Peer review of: The Rad53CHK1/CHK2-Spt21NPAT and Tel1ATM axes couple glucose tolerance to histone dosage (2020)

Reviewers' comments: 
 
Reviewer #1 (Remarks to the Author): 
 
In this manuscript entitled: “The Rad53CHK1/CHK2-Spt21NPAT and Tel1ATM axes couple glucose 
tolerance to histone dosage and subtelomeric silencing”, the authors investigate the links between 
histone homeostasis and glucose metabolism. They show that Rad53 and Spt21 regulation of 
histone levels mediate tolerance to glucose restriction. Additionally, the authors explore potential 
mechanisms including subtelomeric silencing and acetyl-CoA regulation to expand on the initial 
findings. 
 
The following points should be considered when revising this manuscript: 
 
Major: 
 
1. Overall, the narrative of the manuscript is confusing. The results section is clearly defined in 
different sections that make sense and are well written independently, but are poorly linked with 
each other. Additionally, in the introduction, the authors jump back and forth between a few 
subjects without a clear argument or a direct link to the results section. For example, there is a 
substantial section describing DNA damage and DDR while the rest of the paper explores non-DDR 
functions of Rad53. Finally, the claims made throughout lines 12 onwards in the abstract are too 
broad and only partly supported by the data presented. 
 
2. The authors claim in Figure 4 that hyper-acetylation of histones leads to glucose dependence by 
limiting Ac-CoA. While it is shown that Rad53 deletion leads to reduction of Ac-CoA, it is also 
necessary to show what happens to the TCA metabolites and Ac-CoA following Spt21S276A-driven 
histone acetylation. In line with this, figure S4A should be included as part of the main figure. 
Furthermore, although acetate alleviated the glucose consumption, no report of the levels of TCA 
cycle intermediates or Ac-CoA following the rescue with acetate were presented. Metabolomics 
experiments using labelled acetate should be used to show the incorporation of acetate into Ac-
CoA. 
 
3. Overall, the section on subtelomeric silencing on page 6 is confusing, and mainly correlative 
evidence is used to draw conclusions that are not clearly supported by data. Firstly, the authors 
show global mRNA alterations that are histone dependent, none of which are pursued any further. 
Additionally, subtelomeric derepression appears to be a general response to different cellular 
stress, the implications specific to glucose starvation are not controlled appropriately. In line with 
this, there are a few mentions of subtelomeric gene repression being independent from the carbon 
source, which somehow contradicts the general model presented without any further justification. 
 
 
Minor: 
 
1. In various experiments throughout the manuscript, the authors use EtOH or low glucose 
conditions interchangeably. It would be essential for the interpretation of those experiments to 
show some evidence that the metabolic changes observed are similar under both conditions. 
 
2. The panels presenting yeast growth experiments are not clearly explained in the figure legends 
and are unclear to a reader with limited knowledge about yeast as a model. Additionally, 
quantification of those growth assays would add more robustness to the data. 
 
3. In different experiments, 0.01% or 0.02% glucose conditions are used inconsistently without 
any justification. 
4. The use of superscript to identify the human homolog of the genes/proteins mentioned is useful 
and should be used across the whole manuscript. 
 
5. Western blots with accompanying bar chart quantification should be described as such in the 
figure legends. 
 
6. Figure 2D: Which “arrows” is the figure legend referring to? 
 
7. There are a few statements that are very generic or confusing, consider rewording those to be 
more relevant to the subject: 
o Page 3, line 19: “Metabolic adaption and reprogramming involve transcriptional programs” 
o Page 3, line 7: “Metabolic pathways and cell division […] metabolite pools” 
o Page 3, line 31: “Reactive metabolites are a major source of endogenous DNA lesions” 
 
8. Apart from the RNA-Seq and metabolite analysis, the statistics presented in the figures are not 
described. All statistical methods should be described clearly in the methods and figure legends. 
 
 
 
Reviewer #2 (Remarks to the Author): 
 
It has been known that histone dosage is connected to the DNA damage response through the 
DNA damage signaling checkpoint kinase Rad53. Here the authors extend those observations by 
showing that Rad53 mutants have metabolic changes that render them sensitive to glucose. They 
explore the reasons for the glucose sensitivity and link this to changes in histone modification and 
gene silencing of subtelomeric chromosome regions. Finally, the authors suggest that in cancer 
cells, the DDR kinases may coordinate epigenetic modifications to metabolism in a similar way and 
may influence carbon-directed cancer treatments. 
 
This is an extremely complex topic and the yeast system lends itself well to deciphering the key 
components of the process of linking DNA repair and carbon metabolism to histone levels. The 
work is well done but the presentation is very dense, in both the text and the figures. Even the 
abstract and introduction are difficult to get through because so many points of information are 
presented, the direct flow of ideas gets lost. The focus seems to be on glucose tolerance and the 
DNA damage response, but this is not apparent in the abstract until halfway through it. 
 
It seems that the TEL1 mutant TEL1-hy909 is key to some of the studies and really separates 
subtelomeric repression from histone control. This should be emphasized more and perhaps merits 
its own subsection in the Main results section. 
 
Figure images of drop tests. The growth differences on limiting glucose are more apparent in the 
online images than the printed images. The images seem to be too contrasty. 
Figure 1C. Why is the effect of limiting glucose more dramatic for H4 levels than H3 levels? 
Figure 2F. What is the difference between the two red samples for 14R? Why do they give different 
P values compared to sml1∆? 
Figure S4. It is interesting that the THO/TREX complex comes up in the screen. It was reported by 
Schneiter et al that acc1 is synthetic lethal with hpr1, perhaps linking histone levels or 
modifications with DNA damage and subcellular localization of damage. Similarly, Fan et al found 
that hht1-hhf1 combined with hpr1 had growth defects, again linking histone levels with DNA 
damage from this mutant. 
 

Reviewer #3 (Remarks to the Author): 
 
This work present evidence in budding yeast that a basal level of Rad53 downregulates the histone 
level through the inhibition of Spt21. Upon Rad53 loss, the level of histone increases leading to 
glucose dependency. By the elegant use of several separation function mutants, the authors show 
that maintaining a physiological level of histone by Rad53-Spt21 is required for glucose tolerance 
via two parallel pathways : derepression of subtelomeric genes and acetyl-coA regulation by 
histone acetylation. These results are important, providing a new link between central DDR factors 
and metabolic homeostasis. The following points should be addressed in a revised version of the 
manuscript. 
- It is not clear how the two pathways downstream of histone dosage are independent since an 
aberrant level of histone acetylation is expected to alleviate TPE. 
- It was previously shown that an inhibition of the TOR nutrient sensing pathway counteracted TPE 
through a mpk1-dependant Sir3 phosphorylation. Even if the authors mention that TOR inhibition 
does not lead to histone increase, the authors should explore deeper the connection between the 
Razd53-Spt21 and the Tor-mpk1-Sir3 pathway upon glucose starvation and Tor inhibition by 
rapamycin. 
- It is unclear from the presented data whether Rad53 activation upon DNA damage will further 
potentiate the spt21 pathway of histone regulation.