Supplemental Material for Shen and Skibbens, 2020
Donglai Shen
Robert V. Skibbens
10.25386/genetics.10283972.v1
https://gsajournals.figshare.com/articles/dataset/Supplemental_Material_for_Shen_and_Skibbens_2020/10283972
<p></p><p><b>Supplemental
Figure 1. Chl1 DNA helicase does not function in hyperthermic-induced rDNA
hypercondensation.</b>
A) Flow cytometer data of DNA content. Log phase cultures were synchronized in
G1 using alpha factor and aliquots released into either 23°C or 37°C fresh
media supplemented with nocodazole and incubated for 3 hours. B) Chromosomal
mass and rDNA loop structures detected using DAPI. Yellow arrows indicate long
rDNA loops. Yellow arrowheads indicate short rDNA loops. All field of views are
shown at equal magnification. C) Quantification of condensed rDNA loop lengths
in wildtype (YPH499) and <i>chl1</i> deletion mutant cells (YBS1141). Data
shown was obtained from 3 biological replicates with at least 100 cells for
each strain analyzed per replicate. Error bars represent standard deviation of
each sample. Statistical analysis was performed using Tukey HSD one way ANOVA.
P-Value = 0.001 indicates a significant difference between the average loop
lengths of wildtype cells at 23°C versus 37°C. P-Value = 0.001 indicates
significant differences between the average loop lengths of <i>chl1</i> mutant
cells at 23°C versus 37°C. P-Value = 0.899 indicates there is no significant
difference between the average loop lengths of wildtype versus <i>chl1</i>
mutant cells at 23°C. P-Value = 0.882 indicates there is no significant
difference between the average loop lengths of wildtype versus <i>chl1</i> mutant
cells at 37°C. Statistically significant differences (*) are based on P <
0.05. Wildtype rDNA loop measurements
adapted from Figure 1E, Shen and Skibbens 2017a, under the terms of the
Creative Commons Attribution-NonCommercial-NoDerivatives License.</p>
<p> </p>
<p><b>Supplemental
Figure 2. rDNA hypercondensation can be induced prior to anaphase onset.</b> A) Flow cytometer data
of DNA content. Log phase cultures of <i>cdc23-1</i> were synchronized in G1
using alpha factor and aliquots released into fresh medium along or medium supplemented
with DMSO (+DMSO) or nocodazole (+NZ) and incubated for 3 hours at 37°C. B)
Chromosomal mass and rDNA loop structures were detected using DAPI. All field
of views are shown at equal magnification. Red arrows indicate distorted
chromatin structure. Yellow arrows indicate hypercondensed rDNA loops.
Hypercondensed rDNA loops are clearly present in cells that harbor
temperature-sensitive alleles of <i>cdc23-1</i> (FLY83/H23C1AX) at all three
conditions. </p>
<p> </p>
<p><b>Supplemental
Figure 3. Independent confirmation that Hsp90 protein </b><b>promotes
hyperthermic-induced rDNA hypercondensation and the impact of </b><b>Hps90
inhibitors.</b>
A) Flow cytometer data documents DNA content for wildtype (BY4741)
and <i>hsc82</i> (YDS210) null cells. Log phase cells were synchronized in G1
using alpha factor, then released into 37°C fresh medium supplemented with
nocodazole for 3 hours to arrest cells in preanaphase. Wildtype cells were
further treated with Geldanamycin (GA) or Radicicol (RD), in addition to NZ,
which is reflected by duplicated Log and G1 DNA profiles for those treatments.
B) Chromosomal mass and rDNA loop structures detected using DAPI. White arrows
indicate the track of rDNA long loops, arrowheads indicate rDNA short loops. C)
Quantification of loop lengths of condensed rDNA in wildtype (BY4741) and <i>hsc82</i>
null cells (YDS210). At least 100 cells for each strain were analyzed. Error
bars represent standard deviation of each sample. Statistical analysis performed
using Tukey HSD one-way ANOVA. P-Value = 0.001 indicates significant differences
between the average loop lengths of wildtype cells versus the <i>hsc82 </i>mutant
cells at 37°C. Statistically significant differences (*) are based on P <
0.05.</p>
<p> </p>
<p><b>Supplemental
Figure 4. Geldanamycin inhibits Hsp90 ATPase activity, resulting in Chl1
degradation.</b>
Cells (YBS1129) expressing MYC-tagged Chl1 were exposed to short (3 hours)
versus long (24 hours) incubations in 37°C fresh medium supplemented with
nocodazole and either vehicle (DMSO) or 40µM GA, post G1
synchronization/release. As previously reported, cells exposed to 37°C in
medium supplemented with nocodazole (NZ) and Geldanamycin (GA) for short
durations (3 hrs) retain relatively high levels of Chl1. In contrast, cells
exposed to GA for an extended period of time (24 hrs), contain dramatically
reduced levels of Chl1, compared to DMSO vehicle. Phosphoglycerate kinase (PGK)
levels are shown as an internal loading control.</p>
<p> </p>
<p><b>Supplemental
Figure 5. Diploidization of hmo1 strain obtained from the yeast deletion
collection. </b>A)
Chromosomal mass and rDNA loop structures detected using DAPI. The majority of
DNA masses exhibit two rDNA loops. B) Flow cytometer of DNA content. Log phase
wildtype (YDS202) and<i> hmo1</i> null strains (obtained from yeast deletion
collection – a generous gift from Prof Greg Lang) were split and equal portions
placed in fresh medium supplemented with nocodazole and incubated for 3 hours
at either 23°C or 37°C. </p><br><p></p>
2020-01-23 14:02:08
Cohesin
condensin
Hsp90/Hsp82/Hsc82
Chaperone
HMG proteins
rDNA
Chromatin
Cell Biology
Genetics
Physiology