PHD1-dependent hydroxylation of RepoMan (CDCA2) on P604 modulates the control of mitotic progression
- Division of Molecular Cell and Developmental Biology, School of Life Sciences, University of Dundee, United Kingdom
- Department of Biochemistry, Cell and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, United Kingdom
- Cancer Research, School of Medicine, University of Dundee, United Kingdom
Figures
Figure 1
RepoMan localisation across the cell cycle.
(A) Immunofluorescence analysis of endogenous RepoMan in HeLa cells along the cell cycle. CDCA2 antibody was used to detect endogenous RepoMan. DNA was stained with DAPI. Scale bar represents 5 μm. (B) Time lapse of YPF-RepoMan in cells treated with thymidine for 24 hr and release in normal media. DNA was stained with sirDNA. YFP-RepoMan in green and DNA in red. Scale bar represents 5 μm.
Figure 2
Endogenous RepoMan interacts with EGFP-PHD1.
(A) Asynchronous HeLa cells were transiently transfected with 1 µg of EGFP-PHD1 expression vector and after 48 hr were fixed with 4% PFA and subjected to immunofluorescence using CDCA2 and GFP antibodies (endogenous RepoMan and EGFP-PHD1, respectively). DNA was stained with DAPI. The images show the localisation of endogenous RepoMan and EGFP-PHD1 along the cell cycle: interphase, prometaphase, metaphase, and anaphase. Scale bars represent 5 µm. (B) Colocalisation of GFP-PHD1 with endogenous RepoMan is shown. The graph displays the median of Pearson’s correlation coefficient measured (using Costes’ automatic threshold) in asynchronous cells captured in different stages of the cell cycle (interphase, (pro)metaphase, and anaphase). Unpaired t-test p=0.0531 interphase vs (pro)metaphase, p<0.0001 anaphase vs (pro)metaphase, p<0.0001 anaphase vs interphase. (C) Short sequence similarity between HIF1α and RepoMan around prolines 564 and 604 (PYIP, PSIP), respectively. (D) Co-immunoprecipitation between EGFP-PHD1 and endogenous RepoMan. Asynchronous HeLa cells were transiently transfected with EGFP-PHD1. After 48 hr cells were lysed and subject to immunoprecipitation using GFP trap magnetic beads and analysing by western blot.
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Figure 2—source data 1
TIFF files containing full western blots for data presented in Figure 2.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig2-data1-v1.zip
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Figure 2—source data 2
PDF file containing full western blots for data presented in Figure 2, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig2-data2-v1.pdf
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Figure 2—source data 3
Data utilised to generate the graph in Figure 2B.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig2-data3-v1.pdf
Figure 3 with 1 supplement
PHD1 regulates phosphorylation of H3T3 during prometaphase.
(A) Schematic model of the role of PP1-RepoMan during prometaphase. (B) HeLa cells were transfected with siControl, or siRNA RepoMan. Cells were arrested in prometaphase with nocodazole 100 ng/mL for 16 hr and released from the arrest for 1 hr in normal media before fixation and stained with pH3T3 and DAPI. (C) Cells were treated as in (B) and harvested for western blot analysis. (D) Asynchronous or prometaphase-arrested HeLa cells were treated with FG4592 50 µM for 2 hr or DMSO. In case of mitotic cells, they were treated 1 hr before the release and during the release for another hour. Cells were lysed and subject to western blot. (E) Asynchronous or prometaphase arrested HeLa cells were treated with 100 µM fumarate for 1 hr. In case of mitotic cells, they were treated for 1 hr during the release. Cells were lysed and subject to western blot. (F) Immunofluorescence images of HeLa cells arrested in prometaphase. HeLa cells were transfected with siControl, siPHD1, siPHD2 (as in B). Prometaphase-arrested cells were released for 1 hr before fixation. Image scale bars represent 5 µm. (G) Graph displays the normalised pH3T3 intensity of total cells per condition from 3 independent experiments (n = 48 si control, n = 62 si PHD1, n = 47 siPHD2). pH3T3 intensity of each condition (siPHD1 and siPHD2) was normalised to the intensity values of pH3T3 in the siControl. The average of 3 independent experiments is shown. Unpair t-test ****p<0.0001, sicontrol vs siPHD1 and siPHD1 vs siPHD2; n.s, sicontrol vs siPHD2 p = 0.8963. (H) Western blot analysis of HeLa cells treated as in (F) and including asynchronous cells transfected with the control siRNA.
