Figure S2 Acetylation of H4K12 at native telomeric hetero- chromatin in galactose condition. ChIP experiments were performed as Figure 3 except that the cells were cultured in galactose medium. Found at: doi:10.1371/journal.pgen.1001272.s002 (0.06 MB PDF) Figure S3 Histone N terminal lysine acetylation profile at HMR and HMR-proximal regions. (A) Schematic diagram of the HMR region and subtelomeric region of the right arm of chromosome III. Primer sets used for the following ChIP analysis were shown on the top. (B) Relative abundance of H4K5 acetylation, K8 acetylation, K12 acetylation and K16 acetylation at HMR and HMR-proximal regions. The relative abundance of lysine acetylation was corrected by H4 occupancy. Grey areas were predicted silenced chromatin. Found at: doi:10.1371/journal.pgen.1001272.s003 (0.21 MB PDF) Figure S4 Contribution of Esa1 and Hat1 to chromatic H4K12 acetylation (A) Schematic diagram of the subtelomeric region of the right arm of chromosome XIV (TELXIV-R). Primer sets used for the following ChIP analysis were shown on the top. (B) ChIP analysis of Esa1-myc at subtelomeric region of TELXIV-R. The locations of primer sets were depicted in (A). (C) Histone acetylation assay. Recombinant Esa1 was incubated with native nucleosomes derived from sas2Deaf4D cells. Reaction products were loaded onto 15% SDS-page gel and detected using indicated antibodies. (D) Western-blot of chromatin derived from wild-type and hat1D cells. Antibodies used were indicated on the right. (E) ChIP of H4K12ac at subtelomere XIV-R in wild-type and hat1D cells. Found at: doi:10.1371/journal.pgen.1001272.s004 (0.25 MB PDF) Figure S5 H4K12R mutation does not affect the perinuclear localization of telomeres. Sir3-myc (used to represent telomeres) and nuclear pores were stained by rabbit anti-myc antibody and mouse anti-mAb414 antibody, respectively. Found at: doi:10.1371/journal.pgen.1001272.s005 (0.66 MB PDF) Figure S6 H4K12 regulates subtelomeric binding of Sir proteins. (A) Relative abundance of Sir2-myc at all telomeres in isogenic wild-type and H4K12R strains. Regions at ,0.5 kb from each telomere were subjected to real-time PCR analysis. PCR data References 1. Rusche LN, Kirchmaier AL, Rine J (2003) The establishment, inheritance, and function of silenced chromatin in Saccharomyces cerevisiae. Annu Rev Biochem 72: 481–516. 2. Luo K, Vega-Palas MA, Grunstein M (2002) Rap1-Sir4 binding independent of other Sir, yKu, or histone interactions initiates the assembly of telomeric heterochromatin in yeast. Genes Dev 16: 1528–1539. 3. Imai S, Armstrong CM, Kaeberlein M, Guarente L (2000) Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403: 795–800. 4. Hecht A, Laroche T, Strahl-Bolsinger S, Gasser SM, Grunstein M (1995) Histone H3 and H4 N-termini interact with SIR3 and SIR4 proteins: a molecular model for the formation of heterochromatin in yeast. Cell 80: 583–592. 5. Carmen AA, Milne L, Grunstein M (2002) Acetylation of the yeast histone H4 N terminus regulates its binding to heterochromatin protein SIR3. J Biol Chem 277: 4778–4781. 6. Kimura A, Umehara T, Horikoshi M (2002) Chromosomal gradient of histone acetylation established by Sas2p and Sir2p functions as a shield against gene silencing. Nat Genet 32: 370–377. 7. Suka N, Luo K, Grunstein M (2002) Sir2p and Sas2p opposingly regulate acetylation of yeast histone H4 lysine16 and spreading of heterochromatin. Nat Genet 32: 378–383. 8. Katan-Khaykovich Y, Struhl K (2005) Heterochromatin formation involves changes in histone modifications over multiple cell generations. Embo J 24: 2138–2149. 