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. 2018 Jul 17;9(1):2782.
doi: 10.1038/s41467-018-05177-6.

The Replication Initiation Determinant Protein (RepID) Modulates Replication by Recruiting CUL4 to Chromatin

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Free PMC article

The Replication Initiation Determinant Protein (RepID) Modulates Replication by Recruiting CUL4 to Chromatin

Sang-Min Jang et al. Nat Commun. .
Free PMC article

Abstract

Cell cycle progression in mammals is modulated by two ubiquitin ligase complexes, CRL4 and SCF, which facilitate degradation of chromatin substrates involved in the regulation of DNA replication. One member of the CRL4 complex, the WD-40 containing protein RepID (DCAF14/PHIP), selectively binds and activates a group of replication origins. Here we show that RepID recruits the CRL4 complex to chromatin prior to DNA synthesis, thus playing a crucial architectural role in the proper licensing of chromosomes for replication. In the absence of RepID, cells rely on the alternative ubiquitin ligase, SKP2-containing SCF, to progress through the cell cycle. RepID depletion markedly increases cellular sensitivity to SKP2 inhibitors, which triggered massive genome re-replication. Both RepID and SKP2 interact with distinct, non-overlapping groups of replication origins, suggesting that selective interactions of replication origins with specific CRL components execute the DNA replication program and maintain genomic stability by preventing re-initiation of DNA replication.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
RepID is required for CUL4 loading on chromatin. a Levels of RepID, cullins and their complexes from wild-type (WT) or RepID-depleted (KO) U2OS, HCT116 and K562 cancer cell lines. Histone H3 and α-tubulin were used as loading controls. WCL, whole cell lysates; CB, chromatin-bound proteins. b Quantification of relative intensities of chromatin-bound protein signals analyzed as shown in a after normalization with respect to histone H3 signal intensities in each cell line. CUL4 was indicated as bold. Error bars represent standard deviations from three independent experiments. c Construction of RepID fragments. d Soluble nuclear and chromatin-bound fractions were extracted from U2OS cells transfected with RepID fragments as indicated. A pull-down assay using a FLAG antibody was performed and co-precipitated cullins were analyzed by immunoblotting. Yellow asterisks, FLAG-RepID fragments. e Cullin levels in WCL and CB from RepID KO U2OS cells transfected with RepID constructs as indicated. f Quantification of chromatin-bound cullin levels, normalized to chromatin bound levels in RepID KO cells. Error bars represent standard deviations from three independent experiments. g K562 RepID WT and KO cells were fractionated by elutriation and cell cycle status of each fraction (G1, early [ES], middle [MS], and late [LS] S-phase and G2/M) was confirmed by FACS (upper panel). Chromatin-bound proteins were analyzed by immunoblotting (bottom panel). The numbers under the panels represent the intensity ratios for each protein normalized by the intensity of the signal at G1 phase in RepID WT from three independent experiments (bold). h U2OS cells were EdU-labeled, pre-extracted and chromatin-bound RepID (green), chromatin-bound CUL4A (red), DNA content (DAPI) and EdU (magenta) were detected. Cells in ES and LS were identified by EdU staining patterns and G1 and G2 nuclei (EdU negative) were identified by DAPI intensity. To clearly observe colocalization with EdU and CUL4A, magenta was converted to green (left panel). The extent of colocalization between RepID and CUL4A, or between EdU and CUL4A (right panel). Pearson’s correlation coefficients (n = 15) and p-values were calculated using a two-tailed t-test. RepID KO cells were used to estimate background (red line, right panel). Scale bar indicates 10 μm
Fig. 2
Fig. 2
