A novel form of transcriptional silencing by Sum1-1 requires Hst1 and the origin recognition complex - PubMed (original) (raw)

A novel form of transcriptional silencing by Sum1-1 requires Hst1 and the origin recognition complex

A Sutton et al. Mol Cell Biol. 2001 May.

Abstract

In the yeast Saccharomyces cerevisiae, a and alpha mating-type information is stored in transcriptionally silenced cassettes called HML and HMR. Silencing of these loci, maintained by the formation of a specialized type of heterochromatin, requires trans-acting proteins and cis-acting elements. Proteins required for silencing include the Sir2 NAD(+)-dependent deacetylase, Sir3, and Sir4. Factors that bind to the cis elements at HMR and HML and that are important for silencing include the origin recognition complex (ORC). Mutations of any of these Sir proteins or combinations of cis elements result in loss of silencing. SUM1-1 was previously identified as a dominant mutation that restores silencing to HMR in the absence of either the Sir proteins or some of the cis elements. We have investigated the novel mechanism whereby Sum1-1 causes Sir-independent silencing at HMR and present the following findings: Sum1-1 requires the Sir2 homolog, Hst1, for silencing and most probably requires the NAD(+)-dependent deacetylase activity of this protein. Sum1-1 interacts strongly with ORC, and this strong interaction is dependent on HMR DNA. Furthermore, ORC is required for Sum1-1-mediated silencing at HMR. These observations lead to a model for Sum1-1 silencing of HMR in which Sum1-1 is recruited to HMR by binding to ORC. Sum1-1, in turn, recruits Hst1. Hst1 then deacetylates histones or other chromatin-associated proteins to cause chromatin condensation and transcriptional silencing.

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Figures

FIG. 1

SUM1-1 silencing requires HST1. Patches of the indicated strains were replica plated onto an SD plate containing a lawn of MATa his1 cells (AY1283). The plate was incubated for 2 days at 30°C. WT, strain W303-1b; Δ_sir2_, strain YRH15; Δ_sir2 SUM1-1_, strain YRH34; Δ_sir2 SUM1-1_ Δ_hst1_, strain YRH38.

FIG. 2

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 in SUM1-1 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 of MATa 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 SUM1-1 silencing. Strain JLY04 (MAT_α SUM1-1 Δ_sir2 Δ_hst1_) 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.

FIG. 3

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.

FIG. 4

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 Δ_sir2_ strains was measured by a patch-mating assay with WT (W303-1b), Δ_sir2_ (YRH15), Δ_sir2 SUM1-1_ (YRH34), or Δ_sir2 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), Δ_sum1_ (JXY3), SUM1-1 (YRH34), and SUM1-1-myc (YRH36) strains were used.

FIG. 5

HMR_-I is required for Sum1-1 silencing. (A) Strains YRH15 (Δ_sir2), RS1056 (Δ_sir2 SUM1-1_), and YRH07 (Δ_sir2 SUM1-1_ Δ_hmr_-I) were tested for mating by plating 10-fold serial dilutions of saturated cultures onto an SD plate containing a lawn of MATa his1 (AY1283) cells. (B) Strain YRH07 was transformed with either vector (YEp351) or SIR2 (pLP0349) and tested for mating as in Fig. 1.

FIG. 6

Model for Sum1-1 silencing at HMR.

References

1. Bartel P L, Chien C, Sternglanz R, Fields S. Using the two-hybrid system to detect protein-protein interactions. In: Hartley D A, editor. Cellular interactions in development: a practical approach. Oxford, United Kingdom: IRL Press; 1993. pp. 153–179.
1. Brachmann C B, Sherman J M, Devine S E, Cameron E E, Pillus L, Boeke J D. The SIR2 gene family, conserved from bacteria to humans, functions in silencing, cell cycle progression, and chromosome stability. Genes Dev. 1995;9:2888–2902. -PubMed
1. Braunstein M, Rose A B, Holmes S G, Allis C D, Broach J R. Transcriptional silencing in yeast is associated with reduced nucleosome acetylation. Genes Dev. 1993;7:592–604. -PubMed
1. Chi M H, Shore D. SUM1–1, a dominant suppressor of SIR mutations in Saccharomyces cerevisiae, increases transcriptional silencing at telomeres and HM mating-type loci and decreases chromosome stability. Mol Cell Biol. 1996;16:4281–4294. -PMC -PubMed
1. Chu S, DeRisi J, Eisen M, Mulholland J, Botstein D, Brown P O, Herskowitz I. The transcriptional program of sporulation in budding yeast. Science. 1998;282:699–705. -PubMed

A novel form of transcriptional silencing by Sum1-1 requires Hst1 and the origin recognition complex - PubMed (original) (raw)