ClHst1 causes deacetylation at the rDNA repeats.

(A) The relative abundance of acetylated histones was assessed by immunoblotting. Whole-cell lysates were prepared from replicate samples of ClHST1 yeast (LRY2544) untreated or treated with 25 mM nicotinamide (NAM) or 10 µM trichostatin A (TSA) and from Clhst1Δ yeast (LRY2671). Proteins were electrophoretically separated, transferred to membranes, and probed with antibodies against tetra-acetylated H4, H3K9Ac, or H3K56Ac. (B) The acetylation of histones H3 and H4 across the rDNA repeat was examined by chromatin IP. Total H3, tetra-acetylated H4, H4-K16Ac, H3-K9Ac, or H3-K56Ac was immunoprecipitated from ClHST1 (LRY2544) yeast. For each immunoprecipitation, the recovery of probes was normalized to the control locus, ClPRI2. (C–E) The change in acetylation in deacetylase-deficient yeast relative to wild-type untreated yeast was determined. H3-K9Ac, tetra-acetylated H4, H4-K16Ac, or H3-K56Ac was immunoprecipitated from yeast. For each probe, the enrichment was determined relative to that of total H3. This value was then compared to wild-type untreated cells. Yeast strains used were (C) Clhst1Δ (LRY2671), (D) ClHST1 (LRY2544) treated with 25 mM nicotinamide (NAM), or (E) ClHST1 (LRY2544) treated with 10 µM trichostatin A (TSA).