A Novel Form of Transcriptional Silencing by Sum1-1 Requires Hst1 and the Origin Recognition Complex

Hst1 is an NAD⁺-dependent protein deacetylase. (A) NAD⁺-nicotinamide exchange reactions with purified Hst2 (∼200 ng) or 5 μl of total extracts prepared following induction of BL21(DE3) transformed with either pAGN24 (Hst1) or pET28c (vector). Reaction mixtures also contained acetylated BSA (0.2 mg/ml), H4 peptide acetylated at K16 (5 nM), or no acetylated substrate (none). The arrow points to the position of the NAD⁺ that has become radioactive during the exchange reaction. (B) npt1 mutants are defective inSUM1-1 silencing. Strains YRH34 and YRH35 were tested for mating by spotting 10-fold serial dilutions of exponentially growing cultures onto an SD plate containing a lawn ofMAT a his1 (AY1283) cells. The same strains were spotted onto a synthetic complete (SC) plate to show number of cells tested. The npt1 strain grows somewhat more slowly than the NPT1 strain does. (C) The left panel shows that the zinc finger mutant of Hst1 abolishes SUM1-1silencing. Strain JLY04 (MATα SUM1-1Δsir2 Δhst1) was transformed with either pJC10a (hst1-10), pJC7a (HST1), or pRS424 (vector). Transformed strains were tested for mating as described in the legend to Fig. 1. The right panel shows that the zinc finger mutant of Hst1 is defective in MSE-dependent repression. Patches of strain JXY5, transformed with an SPR3-lacZ reporter plasmid (pGK16) and the plasmids described in the left panel, were grown for 2 days on SC −Ura −Trp medium and then tested for β-galactosidase activity using a filter assay.

Sum1-1, but not Sum1, interacts with Orc5 in the two-hybrid assay. (A) Filter assay for lacZ expression of strain L40 transformed with a plasmid containing lexA-ORC5(pTT93) and plasmids containing GAD (pGAD424),GAD-SUM1-1 (pRH02), or GAD-SUM1 (pRH01). (B) Western analysis of extracts from strains used in panel A probed with anti-GAD antibody. The significance of the faint upper band in panel B, which is seen only with GAD-Sum1 and GAD-Sum1-1 fusion proteins, is unknown. β-gal, β-galactosidase.

Sum1-1 coimmunoprecipitates with Orc3. (A) IP analysis with Orc3. Strains YRH15 (WT), YRH20 (SUM1-myc), and YRH21 (SUM1-1-myc) were transformed with plasmid pRH34 (HMR in pRS426). Extracts were prepared and proteins were immunoprecipitated using anti-myc antibodies. Following SDS-polyacrylamide gel electrophoresis, the precipitates were analyzed by immunoblotting and probed with an anti-Orc3 antibody. A portion of the wild-type extract from YRH15 (with plasmid pRH34) was also run on the same gel to show the position of Orc3 as well as a slower-migrating protein that interacts with the Orc3 antibody (20). HC, heavy chain of IgG. (B) A portion (5%) of the precipitated material used in panel A was analyzed by immunoblotting with anti-myc antibodies. The Sum1-myc and Sum1-1–myc proteins always show multiple bands following immunoprecipitation, presumably due to proteolysis. The arrow points to the position of the full-length proteins. (C) Sum1-1–myc functions in vivo. Sum1-1–myc silencing in Δsir2 strains was measured by a patch-mating assay with WT (W303-1b), Δsir2 (YRH15), Δsir2 SUM1-1(YRH34), or Δsir2 SUM1-1-myc (YRH36) strains. (D) Sum1-myc functions in vivo. Strains were transformed with plasmid pJX43 (SMK1 MSE-lacZ) and grown to early log phase in SC −Ura medium. β-Galactosidase activity was determined for three independent transformants for each strain. WT (YRH15),SUM1-myc (YRH20), Δsum1 (JXY3),SUM1-1 (YRH34), and SUM1-1-myc (YRH36) strains were used.

HMR-I is required for Sum1-1 silencing. (A) Strains YRH15 (Δsir2), RS1056 (Δsir2 SUM1-1), and YRH07 (Δsir2 SUM1-1 Δhmr-I) were tested for mating by plating 10-fold serial dilutions of saturated cultures onto an SD plate containing a lawn of MAT a his1 (AY1283) cells. (B) Strain YRH07 was transformed with either vector (YEp351) or SIR2 (pLP0349) and tested for mating as in Fig. 1.