Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - SUVH5 has histone H2A MTase activity in vitro. (A) H2A amino-terminal sequence alignments. Predicted amino acid sequences for mammalian H2A and Arabidopsis H2A (HTA) variants were obtained from GenBank, aligned using ClustalW (http://www.ch.embnet.org/software/ClustalW.html), and colored according to sequence identity or similarity using BoxShade Server (http://www.ch.embnet.org/software/BOX_form.html). Identical residues are highlighted in black and similar residues are highlighted in gray. Arrowheads denote positions of target lysines (K13 and K14) in HTA2 and HTA13. The residues mutated in the HTA13 analysis are boxed. (B) and (C) Histone MTase assays were performed as described in Methods, except that 2 μg of each GST-HTA substrate was used per reaction and samples were resolved on 12.5% SDS-PAGE gels. Coomassie stained gels (upper panel) and their fluorograms (lower panel) are shown. * indicates GST-SUVH5 truncations; • indicates GST-HTA truncations. Positions of protein molecular weight markers in kD are shown along the left margin. (B) SUVH5 methylates a subset of HTA proteins. GST-SUVH5 was incubated with each GST-HTA as indicated. (C) HTA mutational analysis. In vitro MTase assays were done with GST-SUVH5 and GST-HTA13 mutants. WT indicates wild-type GST-HTA13 sequence; KDM indicates K13R K14R double mutation.
• Supplemental Figure 2 - Bisulfite genomic sequencing of DNA methylation patterning on the PAI1 and PAI2 proximal promoters in suvh mutants. Eight independent top-strand clones were sequenced for PAI1 (P1) or PAI2 (P2) from genomic DNA of the indicated mutants as previously described (Melquist and Bender, 2003). The percentage of 5-methyl-cytosines detected out of total available cytosines within the region of PAI sequence identity (344 bp for PAI1 or 338 bp for PAI2) is shown, divided into the contexts CG (black), CNG (white), or other (gray). Data for wild type WS (WT), suvh6, suvh4, suvh4 suvh6, and cmt3 are from previous publications (Luff et al., 1999; Bartee et al., 2001; Malagnac et al., 2002; Ebbs et al., 2005).
• Supplemental Figure 3 - Replicates of ChIP analysis of H3 2mK4 and H3 2mK9 patterning. GelStar-stained PCR products of three independent immunoprecipitations are shown as described in Figure 3 and 4. Data from ChIP1 are shown in Figure 3C (PAI1, PAI2, PAI3, and ACTIN) and Figure 4C (Ta3, Mu1, and ACTIN).
• Supplemental Figure 4 - The suvh5 suvh6 mutant does not display DNA methylation defects. (A) Southern blot assays for PAI DNA methylation patterning. Genomic DNA from the indicated mutants was cleaved with HincII and used in Southern blot analysis with a PAI1 cDNA probe. P1-P4 indicates PAI1-PAI4, P2 indicates PAI2, and P3 indicates PAI3, with bands diagnostic of methylation on PAI-internal sites denoted with asterisks. (B) and (C) Southern blot assays for transposon DNA methylation patterning. DNA from the indicated mutants was cleaved with (B) MspI or (C) HindIII plus MspI and used in Southern blot analysis with (B)a Ta3 probe or(C) a Mu1 probe. Arrowheads along the left margin indicate the positions of fully-cleaved bands. All mutations assayed were in the WS pai1 background, with WT indicating wild type, 5 + 6 indicating a suvh5-1 suvh6-1 double mutant, 4 indicating suvh4R302*, and cmt3 indicating cmt3illa.
• Supplemental Figure 5 - Two suvh5 alleles confer similar loss of transposon non-CG methylation in the absence of SUVH4. (A) and (B)Southern blot assays for transposon DNA methylation patterning. DNA from the indicated mutants was cleaved with (A) MspI or (B) HindIII plus MspI and used in Southern blot analysis with (A) a Ta3 probe or(B) a Mu1probe. Arrowheads along the left margin indicate the positions of fully-cleaved bands. All mutations assayed were in the WS pai1 background, with WT indicating wild type, 4 indicating suvh4R302*, 5-1 indicating suvh5-1, 5-2 indicating suvh5-2, and cmt3 indicating cmt3i11a.
• Supplemental Figure 6 - Replicates of ChIP analysis of H3 1mK9 patterning. GelStar-stained PCR products of three independent immunoprecipitations are shown as described in Figure 6. ChIP1 is shown in Figure 6.
• Supplemental Table 1 - ChIP primer sequences.