H4K12ac Regulates Heterochromatin Plasticity Figure 8. Model for the regulation of telomeric heterochromatin plasticity by histone H4K12 acetylation. In wild-type cells where subtelomeric histone H4K12 is partially acetylated, heterochromatin structure is plastic and accessible to proteins, such as the RNA polymerase machinery and the telomerase machinery. In H4K12 deacetylated strain, heterochromatin becomes aberrantly condensed and less accessible to proteins, thereby inhibiting chromosomal processes. doi:10.1371/journal.pgen.1001272.g008 AQ-C18 column (5 mm, 4.66250 mm, Welch Materials Inc.) guarded by a precolumn (Phenomenex, Security Guard), using a Beckman device (SYSTEM GOLD 125 Solvent Module and SYSTEM GOLD 166 Detector). The eluate was obtained using a flow rate of 1 ml/min and 100% buffer A (10 mM KH2PO4/ H3PO4, pH 3.7) for 50 minutes, followed by a shift to 70% A/ 30% methanol for 10 minutes, and then to 100% buffer A for 10 minutes. Peaks were quantified by measuring their heights with a 32 Karat software V7.0 after identity confirmation by comparison of spikes with different combinations of dCMP and 59-me-dCMP (Hongene Biotechnology Ltd. Shanghai). At least three indepen- dent yeast clones were assayed for each construct. Southern blot Southern blot was performed as described [62]. Yeast genomic DNAs were digested with restriction enzymes as indicated, separated by 1.2% agarose gel electrophoresis, and transferred to Hybond-N membrane (Amersham). The blot was hybridized with a 32P-dCTP incorporated probe as indicated. The radioactive signal was detected by phosphorimager. Micrococcal nuclease sensitivity assay Micrococcal nuclease (MNase) sensitivity assay was performed as described [27]. Briefly, Cells from 50-ml cultures were collected by centrifugation, treated with zymolyase, and digested with micrococcal nuclease (MNase). genomic DNA samples were purified, digested with BamHI, resolved in 1.2% agarose gels. Then the cutting profiles were visualized after hybridization with an internal probe from Ty5-1. Immunofluorescence of yeast cells Cells were grown in YPD medium overnight to a density of ,1– 26107 cells/ml and were fixed for 30 min by incubation with 3.7% formaldehyde. Next, cells were washed with 0.1 M potassium phosphate (pH 6.5) and P solution (1.2 M sorbitol, 1 M K2PO4), and re-suspended in P solution. Cells were subsequently treated with 0.1 mg/ml Zymolyase (20T, MP Biomedicals) for 10 min, washed with P solution, spotted on Poly-L-Lysine pre-treated slides. After rinsing in PBS-T buffer (PBS containing 0.1% Triton X-100 and 1% BSA), slides were incubated overnight with anti-Myc, anti-Rap1 and anti-Nop1 antibody diluted in PBS containing 1% BSA. Slides were then washed with PBS-T and incubated with the appropriate secondary antibodies conjugated to Cy3 or fluorescein isothiocyanate (FITC). The DNA fluorescence signal was detected by DAPI (1 mg/ml in Phosphate Buffered Saline (PBS) solution) staining. Slides were mounted with PBS containing 1 mg/ml p-phenylenediamine, 2.5 mM NaOH, and 90% glycerol. Confocal microscopy was performed on a Leica TCS SP2 microscope with a 636 lamda blue objective (oil). Image processing including similar filtration and threshold levels was standardized for all images. Purification of Esa1 protein N terminal 6xHis tagged Esa1 protein was overexpressed and purified in E. coli according to the manufacturer’s instructions (GE Healthcare). Supporting Information Figure S1 Specificity of the anti-acetyl-H4K12 antibody. ChIP assays were performed on chromatin from wild-type and H4K12R mutant strains. IPs were done with increasing amount of anti-acetyl-H4K12 antibody: 1, 1:200; 2, 1:200; 3, 1:50; 4, 1:25. Found at: doi:10.1371/journal.pgen.1001272.s001 (0.12 MB PDF) PLoS Genetics | www.plosgenetics.org 9 January 2011 | Volume 7 | Issue 1 | e1001272