Figure 7. Histone H4K12 acetylation regulates basal transcrip- tion at telomeric heterochromatin. URA3 gene was artificially inserted into a locus that is proximal to the telomeric TG-tracts of chromosome XIV-R. The mRNA of URA3 in wild-type and mutant strains were measured by real-time PCR and normalized to ACT1. The relative mRNA level of URA3/ACT1 in wild-type strain was set as 1. Therefore, values above ‘‘1’’ indicated higher URA3 expression. doi:10.1371/journal.pgen.1001272.g007 mechanism for the recruitment of NuA4 during DNA damage repair [44]. However, due to the fact that normal telomeres are protected from being recognized as double-strand DNA break and phosphorylated H2A is absent from normal telomeres, we propose that yeast cells take a distinct strategy such as a Rap1-dependent mechanism to recruit NuA4 onto telomeric heterochromatin [45]. Esa1 preferentially acetylated nucleosome on both H4K5 and K12 in vivo [46] and in vitro (Figure S4C). This raised a question of why H4K5 was hypoacetylated at telomeric heterochromatin. We suspected that another histone deacetylase besides Sir2 was involved in the establishment of the acetylation pattern at telomeric heterochromatin. Two candidates are Rpd3 and Hda1, which displayed the highest in vivo activity toward acetylated H4K5 from among H4K5, K8, K12 and K16 [47]. Indeed, our recent study did reveal a genetic interaction between Rpd3L and H4K5 at subtelomeric regions [48]. Moreover, analysis of the CIDMS/MS spectra shows K12 to be the most highly acetylated site (54%) from among H4K5, K8 and K12, followed by K5 (32%), and K8 (24%) [49], suggesting that K12 acetylation covers a much wider range of yeast chromatin than that of K5 or K8 acetylation. NuA4 complex also functions to prevent the Sir complex from spreading out of heterochromatic domains [19,50–52]. Therefore, mutation of ESA1 or other key subunits of the NuA4 complex resulted in gene silencing near heterochromatin [19,50,51] and a modest reduction of silencing within telomeric heterochromatin [53,54]. The histone H4K12 acetylation per se has little effect on heterochromatin boundary activity (Figure 5B and 5C), however, since it is the uniquely acetylated site within heterochromatin, abolishment of histone H4K12 caused a increase of heterochro- matin silencing (Figure 7 and [36]). Chromatin modifications have been implicated in telomere elongation in several organisms [55,56]. Recent study on H4K16 demonstrated the relationship between histone acetylation and telomere regulation [57]. The natural presence of H4K12 acetylation at telomeres and Sir-dependent regulation of telomere replication via H4K12 have provided additional direct evidence supporting the proposal that chromatin modifications affect telomere homeostasis. Elimination of H4K12 acetylation increased H4K12ac Regulates Heterochromatin Plasticity subtelomeric binding of Sir proteins (Figure 5B and 5C), accelerated senescence in est2D cells (Figure 6B) and suppressed homologous recombination within TG-tracts (Figure 6A). By contrast, inactivation of Sas2, the acetyltransferase of H4K16 [38], decreased Sir3 binding at telomere ends and thereby delayed senescence in tlc1D cells through homologous recombination- dependent mechanism [57]. Therefore, H4K12 acetylation and K16 acetylation seem to play opposite roles in the Sir-dependent regulation of homologous recombination at telomeric heterochro- matin. It is quite interesting that H4K12 and K16 are in close proximity to each other but have opposing roles in regulating telomere dynamics through Sir-dependent mechanisms. Finally, a recent paper showed that Esa1 and Rpd3L controlled H4K12 acetylation, which is necessary for cell growth and viability [46]. Although H4K12R does not change the genome-wide transcrip- tion profile [34], it is still possible that H4K12 also fine-tunes the chromatin structure at sequences other than the telomeric heterochromatin. In conclusion, heterochromatin is known as a highly condensed chromatin domain that is transcriptionally silent [1]. However, pioneering studies have recently revealed the dynamic aspect of yeast heterochromatin [11–13,15]. In this study, we have built on these pioneering studies and shown that the H4K12R mutation led to a more condensed telomeric heterochromatin structure (Figure 5) and more static telomere metabolism (Figure 4, Figure 5, Figure 6, Figure 7). Therefore, we propose that H4K12 provides a mechanism for yeast cells to maintain partial plasticity of their telomeric heterochromatin (Figure 8). Interestingly, histone H4K12 acetylation has also been observed at the chromocenter in fly [58]. Mst1, the orthologue of Esa1 in fission yeast, acetylates H3K4 at pericentric heterochromatin to regulate heterochromatin reassembly [59]. TIP60, the orthologue of NuA4 in mammals, physically interacts with Sirt1, the mammalian homologue of Sir2 [60], and associates with tri-methylated H3K9, a hallmark of heterochromatin [61]. Hence, it will be of great interest to determine if histone acetylation also plays a general role in heterochromatin dynamics in other eukaryotes. Materials and Methods Yeast strains and reagents Antibodies, yeast strains and primers used in this study are listed in Tables S1, S2, S3, respectively. Chromatin immuno-precipitation ChIP assays were performed as described [18,19]. Most ChIP products were directly analyzed by real-time Q-PCR using SYBR green as a label (TOYOBO). Alternatively, pellet and whole-cell extract DNAs were analyzed by Q-PCR performed in a linear range with 32P-dATP, electrophoresis through 6% PAGE in Tris- Borate-EDTA buffer, and phosphorimager quantification of radioactive bands in dried gels. The relative enrichment value represented the ratio (IPs/Input) at indicated loci relative to internal control. All ChIP experiments were performed in triplicate on paired isogenic wild-type and mutant strains. HPLC analysis of DNA methylation status After protein expression was induced by galactose for indicated time, total DNA was isolated from yeast cells by glass beads lysis, proteinase K digestion and extraction with QIAGEN Genomic DNA Kit, followed by RNaseA and RNaseT digestion, phenol/ chloroform extraction and re-precipitation. Finally, purified genomic DNA was digested into mononucleoside 59-monophos- phates with Nuclease P1 (Sigma). Nucleosides were separated by an PLoS Genetics | www.plosgenetics.org 8 January 2011 | Volume 7 | Issue 1 | e1001272