acetylated by Piccolo NuA4 in a non-targeted manner [23]. We found that the binding of Esa1 at subtelomeric region was restricted to the distal region because there was no Esa1 binding in the subtelomeric domain 3.2 kb,7.6 kb from the end of chromosome XIV-R (Figure S4A and S4B). A previous study showed that Hat1, another lysine acetyl- transferase in yeast, also possesses H4K12 acetylation activity [24], however it is predominantly localized to the cytoplasm and thought to specifically acetylate free histone H4 [25]. Western- blot results showed that HAT1 deletion did not affect chromatic H4K12 acetylation (Figure S4D). ChIP analyses revealed that the H4K12 acetylation level at the subtelomeric region of ChrXIV-R was not reduced, but rather modestly increased in hat1D cells (Figure S4E). Therefore, these data suggest that Hat1 is likely not needed for dynamic H4K12 acetylation at telomeric heterochromatin. Histone H4K12 acetylation regulates telomere replication Histone acetylation is thought to regulate chromatin assembly [26]. To address whether H4K12R mutation affects subtelomeric nucleosomal organization, the chromatin from wild-type and H4K12R mutant cells was digested with increasing concentrations of micrococcal nuclease (MNase) and the chromatin structure of subtelomere III-L was analyzed by Southern-blot as previously reported [27]. The MNase digestion of the subtelomeric region at specific sites indicated the presence of a regular array of nucleosomes (Figure 4A). There was little difference in subtelo- meric nucleosome organization when comparing wild-type to H4K12R cells (Figure 4A), indicating that H4K12 does not influence the nucleosome organization of subtelomeric DNA on chromosome III-L. Surprisingly, the upper bands representing the undigested telomere-containing DNA, were much shorter in H4K12R cells when compared to wild-type cells (Figure. 4A), indicating that H4K12 acetylation affects the telomere length of chromosome III-L. H4K12ac Regulates Heterochromatin Plasticity To investigate whether H4K12 acetylation regulates telomere length in a universal manner, we examined the average telomere length of wild-type and H4K12R mutant cells. Strikingly, Southern-blot analysis revealed that telomeres in H4K12R mutant cells were much shorter than that in wild-type cells (Figure 4B). Consistently, the ESA1 mutant esa1-338, which lost telomeric H4K12 acetylation (Figure 3), also had shorter telomeres (Figure 4B). However, the telomere length in H4K12R esa1-338 double mutation cells resembled that in esa1-338 cells, rather than that in H4K12R cells (Figure 4B). Because H4K12R mutation didn’t affect Esa1-telomere association (Figure S4B), it remains possible that the shorter telomeres observed in esa1-338 mutant could not be solely attributed to a defect in H4K12 acetylation. Histone H4K12R mutation reduces the accessibility of telomeres to telomerase The major structural components of telomere heterochromatin are Sir2, Sir3 and Sir4 [1]. Previous studies have shown that deletion of SIR3 or SIR4 caused telomere shortening, suggesting that disturbing telomere heterochromatin structure affects telo- mere replication [28]. Because the distal telomeres are devoid of nucleosome structure [29], we proposed that the telomere length defect observed in H4K12R mutant was attributable to a change in telomere heterochromatin structure. To test this possibility and to elucidate the molecular mechanism by which histone H4K12 acetylation affects telomere length, we examined the structural change(s) of telomere heterochromatin in H4K12R mutant cells. Immuno-staining analysis of myc-tagged Sir3 (Sir3-myc) with monoclonal anti-myc antibody revealed that H4K12R mutation did not affect the sub-cellular localization of telomeres (Figure S5), excluding the possibility that H4K12 affects the perinuclear localization of telomeres, which potentially regulates telomerase activity [30]. A distinctive feature of telomeric heterochromatin involves high-order structure, such as a fold-back loop-like structure Figure 4. Histone H4K12 acetylation regulates telomere replication. (A) Sensitivity of subtelomere III-L chromatin to MNase. Chromatin samples from wild-type and H4K12R cells were digested with increasing concentrations of MNase (0 U, 50 U, 150 U and 300 U) for 10 minutes and genomic DNA was purified, digested with BamHI and resolved in 1.2% agarose gel. Then the cutting profile was visualized after hybridization with an internal probe from Ty5-1. This probe abutted the BamHI site selected for the analyses and extended from position 1495 to 1725 of S. cerevisiae chromosome III. A scaled representation of the telomere III-L was shown on the left. Arrows pointed to the main cuts detected in chromatin. Gray circles represented translationally phased nucleosomes. The upper bands represented MNase-undigested telomere containing DNA. (B) Telomere- blot of wild-type and mutant cells. The esa1-338 mutation of ESA1 was a point mutation at position 338 (Glu to Gln), which is the catalytic site of Esa1. Genomic DNA was digested with XhoI, separated by 1.0% agarose gel, transferred to a nitrocellulose membrane and hybridized with a probe for yeast telomeric DNA. Tel: terminal TG-tracts-containing DNA fragments. (C) ChIP experiment of Est2-myc in wild-type and H4K12R cells. The enrichment value represented the ratio (IP/Input) at TELXIV-R relative to ACT1. doi:10.1371/journal.pgen.1001272.g004 PLoS Genetics | www.plosgenetics.org 5 January 2011 | Volume 7 | Issue 1 | e1001272