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Figure 3—source data 1
TIFF files containing full western blots for data presented in Figure 3.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig3-data1-v1.zip
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Figure 3—source data 2
PDF file containing full western blot for data presented in Figure 3, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig3-data2-v1.pdf
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Figure 3—source data 3
Data utilised to generate the graph in Figure 3G.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig3-data3-v1.pdf
Figure 3—figure supplement 1
siPHD1 is effective at depleting PHD1 protein.
(A) Immunofluorescence analysis of siPHD1 knockdown in HeLa cells transiently transfected with EGFP-PHD1. siControl was used as a control. GFP antibody was used for EGFP-PHD1, and DNA was stained with DAPI. Scale bar represents 10 µm. (B) Western blot analysis of siPHD1 or si PHD1 UTR knockdown on HEK293 cells stably expressing GFP-PHD1 or GFP empty vector.
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Figure 3—figure supplement 1—source data 1
TIFF files containing full western blots for data presented in Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
PDF file containing original western blot for Figure 3—figure supplement 1, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig3-figsupp1-data2-v1.pdf
Figure 4 with 1 supplement
P604 in RepoMan is required for correct localisation and function in mitosis.
(A) Western blot analysis of HeLa-YFP-RepoMan-wt or the P604A mutant induction in the presence of 1 µg/mL doxycycline. Cells were transfected with the siRNA of RepoMan and after 24 hr cells were induced with doxycycline for another 24 hr before harvested. Blots were developed with GFP, RepoMan, and actin antibodies. (B) Immunofluorescence of HeLa-YFP-RepoMan-wt or the P604A synchronised in prometaphase with nocodazole after siRM knockdown. Dox was used to induce the expression of the YFP proteins. Anti phH3T3, GFP antibodies were used, and DNA was stained with DAPI. (C) Quantification of (B) in cells treated with siRM, with or without doxycycline induction. The graph displays the distribution of phH3T3 on prometaphase cells. Graph represents the percentage of cells showing phH3T3 localisation as foci (centromeric) or diffuse localisation (along chromosome arms). Average of 4 independent experiments with a total number of cells 46 for siRM (not induced), 46 for siRM+ YFPRMwt, and 52 for siRM+YFP-P604A (9–20 cells per condition per experiment). Error bars represent standard deviation (SD). Unpair t-test, control vs wt ***p = 0.0002 ,** wt vs P604A p=0.0087 and n.s , P604A vs control p = 0.1254.
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Figure 4—source data 1
TIFF files containing full western blots for data presented in Figure 4.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig4-data1-v1.zip
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Figure 4—source data 2
PDF file containing full western blots images for data presented in Figure 4, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig4-data2-v1.pdf
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Figure 4—source data 3
Data utilised to generate the graph in Figure 4C.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig4-data3-v1.pdf
Figure 4—figure supplement 1
siRepoMan is effective at depleting endogenous but not exogenous RepoMan.
HeLa cells were transfected with siRNA control or siRNA against endogenous RepoMan (siRM). 24 hr later, cells were incubated with thymidine 2 mM±doxycycline for 24 hr. After the incubation time, cells were released from the thymidine for 2 hr in normal media and nocodazole±doxycycline was added for 14–16 hr. Cells were lysed, and protein extracts analysed by western blot.
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Figure 4—figure supplement 1—source data 1
TIFF files containing full western blots for data presented in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
PDF file containing original western blot for Figure 4—figure supplement 1, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig4-figsupp1-data2-v1.pdf
Figure 5 with 3 supplements
RepoMan-P604 hydroxylation is required for the recruitment of B56γ in prometaphase cells.
