Article Figures & Data
Figures
- Figure 1.
Rb1-mutant MEFs exhibit increased γH2AX foci that are enriched at pericentromeric DNA. A, immunofluorescence microscopy of γH2AX staining is shown in red and cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Red arrows, colocalization; white arrows, lack of colocalization; scale bars, 10 μm. B, the quantity of foci per cell was determined for each genotype and compared using a χ2 test. Rb1+/+, n = 103; Rb1ΔL/ΔL, n = 114; Rb1−/−, n = 63. C, the proportion of cells with γH2AX foci colocalizing with DAPI-rich foci was determined by confocal 3D rendering and compared using a χ2 test. D, ChIP-seq analysis was performed for γH2AX, and tracks comparing abundance at Fra16d among different Rb1 genotypes are shown. A red box indicates this locus on a chromosome 8 ideogram. E, heatmap to show log2 ratios of the abundance of Rb1-mutant γH2AX precipitable reads per million mapped reads versus wild-type γH2AX precipitated reads. ERV, endogenous retrovirus. F and G, ChIP-qPCR for γH2AX to quantitate major satellite (major sat.) repeats. *, P < 0.05 using a t test; n = 3.
- Figure 2.
Replication abnormalities in Rb1-mutant cells. A, fluorescence microscopy of a replicating fork is shown by CIdUrd labeling (P1, green) and IdUrd (P2, red), and light blue bars mark unlabeled DNA. B, fork rates were measured for Rb1+/+ (n = 718) and Rb1ΔL/ΔL (n = 855). Median values werecompared using the Mann–Whitney U test. C, diverging forks, as shown in A, were measured and the percentage asymmetry was determined, Rb1+/+ (n = 141) and Rb1ΔL/ΔL (n = 174). Median values were compared as in B. D, extracts were blotted for RPA and the indicated phosphorylated RPA-S33. Relative intensity of RPA pS33 is shown below each lane. E, ChIP-qPCR analysis of RPA at major satellite repeats in Rb1+/+ and Rb1ΔL/ΔL MEFs. *, P < 0.05 using a t test; n = 3. F–H, ChIP-qPCR analysis of PCNA, MCM3, and DNA Polδ occupancy at major satellites.
- Figure 3.
pRB, E2F1, and Condensin II localize to pericentromeric DNA. A, ChIP-qPCR for major satellite repeats using antibodies against pRB and CAP-D3. *, P < 0.05 using a t test; n = 3. B, chromatin was precipitated with E2F1 and CAP-D3 antibodies and major satellite repeats were amplified. C, ChIP-qPCR using a CAP-D3 antibody to precipitate major satellite repeats in Rb1+/+, Rb1−/−, and Rb1ΔL/ΔL MEFs. D, ChIP-seq was performed for CAP-D3 using chromatin from Rb1+/+ and Rb1ΔL/ΔL MEFs and tracks are shown for selected genomic regions. E2F binding sites present in the promoters of Ccne1, Mcm3, and Rbl1 are indicated by red boxes, and regions of significant enrichment (MACS peaks) are indicated in green.
- Figure 4.
pRB, E2F1, and Condensin II form a complex on pericentromeric DNA. A, nuclear extracts (NE) were mixed with streptavidin beads and a biotinylated major satellite (maj. sat.) repeat containing probe. Precipitated proteins were analyzed by SDS-PAGE and Western blotting. B, wild-type nuclear extracts were mixed with the indicated probes and/or competitor DNA. C, RB1-deficient C33A cells were transfected with HA-E2F1 and HA-DP1 expression vectors and nuclear extracts were prepared. Western blots of relevant proteins are shown. D, nuclear extracts were mixed with streptavidin beads and biotinylated major satellite probes, either with or without GST-RBLP. Associated proteins were precipitated and analyzed by Western blotting. E and F, ChIP-qPCR for E2F1 was performed, and major satellite repeat DNA was amplified by real-time PCR. *, P < 0.05 using a t test; n = 3. F, ChIP-qPCR using a CAP-D3 antibody was performed to precipitate major satellite repeats from Rb1+/+, Rb1ΔG/ΔG, and Rb1ΔS/ΔS MEFs.
- Figure 5.
