Persistence of an alternate chromatin structure at silenced loci in vitro

Abstract

In Saccharomyces cerevisiae, transcriptional repression at the HM mating-type loci and telomeres results from the formation of a heterochromatin-like structure. Silencing requires at least three Sir proteins (Sir2p-4p), which are recruited to chromatin by silencers at the HM loci and TG1-3 tracts at telomeres. Sir proteins and telomeres colocalize at the nuclear periphery, suggesting that this subnuclear position may also contribute to transcriptional repression. To evaluate the contribution of nuclear context to silencing, we developed methodology to isolate silent chromatin for analysis in vitro. Site-specific recombination was used in vivo to produce DNA rings from the silent HMR locus, and differential centrifugation was used to isolate the rings from whole-cell lysate. The partially purified rings retained many of the intracellular hallmarks of transcriptionally repressed domains. Specifically, rings from repressed strains were resistant to restriction endonuclease digestion, bore an altered DNA topology, and were associated with Sir3p. The recombination approach also was used to form rings from HMR that lacked silencers. Despite the uncoupling of these cis-acting regulatory elements, similar but nonidentical results were obtained. We conclude that an alternate chromatin structure at silent loci can persist in vitro in the absence of silencers and nuclear compartmentalization.

Figure 1

Excision cassettes for the formation of extrachromosomal DNA rings. RS sites are shown as half-filled boxes. In strain YCL2, the entire HMR locus is flanked by RS sites, including the E and I silencers (shown as ovals). In strain THC40, only the a1 and a2 genes of the locus are flanked by RS sites. The excision cassette in this strain also contains a small piece of the LYS2 gene (shaded box). THC9 differs from THC40 in that the region between the RS sites is replaced entirely by a fragment of the LYS2 gene. The same fragment can be excised from the LYS2 locus in strain THC16. Strain constructions are described in detail in ref. (25). Ba, BsaAI; Bg, BglII; Bc, BclI; Bt, BstBI; Av, AvaII.

Figure 2

DNA content in pellet P2. Chromatin rings were isolated in RI buffer containing 150 mM KCl and 10% glycerol. Samples were deproteinized and affixed to nylon membrane by dot blotting. Ring rKWD50N was excised from the HMR locus in strains THC9 (Left) and AA4 (Center) and from the LYS2 locus in strain THC16 (Right). Each strain was transformed with pSIR3-HA and a GAL1∷R recombinase expression vector, pRS415-RecR. Blots were hybridized with randomly primed probes specific for rKWD50N or the entire 2-μm plasmid.

Figure 3

Restriction endonuclease accessibility of partially purified chromatin rings. Samples were prepared from strain YCL2, which was transformed with pRS415-RecR and either pSIR3-HA or pRS416 to yield SIR3 and sir3 strains, respectively. Chromatin rings were isolated in RI buffer containing 150 mM KCl and 10% glycerol. After digestion and electrophoresis, blots were hybridized with either a probe to detect the rHMR ring (a) or a probe to detect the entire 2-μm plasmid (b). Linearized, closed-circular, and nicked species are denoted as C (cut), U (uncut), and N (nicked), respectively. Enzymes corresponding to unique sites in rHMR were used as indicated above each lane (see Fig. 1 for map and legend). In lane 1, no enzyme (No) was used. The extent of enzymatic digestion is reported as the percentage of band C relative to the sum of bands C and U in each lane. The 2-μm plasmid contains no BglII sites and one BclI site. There are multiple sites for AvaII and BstBI yet singly cut species are observed because of partial cleavage of the chromatin templates.

Figure 4

Topoisomer analysis of partially purified chromatin rings. Electrophoresis was performed in buffer containing 1.5 μg/ml chloroquine. Samples were obtained from strains described in Fig. 3. (Lanes 1 and 2) DNA isolated directly from intact cells. (Lanes 3 and 4) DNA isolated via P2 pellets that were obtained in RI buffer containing 150 mM KCl and 10% glycerol. Topoisomer bands were quantified by phosphorimaging (Bio-Rad), and the centers of the topoisomer distributions (marked with filled arrows) were determined by the Gaussian method (44).

Figure 5

Analysis of rings lacking silencers. Samples were prepared from strain THC40, which was transformed with pRS415-RecR and either pSIR3-HA or pRS416. P2 pellets were obtained in RI buffer containing either 100 mM KCl and 20% glycerol (a) or 150 mM KCl and 10% glycerol (b). Enzymes corresponding to unique sites in rA1A2 were used (see Fig. 1 for map and legend). (c) Topoisomer distributions of uncut samples were determined by using electrophoresis in buffer containing 2.0 μg/ml chloroquine.

Figure 6

Association of Sir3p with rings lacking silencers. Samples were obtained from strains described in Fig. 2. An anti-HA antibody was used to detect epitope-tagged Sir3p. (a) Resupended P2 preparations were subjected to Western blot analysis. Mobility of size markers is shown at left, in kDa. Approximately equal amounts of protein were loaded in each lane, as judged by comparing the levels of a 71-kDa crosshybridizing species. wce, whole-cell extract of strain in lane 1. (b) Resuspended P2 preparations were subjected to immunoprecipitation by anti-HA antibody and PCR analysis of precipitates. Strain THC9 transformed with pSIR3-HA was used for panels 1 and 3. Strain THC16 transformed with pSIR3-HA was used for panel 2. PCR primers were specific for the circular form of the rKWD50N excision cassette (panels 1 and 2) or pSIR3-HA (panel 3). Lanes 1, 4, and 7 were PCR products of resuspended P2. Lanes 2, 5, and 8 and 3, 6, and 9 were PCR products of immunoprecipitations performed with or without anti-HA antibody, respectively.