Quantitative fitness analysis shows that NMD proteins and many other protein complexes suppress or enhance distinct telomere cap defects

Abstract

To better understand telomere biology in budding yeast, we have performed systematic suppressor/enhancer analyses on yeast strains containing a point mutation in the essential telomere capping gene CDC13 (cdc13-1) or containing a null mutation in the DNA damage response and telomere capping gene YKU70 (yku70Δ). We performed Quantitative Fitness Analysis (QFA) on thousands of yeast strains containing mutations affecting telomere-capping proteins in combination with a library of systematic gene deletion mutations. To perform QFA, we typically inoculate 384 separate cultures onto solid agar plates and monitor growth of each culture by photography over time. The data are fitted to a logistic population growth model; and growth parameters, such as maximum growth rate and maximum doubling potential, are deduced. QFA reveals that as many as 5% of systematic gene deletions, affecting numerous functional classes, strongly interact with telomere capping defects. We show that, while Cdc13 and Yku70 perform complementary roles in telomere capping, their genetic interaction profiles differ significantly. At least 19 different classes of functionally or physically related proteins can be identified as interacting with cdc13-1, yku70Δ, or both. Each specific genetic interaction informs the roles of individual gene products in telomere biology. One striking example is with genes of the nonsense-mediated RNA decay (NMD) pathway which, when disabled, suppress the conditional cdc13-1 mutation but enhance the null yku70Δ mutation. We show that the suppressing/enhancing role of the NMD pathway at uncapped telomeres is mediated through the levels of Stn1, an essential telomere capping protein, which interacts with Cdc13 and recruitment of telomerase to telomeres. We show that increased Stn1 levels affect growth of cells with telomere capping defects due to cdc13-1 and yku70Δ. QFA is a sensitive, high-throughput method that will also be useful to understand other aspects of microbial cell biology.

Figure 1. Cell fitness determination from growth on agar plates for quantitative fitness analysis (QFA).

A) Time course images of eight independent upf2Δ ura3Δ, yku70Δ ura3Δ and upf2Δ yku70Δ strains at the indicated temperatures; B) Cell density of individual replicate cultures was determined after image-analysis. The logistic growth model is fitted to each culture density time-series. The same data are plotted on linear or logarithmic scales on left and right respectively. C) Average values for Maximum Doubling Rate, Maximum Doubling Potential and Fitness (MDR, MDP and F respectively; see Text S1, experimental procedures), determined from the fitted curves. Data for yku70Δ ura3Δ is presented here to illustrate epistasis between yku70Δ and upf3Δ, however this is not how epistasis was calculated (see Figure 2 and Text S1, experimental procedures).

Figure 2. Fitness of yku70Δ strains at high temperature.

The yeast genome knock out collection was crossed to the yku70Δ mutation, or as a control to the ura3Δ mutation. 8 replicate crosses were performed and for each, the fitness of all double mutant cultures measured as in Figure 1. Growth of yku70Δ yfgΔ (“your favourite gene deletion”) double mutants was measured at 37.5°C and ura3Δ yfgΔ strains at 37°C. Gene deletions that significantly enhance (green) or suppress (red) the yku70Δ defect, in comparison with the ura3Δ mutation are indicated. Those marked by open circles have p-values <0.05 and those filled circles have FDR corrected p-values (q-values) <0.05. The line of equal growth (dashed grey) and a population model of expected fitness (solid grey) are indicated. The average position of his3Δ strains are indicated by solid light-blue lines on each axis, as proxy for “wild-type” growth.

Figure 3. Genetic interaction strength (GIS) comparison between cdc13-1 and yku70Δ.

Genes that significantly interacted with cdc13-1 or yku70Δ are shown, most genes did not interact and would be placed in the centre of the plot. Genes encoding components of selected protein complexes (or proteins which work closely together towards the same function) are indicated by colour-co-ordinated text and symbols. Genes that interact with both cdc13-1 and yku70Δ are open white circles, those that interact with just one mutation are filled white circles. Different regions of the plot are indicated on the top right and borders between regions are intentionally blurred/overlapping as there are not precise cut-offs. An arbitrary GIS cutoff of +/−0.5 is indicated by the black dashed rectangle. Also see Figure S3 for further analysis of these data.

