Changes in RENT component recruitment at NTS regions due to loss of H3 methylases or demethylases. (A) Schematic diagram of an rDNA unit embedded within a tandem array on chromosome XII. The 35S pre-rRNA encoding the 18S, 5.8S and 25S rRNAs is separated by NTS1 and NTS2. The locations of RFB (double triangle), replication origin ARS (oval), 5S rRNA gene (triangle), and 35S transcription start site (bent arrow) are shown. The bars and numbers below the NTS regions indicate the positions of the ChIP PCR products in (B) and (E) and those used in all subsequent ChIP experiments. (B) The association of Net1 and Sir2 with rDNA regions was analyzed by ChIP using immunoglobulin G (IgG)-Sepharose or a Sir2 antibody in WT or the indicated deletion strains carrying TAP-tagged NET1. The upper bands in each pane indicate PCR products amplified by the primer sets shown in (A) and the lower bands marked by the asterisks are internal controls amplified from untranscribed regions on chromosome V. The bottom panels show PCR products from the input DNA. (C) Quantitation of the ChIP results in (B). Error bars indicate the SD from three PCRs performed using two independent chromatin preparations, and asterisks indicate statistically significant differences compared with WT (*, P <0.05; **, P <0.01). (D) URA3-based silencing assays at the rDNA region were performed in WT and H3K4A, H3K36A and H3K79A mutant strains. (E) The association of Net1 and Sir2 with the rDNA regions was analyzed in WT and H3K4A, H3K36A and H3K79A mutant cells as shown in (B). Each quantified result is shown on the right. Error bars show the SD from three PCRs with two independent chromatin preparations, and asterisks indicate statistically significant differences compared with WT (*, P <0.05; **, P <0.01). Chr, chromosome; FOA, 5-fluoroorotic acid; IgG, immunoglobulin G; IP, immunoprecipitation; WT, wild type; ARS, autonomously replicating sequence.