H4K12ac Regulates Heterochromatin Plasticity Figure 2. Histone H4K12 acetylation survives heterochromatin formation. (A) ChIP analyses of H4K5 acetylation, K8 acetylation, K12 acetylation and K16 acetylation in the time course of HO induction. Shown were the real-time Q-PCR results of the ChIP products. Error bars represented standard error of the mean for three independent experiments. The relative abundance indicated the enrichment value (IP/Input) at SubTel relative to HMR. Values that were greater than 1 indicated more enrichment at the SubTel than background (HMR). (B) Whole-cell extracts from NSY429 strains expressing wild-type histone H4 (lane 1) and arginine substitution mutations of H4 Lys5 (lane 2), Lys8 (lane 3), Lys12 (lane 4) or Lys16 (lane 5) were subjected to western blot analysis with the specific antibodies indicated on the right. (C) ChIP assays were performed as in (A) except that chromatin from strains that contained either wild-type or H4 lysine substituted mutants was used. Q-PCR with 32P-ATP incorporation was done with primer pairs directed against GIT1 and GAL regions. PCR products were separated by 6% TBE gel and product abundance was detected using a PhosphorImager. doi:10.1371/journal.pgen.1001272.g002 Figure 3. Acetylation of H4K12 at native telomeric heterochromatin. Relative abundance of H4K5 acetylation, K8 acetylation, K12 acetylation, K16 acetylation, histone H4, Sir2-myc and Esa1-myc at 32 telomeres. Real-time PCR was done with primer pairs against regions ,0.5 kb from each telomere. PCR data for acetylations were normalized to an HMR region (region D in Figure S3); PCR data for Sir2-myc were normalized to ACT1; PCR data for Esa1-myc were normalized to a subtelomeric region ,4 kb from the end of chromosome III-R (Stel). The relative abundance of lysine acetylation was normalized to H4 occupancy levels. doi:10.1371/journal.pgen.1001272.g003 PLoS Genetics | www.plosgenetics.org 4 January 2011 | Volume 7 | Issue 1 | e1001272