(A) Schematic representation of RepoMan proline 604 proximity to B56-PP2A binding site (LSPIxE). (B) Proximity ligation assay (PLA). Graph represents the number of PLA foci per cell in the indicated conditions (monastrol arrested or asynchronous). Quantified PLA foci are located in the YFP area (RepoMan localisation) of the mitotic cells (phSer10 positive cells). Endogenous RepoMan was knocked down using siRM. Median is shown in red. Statistical analysis was performed using an unpaired t-test, and the p-value comparing wt vs mutant under monastrol treatment is <0.0001. Number of cells per condition, in the same order as shown, are: 229, 301, 237, 71, 130, and 139. Before PLA reaction, cells were fixed with PFA 4% and stained with Anti-GFP+B56γ antibodies or only GFP antibody was used as a negative control. After PLA reaction cells were stained with anti-phH3Ser10 as a mitotic marker. (C) Co-immunoprecipitation between YFP-RepoMan-wt or P604A mutant with endogenous B56γ. Endogenous RepoMan was knocked down using siRM. 16 hr after transfection, YFP-RepoMan was induced with 1 µg/mL doxycycline and cells were synchronised in prometaphase. Cells lysates were subject to immunoprecipitation using GFP trap magnetic beads and analysed by western blot. (D) Co-immunoprecipitation between endogenous RepoMan and endogenous B56γ in cells depleted or not of PHD1 using siRNA. HeLa cells were transfected with siRNA (PHD1 or ctl), 24 hr later cells were incubated with thymidine 2 mM for another 24 hr. After the incubation time, cells were released from the thymidine block, for 2 hr in normal media and arrested in prometaphase with nocodazole for 14 hr. Cells were harvested by shake-off and lysed for immunoprecipitation of endogenous RepoMan. (E) Western blot analysis of HeLa-YFP-RepoMan-wt or P604A/mCherry B56γ cell line. Cells were depleted of endogenous RepoMan, and the indicated YFP-RepoMan variants were induced with doxycycline. GFP, CDCA2 (RepoMan), and actin as a loading control were utilised. (F) Immunofluorescence showing the recruitment of mCherry B56γ through YFP-RepoMan-wt or P604A at ectopic foci on chr 1 in prometaphase-arrested cells. GFP, mCherry, and Flag antibodies were used to detect YFP, B56γ, and dCas9, respectively. DAPI shows DNA. Graphs represent B56γ levels (G) and GFP levels (G). Average of 3 independent experiments, 58 (wt) 60 (P604A) cells. Scale bar represents 5 µm. Mann-Whitney test was applied; p-value<0.0001.
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Figure 5—source data 1
TIFF files containing full western blots for data presented in Figure 5.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig5-data1-v1.zip
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Figure 5—source data 2
PDF file containing full western blot images for data presented in Figure 5, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig5-data2-v1.pdf
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Figure 5—source data 3
Data utilised to generate the graph in Figure 5B.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig5-data3-v1.xlsx
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Figure 5—source data 4
Data utilised to generate the graph in Figure 5G.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig5-data4-v1.pdf
Figure 5—figure supplement 1
Wild-type (wt) and P604A mutant RepoMan can load to chromatin.
(A) Western blot analysis of endogenous RepoMan levels in the presence of prolyl-hydroxylases (PHD) inhibitor FG4592 50 µM for the indicated times. DMSO was used in time 0 as a control for 24 hr. MG132 20 µM was added for 3 hr. CDCA2, HIF1α, and Actin antibodies were used. (B) Cells were transfected with siRNA control or siRM. 16 hr later, cells were incubated with thymidine 2mM±doxycycline to induce YFP constructs or not (siCtl – dox) for 24 hr. After the incubation time, cells were released from the thymidine block for 6 hr in normal media. Cells were lysed and analysed by western blot. (C) Both RepoMan-wt and P604A mutant can load to chromatin during pro(metaphase). Live-cell images of YFP-RepoMan-wt (upper) or P604A mutant in metaphase. Cells were depleted of endogenous RepoMan with siRNAs. Cells were incubated with thymidine plus doxycycline for 24 hr and released in fresh media+doxycycline. Before imaging DNA was stained with sirDNA for 20 min in L15 media, after the incubation time, the media containing sirDNA was removed and cells were incubated in L15 media for cell imaging. Scale bar represents 5 µm. (D) Graph depicting tracking of YFP-RepoMan-wt or P604A mutant over chromatin during mitosis. Cells were treated as in (C). The graph represents the ratio between RepoMan/DNA intensity over mitosis, values of 2 independent experiments (total number of cells between 2 experiments: RepoMan-wt 29 and RepoMan P604A 24). The values were normalised to the RepoMan/DNA intensity during interphase 2 frames before cells enter in mitosis. Error bars represent SEM.
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Figure 5—figure supplement 1—source data 1
TIFF files containing full western blots for data presented in Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
PDF file containing original western blot for Figure 5—figure supplement 1, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig5-figsupp1-data2-v1.pdf
Figure 5—figure supplement 2
Wild-type (wt) and P604A RepoMan cells are synchronised by monastrol.
