Histone H3G34R mutation causes replication stress, homologous recombination defects and genomic instability in S. pombe
- St. Jude Children’s Research Hospital, United States
- Fox Chase Cancer Center, United States
- Icahn School of Medicine at Mount Sinai, United States
- University of Edinburgh, Scotland
Figures
Figure 1 with 2 supplements
Post translational modification of H3K36 is altered in H3-G34R mutants.
(a) Scheme of the histone H3 (hht) and histone H4 (hhf) genes in S. pombe highlighting the H3 gene (hht2) in which mutations were engineered (blue), and representation of the H3-WT and H3-G34R …
Figure 1—figure supplement 1
Characterization of the H3-G34R mutants.
(a) Clustal W alignment of histone H3 protein sequences for fission yeast (H3pom), human histone H3.3 (product of H3F3A) and histone H3.1 (product of HIST1H3A). The G34 residue mutated in this …
Figure 1—figure supplement 2
Characterization of cell cycle regulation of H3K36 methylation during the cell cycle.
(a) Thermosensitive cell cycle mutations were incorporated into H3-WT and H3-G34R strains and cells were maintained at permissive temperature (P) or shifted to restrictive temperature (R) before …
Figure 2 with 1 supplement
H3-G34R mutants show a distinct transcriptional profile to set2Δ cells.
Schematic of genes that are (a) differentially-regulated, or (b) similarly down-regulated or up-regulated comparing either H3-G34R or set2Δ cells with H3-WT cells in RNA-seq analyses. Triplicate …
Figure 2—figure supplement 1
Heterochromatin integrity is maintained in H3-G34R cells.
(a) Real-time (RT) PCR analysis of pericentromeric imr, dg, and dh expression as well as the subtelomeric tlh expression in the indicated strains. Transcripts were compared relative to adh1+ …
Figure 3
H3-G34R cells exhibit genomic instability.
(a) Frequency of cells that lose the non-essential minichromosome Ch16 in H3-WT, H3-G34R, set2Δ and swi6Δ cells. Asterisk indicates significant difference from H3-WT (p<0.05). Mean ± SEM from 4 …
Figure 4
H3-G34R mutants show DNA damage sensitivity upon replication stress.
(a) 5-fold serial dilutions showing the effect of methyl methanesulfonate (MMS), camptothecin (CPT), and hydroxyurea (HU) on the indicated strains. (b) Effect of γ−irradiation (IR) exposure on …
Figure 5
H3-G34R cells have intact DNA replication checkpoints.
(a) Scheme depicting major kinases involved in checkpoint control and in brackets, their mammalian homologs. (b) Western blot analysis of γ-H2A phosphorylation in WT, G34R, and set2Δ cells. Cells …
Figure 6 with 3 supplements
H3-G34R cells show defects in homologous recombination pathways.
(a) Scheme depicting DNA damage repair pathways. (b) Cells of indicated genotypes were cultured at 25°C in the presence or absence of 11 mM HU for 6.5 hr and then washed and either kept at 25°C …
Figure 6—figure supplement 1
Characterization of H3-G34R and set2Δ cells in homologous recombination-directed repair.
(a) Schematic illustrating the experimental set up for the HR efficiency assay (see Figure 6f). All cells contained a point mutant in leu1 gene (leu1-32) and were transformed with either plasmid DNA …
Figure 6—figure supplement 2
FACS analysis of cdc10-M17 arrested and released cells (G1) on the left, or mitotically arrested and released cells (nda3-km311) on right.
https://doi.org/10.7554/eLife.27406.013
Figure 6—figure supplement 3
Localization of homologous recombination-directed repair proteins in H3-G34R cells.
(a) Representative images showing foci formation of RPA (Rad11-GFP) or Rad52-YFP in either H3-WT or H3-G34R backgrounds. Cells were either treated in the presence (right) or absence (left) of 0.05% …
Figure 7
H3-G34R mutants exhibit delayed recovery from replicative stress.
(a) Cell proliferation of H3-WT, H3-G34R, and cds1Δ cells was analyzed after synchronization in 11 mM HU for 4 hr. Cells were counted at 30 min intervals following release. Results represent average …
Figure 8
Model illustrating how HR defects in H3-G34R mutants may compromise genomic integrity.
Model illustrates the key defects in H3-G34R cells and how these may contribute to genomic instability. Replication forks provide an endogenous source of DNA damage, as they are fragile structures …
Additional files
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Supplementary file 1
Peptides used for antibody characterization and mass spectrometry calibration.
- https://doi.org/10.7554/eLife.27406.017
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Supplementary file 2
Detection parameters of unique tryptic peptides from S. pombe H3.
- https://doi.org/10.7554/eLife.27406.018
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Supplementary file 3
Gene expression changes in H3-G34R and set2Δ relative to H3-WT.
- https://doi.org/10.7554/eLife.27406.019
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Supplementary file 4
S. pombe strains.
- https://doi.org/10.7554/eLife.27406.020
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Supplementary file 5
Primers used for real time PCR analyses
- https://doi.org/10.7554/eLife.27406.021
