Regulation of nuclear positioning and dynamics of the silent mating type loci by the yeast Ku70/Ku80 complex - PubMed (original) (raw)

Regulation of nuclear positioning and dynamics of the silent mating type loci by the yeast Ku70/Ku80 complex

Kerstin Bystricky et al. Mol Cell Biol. 2009 Feb.

Abstract

We have examined the hypothesis that the highly selective recombination of an active mating type locus (MAT) with either HMLalpha or HMRa is facilitated by the spatial positioning of relevant sequences within the budding yeast (Saccharomyces cerevisiae) nucleus. However, both position relative to the nuclear envelope (NE) and the subnuclear mobility of fluorescently tagged MAT, HML, or HMR loci are largely identical in haploid a and alpha cells. Irrespective of mating type, the expressed MAT locus is highly mobile within the nuclear lumen, while silent loci move less and are found preferentially near the NE. The perinuclear positions of HMR and HML are strongly compromised in strains lacking the Silent information regulator, Sir4. However, HMLalpha, unlike HMRa and most telomeres, shows increased NE association in a strain lacking yeast Ku70 (yKu70). Intriguingly, we find that the yKu complex is associated with HML and HMR sequences in a mating-type-specific manner. Its abundance decreases at the HMLalpha donor locus and increases transiently at MATa following DSB induction. Our data suggest that mating-type-specific binding of yKu to HMLalpha creates a local chromatin structure competent for recombination, which cooperates with the recombination enhancer to direct donor choice for gene conversion of the MATa locus.

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Figures

FIG. 1.

FIG. 1.

MAT adopts a central nuclear position, while HM loci assume a peripheral, Sir4-dependent location. (A) Schematic representation of Saccharomyces cerevisiae Chr3. GFP-LacI or GFP-TetR fusions allow visualization of the lacO and tetO arrays inserted near one of the three mating type loci. Distances are indicated in kb from the left telomere. (B) Positions were mapped relative to the NE in strains of GFP-tagged MAT loci (white), as well as HML and HMR loci in wt (black) and sir4 (green) cells. Data are represented in bar graphs as the percentage of spots in one of three concentric zones of equal surface area. The numbers of G1-phase MATa cells analyzed for the MAT, HML, and HMR loci were as follows: 80 for MAT, 181 (wt) and 135 (sir4) for HML, and 122 (wt) and 146 (sir4) for HMR. The numbers of G1-phase _MAT_α cells analyzed for the MAT, HML, and HMR loci were 69 for MAT, 103 (wt) and 87 (sir4) for HML, and 172 (wt) and 67 (sir4) for HMR. Confidence values (P) for the χ2 analysis between random (33% in each zone) and test distributions are summarized in Fig. 2. *, value significantly different from random (P < 0.05). Bar, 2 μm.

FIG. 2.

FIG. 2.

Summary of HML, MAT, and HMR locus positioning data from comparison of G1- and S-phase cells. Shown are the percentages of total cells counted in three equal-surface concentric nuclear zones of the indicated tagged loci in the wt and indicated mutant strains in the G1 and S phases. n, number of cells analyzed. Confidence values (P) using a proportional analysis between two test frequencies or a χ2 analysis between a predicted random distribution (33% in each zone) and test distributions are given for S- and G1-phase values. Bold numbers indicate P < 0.05.

FIG. 3.

FIG. 3.

Anchoring of HML is increased in the absence of yKu70 and of the RE. Positions relative to the NE (percentage in zone 1) (Fig. 1) in MATa wt (light gray), yku70 (black), yku70 sir4 (white), and Δ_re_ (dark gray) strains GFP tagged at HML, HMR, or HML in the absence of the silenced HML locus (Tel3LΔ_hml_). The confidence values (P) for the proportional analysis between two test frequencies are given. For complete statistical data, see Fig. 2.

FIG. 4.

FIG. 4.

Influence of lac or tet operator repeat insertion site distance to the telomere on peripheral anchoring. The degree of peripheral anchoring of the HM loci compared with previously analyzed zone 1 values for telomere proximal sites (7, 20, 21, 51) in wt, yku70, and sir4 mutant strains. lac or tet operator insertion site distances in kb from the respective chromosome end plotted against scored positions relative to nuclear pores. HML_α values are in blue and are marked by asterisks. The left arm of Chr3 bearing the HML deletion (Tel3LΔ_hml) is in red and is labeled 3LΔ. 6Rt indicates Tel6R bearing a truncation of 5 kb.

FIG. 5.

FIG. 5.