RepID-dependent CUL4 recruitment on chromatin is required for cell cycle progression. a siRNAs for CUL4A, CUL4B or CUL4A + B were transfected into RepID WT and KO U2OS cells. After 5 days, cells were labeled by EdU for 30 min, collected and analyzed by flow cytometry. Percentages of cells in each cell cycle phase are indicated (upper panel). Graphs depicting the distributions of G1, S and G2/M phase cells in control vs. CUL4A + B siRNA transfected cells are shown in the bottom panel. Error bars represent standard deviations from three independent experiments. b Chromatin-bound proteins were isolated from cells collected as in a and analyzed by immunoblotting. The numbers under the panels represent the intensity ratios compared to the intensity of the signal in control-siRNA transfected RepID WT cells. c CUL4A + B-siRNA transfected RepID WT and KO U2OS cells were labeled with EdU for 30 min, and immunofluorescence analysis was performed using CUL4A, γH2AX, phospho-RPA, or PCNA antibodies after pre-extraction (upper and middle panel). Scale bar indicates 10 μm. The intensity of each protein was determined in G1 (EdU negative) and ES (EdU positive) using the Pearson’s correlation coefficient. p values were calculated using a two-tailed t-test (n = 10) (bottom panel). d Mitotic HCT116 RepID WT or KO cells were collected after released from a nocodazole block, reseeded in fresh medium and harvested at the indicated time points after incubation with EdU for 30 min. Cells were then analyzed by flow cytometry, and the percentage of cells in each cell cycle phase is indicated as in a. e Aliquots of cells in d were analyzed by immunoblotting after extraction of chromatin fractions. f Graphs representing the percentages of cells in G1, G2/M phase and the ratio of cells in S phase vs. G1 at the indicated time points from three independent experiments. g Schematic of the expected cell cycle patterns in relation to the abundance of chromatin-bound CUL4 (High to Low) in RepID WT, RepID KO, and CUL4A + B-siRNA transfected RepID KO cells
Fig. 3
Fig. 3
CUL4A colocalizes with distinct replication origins and different DCAFs during S phase. a CDT2-siRNA transfected RepID WT and KO U2OS cells were labeled with EdU for 30 min and analyzed by flow cytometry. Percentages of cells in each cell cycle phase are indicated in the flow cytometry plots (upper panel) and histograms (bottom panel). Fold changes were based on the values from control-siRNA transfected cells. Data are representatives of three independent experiments. b Left, table indicating colocalizations between CUL4A ChIP-Seq and replication origins (^: intersection, -: subtraction). Right, Venn diagram comparing CUL4A, CDT2 and RepID ChIP-Seq results. Three subgroups indicate CUL4A binding sites overlapping with RepID (Group 2), CDT2 (group 3), RepID and CDT2 (group 4) while three subgroups represent exclusive binding sites for CUL4A (group 1), CDT2 (group 5) and RepID (group 6). c Heat maps showing colocalization between the six subgroups defined in b and sites enriched in methylated histone H3 (H3K4me3, top panel and H3K9me3, bottom panel). AMI value (red) and colocalized percentage (black) are indicated. Twenty kb windows were used for the analysis. d A centered bar chart representing the AMI ratio from c between H3K4me3 and H3K9me3
Fig. 4
Fig. 4
RepID depletion confers resistance to the neddylation inhibitor MLN4924. a U2OS WT or RepID KO cells were incubated with or without 250 and 500 nM MLN4924 for 2 days. Cells were labeled with EdU for 30 min prior to collection and analyzed by FACS. Percentage of re-replicating cells is indicated. b Bar chart depicting the cell cycle distribution of cells collected in a. c Chromatin-bound (CB) proteins isolated as in a and analyzed by immunoblotting using antibodies directed against CRL4 substrates including CDT1, SET8, and p21. d WT, RepID KO U2OS cells and RepID KO U2OS cells stably transfected with RepID variants were treated with 500 nM MLN4924 for 2 days and labeled with EdU for 30 min prior to collection and analyzed by FACS. Percentage of subG1 and re-replicating cells are indicated (upper panels). Percentages of chromatin-bound CDT1 in re-replicating cells are indicated (red, bottom panel). e Graphs depicting the percentage of chromatin-bound CDT1 in cells in G2/M phase (left panel) or in re-replicating cells (right panel). The intensity of chromatin-bound CDT1 in specific cell cycle fractionated cells isolated as in d was measured by immunoblotting (bottom panel). The numbers under the panel represent the ratios of CDT1 intensities relative to the intensity of the signal in MLN4924-treated RepID WT cells after normalization with histone H3 from three independent experiments