Rb1 heterozygous MEFs have elevated γH2AX and underloading of CAP-D3 at major satellites. A, immunofluorescence microscopy of γH2AX staining in Rb1+/+, Rb1ΔL/+, and Rb1+/− MEFs is shown. Scale bars, 10 µm. B, the quantity of γH2AX foci per cell was determined for Rb1+/+, Rb1ΔL/+, and Rb1+/− MEFs and compared with homozygous mutants from Fig. 1. The quantity of foci was compared using a χ2 test. Rb1ΔL/+, n = 114; Rb1+/−, n = 63. C, quantification of DAPI foci colocalization with γH2AX foci by confocal microscopy. The proportion of colocalization was compared using a χ2 test. D, γH2AX levels were detected by Western blotting and Coomassie staining of histones was used as a loading control. Relative intensity of γH2AX is shown below. E, RPA32 and RPA32 phospho serine 33 levels were detected by Western blotting. Relative intensity of RPA pS33 is shown below. F, heatmap depicting log2 ratios of γH2AX precipitated sequence tags in the indicated mutants relative to wild-type control. G, ChIP-qPCR using a CAP-D3 antibody in Rb1+/+ and Rb1ΔL/+ MEFs was used to detect major satellite repeats. *, P < 0.05 using a t test; n = 3.
- Figure 6.
Normal RB1+/− cells have elevated γH2AX foci, mitotic errors, and aneuploidy. A, immunofluorescence microscopy for γH2AX in control and RB1+/− patient fibroblasts. Scale bars, 10 μm. B, quantitation of γH2AX foci in control and each patient fibroblast isolate. Foci were compared for patient fibroblasts against pooled control data (IMR90, BJ, and WI-38) using a χ2 test. Pooled control, n = 246; GM_01408, n = 79; GM_01123, n = 93; GM_06418, n = 71. C, Western blotting was used to detect phosphorylated RPA pS33 and total RPA32. Relative intensity of RPA pS33 is displayed. D, cells were transduced with H2B-GFP, and video microscopy was performed to capture phase contrast and GFP images over 15 hours. The left-most image shows the onset of prophase, and the middle image is of the metaphase plate just before the onset of anaphase (elapsed time since the onset of prophase is shown in yellow). The right-most image shows cells in telophase. Scale bars, 50 μm. E, summary of video microscopy data showing the number of divisions observed along with the number that contained anaphase bridges. Similarly, the number of cells observed to complete prophase and metaphase is shown along with the average length of these two phases. Each patient fibroblast is compared with pooled control data. #, P < 0.05, χ2 test; †, P < 0.05, t test. F–I, quantitation of genomic abnormalities in mesenchymal-derived cancer cell lines that are wild-type (n = 15), hemizygous (n = 10), or null (n = 12) for RB1. Means were compared using a t test. *, P < 0.05. NS, not significant.
- Figure 7.
Haploinsufficiency of Rb1 contributes to aneuploidy. A, Kaplan–Meier survival proportions are shown for Rb1ΔL/+;Trp53−/− mice (n = 52). Previously reported data for Rb1ΔL/ΔL;Trp53−/− and Trp53−/− mice is included as a comparison. Asterisks indicate populations that are significantly different from the Trp53−/− control (log-rank test, P < 0.05). B, Rb1 tumor genotypes (Tu) were determined by PCR; matched tail DNA (T) is shown as a comparison. C, representative images of H&E-stained tumors from Trp53−/− control, Rb1ΔL/ΔL;Trp53−/−, and Rb1ΔL/+;Trp53−/− compound mutants. Scale bars, 100 μm. D, control, or tumor DNA, was used for array comparative genomic hybridization (CGH). Representative graphs show log2 ratio values plotted against chromosome number. Individual chromosomes are shown in different colors. E, whole-chromosome changes (among autosomes) for Rb1ΔL/+;Trp53−/− tumors is plotted against their genotype. Previously reported control, Trp53−/−, and Rb1ΔL/ΔL;Trp53−/− data are shown for comparison. The control male versus control male hybridization is shown in blue; the male versus female hybridizations are shown in yellow. The means were compared between genotypes using a t test, *, P < 0.05. NS, not significant.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Methods - PDF file 125K, Detailed methods for ChIP, microscopy, and computational analysis. Lists of cell lines and antibodies used are included
- Supplementary Figure Legends - PDF fie 148K, Description of additional genotypes of cells analyzed for gammaH2AX distribution and detailed instability phenotypes of heterozygous cells
- Supplementary Figures - PDF file 3766K, Analysis of gammaH2AX distribution, chromosomal instability phenotypes in heterozygous cells, and verification of genotypes in human cells
Volume 4, Issue 7
