of pericentromeric heterochromatin, and smaller patches of H3K9m2 on repeats and transposons found in the euchromatic arms of the chromo- somes. Future studies will hopefully uncover the mechanistic differences between these two patterns of epigenetic silencing. Methods Native chromatin immunoprecipitation The entire shoots of three week old Arabidopsis plants (Col-0 ecotype), grown under continuous light, were harvested, frozen in liquid nitrogen and ground to powder (1 g). Plant tissue was resuspended in 10 ml of HBM buffer (25 mM Tris-Cl pH 7.6, 440 mM sucrose, 10 mM MgCl2 and 0.1% Triton-X, 10 mM b- mercaptoethanol, 2 mM spermine, 1 mM PMSF, 1 ug/ml pepstatin and EDTA-free protease inhibitor cocktail (Roche), homogenized and filtered through Miracloth (Calbiochem). After spinning at 3000 rpm for 5 min (SS-34, Sorvall), the pellet was resuspended in 5 ml of NIB buffer (20 mM Tris-Cl pH 7.6, 250 mM sucrose, 5 mM MgCl2, 5 mM KCl, 0.1% Triton-X, 10 mM b-mercaptoethanol), applied to a 15/50% Percoll (GE Healthcare) gradient in NIB and spun 2000 rpm for 20 min (SS- 34, Sorvall). Isolated nuclei were washed two times in NIB buffer and flash frozen in liquid nitrogen in HBC buffer (25 mM Tris-Cl pH 7.6, 25 mM Tris-Cl pH 7.6, 440 mM sucrose, 10 mM MgCl2 and 0.1% Triton-X, 10 mM b-mercaptoethanol, 20% glycerol). Nuclei from 1/5 of each preparation were treated with four ul of RNAse A, 10 ug/ul, (Qiagen) and used for Micrococcal Nuclease (Takara) digestion for 6 minutes (final concentration 0.2 U/ul) in digestion buffer (16 mM Tris-Cl, pH 7.6, 50 mM NaCl, 2.5 mM CaCl2, 0.01 mM PMSF and EDTA-free protease inhibitor cocktail (Roche) and stopped with 10 mM EDTA. Mononucleo- somes were released by treating nuclei with 0.1% Triton-X for 1– 2 hours in the cold, and then pelleting the debris by centrifuging at 3500 rpm for 3 min (Eppendorf, 5415R). 500 ul of supernatant was applied to 50 ul of Dynabeads Protein A (Invitrogen) that were preincubated with 2.5 ug of the appropriate antibody (#1220, monoclonal anti-H3K9m2 antibody, Abcam; #1791, polyclonal anti-H3 antibody, Abcam) in buffer (20 mM Tris-Cl, pH 7.6, 50 mM NaCl, 5 mM EDTA and 0.1% Triton) and incubated overnight. Beads were washed (10 min incubation in the cold) with 500 ul of the following buffers: 50 mM Tris-Cl pH 7.6, 10 mM EDTA, 0.1 mM PMSF, protease inhibitor cocktail (Roche) with changing concentration of NaCl to 50 mM, 100 mM and 150 mM, subsequently. Final wash was done in PLoS ONE | www.plosone.org 8 September 2008 | Volume 3 | Issue 9 | e3156