Fig. 5
Fig. 5
SKP2 inhibitors synergize with RepID depletion to kill cancer cells. a HCT116 cells released from nocodazole were collected every 3 h for up to 33 h, and chromatin-bound cullins were analyzed using immunoblotting. b Graphs showing relative intensities of cullins and SKP2 calculated from blots derived from (a) and normalized using histone H3 levels. Error bars represent standard deviation from three independent experiments (*p-value < 0.05, **p < 0.01, ***p < 0.001, no asterisk: n.s.). c Table depicting the colocalization between SKP2 ChIP-seq peaks and replication origins in HCT116 WT or RepID KO cells. d Heat map showing the colocalizations between origin-associated SKP2 and the H3K4me3 or H3K9me3 peaks (AMI, red; colocalized percentage, black; ^, intersection). e Bar graph representing the AMI ratios from d. f Heat maps for colocalizations between RepID and SKP2 or CDT2 peaks (first panel), between SKP2 and RepID or CDT2 peaks (second panel), between the origin-associated RepID and the origin-associated SKP2 or origin-associated CDT2 (third panel), and between the origin-associated SKP2 and origin-associated RepID or origin-associated CDT2 (fourth panel). window scale, 20 kb. g WT, RepID KO U2OS cells and RepID KO U2OS cells transfected with the specified RepID fragments were treated with 50 μM SKP2 inhibitor for 2 days and labeled with EdU. Cell cycle (upper panel) and chromatin-bound CDT1 (bottom panel) were analyzed as in Fig. 4d. h Top, a schematic of the experimental procedure. Mitotic HCT116 WT or RepID KO cells were released in fresh medium and collected every 3 h with MLN4924 or SKP2 inhibitors added during the last 3 h prior to collection. The time zero samples were mitotic cells without drug exposure. Chromatin-bound CDT1 was analyzed and normalized using histone H3. Fold changes were calculated using the highest CDT1 intensity (bold) in each group from three independent experiments. i, j Colony assay with HCT116 cells (i) and U2OS cells (j). bar charts, colony number. Ratios for the average size of colonies in each group relative to the size of colonies in untreated cells are provided above the bars. Error bars represent standard deviations from three independent experiments
Fig. 6
Fig. 6
Model depicting RepID roles in regulating CRL4 activity and RepID-independent SCF functions during replication. In RepID wild-type (RepID WT) cells, CUL4 is recruited to chromatin by interacting with RepID (architectural DCAF) during G1 in the PCNA-free stage. During S-phase, PCNA is loaded and the CRL4-PCNA complex induces CDT1 ubiquitination using CDT2 (functional DCAF) by associating with early replication origins. Additional CUL4 can be recruited by PCNA during the G1/S transition and early S-phase. The CRL4-PCNA complex associates with CDT2 to degrade CDT1 later during S-phase. SCF is also recruited to prevent CDT1 accumulation at late replication origins. In cells expressing RepID, the SKP2 inhibitor has little effect because of the major role of CRL4 in degrading CDT1. In RepID depleted (RepID KO) cells, CUL4 loading to chromatin is compromised because RepID is not available to recruit it to chromatin during the G1 phase. During S-phase, some PCNA-dependent CUL4 is recruited, but CDT1 degradation is reduced because of low chromatin-bound CUL4 levels. Thus, in RepID KO cells, SCF recruitment to late replication origins compensates for the lack of CRL4 and have a greater role in targeting CDT1. Because SCF now acts as the major ubiquitin machinery for CDT1 degradation, the SKP2 inhibitor shows increased potency, and treatment leads to increased CDT1 levels and re-replication-mediated cancer cell death

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