H4K12ac Regulates Heterochromatin Plasticity Figure 1. De novo telomeric heterochromatin assembly on a short telomere. (A) Schematic representation of the de novo telomeric heterochromatin formation system [16]. The ADH4 locus was replaced in a haploid yeast strain with a 6-kb fragment consisting of the ADE2 selection marker, 81-bp telomeric DNA sequence and the recognition site for the HO endonuclease. The LYS2 gene was placed approximately 10 kb from the natural telomere VII-L. HO was induced by the addition of galactose to the media. ADE2 was a gene adjacent to the telomere seed. The black line indicated the primer set (SubTel) used for ChIP analysis. (B) De novo telomere addition analysis [16]. After HO induction, yeast cells were harvested at time points as indicated. Genomic DNA was isolated, digested with BstXI and subjected to Southern-blot analysis. The blots were hybrid with a telomere seed-proximal probe. (C–F) ChIP analyses of Rap1 (C), Sir2-myc (D), Sir3-myc (E) and Sir4-myc (F) in the time course of HO induction. Shown were the real-time Q-PCR results of the ChIP products. X axis indicated the distance from the 81-bp telomere seed. Y axis indicated the timed intervals after HO induction. Z axis indicated the enrichment values (IP/Input) at indicated regions relative to ACT1. Values that were greater than 1 indicated more enrichment at indicated regions than background. (G) mRNA analysis of ADE2 upon HO induction. The mRNA level of ADE2 was normalized by ACT1. The relative mRNA before galactose addition was set as 1. doi:10.1371/journal.pgen.1001272.g001 PLoS Genetics | www.plosgenetics.org 3 January 2011 | Volume 7 | Issue 1 | e1001272