Yeast heterochromatin is a dynamic structure that requires silencers continuously

Abstract

Transcriptional silencing of the HM loci in yeast requires cis-acting elements, termed silencers, that function during S-phase passage to establish the silent state. To study the role of the regulatory elements in maintenance of repression, site-specific recombination was used to uncouple preassembled silent chromatin fragments from silencers. DNA rings excised from HMR were initially silent but ultimately reactivated, even in G(1)- or G(2)/M-arrested cells. In contrast, DNA rings bearing HML-derived sequence were stably repressed due to the presence of a protosilencing element. These data show that silencers (or protosilencers) are required continuously for maintenance of silent chromatin. Reactivation of unstably repressed rings was blocked by overexpression of silencing proteins Sir3p and Sir4p, and chromatin immunoprecipitation studies showed that overexpressed Sir3p was incorporated into silent chromatin. Importantly, the protein was incorporated even when expressed outside of S phase, during G(1) arrest. That silencing factors can associate with and stabilize preassembled silent chromatin in non-S-phase cells demonstrates that heterochromatin in yeast is dynamic.

Figure 1

Excision of chromosomal fragments by site-specific recombination. RS target sites (half-filled boxes) for the Zygosaccharomyces rouxii R recombinase (Matsuzaki et al. 1990) were built into the HMR locus, either within the E and I silencers (A), or just beyond the E and I silencers (B) to produce rings that either lacked or contained the elements, respectively. In most laboratory strains, HMR contains the a1 and a2 mating-type genes (HMRa), whereas HML contains the α1 and α2 mating-type pair (HMLα). A third mating-type locus, MAT, contains an active copy of either pair of mating-type genes. In the various experiments described herein, either a mating type sequences, α mating type sequences, or a composite of the two were positioned at HMR for subsequent excision. All excision cassettes are named according to the rings they produce (e.g., locus hmr::rHMRa shown in A produces ring rHMRa).

Figure 2

Maintenance of silencing requires silencers. DNA rings were excised from HMR by induction of recombination in cell cycle-arrested cultures. Strains THC78 hmr::rHMRa Δhml::kanMX Δsir3::HIS3 (lane 1) and THC43 hmr::rHMRa (lanes 2–4) produced rings that lacked silencers. Strains THC79 hmr::rHMRa+EI Δhml::kanMX Δsir3::HIS3 (lane 5) and THC37 hmr::rHMRa+EI (lanes 6–8) produced rings that contained silencers. HMLα was deleted in the sir3 strains; otherwise, expression of the locus would have prevented arrest by α-factor. MATa was deleted from all of the strains so that HMRa-derived rings provided the sole source of a1 mRNA. In cells arrested with α-factor or nocodazole, nucleic acids were harvested at timed intervals following galactose addition. (A) Northern analysis of a1 gene silencing in cells arrested by α factor in G₁. Blots were hybridized sequentially with probes to a1 and the ACT1 gene. When normalized to ACT1, the levels of a1 mRNA in lanes 1 and 4 are comparable. (B) Chloroquine gel electrophoresis of DNA rings from α-factor-arrested cells. Centers of the topoisomer distributions were determined by the Gaussian method and marked with an arrowhead for clarity. Changes in DNA supercoiling were attributable to changes in chromatin structure and not due to the mechanics of transcription because rings that lacked promoters also bore SIR-dependent DNA topology changes (Cheng et al. 1998). (C) Analysis of a ring lacking silencers from cells arrested at G₂/M with nocodazole.

Figure 3

UASα maintains silencing in the absence of silencers. Following treatment with α-factor, DNA rings were formed in the following isogenic strain pairs: THC59 Δhmr::rHMRα Δsir3::HIS3 (lane 1), THC62 Δhmr::rHMRα (lanes 2–4); THC75 Δhmr::rHMRa/α Δsir3::HIS3 (lane 5), THC77 Δhmr::rHMRa/α (lanes 6–8); THC68 Δhmr::rHMRα(rap1pm) Δsir3::HIS3 (lane 9), THC69 Δhmr::rHMRα(rap1pm) (lanes 10–12). Diagrams of the excision cassettes are shown above each panel with UASα depicted as a green box. DNA rings were examined as in Fig. 2. Asynchronous cultures of THC59, THC75, and THC68 were used as nonsilent control because these sir3 strains contain HMLα and do not respond to α-factor. However, the supercoiling of rings in sir3 strains was influenced negligibly by α-factor arrest (data not shown).

Figure 4

UASα contributes to silencing of the chromosomal HMLα locus. Strains THC74 hml::URA3P–ADE2 and THC76 hml(rap1pm)::URA3P–ADE2 were plated on SC media containing minimal adenine after overnight growth on nonselective media. Red or pink pigmentation corresponds to full or partial repression of ADE2, respectively. Unpigmented (white) colonies correspond to complete ADE2 derepression. Previously, the URA3P–ADE2 construct was used to monitor transcriptional repression at HMRa (Rivier et al. 1999). The URA3 promoter is up-regulated when uracil is omitted from the growth media. On SC plates lacking uracil, more frequent derepression was observed in the UASα mutant strain (data not shown).

Figure 5

Stabilization of silent chromatin rings by elevated SIR gene dosage. Strain THC43 hmr::rHMRa was transformed with single copy, CEN-based vectors pJR910 (SIR1, lanes 4–6), pJR69 (SIR2, lanes 7–9), pJR273 (SIR3, lanes 13–15, 23–26), pJR368 (SIR4, lanes 16–18), or pRS416 (empty vector, lanes 1–3, 10–12, 19–22). In each case, the gene of interest was transcribed from its own promoter. Supercoiling of DNA rings (A) and RNA levels (B) were examined as in Fig. 2.

Figure 6

Stabilization of silent chromatin rings by elevated SIR3 expression in G₁-arrested cells. Strains THC43 hmr::rHMRa (lanes 1–3), THC57 hmr::rHMRa ura3-1::GAL10P–SIR3::URA3 (lanes 4–6), and THC70 hmr::rHMRa Δsir3::HIS3 ura3-1::GAL10P–SIR3HA::URA3 (lanes 7–9) containing recombinase expression vector pHM153 were arrested with α-factor. Subsequently, expression of the R recombinase gene and SIR3 were induced simultaneously with galactose. Supercoiling of DNA rings was examined as in Fig. 2.

Figure 7

Incorporation of Sir3p into silent chromatin in G₁-arrested cells. Strains THC57 ura3-1::GAL10P–SIR3::URA3 (lanes 1,5), THC67 ura3-1::GAL10P–SIR3HA::URA3 (lanes 2,3,6,7), and THC70 Δsir3::HIS3 ura3-1::GAL10P–SIR3HA::URA3 (lanes 4,8) were arrested with α-factor. Following induction of SIR3 expression with galactose, cells were treated with formaldehyde. Processed samples were immunoprecipitated with anti-HA antibody, and the precipitated material was probed for the presence of DNA with PCR using primer pairs specific for the a1, GAL1, ACT1, and PHO5 genes. The MATa locus was deleted from all of these strains so that HMRa provided the sole copy of a1 DNA. The positions of DNA mobility standards are marked at left.