Author Response

Reviewer #1 (Public Review):

“I recommend that the authors revisit their calculation methods to provide a more convincing conclusion on the presence of positive epistasis for fitness in their dataset.”

The reviewer is right that the present description of the fitness calculation can be found insufficient. Below we provide relevant derivation. It will be included in our planned revision, probably as a supplementary text:

Expected fitness effect of multiple mutations

Fitness is the number of offspring divided by the number of progenitors, w=No/Np. This can be the number of cells left by one cell (including itself in the case of budding cells) over a unit of time.

Assume that an organism carries multiple mutations—α, β, … ω—which are in heterozygous loci, their wild-type counterparts are marked universally with +. The fitness effect of a single mutation is wα/+, and so on. Fitness can be converted to relative fitness, i.e., expressed as a quotient of the wild-type fitness, wα/+/w+/+, and so on. Under the multiplicative model of mutation accumulation, an expected joint effect of multiple mutations on relative fitness is a product of individual quotients:

wexp/w+/+ = (wα/+/w+/+) (wβ/+/w+/+) … (wω/+/w+/+).

When a population is continuously growing, log-transformation of fitness is typically applied as it equates the rate of growth. In particular, it could be the number of doublings completed over a unit of time:

log2(wexp/w+/+) = log2[(wα/+/w+/+) (wβ/+/w+/+) … (wω/+/w+/+)].

After replacing the above log multiplicative formula with its log additive equivalent, all its terms can be normalized by dividing by log2 fitness of the wild type which turns them into relative doubling rates, for example, log2(wα/+)/log2(w+/+)=rDRα/+. The joint effect of multiple mutations is then equal to

rDRexp = 1 + (rDRα/+ ̶ 1) + (rDRβ/+ ̶ 1) + … + (rDRω/+ ̶ 1)

or

rDRexp = 1 + ∑d

where d=rDR ̶ 1 (see Fig. 2B in the main text).

Reviewer #2 (Public Review):

“The initiation and interpretation of the results were apparently performed in a vacuum of a century of work on genomic balance.”

Indeed, we neither introduce nor discuss results obtained with organisms other than the budding yeast. We accept that researchers working with other beasts may expect to see such considerations. We will introduce them into a revised submission, to the extent allowable for non-review articles. The suggestions provided by the reviewer will be followed.

“If there is an increase in the general transcriptome size, then there might not be much reduction of the proteosome subunits as claimed and the increases might be somewhat less than indicated.”

- together with –

“A second experiment that would clarify the results would be to perform estimates of the general transcriptome size. If the general transcriptome size is actually increased, the claims of reduced expression of the proteosome might need to be revised.”

Multicellular organisms may be well heterogenic across their bodies, in terms of the cell number and (transcriptome) composition. We believe that any proper sampling of their mRNA requires careful absolute, and not only relative, quantification. We worked with clones prepared to be mostly homogeneous (about two-three divisions under conditions promoting strong growth). We maintain that we are allowed to rely on chromosomal averages expressed as proportions of the total mRNA. Our monosomic counts were very strictly around 50% of those predicted for euploids, we do not see any danger of erroneous sampling or calculation in our wonderfully simple case. Regarding the problem of a possible general increase in the transcriptome size which would help to compensate for the decrease in the proteasomic mRNAs, we adopted a strictly linear interpretation. That is, in a doubled transcriptome not only mRNAs for the proteasome but also all other proteins (prey species for the proteasome) would be doubled and thus no relief in proteolysis would happen. We follow here previous findings that in yeast the fractions of individual mRNAs are reflected in the fractions of ribosome-bound mRNA fragments and (at least roughly) mature proteins (e.g., the cited by us Larrimore et al. 2020).

“The claim of Torres et al that there are no global modulations in trans is counter to the knowledge that transcription factors are typically dosage sensitive and have multiple targets across the genome … Taken as a whole it would seem to suggest that there are many inverse relationships of global gene expression with chromosomal dosage in both yeast disomies and monosomies.”

Well, the debate about mRNA compensation in relation to aneuploidy in yeast has been intense and sometimes heated. Disparate claims can be found but we are left with an overwhelming impression that the relation between the total amount of mRNA and the number of chromosome copies is pretty much (perhaps not ideally) linear. We will consider our wording again. We will admit that such a rigid relation is somewhat unusual compared to other eukaryotes. But again, we see strict halves of mRNA for the monosomic chromosomes.

“To clarify the claims of this study, it would be informative to produce distributions of the various ratios of individual gene expression in monosomy versus diploid as performed by Hou et al. 2018.”

Such distributions are already prepared and will be likely presented in a revised msc.

“The authors claim there are no genes that are compensated on the varied chromosome but considering how many genes are upregulated across the genome, it would seem that a subset are probably upregulated on the cis chromosome as well and approach the diploid level, i.e. are dosage compensated.”

Perhaps we misstated our conclusions somewhere but it was obvious to us that some genes were upregulated on the cis chromosome (monosomic), some other were downregulated, the net result was the average 50% (Fig. 3).

Reviewer #3 (Public Review):

“1) In Figure 3b (and line 179) … What the data really show is that the level of overexpression is not correlated with the fitness effect of the deletion (since all the p values are not significant). The authors need to correct their conclusions.”

That’s right! We already mended it as we are preparing for revision.

“2) Why are some monosomic strains removed from the transcriptomics analysis, especially when the chromosome IV and XV strains show very strong positive epistasis? The authors need to provide an explanation here.”

We run out of money. In more scientific terms, we believe our sample of eight strains is unbiased and sufficient. It was truly randomly chosen. We were glad to see that it covers both slightly and most strongly affected (with profound epistasis) monosomics. All of them displayed parallel shifts in the transcriptome (RP up, proteasome down). We judged we could stop here, the additional five strains would be unlikely to change our main conclusions.

“3) The authors stated that diploidy observed in chromosome VII and XIII strains were due to endoreplication after losing the marked chromosomes (lines 97 and 117). Isn't chromosome missegregation an equally possible explanation? Since monosomic cells are generated by chromosome missegregation during mitosis, another chromosome missegregation event may occur to rescue the fitness (or viability) of monosomic cells in these strains.”

We believe that it happened in this way as the reviewer suggests, at least in most cases. By “endoreduplication”, we understand any event making two chromosomes of one, not necessarily additional DNA replication. We will check our text to make it clear in this respect.

Figures and data

Diploid yeast strains lacking single chromosomes (monosomics).

Contribution of positive epistasis to fitness of monosomics.

Absence of transcriptional compensation in monosomic strains.

Parallel and divergent shifts in transcriptomes of monosomic strains.

Parallel and divergent shifts in transcriptomes of monosomic strains.

DNA whole genome sequencing coverage after the postulated endoreduplication.