Figure 4. Confirmation of genetic interactions in an alternative genetic background.

A selection of genes identified by QFA were combined with either the yku70Δ or cdc13-1 mutations in the W303 genetic background and assessed for growth by manual spot test. Strains were cultured to saturation in 2 ml YPD at 23°C, a six-fold dilution series generated and spotted onto YPD. Strains were incubated at the indicated temperatures for three days before being photographed. All plates contained the reference strains 640 (wild type), 1108 (cdc13-1) and 1412 (yku70Δ), indicated as red cultures in the key. The “Wild Type” single mutant strains assessed were: 6656, 6811, 3622, 3653, 6620, 1273, 659, 6862, 6927 6951, 6963, 6692 and 6632. The cdc13-1 double mutant strains assessed were: 6810, 6814, 3624, 3655, 6614, 1296, 1258, 6860, 6928, 6865, 6967, 6694 and 6396. The yku70Δ double mutant strains assessed were: 6808, 6812, 4290, 4296, 6628, 1409, 1284, 2413, 2415, 6968, 6971, 6776 and 6763. Growth at other temperatures is shown in Figure S2.

Figure 5. UPF2 influences telomere capping through STN1 and telomerase recruitment.

A) Transcript levels of four telomere-binding factors measured in upf2Δ and ebs1Δ mutants. Four strains of each genotype were grown exponentially in liquid culture at 23°C. RNA was isolated and transcript levels were determined by SYBR Green RT-PCR. Each measurement was performed in triplicate and error bars indicate standard deviation from four independent measurements. RNA concentrations of the samples were normalized to the loading control BUD6. A single wild type sample was given the value of 1 and all other values were corrected relative to this. Strains measured are 640, 2824, 3001, 4763, 4764, 4765, 4766, 4780, 4781, 4782, 4783 and 4784; B) Western blot analysis of Stn1 protein levels using antibodies against Stn1-13Myc tagged strains. Strains shown are 5757, 5758, 5759, 5760, 5761, 5763, 5764, 5765 and 5766; C) Growth analysis of yku70Δ or cdc13-1 mutants over-expressing STN1 using the centromeric plasmid pVl1045. The empty vector Ycplac111 was used as a control. Strains 5046, 5047, 5051 and 5052 were spot tested on –LEU medium; D) upf2Δ and ebs1Δ mutants were combined with yku70Δ or cdc13-1 mutations in the W303 genetic background and assessed for growth by spot test. Strains shown are 640 and 3001 (wild type), 2764 and 2824 (ebs1Δ), 2787 and 4309 (yku70Δ), 2889 and 2890 (ebs1Δ yku70Δ), 5007 and 5008 (nmd2Δ yku70Δ), 5251 and 5242 (ebs1Δ nmd2Δ yku70Δ), 1195 and 4557 (cdc13-1), 4576 and 4577 (ebs1Δ cdc13-1), 4624 and 4625 (nmd2Δ cdc13-1), 5238 and 5239 (ebs1Δ nmd2Δ cdc13-1); E) ChIP analysis of Est2-13Myc association to the VI-R telomere and the internal locus PDI1 on Chromosome III using primers previously described . Duplicate cultures were grown and harvested in exponential phase. Individual ChIP samples were measured in triplicate and group means are shown with 95% confidence bars derived from a two-way ANOVA. Strains shown are 6977 (Est2-13Myc), 6978 (nmd2Δ Est2-13Myc), 6979 (nmd2Δ yku70Δ Est2-13Myc) and 6980 (yku70Δ Est2-13Myc).

Figure 6. Model of telomere capping activities influenced by NMD.

Disruption of NMD activity results in higher Stn1 transcript and protein levels (Figure 5). Stn1 promotes capping directly and is thought to oppose telomerase recruitment through interaction with Cdc13. Since telomerase has telomere capping function, Stn1 therefore both promotes and inhibits telomere capping.