Identification of epigenetic modulators as determinants of nuclear size and shape
- National Cancer Institute, United States
- Department of Molecular Biology, University of Wyoming, United States
- High Throughput Imaging Facility (HiTIF), National Cancer Institute, NIH, United States
Figures
Figure 1 with 5 supplements
A high-throughput image-based screen for nuclear size and shape determinants.
(A) Schematic overview of the nuclear morphology screen to identify genes required for proper nuclear shape and size. Cells were cultured in the presence of siRNAs targeting specific genes in …
Figure 1—figure supplement 1
Reproducibility of nuclear morphology screen using human fibroblast cells.
(A) The initial screen was performed in two biological replicates on different days. Nuclear shape Z-scores of the two replicates (Replicate 1 and Replicate 2) of the nuclear shape screen. …
Figure 1—figure supplement 2
A schematic overview of nuclear shape and nuclear size image analysis pipeline and generation of Z-scores.
Independent Z-scores for nuclear size or nuclear shape were generated first by measuring nuclear circularity or area, respectively. Nuclear shape was measured by calculating circularity (circularity …
Figure 1—figure supplement 3
Nuclear shape hits that affected cell number.
(A) Z-scores of the top nuclear shape determinants which had low cell number. Error bars indicate the standard deviation. (B) Montage of nuclear shape determinants with low cell number revealed …
Figure 1—figure supplement 4
Nuclear shape Z-scores in comparison to nuclear shape, area, and perimeter raw scores of fibroblast hits.
(A) Nuclear shape hits were calculated by scoring circularity (circularity = 4πArea/perimeter2). Z-scores were generated to compare hits across the screen. Error bars indicate the standard …
Figure 1—figure supplement 5
Single cell values of nuclear shape hits compared to control cells.
(A–J) Frequency distribution of nuclear shape in cells treated with scrambled control or indicated siRNA. siRNA-treated samples generally show a shift of the entire distribution rather than loss or …
Figure 2 with 1 supplement
Identification of nuclear size determinants in human fibroblast cells.
(A) A diagram of the relationship between circularity used to calculate nuclear shape and nuclear perimeter and area revealing the possibility that nuclear size might affect nuclear shape. Nuclear …
Figure 2—figure supplement 1
Nuclear morphology data using human fibroblast cells reveal lack of correlation between lamin expression and nuclear morphology features.
(A) The fibroblast screen was performed in two biological replicates on different days. Lamin A/C expression Z-scores compared to nuclear size Z-scores in human fibroblast cells. Spearman’s …
Figure 3 with 7 supplements
Cell-type specificity of effector hits.
(A) Little overlap of hits for nuclear shape changes in immortalized human fibroblast cells compared to nuclear shape hits for the breast epithelial cell line MCF10AT. The MCF10AT screen was …
Figure 3—figure supplement 1
Validation of nuclear shape hits in fibroblast and MCF10AT cell lines.
Nuclear shape changes were measured by calculating mean circularity values on a per well basis. (A–J) Cells were treated with single siRNAs to the indicated target previously identified nuclear …
Figure 3—figure supplement 2
Validation of knockdown efficiency in both fibroblast and MCF10AT cell lines.
(A–H) Knockdown efficiency of indicated target gene in fibroblast and MCF10AT cells treated with a single siRNA. Error bars indicate standard deviation.
Figure 3—figure supplement 3
Identification of nuclear size determinants in MCF10AT cells.
(A) The MCF10AT screen was performed in two biological replicates on different days. Representation of normal nuclei and hits with enlarged nuclei identified by high-throughput screening in MCF10AT …
Figure 3—figure supplement 4
Functional protein association analysis using STRING for nuclear shape hits (A) in human fibroblast cells, (B) in MCF10AT cells.
Figure 3—figure supplement 5
Functional protein association analysis using STRING for nuclear size hits (A) in human fibroblast cells, (B) in MCF10AT cells.
Figure 3—figure supplement 6
Correlation plots of nuclear morphology data using MCF10AT human breast epithelial cells.
(A) The MCF10AT screen was performed in two biological replicates on different days. Lamin A/C expression Z-scores compared to lamin B1 Z-scores in MCF10AT cells. Spearman’s coefficient (r=0.8903). …
Figure 3—figure supplement 7
Identification of nuclear shape determinants in MCF10AT cells.
(A) The MCF10AT screen was performed in two biological replicates on different days. Representation of normal nuclei and nuclear shape hits identified by high-throughput screening in MCF10AT cells. …
Figure 4 with 1 supplement
In vitro binding of lamin A to histone H3.
(A) A diagram of constructs used in binding assays. All histone pulldown assays were performed at least three times. (B) Colloidal staining of purified recombinant proteins and histone pulldown …
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Figure 4—source data 1
Source data for Figure 4B.
- https://cdn.elifesciences.org/articles/80653/elife-80653-fig4-data1-v2.zip
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Figure 4—source data 2
Source data for Figure 4C.
