[31–33] and such structure plays a role in telomere length homeostasis [28]. It was possible that H4K12R mutation affected the access of telomerase machinery to telomere end and thereby impaired telomere replication. To test this idea, we performed a ChIP experiment to compare the telomere binding of Est2, the catalytic subunit of telomerase, in wild-type and H4K12R cells. Interestingly, the telomere association of Est2 was greatly reduced by H4K12R mutation (Figure 4C), supporting our idea that H4K12 influences the recruitment of telomerase to telomeres. Histone H4K12 acetylation suppresses the aberrant accumulation of Sir proteins at telomeric heterochromatin The formation of heterochromatin in yeast is dependent on Sir proteins [1]. To determine if H4K12 acetylation directly modulated the binding of Sir proteins, we carried out ChIP to compare the chromatin binding of Sir2 and Sir3 in wild-type and histone mutant cells at regions 0.7 kb,12 kb from the end of chromosome XIV-R (TELXIV-R) (Figure 5A–5C). We found that Sir proteins in wild-type cells were present only in telomeric heterochromatin as far as 3 kb from the end of the chromosome. In H4K16R mutant cells, the abundance of Sir proteins was reduced within heterochromatin but was greatly increased at heterochromatin adjacent regions (Figure 5B and 5C), a result that is representative of a typical heterochromatin over-spreading phenotype [6,7]. Notably, compared to a H4K5R or H4K8R mutation that did not alter the binding of Sir proteins at TELXIV- R telomere (Figure 5B and 5C), the H4K12R mutation resulted in 2.52-fold more Sir2 binding and 3.09-fold more Sir3 binding 0.7 kb from the telomere end, but not at regions farther from the chromosome end. We also examined Sir2 binding at all other telomeres and found that the H4K12R mutation resulted in enhancement of Sir2 binding at most telomeres, especially at those where Sir2 was already abundant (Figure S6A). Because the H4K12R mutation does not perturb the transcriptional profile of H4K12ac Regulates Heterochromatin Plasticity the genome [34], and the expression and nuclear distribution of Sir proteins were not affected by the H4K12R mutation (Figure S5 and Figure S7), we therefore favor a model where the acetylation status of H4K12 directly affects Sir protein binding. We also analyzed the heterochromatin structure in H4K12Q mutant. Presumably, a lysine to glutamine mimicked a hyperacetylated state of the corresponding lysine. However, we found that the abundance and distribution of Sir2 and Sir3 in H4K12Q cells were largely similar to that in H4K12R cells (Figure S6B and S6C). We proposed that glutamine does not accurately represent the hyperacetylated state of H4K12 in this case. Together, these data suggest that the H4K12 acetylation suppresses the over- congregation of Sir proteins at telomeric heterochromatin. To investigate if the regulation of telomere replication by H4K12 acetylation is dependent on the telomeric heterochromatin structure, we detected the effect of SIR2 deletion on the telomere length in H4K12R mutant cells. As shown in Figure 5D, sir2D cells exhibited a modest reduction of telomere length, which is much longer than that observed in H4K12R mutant cells. Interestingly, deletion of SIR2 efficiently rescued the severe telomere-shortening phenotype in H4K12R mutant cells (Figure 5D), indicating that H4K12 acetylation regulates telomere length though the Sir2 pathway. In addition, telomere length in H4K5R, K8R or K16R cells was indistinguishable from that in wild-type cells (Figure 5D), consistent with their already defined effect on the telomeric association of Sir proteins. Taken together, these data strongly supported the notion that H4K12 acetylation regulates telomere replication directly via modulating telomeric heterochromatin structure. Histone H4K12 acetylation regulates telomere recombination Telomeres are hotspots for recombination when they are deprotected. Telomerase-negative yeast cells could undergo homologous recombination on Y’ or TG1–3 telomeric sequences, Figure 5. Regulation of telomere structure by histone H4K12 acetylation. (A) Schematic diagram of the subtelomeric region of the right arm of chromosome XIV. Distance from each primer set to chromosome end was shown on the top. (B) and (C) ChIP analysis of Sir2-myc (B) and Sir3-myc (C) in wild-type and histone H4 mutant cells. The Q-PCR data were normalized to ACT1. (D) Telomere-blot of wild-type and histone mutant cells. doi:10.1371/journal.pgen.1001272.g005 PLoS Genetics | www.plosgenetics.org 6 January 2011 | Volume 7 | Issue 1 | e1001272