Saccharomyces cerevisiae Esc2p interacts with Sir2p through a small ubiquitin-like modifier (SUMO)-binding motif and regulates transcriptionally silent chromatin in a locus-dependent manner

Abstract

Saccharomyces cerevisiae Esc2p is a member of a conserved family of proteins that contain small ubiquitin-like modifier (SUMO)-like domains. It has been implicated in transcriptional silencing and shown to interact with the silencing protein Sir2p in a two-hybrid analysis. However, little is known about how Esc2p regulates the structure of silent chromatin. We demonstrate here that ESC2 differentially regulates silent chromatin at telomeric, rDNA, and HM loci. Specifically, ESC2 is required for efficient telomeric silencing and Sir2p association with telomeric silent chromatin and for silencing and maintenance of silent chromatin structure at rDNA. On the other hand, ESC2 negatively regulates silencing at HML and HMR and destabilizes HML silent chromatin without affecting Sir2p association with chromatin. We present evidence that Esc2p is associated with both transcriptionally silent and active loci in the genome, and the abundance of Esc2p is not correlated with the chromatin state at a particular locus. Using affinity pull-down analyses, we show that Esc2p and Sir2p interact in vivo, and recombinant Esc2p and Sir2p interact directly. Moreover, we dissect Esc2p and identify a putative SUMO-binding motif that is necessary and sufficient for interacting with Sir2p and SUMO and is required for the function of Esc2p in transcriptional silencing.

FIGURE 1.

Deletion of ESC2 differentially affects transcriptional silencing at telomeric, rDNA, and HM loci and increases telomere length. A, schematic of Esc2p. The SUMO-like domains and SUMO-binding motifs are indicated by open boxes and by diamonds, respectively. The zigzag line indicates a long helical region conserved among Esc2p and its close relatives (13). B, effect of esc2Δ on transcriptional silencing. Shown are growth phenotypes of strains 1–10 under different conditions. Strains 1–10 are, in order, CCFY101, YQY498, YHK53, YHK56, JS125, YQY495, YYZ208, YQY610, YXB61-II, and YQY603. Cells were grown to late log phase, and serial 10-fold dilutions were spotted on synthetic complete medium (SC), −Ura (SC lacking uracil), SC supplemented with 1 mg/ml FOA (FOA), and SC with 0.01% MMS (MMS). Two independent clones of each esc2Δ strain were tested. Strains 1 and 2 were also spotted on SC lacking tryptophan (−Trp). The plates were incubated for 3 days. Silencing reporter constructs are indicated on the left. Tel VR, right telomere of chromosome V. HMRΔe, HMR deleted for the Rap1p site in HMR-E silencer. Tel VIIL, left telomere of chromosome VII. NTS2, nontranscribed spacer 2 in the rDNA array. mURA3, URA3 driven by a minimal TRP1 promoter. HMRΔI, HMR deleted for the HMR-I silencer. C, ESC2 deletion increases telomere length. Genomic DNAs from YHK53 (ESC2) and YHK56 (esc2Δ) bearing Tel VIIL-URA3 (top) were isolated, digested with PstI, and fractionated by gel electrophoresis. The Tel VIIL-URA3 fragment was detected by Southern blotting with a URA3 probe. Two independent clones of each strain were examined. M, DNA markers.

FIGURE 2.

Esc2p is associated with the genome and regulates Sir2p association with silent loci. A, effect of esc2Δ on Sir2p association with chromatin. The abundance of the indicated sequences in YQY616 (ESC2 SIR2-Myc) or YQY616 (esc2Δ SIR2-Myc) was measured by PCR before (input) and after chromatin IP (α-Sir2p-Myc) with an α-Myc antibody. The gel picture of PCR products from one of three independent experiments is shown. The data were quantified and plotted in B. The value for HMR in ESC2 cells was taken as 1. No Ab, samples from mock ChIP without using antibody. C, ChIP analysis of Esc2p association with the genome. Three independent ChIP experiments were performed with an α-Myc antibody in YKA21 carrying ESC2-Myc. Data from one experiment are shown on the left. Data were quantified and plotted on the right. The value for ACT1 was taken as 1.

FIGURE 3.

Effect of esc2Δ on silent chromatin structure at rDNA. Top, rDNA array in JS125 (ESC2 SIR2), YQY541 (sir2Δ), YQY495 (esc2Δ), and YQY618 (trp1Δ). The gene encoding the 35S precursor rRNA is indicated by a black arrow. NTS, nontranscribed spacer. In each strain, a Ty1-mURA3 silencing reporter was integrated at one of the nontranscribed spacers. Arrowhead, long terminal repeats of Ty1. The URA3 coding region is indicated by an open arrow. Bottom, mapping chromatin around the URA3 promoter. DNA isolated from MNase-treated chromatin in each strain was digested with AlwnI and then subjected to gel electrophoresis and Southern blotting using a probe abutting the AlwnI site within URA3. N, naked genomic DNA.

FIGURE 4.