- https://cdn.elifesciences.org/articles/80653/elife-80653-fig4-data2-v2.zip
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Figure 4—source data 3
Source data for Figure 4D.
- https://cdn.elifesciences.org/articles/80653/elife-80653-fig4-data3-v2.zip
Figure 4—figure supplement 1
Lamin C inhibits lamin A-H3 interactions.
(A) Diagram of mature lamin A and progerin constructs used in the binding assays. All histone pulldown assays were performed at least three times. (B) GST pulldown assay with wild-type GST-lamin A …
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Figure 4—figure supplement 1—source data 1
Source data for Figure 4—figure supplement 1B.
- https://cdn.elifesciences.org/articles/80653/elife-80653-fig4-figsupp1-data1-v2.zip
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Figure 4—figure supplement 1—source data 2
Source data for Figure 4—figure supplement 1C.
- https://cdn.elifesciences.org/articles/80653/elife-80653-fig4-figsupp1-data2-v2.zip
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Figure 4—figure supplement 1—source data 3
Source data for Figure 4—figure supplement 1D.
- https://cdn.elifesciences.org/articles/80653/elife-80653-fig4-figsupp1-data3-v2.zip
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Figure 4—figure supplement 1—source data 4
Source data for Figure 4—figure supplement 1E.
- https://cdn.elifesciences.org/articles/80653/elife-80653-fig4-figsupp1-data4-v2.zip
Figure 5
Specificity of lamin A binding histone modifications.
(A) In vitro peptide binding array assay using GST-lamin A (506–646). Intensity of signal indicates binding. The peptide binding assay was performed three times. (B) Peptide binding assays for …
Figure 6 with 5 supplements
Expression of histone H3.3 mutants affect nuclear shape.
(A) Wild-type (WT) and mutant histone expression constructs . Mutant histone expression experiments were performed with three biological replicates. (B) Stable expression of indicated H3.3 mutants …
Figure 6—figure supplement 1
Histone H3.3 total expression relative to wild-type H3.3-V5 and H3.3-V5 mutant expression.
Correlation plot of total histone H3.3 expression relative to (A) V5 alone, (B) wild-type H3.3-V5, (C) H3.3K9M-V5 expression, (D) H3.3K27M-V5 expression, (E) H3.3K36M-V5 expression. Values …
Figure 6—figure supplement 2
Histone H3.1 mutants display a lesser nuclear morphology phenotype compared to wild-type H3.1.
(A) Cells expressing wild-type histone H3.1, H3.1K9M, or H3.1K27M mutants showed little change in nuclear shape scores. The mean is indicated by the horizontal line. Mutant histone expression …
Figure 6—figure supplement 3
Nuclear shape score relative to wild-type H3.3-V5 and H3.3-V5 mutant expression.
Correlation plot of nuclear shape scores relative to (A) wild-type H3.3-V5 expression, (B) H3.3K9M-V5 expression, (C) H3.3K27M-V5 expression, (D) H3.3K36M-V5 expression. Values determined by …
Figure 6—figure supplement 4
Lamin A/C expression relative to wild-type H3.3-V5 and H3.3-V5 mutant expression.
Correlation plot of lamin A/C expression relative to (A) wild-type H3.3-V5, (B) H3.3K9M-V5 expression, (C) H3.3K27M-V5 expression, (D) H3.3K36M-V5 expression. Values determined by quantitative …
Figure 6—figure supplement 5
Single cell analysis of nuclear size and nuclear shape in cells expressing wild-type and mutant histone H3.3.
Correlation plot of nuclear size values relative to nuclear shape values in cells expressing (A) V5 alone, (B) wild-type H3.3-V5, (C) H3.3K9M-V5, (D) H3.3K27M-V5, (E) H3.3K36M-V5. Values determined …
Additional files
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Supplementary file 1
High-throughput screening targets and hits.
(A) lists the genes targeted in the screen. (B) is a file describing the plasmids generated during this study. (C) lists the antibodies used in this study. (D) is a comparative list of nuclear shape Z-score, nuclear shape raw score, nuclear area, and nuclear perimeter measurements.
- https://cdn.elifesciences.org/articles/80653/elife-80653-supp1-v2.xlsx
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Supplementary file 2
Nuclear shape hits.
(A) lists hits altering nuclear shape in fibroblast cells. (B) is a list of hits resulting in lower lamin A/C expression. (C) is a list of hits resulting in lowered lamin B1 expression.
- https://cdn.elifesciences.org/articles/80653/elife-80653-supp2-v2.xlsx
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Supplementary file 3
Screen validation.
(A) lists validation results for the nuclear shape screen in fibroblast cells. (B) lists validation results for hits increasing nuclear size in fibroblast cells. (C) lists validation results for hits decreasing nuclear size in fibroblast cells. (D) identifies nuclear shape hits in MCF10AT cells. (E) lists lamin A interacting peptides.
- https://cdn.elifesciences.org/articles/80653/elife-80653-supp3-v2.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/80653/elife-80653-mdarchecklist1-v2.pdf
