Author Summary The genetic material in eukaryotes is packaged into chromatin. The chromatin structure is orchestrated such that euchromatic regions are relatively uncondensed and accessible to factors that bind DNA, whereas heterochro- matic regions are densely packaged into higher-order conformations. The compact nature for heterochromatin may endanger normal DNA metabolism, such as DNA replication and recombination. We found that targeted histone acetylation provided a way for cells to maintain a relatively plastic heterochromatin structure that is neces- sary for DNA metabolisms within telomeric heterochroma- tin. Therefore, although heterochromatic domains are largely silenced, they are not as static as we previously assumed, and the dynamic aspect of heterochromatin is directly attributable to changes in its own chemical properties. was not affected. Technically, we used a galactose-inducible HO endonuclease to cut a chromosome to expose a pre-inserted 81-bp telomeric ‘‘seed’’ for further telomere addition [16] (Figure 1A). Chromosome ‘‘healing’’ was hardly observed in the first 6 hours upon HO induction. Later, the telomere seed was gradually extended in a telomerase dependent manner ([16] and Figure 1B). The recruitment of Rap1 and Sir proteins onto regions 100-bp to 5.0-kb from the telomere seed upon HO induction was then monitored by chromatin immunoprecipitation (ChIP). High occupancy of Rap1 was observed at the 81-bp telomere seed prior to HO induction (Figure 1C), indicating that the 81-bp telomeric DNA tract is sufficient for Rap1 recruitment. Upon HO induction, the Rap1 occupancy at the newly formed telomere was barely altered in the first 6 hours and then gradually increased along with telomere elongation (Figure 1B and 1C). Notably, upon HO induction, Sir proteins were recruited to the new telomere within the first 6 hours in an extremely efficient manner (Figure 1D–1F). Once occupying a newly formed telomere, Sir proteins began to spread into the lateral regions up to 3 kb proximal to the newly formed telomeres (Figure 1D–1F). Consistently, the expression of ADE2, a gene near the telomere seed, was gradually reduced upon HO induction (Figure 1G). Therefore, telomeric heterochromatin was quickly assembled at the 81-bp telomeric seed after HO induction. Histone H4K5 and K12 acetylation partially survived the formation of telomeric heterochromatin Since histone H4 hypoacetylation was required for the onset of telomere silencing [1,4,5] and K16 seemed to be the unique deacetylation target for Sir2 on H4 [3], we were curious about the deacetylation kinetics of different lysines on H4 (K5, K8, K12 and K16) during heterochromatin formation. To avoid recombination- induced histone acetylation, we deleted the RAD52 gene in our strains [17]. The result showed that, prior to HO cleavage, all lysine acetylation was robust at the subtelomeric region (indicated as SubTel in Figure 1) (Figure 2A). Upon HO induction, H4K8 and K16 acetylation levels decreased most substantially and were largely eliminated by 6 hours (Figure 2A), a time point when Sir proteins saturated SubTel (Figure 1D–1F). In contrast, H4K5 and K12 acetylation, especially K12 acetylation, decayed more slowly at SubTel during HO induction, and were still detectable after 24- hour HO induction (Figure 2A). These observations raise a possibility that H4K5 and K12 acetylation may partially survive the formation of new telomeric heterochromatin. H4K12ac Regulates Heterochromatin Plasticity To exclude the possibility that anti-acetyl-lysine antibodies cross-reacted with other subtelomeric factors, we compared the levels of H4 lysine acetylation in a wild-type strain to that of ‘‘unacetylated strains’’ where the corresponding lysines were mutated to arginines, respectively, thereby preventing anti-acetyl lysine antibodies from recognizing corresponding residues. In the western-blot experiment, we found that chromatin derived from strains with the lysine to arginine mutation was not detected by the corresponding anti-acetyl lysine antibodies (Figure 2B). In the ChIP experiment, we found that in the wild-type strain, all antibodies enriched GIT1, a typical hyperacetylated region [18,19] (Figure 2C). In contrast, lysine to arginine mutations eliminated corresponding anti-acetyl lysine antibodies from enriching any chromatin fragments (Figure 2C). Additionally, increasing the amount of anti-acetyl-H4K12 antibody in the immunoprecipita- tion (IP) procedure couldn’t recover chromatin in the H4K12R strain (Figure S1). These data are consistent with previous report [20], and indicate that all anti-acetyl-H4 lysine antibodies perform appropriately in our ChIP experiments. Histone H4K12 is modestly acetylated at telomeric heterochromatin To determine if the acetylation of H4K5 and K12 was generally associated with native telomeres that were not undergoing a double-strand break response [21], we performed ChIP to examine the acetylation profile of H4 lysines at all yeast telomeres. Sir2 binding was firstly mapped (Figure 3, pink bars) so as to mark the silencing status of the individual telomeres. We found that the abundance of Sir2 at different telomeres varied (Figure 3). Low amounts of Sir2 were mostly observed at telomeres that contain Y’ elements (http://www.yeastgenome.org/ and Figure 3), support- ing an anti-silencing role for these Y’ elements [22]. The acetylation levels at subtelomeric regions were normalized to an HMR region, where we detected lowest level of acetylation (Figure S3, region D). Figure 3 showed that H4K12 acetylation was observed at nearly all telomeres in a manner that was independent of Sir2 binding. In contrast, the relative amount of H4K5, K8 or K16 acetylation at telomeres seemed to correlate in an opposing manner to the level of Sir2 binding (Figure 3). For example, at the end of left arm of chromosome II where there was little Sir2 binding and all lysine acetylations were robust. In contrast, at the end of right arm of chromosome II where there was abundant Sir2, only H4K12 acetylation was detectable (Figure 3). Essentially identical results were also obtained when yeast were grown in galactose culture (Figure S2). Furthermore, H4K12 acetylation was also detected at some regions of yeast HM heterochromatin (Figure S3). Taken together, these data suggest that H4K12 acetylation exists within yeast heterochromatin. Esa1 is responsible for H4K12 acetylation at telomeric heterochromatin We next set out to ascertain how the subtelomeric H4K12 acetylation pattern was established. Genome-wide nucleosome H4K5, K8 and K12 acetylation in budding yeast is catalyzed by Piccolo NuA4 in a non-targeted manner [23]. We found that the subtelomeric H4K12 acetylation was sensitive to esa1-338 mutation (Figure 3, white bars). Since Esa1 is the catalytic subunit of NuA4 complex, these results support the idea that NuA4 is responsible for heterochromatic H4K12 acetylation. Interestingly, ChIP analysis revealed that Esa1 was specifically enriched at Sir2- abundant subtelomeres (Figure 3, red bars). Thus, heterochro- matic H4K12 was acetylated by NuA4 in a targeted manner while the euchromatic H4K12, as the case of other H4 lysines, was PLoS Genetics | www.plosgenetics.org 2 January 2011 | Volume 7 | Issue 1 | e1001272