ESC2 negatively regulates the stability of silent chromatin at HML. A, strategy for examining the topology of chromosomal DNA. Two FRT sequences (filled arrows) are inserted to flank the chromosome region of interest. Recombination between FRTs by the site-specific recombinase Flp1p excises the region as a minichromosome circle. After deproteinization, the topology of the DNA circle can be examined by electrophoresis in the presence of a DNA intercalator. Filled circles, nucleosomes. B and C, effect of esc2Δ on the supercoiling of HML DNA. Top, strategy for examining the topology of HML DNA with or without silencers. Two FRT sites are inserted in direct orientation at HML to flank a region that includes (B) or excludes (C) the silencers. Recombination between the FRTs leads to the excision of a minichromosome that has captured HML silent chromatin (labeled SIR⁺). Cell cycle progression would convert the silenced SIR⁺ circle free of silencers into an active circle (labeled sir⁻) but does not affect the SIR⁺ circles associated with silencers (25). The filled and shaded circles indicate nucleosomes in silent and active chromatins, respectively. Middle, cells of the indicated strains were grown in YPR to early log phase, at which time galactose was added. After 2.5 h of incubation, DNA was isolated and subjected to gel electrophoresis in the presence of 20 μg/ml chloroquine. Under this condition, more negatively supercoiled circles migrate more slowly. The positions of nicked (N) and linear (L) circles are indicated. Samples in lanes 1–7 were from YXB10, YQY537, YXB10s, YQY539, YXB6, YQY585, and YXB6s, respectively. Topoisomers of the HML circle associated with silent or active chromatin are labeled as SIR⁺ or sir⁻, respectively. A densitometer scan of each lane is shown at the bottom. The distribution center of topoisomers in each sample is indicated by a dot. The asterisk indicates a gel artifact. D, deletion of ESC2 enhanced the stability of HML silent chromatin. Top, scheme of the experiment. Silencer-free HML circles were excised from ESC2 (YXB6) or esc2Δ (YQY585) cells arrested in G₁ phase by α-factor treatment. The purpose of excising HML circles from non-cycling cells (arrested in G₁) instead of asynchronously growing cells is to prevent the disruption of silent chromatin on the circle by cell cycle progression during the 2.5-h induction of circle excision. The supercoiling of the HML circles were then followed through subsequent cell growth in YPD. DNA samples taken at 0, 1, 2, 4, and 5 h after release from G₁ arrest were subjected to electrophoresis in the presence of 16 μg/ml chloroquine.

FIGURE 5.

Esc2p directly interacts with Sir2p. A, Esc2p and Sir2p interaction in vivo. Proteins isolated from strain YJS537 with (+Esc2p-His-HA) or without (−Esc2p-His-HA) the pBG-ESC2-His-HA plasmid were added to Ni²⁺-nitrilotriacetic acid resin. The resin was then washed and eluted by boiling. The input (lanes 1 and 2) and eluate (1′ and 2′) protein samples were subjected to SDS-PAGE and Western blotting. The blot was then separately probed with the α-Myc and α-HA antibodies. Only the relevant parts of the blots are shown. A similar experiment was done with YXB76 with untagged SIR2 (lanes 3 and 3′) and YJS537 with pBG-CYT2-His-HA (lanes 4 and 4′). Samples in lanes 5 and 5′ were the same as those in lanes 2 and 2′, respectively. B, interaction between recombinant Esc2p and Sir2p. Recombinant GST-Sir2p or GST was pulled down by glutathione-Sepharose beads with bacteria lysate containing His-HA-Esc2p. After SDS-PAGE, GST-Sir2p and GST were detected by Coomassie staining, and His-HA-Esc2p was detected by immunoblotting with α-HA antibody.

FIGURE 6.

Esc2p interacts with Sir2p and SUMO through a SUMO binding motif. A, two-hybrid analyses of interactions between GAD-Sir2p and GBD-fused Esc2p alleles. The Esc2p alleles tested are shown on the left. The symbols used here are similar to those in Fig. 1A. Note that of the four SBMs of Esc2p, only SBM1 is indicated for clarity. Growth of the host PJ69–4α on −Ura−Leu−His+3AT medium indicates positive interaction, and that on −Ura−Leu−Ade indicates strong interaction (19). Two independent transformants were tested for each two-hybrid analysis. B, Western blotting analysis of GBD-fused Esc2p alleles. Equal amounts of protein extracts from PJ69–4α containing individual plasmids 1–18 (A) were run on SDS-PAGE and probed with an anti-GBD antiserum. C, Esc2p-(116–135) is necessary and sufficient for SUMO-binding. Growth phenotypes of PJ69–4α bearing the indicated plasmids are shown. The four SBMs in Esc2p are indicated by diamonds. D, Esc2p-Sir2p interaction is independent of SIR3 and SIR4. Growth phenotypes of YLO55 (sir3Δ sir4Δ) bearing the indicated plasmids are shown.

FIGURE 7.

Targeted silencing by Esc2p is mediated by SBM1. Top, the silencing reporter construct in YSB35. YSB35 bearing plasmids 1–20 (shown on the left) were tested. Lack of growth on −Ura−Trp medium indicates silencing of TRP1. 10-Fold serial dilutions of the cultures were spotted. Two independent transformants were tested.

FIGURE 8.

The SBM1 of Esc2p is required for telomeric silencing but not cellular tolerance of genotoxic stress. Shown are growth phenotypes of strain YHK56 (esc2Δ) bearing plasmids 1′, 2′, or 18′ on −Leu, −Leu+FOA, and −Leu+MMS media. The Tel VIIL-URA3 construct in YHK56 is shown at the top.