9. Bi X, Broach JR (2001) Chromosomal boundaries in S. cerevisiae. Curr Opin Genet Dev 11: 199–204. H4K12ac Regulates Heterochromatin Plasticity were normalized to ACT1. (B) and (C) ChIP of Sir2-myc (B) and Sir3-myc (C) in wild-type and H4K12Q mutant cells. Found at: doi:10.1371/journal.pgen.1001272.s006 (0.08 MB PDF) Figure S7 Histone mutations do not affect the expression of silencing-related genes. (A) and (B) Western-blot of Sir2-myc (A) or Sir3-myc (B) in wild-type and histone mutants. Sir2 and Sir3 levels were detected using an anti-myc antibody and were normalized to anti-tubulin protein level. (C) Quantitative PCR of relative mRNA levels in a cluster of silencing-related genes in the indicated histone mutant strains. Shown are the average expression ratios (normalized by that of ACT1) relative to the wild-type. A log2 ratio less than zero indicates repression of transcription, whereas greater than zero indicates enhancement of transcription. Error bars represent standard error of the mean for three independent RNA purifications. (D) Immunolocalization of Sir2-myc and Nop1 in wild-type and H4K12R cells. Sir2-myc and Nop1 (indicating the nucleolus) were stained by rabbit anti-myc antibody and mouse anti-Nop1 antibody, respectively. Found at: doi:10.1371/journal.pgen.1001272.s007 (1.04 MB PDF) Table S1 Antibodies used in this study. Found at: doi:10.1371/journal.pgen.1001272.s008 (0.04 DOC) Table S2 Yeast strains used in this study. Found at: doi:10.1371/journal.pgen.1001272.s009 (0.09 DOC) Table S3 Primers used for real-time PCR. Found at: doi:10.1371/journal.pgen.1001272.s010 (0.07 DOC) MB MB MB Author Contributions Conceived and designed the experiments: BOZ JQZ. Performed the experiments: BOZ XHF WD. Analyzed the data: BOZ XHF BAL JQZ. Contributed reagents/materials/analysis tools: BOZ SSW YZ. Wrote the paper: BAL JQZ. Assisted in constructing yeast strains, checking the specificity of antibodies, and detecting the expression, cellular localization, and telomere binding of Sir proteins: SSW. Assisted in strain construction: YZ. 10. Smith ER, Eisen A, Gu W, Sattah M, Pannuti A, et al. (1998) ESA1 is a histone acetyltransferase that is essential for growth in yeast. Proc Natl Acad Sci U S A 95: 3561–3565. 11. Cheng TH, Gartenberg MR (2000) Yeast heterochromatin is a dynamic structure that requires silencers continuously. Genes Dev 14: 452–463. 12. Sekinger EA, Gross DS (2001) Silenced chromatin is permissive to activator binding and PIC recruitment. Cell 105: 403–414. 13. Chen L, Widom J (2005) Mechanism of transcriptional silencing in yeast. Cell 120: 37–48. 14. Andrau JC, van de Pasch L, Lijnzaad P, Bijma T, Koerkamp MG, et al. (2006) Genome-wide location of the coactivator mediator: Binding without activation and transient Cdk8 interaction on DNA. Mol Cell 22: 179–192. 15. Teytelman L, Eisen MB, Rine J (2008) Silent but not static: accelerated base-pair substitution in silenced chromatin of budding yeasts. PLoS Genet 4: e1000247. doi:10.1371/journal.pgen.1000247. 16. Diede SJ, Gottschling DE (1999) Telomerase-mediated telomere addition in vivo requires DNA primase and DNA polymerases alpha and delta. Cell 99: 723–733. 17. Tamburini BA, Tyler JK (2005) Localized histone acetylation and deacetylation triggered by the homologous recombination pathway of double-strand DNA repair. Mol Cell Biol 25: 4903–4913. 18. Meneghini MD, Wu M, Madhani HD (2003) Conserved histone variant H2A.Z protects euchromatin from the ectopic spread of silent heterochromatin. Cell 112: 725–736. 19. Zhou BO, Wang SS, Xu LX, Meng FL, Xuan YJ, et al. (2010) SWR1 complex poises heterochromatin boundaries for antisilencing activity propagation. Mol Cell Biol 30: 2391–2400. PLoS Genetics | www.plosgenetics.org 10 January 2011 | Volume 7 | Issue 1 | e1001272