The demethylase activity of Jhd2 is required for the regulation of rDNA silencing. (A) Schematic diagram of the ADE2 reporter integrated at TEL05R. (B) ADE2-based telomeric silencing assays were performed for the indicated strains. ADE2-TEL05R strains contain an ADE2 reporter as shown in (A). (C) Schematic representation of the URA3 reporter integrated at TEL07L. The bars and numbers below the subtelomeric regions indicate the relative positions of the ChIP PCR products in (E) and the distances from telomeric DNA sequences (TG1–3). (D) URA3-based telomeric silencing assays for the indicated strains. (E) ChIP analyses were performed using antibodies against Sir2 or Rap1 for the indicated strains with a URA3 reporter. Cells grown to an A600 of 0.1 in synthetic complete (SC) medium were transferred to SC medium containing 0.1% 5-fluoroorotic acid (FOA). Error bars indicate the standard deviation (SD) calculated from PCRs performed using three independent chromatin preparations. Asterisks indicate statistically significant differences determined by pairwise comparisons between wild type (WT) and each of the indicated mutants using a two-tailed Student’s t test (*, P <0.05; **, P <0.01). (F) Schematic of the URA3 reporter integrated at the NTS1 region of rDNA. (G, I) URA3-based rDNA silencing assays for the indicated strains. For the silencing assay in (I), strains containing empty vector (pRS424), constructs expressing hemagglutinin (HA)-tagged WT Jhd2, or the H427A mutant were grown in SC medium lacking Trp and then serially diluted and spotted onto SC-Trp or SC-Trp medium containing 0.1% FOA. (H) Strains used in (I) were grown in SC medium lacking Trp and histone H3K4me3 levels were analyzed using immunoblotting assays with an anti-H3K4me3 antibody. As controls, anti-HA and anti-H3 antibodies were used to determine the protein levels of Jhd2 and histone H3, respectively. bp, base pair; ChIP, chromatin immunoprecipitation; Chr, chromosome; FOA, 5-fluoroorotic acid; IP, immunoprecipitation; kb, kilobase; SC, synthetic complete; WT, wild type.