A Silencing Pathway to Induce H3-K9 and H4-K20 Trimethylation at Constitutive Heterochromatin. linked to transcription, which may be associated with transcriptional attenuation (10, 11). In addition, in mammals, mono- and tri-methylated H4 K20 localize respectively to the transcriptionally silent X chromosome Barr body and pericentromeric heterochromatin (12). In addition to the role of H4 K20 methylation, the residue itself may be linked to heterochromatin function as part of a patch of basic amino acids (K16RHRK20). In budding yeast, this patch, in particular the RHR motif, recruits or regulates several chromatin proteins, including Choline Fenofibrate Isw2 ATP nucleosome remodeling complex, Sir2/3/4 deacetylase complex, and Dot1 methylase (13, 14, 15). Lysine 20, however, has been less well studied in budding yeast, and its role and modifications have not Choline Fenofibrate been FCRL5 elucidated. While lysine methylation associated with active transcription (H3 K4, K36, K79) is conserved from budding Choline Fenofibrate yeast to humans, lysine methylation associated with gene repression (H3 K9 and K27, and H4 K20), is generally thought to be absent in (16C18). However, intriguingly, mass spectrometry suggested that H4 K20me1 exists in budding yeast in low abundance (19). Because of the important role of H4 K20 in histone-protein interactions, and the conservation of its methylation throughout higher organisms, we sought to confirm the presence of H4 K20 methylation in was amplified by the Expand High Fidelity PCR System (Roche), cloned into pBM272 (GAL promoter, CEN, ARS, and demethylase deletion strains were created by standard gene knockout methods. Overexpression strains were created by transforming pBM272 or FLEXGene Collection plasmids (pBY011) containing galactose-inducible genes into yeast or by integrating galactose-inducible promoters into the genome. Strains with subtelomeric and reporters plus wild-type or mutant histones were created as follows. pRM204 with wild-type or mutant H3/H4 genes were transformed into UCC1369 to create YCE UA1 to UA9 after which strains were grown on SC media lacking tryptophan to select for pRM204 and dilute out the original histone plasmid. Plasmids were similarly transformed into UCC7262 to make YCE UC1 to UC9, and UCC7266 to make YCE UD1 to UD9. UCC1369, UCC7262, and UCC7266 are reported elsewhere (23). All other mutant histone strains were created as follows. pRM204 (or derivatives of this) containing wild-type, Choline Fenofibrate FLAG-tagged, or mutant H3/H4 genes were transformed into the H3/H4 shuffling strain FY1716 after which SC media containing 5-FOA was used to select against the original FY1716 histone plasmid. Strains with either WT or K20 substitutions of H4 integrated into the genome were made as previously described (24). Whole-cell extract (WCE) preparation & FLAG-affinity purification Yeast were grown in YPD (or YP+Galactose for overexpressions) to mid-log phase, resuspended in TENG-300 buffer (50mM Tris-Cl pH 7.5, 300mM NaCl, 0.5% NP-40, 1mM EDTA, 10% glycerol, 0.5mM PMSF, protease inhibitors), beat with silica beads, and sonicated after which lysates were cleared by centrifugation at 14krpm. Bradford assays determined protein concentrations. Anti-FLAG-agarose beads (Sigma) were incubated with WCEs overnight and then washed with TENG-300. FLAG peptides (Sigma) then competed off FLAG-tagged proteins. Western analyses Samples were run on polyacrylamide gels, transferred to PVDF, and probed with antibodies followed by incubation with chemiluminescence reagent. Signals were visualized with a Fujifilm LAS-3000 Image Reader. Supplementary table S2 lists antibodies used in this study. Roche supplied calf thymus histone H4. Dot blots & peptide competitions Peptides matching the first thirty amino acids of budding yeast histone H4 plus a C-terminal cysteine were synthesized with no modifications, acetylated K16, monomethylated K20, or both modifications (Baylor College of Medicine Protein Chemistry Core Laboratory). Peptides matching the higher eukaryote histone H4 lysine 20 epitope without modifications or with mono, di, or trimethylated.