Cell cycle distribution of the experiment shown. The number of cells is represented in the y axis and DAPI content in the x axis, from n (G1) to 2n (G2/mitosis), marked with dashed lines. First line of graph shows asynchronous cells and the graph on the bottom shows monastrol-treated cells. The whole cell population from each well was analysed. The number of cells per sample: asynchronous 3668, 7725, 7621; monastrol 5089, 5197, 6820. One representative experiment is shown.
Figure 5—figure supplement 3
PHD1 is required for the interaction between GFP-RepoMan and endogenous B56γ in mitosis.
HeLa cells were transfected with siRNA (PHD1 or ctl). Next day cells were transfected with 5 µg of EGFP-RepoMan for 4 hr. After the incubation time, cells were incubated with thymidine 2 mM for 24 hr, released for 2 hr and incubated with nocodazole 100 ng/mL for 14 hr. Cells were harvested by shake-off and resuspended in lysis buffer. Lysates were subjected to GFP Trap IP, and the interaction of overexpressed EGFP-RepoMan with endogenous B56γ was analysed.
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Figure 5—figure supplement 3—source data 1
TIFF files containing full western blots for data presented in Figure 5—figure supplement 3.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig5-figsupp3-data1-v1.zip
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Figure 5—figure supplement 3—source data 2
PDF file containing original western blot for Figure 5—figure supplement 3, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig5-figsupp3-data2-v1.pdf
Figure 6 with 1 supplement
P604 of RepoMan is required for normal mitotic progression.
(A) Schematic representation of the experimental design. (B) Representative immunofluorescence images of YFP-RepoMan arrested in prometaphase with monastrol. Cells were released for 30 min. Monopolar or bipolar cells are shown for either RepoMan-wt or P604A. Cells were fixed and stained with MT and GFP antibodies, and DAPI shows the DNA. Scale bar represents 5 µm. (C) Quantification of chromosome alignment in bipolar spindles after release of 1 hr from monastrol arrest. The graph represents the percentage of cells with aligned or unaligned chromosomes in bipolar spindles with respect to the total number of bipolar mitotic cells from 3 independent experiments. Total cells analysed: Ctl 160, RM-wt 196, RM P604A 187. Error bars represent SEM. Unpaired t-test unaligned wt vs mut p=0.0105. (D) Time lapse of YFP-RepoMan-wt or P604A cells arrested in prometaphase with monastrol and released into fresh media. (YFP-RepoMan) green and DNA (magenta). Representative images are shown. Scale bar represents 5 μm. (E) The graph shows the percentage of normal vs defective mitosis over the total mitotic cells per condition per experiment. (Total number of cells analysed: 57 cells RepoMan-wt and 55 cells for RepoMan-P604A from 3 independent experiments). Error bars represent SD. Unpaired t-test, p=0.0058, defects wt vs mut.
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Figure 6—source data 1
Data utilised to generate the graph in Figure 6C.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig6-data1-v1.pdf
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Figure 6—source data 2
Data utilised to generate the graph in Figure 6E.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig6-data2-v1.pdf
Figure 6—figure supplement 1
Efficient depletion of endogenous RepoMan and rescue with exogenous RepoMan-wt and P604A.
HeLa cells were transfected with siRNA control or siRM. 16 hr later, cells were incubated with thymidine 2 mM±doxycycline to induce YFP constructs or not (siCtl – dox) for 24 hr. After the incubation time, cells were released from the thymidine block for 9 hr in media containing monastrol with or without doxycycline as indicated. After the incubation time, cells were lysed and extracts analysed by western blot.
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Figure 6—figure supplement 1—source data 1
TIFF files containing full western blots for data presented in Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
PDF file containing original western blot for Figure 6—figure supplement 1, indicating relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108131/elife-108131-fig6-figsupp1-data2-v1.pdf
Figure 7
Schematic model of RepoMan hydroxylation during prometaphase.
PHD1 hydroxylates RepoMan at proline 604. During prometaphase, this modification is important for the binding of PP2A-B56γ to RepoMan. PP2A-B56γ has a crucial role in the loading of RepoMan to chromatin during prometaphase, leading PP1-RepoMan to dephosphorylate phH3T3 from chromosome arms. The resulted enrichment on the phH3T3 at the centromere assures the correct localisation of the CPC (chromosomal passenger complex) important for the proper mitosis progression.
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PHD1-dependent hydroxylation of RepoMan (CDCA2) on P604 modulates the control of mitotic progression
eLife 14:RP108131.
https://doi.org/10.7554/eLife.108131.3