Gene conversion takes place in the nuclear lumen. (A) Spontaneous and damage-induced foci of Rad52-GFP were scored mainly in S-phase cells in asynchronous (asynchr) cultures of GA-5196. Cultures were grown to exponential phase and then cultured for 60 min or 120 min in the presence of DNA-damaging agents as indicated by the number of foci counted: spontaneous, n = 244; HU treated, n = 192 and n = 96 for 60 and 120 min, respectively; MMS treated, n = 249 and n = 268 for 60 and 120 min, respectively. For treatment in synchronized cultures (synchr), GA-5239 cells were grown to exponential phase and arrested in G1 by incubation with α-factor for 1.5 h. The cells were subsequently released into SC medium (spontaneous, n = 152) or into MMS (n = 130 and n = 105 for 60 and 120 min, respectively), prior to scoring Rad52 focus location. (B) Positions relative to the NE of MATa wt cells before (0 min; white) and during (60 min; black) gene conversion. (C) Positions relative to the NE of HML in MATa wt and yku and _MAT_α wt cells before (0 min; white) and during (60 min; black) gene conversion. Asterisks indicate statistically significant difference between distributions at 0 and 60 min (P = 4.5 × 10−3). Cells were grown in lactate-containing medium, and at 0 min, galactose was added to induce expression of the HO endonuclease from a plasmid. Gray bars indicate the position of HML relative to the NE in cells not bearing the HO plasmid but grown in galactose. For complete statistical data, see Table 3.

FIG. 6.

FIG. 6.

Mating-type-specific binding of yKu70/80 to the silenced HML locus, but not to the RE. ChIP was performed as described in Materials and Methods using strains bearing Myc-tagged copies of yKu70 (GA-3339; yKu70-Myc) and yKu80 (GA-1009; yKu80-Myc) and GA-3340 (Mcm1-Myc). (A) Real-time QPCR was performed with probe/primer sets that amplify the indicated loci of Chr3, a telomeric locus, and the SMC2 control locus on Chr6. Enrichment relative to the SMC2 control locus and a negative control ChIP (B, C, E, and F) or relative to a nontagged strain (D) as the mean ± standard error of the mean are shown. For calculation of enrichment, see Materials and Methods. (B) yKu70 binds to a telomere, HML, and HMR, but not the RE in exponentially growing MATa and _MAT_α cells. yKu70 has a preference for HML in MATa, but not in _MAT_α, cells. (C) Mcm1, but not yKu70 and yKu80, binds the RE in exponentially growing MATa cells. yKu70 and yKu80 bind to a telomere and HML, while Mcm1 does not. (D) yKu70 binding is abolished at both telomeric and HML loci in the absence of Sir4. ChIP was performed as described in Materials and Methods using a wt strain and an isogenic strain with sir4 deleted, each bearing a Myc-tagged copy of yKu80. (E) ChIP was performed for yKu70-Myc on exponentially growing MATa cells (GA-3339) before and after 30 min and 60 min of Gal1::HO expression. Efficiency of DSB induction at the MAT locus was determined by PCR using primers that span the HO cut site at MAT and primers that anneal to the SMC2 control locus on Chr6. DSB induction monitored as loss of the MAT product relative to the SMC2 signal yielded cleavage efficiencies of 86% and 96% at 0.5 h and 1 h after HO induction, respectively. At 60 min after HO induction, switching is nearly complete and yKu70 binding resembles that observed in the MATa strain (data not shown). (F) Mcm1 binds to the RE before and after HO induction, but yKu70 does not. Results are shown as in panel E, except that yKu70-Myc MATa, and Mcm1-Myc MATa cells (GA-3340) were used. Cleavage efficiencies were 86% and 96% (yKu70-Myc _MAT_a) and 60% and 82% (Mcm1-Myc _MAT_a) at 0.5 h and 1 h after HO induction, respectively.

FIG. 7.

FIG. 7.

Movement of the left arm of Chr3 is constrained, but is relieved by yku70 mutation. (A) Time-lapse microscopy was performed by taking single-frame images every 1.5 s on a Zeiss LSM 510 confocal microscope. Representative single-frame images of GFP-tagged HML in wt, sir4, and yku70 MATa strains relative to the NE visualized by GFP-Nup49 pore components. The path obtained by aligning the spot position of 100 frames was superposed in red on the respective right panel. Bar, 1 μm. (B) MSD for MAT, HML, and HMR in wt MATa and MAT_α strains using MSD, where d represents the spot-to-spot distance for each frame as a function of the time interval (Δ_t = 1.5 to 100 s). For each trace, six to eight movies were averaged. (C). Normalized MSD values for wt and mutant strains. The MSD (in μm2) from time-lapse series for HML in MATa and MAT_α wt, sir4, yku70, Δ_re, and Δ_re_ y_ku70_ strains and for Tel3L in the absence of the HML locus (Δ_hml_) in wt and yku70 strains as a function of the time interval (Δ_t_ = 1.5 to 100 s) was calculated. The MSD at Δ100 s was calculated for all movies of one genotype and set to 1 for the wt. The increase (fold) in MSD at 100 s relative to the MSD of each wt strain is shown for the MSD of mutant strains (thick bars) and all individual movies recorded (open circles). (D) Same as panel C, but the movement of the tagged HMR locus was monitored. The MSD (in μm2) from time-lapse series for HMRa in MATa wt, sir4, yku70 and sir3 strains as a function of the time interval (Δ_t_ = 1.5 to 100 s) was calculated and is presented after normalization to the wt.

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