thus generating Type I or Type II post-senescence survivors, respectively [35]. Since Type II survivors grew much faster than Type I, liquid culturing of post-senescent est2D cells yielded primarily Type II survivor cells [35] (Figure 6A). Interestingly, deletion of the catalytic subunit of telomerase EST2 in the H4K12R mutant background eventually led to the generation of a population of cells that contained amplified Y’ telomeric sequence (Figure 6A), a hallmark of Type I survivor cells. Therefore, we conclude that H4K12 acetylation suppresses homologous recom- bination of TG1–3 tracts during the creation of telomerase-null post-senescent survivors. Since histone H4K12 acetylation affected both telomere length and recombination, we wondered whether it regulated the senescence rate of telomerase inactive cells. Therefore, we compared the growth potential of est2Dand H4K12R est2D cells. The result showed that H4K12R mutation greatly accelerated the senescence rate of est2D cells (Figure 6B). Further deletion of SIR2 could suppress the accelerated senescence rate of H4K12R est2D cells (Figure 6B). These data suggested that H4K12 acetylation delays senescence driven by Sir-dependent telomere dysfunction. Histone H4K12 acetylation facilitates basal transcription at telomeric heterochromatin Previous work had shown that histone H4 mutations that led to increased telomere position effect (TPE) were usually associated with a dramatic decrease of K12 acetylation [36]. To further address this point, we carried out TPE assay [37] to evaluate the effects of histone mutations on the transcriptional state at heterochromatin. A URA3 gene was inserted into a locus that was proximal to the right telomeric TG-tracts of chromosome XIV. Strains were then tested for the relative URA3 expression in histone mutant strains compared with wild-type level. In agreement with a previous report [38], H4K16R mutation markedly reduced telomere silencing (Figure 7). Notably, H4K12R, but not K5R or K8R cells, had less URA3 expression than wild-type cells (Figure 7), suggesting that H4K12R mutation enhances telomere silencing. H4K12R mutation in sir2D back- ground had similar URA3 expression to that in sir2Dcells (Figure 7), indicating that the increased silencing by H4K12R mutation is H4K12ac Regulates Heterochromatin Plasticity Sir-dependent. Therefore, we concluded that H4K12 acety- lation contributes to the basal transcription within telomeric heterochromatin. Discussion In this study, we have employed the de novo telomere addition assay [16] as a de novo telomeric heterochromatin formation system, to monitor chromatin dynamics occurred on a newly- formed telomere (Figure 1). Compared with the traditional Sir3- induction system [39–41], this system works in a more physiolog- ical condition, with normal Sir proteins level and much less transcriptional changes across the genome. Hence, the kinetics of multiple events in the course of telomeric heterochromatin formation can be more accurately followed. The experimental observations we have made in this study establish histone H4K12 acetylation as an important component of yeast telomeric heterochromatin. We have provided phenotyp- ic, genetic and mechanistic evidence to support the presence of H4K12 acetylation inside telomeric heterochromatin. By using stringently controlled ChIP analyses, we detected H4K12 acetyla- tion at most telomeres (Figure 3). Compared with euchromatic H4K12 acetylation, the level of heterochromatic H4K12 acety- lation was relatively low but was greatly elevated by SIR2 deletion (Figure S3), a phenomenon also observed in earlier reports [20,42]. Therefore, H4K12 acetylation, as is the case of acetyla- tion of other H4 lysines, is suppressed by heterochromatin structure. Genetically, the H4K12R mutation, which mimicked an unacetylated state of K12, increased Sir protein binding at telomeric heterochromatin and altered several dynamic telomere- related chromosomal processes (Figure 4, Figure 5, Figure 6, Figure 7). Heterochromatic H4K12 acetylation coincides with H4K12 as a memory mark for the heritable chromatin structure in yeast [36]. Mechanistically, Esa1, the catalytic subunit of NuA4 HAT, bound to silenced telomeres and was responsible for H4K12 acetylation (Figure 3). Since Arp4, another subunit of NuA4 complex, is also enriched at heterochromatic domains [43], it is possible that the whole NuA4 complex binds to telomeric heterochromatin and plays a direct role in the acetylation of H4K12. Earlier work suggested a phosphorylated H2A-dependent Figure 6. Histone H4K12 acetylation regulates telomere recombination. (A) Telomere-blot of est2D and est2D H4K12R cells. Cells were continuously passaged for the indicated number of days and the genomic DNA was subjected to telomere-blot. Y’: Y’ elements. (B) Senescence rates were measured in liquid culture by serially passaging strains of the indicated genotypes. doi:10.1371/journal.pgen.1001272.g006 PLoS Genetics | www.plosgenetics.org 7 January 2011 | Volume 7 | Issue 1 | e1001272