Elimination of subtelomeric repeat sequences exerts little effect on telomere functions in Saccharomyces cerevisiae

Reviewer #1 (Public Review):

The authors have generated a set of yeast S. cerevisiae strains containing different numbers of chromosomes. Elimination of telomerase activates homologous recombination (HR) to maintain telomeres in cells containing the original 16 chromosomes. However, elimination of telomerase leads to circularization of cells containing a single or two chromosomes. The authors examined whether the subtelomeric sequences X and Y' promote HR-mediated telomere maintenance using the strain SY12 carrying three chromosomes. They found that the subtelomeric sequences X and Y' are dispensable for cell proliferation and HR-mediated telomere maintenance in telomerase-minus SY12 cells. They conclude that subtelomeric X and Y' sequences do not play essential roles in both telomerase-proficient and telomerase-null cells and propose that these sequences represent remnants of genome evolution.
Interestingly, telomerase-minus SY12 generate survivors that are different from Type I or Type II survivors.

Strengths: The authors examined whether the subtelomeric sequences X and Y' promote HR-mediated telomere maintenance using the strain SY12 carrying three chromosomes.

Weaknesses:
It is not determined how atypical survivors or Type X survivors are generated in telomerase-deficient SY12 cells.
Survivor generation of each type (Type I, Type II, Type X or atypical and circularization) is not quantitated.

Reviewer #2 (Public Review):

Summary:
In this work, Hu and colleagues investigate telomerase-independent survival in Saccharomyces cerevisiae strains engineered to have different chromosome numbers. The authors describe the molecular patterns of survival that change with fewer chromosomes and that differ from the well-described canonical Type I and Type II, including chromosome circularization and other atypical outcomes. They then take advantage of the strain with 3 chromosomes to examine the effect of deleting all the subtelomeric elements, called X and Y'. For most of the tested phenotypes, they find no significant effect of the absence of X- and Y'-element, and show that they are not essential for survivor formation. They speculate that X- and Y'-elements are remnants of ancient telomere maintenance mechanisms.

Strengths:
This work advances our understanding of the telomerase-independent strategies available to the cell by altering the structure of the genome and of the subtelomeres, a feat that was enabled by the set of strains they engineered previously. By using strains with non-standard genome structures, several alternative survival mechanisms are uncovered, revealing the diversity and plasticity of telomere maintenance mechanisms. Overall, the conclusions are well supported by the data, with adequate sample sizes for investigating survivors. The molecular analyses mostly based on Southern blots are also very well-conducted.

Weaknesses:
The qualification of survivor types mostly relies on molecular patterns in Southern blots. While this is a valid method for a standard strain, it might be more difficult to apply to the strains used in this study. For example, in SY8, SY11 and SY12, the telomere signal at 1-1.2 kb can be very faint due to the small number of terminal Y' elements left. As another example, for the Y'-less strain, it might seem obvious that no Type I survivor can emerge given that Y' amplification is a signature of Type I, but maybe Type-I-specific molecular mechanisms might still be used. To reinforce the characterization of survivor types, an analysis of the genetic requirements for Type I and Type II survivors (e.g. RAD51, RAD54, RAD59, RAD50) could complement the molecular characterization in specific result sections.

In the title, the abstract and throughout the discussion, the authors chose to focus on the effect of X- and Y'-element deletion on different phenotypes and on survivor formation, as the main message to convey. While it is a legitimate and interesting message, other important results of this work might benefit from more spotlight. Namely, the observation that strains with different chromosome numbers show different survivor patterns and that several survival strategies beyond Type I and II exist and can reach substantial frequencies depending on the chromosomal context.

In SY12 strain, while X- and Y'-elements are not essential for survivor emergence, they do modulate the frequency of each type of survivors, with more chromosome circularization events observed for SY12Y∆, SY12XY∆ and SY12XY∆+Y strains. This result should be stated and discussed, maybe alongside the change in survivor patterns in the other SY strains, to more accurately assess the roles of these subtelomeric elements.

Reviewer #3 (Public Review):

Summary:
This study investigates subtelomeric repetitive sequences in the budding yeast Saccharomyces cerevisiae, known as Y' and X-elements. Taking advantage of yeast strain SY12 that contains only 3 chromosomes and six telomeres (normal yeast strains contain 32 telomeres) the authors are able to generate a strain completely devoid of Y'- and X-elements.

Strengths: They demonstrate that the SY12 delta XY strain displays normal growth, with stable telomeres of normal length that were transcriptionally silenced, a key finding with wide implications for telomere biology. Inactivation of telomerase in the SY12 and SY12 delta XY strains frequently resulted in survivors that had circularized all three chromosomes, hence bypassing the need for telomeres altogether. The SY12 and SY12 delta XY yeast strains can become a useful tool for future studies of telomere biology. The conclusions of this manuscript are mostly well supported by the data and are important for researchers studying telomeres.

Weaknesses: A weakness of the manuscript is the analysis of telomere transcriptional silencing. They state: "The results demonstrated a significant increase in the expression of the MPH3 and HSP32 upon Sir2 deletion, indicating that telomere silencing remains effective in the absence of X and Y'-elements". However, there are no statistical analyses performed as far as I can see. For some of the strains, the significance of the increased expression in sir2 (especially for MPH3) looks questionable. In addition, a striking observation is that the SY12 strain (with only three chromosomes) express much less of both MPH3 and HSP32 than the parental strain BY4742 (16 chromosomes), both in the presence and absence of Sir2. In fact, the expression of both MPH3 and HSP32 in the SY12 sir2 strain is lower than in the BY4742 SIR2+ strain. In addition, relating this work to previous studies of subtelomeric sequences in other organisms would make the discussion more interesting.