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First published online 14 November 2006
doi: 10.1242/jcs.03262

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The DNA helicase ChlR1 is required for sister chromatid cohesion in mammalian cells

¹ Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA
² Department of Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA
³ Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, TN 38105, USA

View larger version (57K): [in this window] [in a new window]   Fig. 1. Anti-peptide ChlR1 2075 antibody specifically immunoprecipitates ChlR1 protein. FLAG-ChlR1 was in vitro translated using a TNT kit (Promega) and the protein was used for immunoprecipitation with ChlR1 2075 or previously characterized Hel1 antibody (Amann et al., 1997). (B) Cells were transfected with FLAG-ChlR1 and metabolically radiolabeled. Whole cell lysate was used for immunoprecipitation with either ChlR1 2075 antisera or Hel1 (Amann et al., 1997). A band corresponding to the predicted molecular mass of ChlR1 (~102 kDa) was specifically immunoprecipitated with both antibodies. (C) HeLa cell lysates were used to immunoprecipitate endogenous ChlR1 protein with ChlR1 2075 antibody followed by detection by western analysis with the Hel1 antibody.

View larger version (51K): [in this window] [in a new window]   Fig. 2. ChlR1 localizes to chromatin, spindle poles and the midbody. Confocal images of ChlR1 localization in RPE-1 cells at different stages of mitosis. (A) ChlR1 was detected using antibody 2075 (green), -tubulin was stained red and DNA stained blue; merged images are shown on the right. Bar, 8 µm. Spindle poles and midbody structures are indicated by arrows in prophase and telophase, respectively. (B) Top panels: RPE1 cells were transfected with 20 nM ChlR1-specific siRNA and stained for ChlR1 protein (green) and DNA (blue). Image shown is a cell at metaphase with no detectable ChlR1-specific staining at the spindles poles. Bottom panels: ChlR1 2075 antibody was incubated with 50 µg/ml ChlR1 peptide (used to raise antibody) for 15 minutes at room temperature and then used to detect ChlR1 in RPE1 cells. Incubation of ChlR1 antiserum with peptide significantly reduced specific fluorescence (a telophase cell is shown).

View larger version (26K): [in this window] [in a new window]   Fig. 3. ChlR1 co-localizes with -tubulin and Aurora B at the centrosomes and midbody, respectively. RPE1 cells were fixed as previously described and stained for (A) ChlR1 (green), -tubulin (red), -tubulin (cyan) and DNA (blue) in metaphase, and (B) ChlR1 (green), Aurora B (red) and DNA blue in telophase; merged images are shown on the right. Bar, 8 µm for all parts of the figure.

View larger version (40K): [in this window] [in a new window]   Fig. 4. shRNA-mediated depletion of ChlR1 results in mitotic delay. (A) RT-PCR results with ChlR1-specific (top panel) or actin-specific (lower panel) primers from DNase-treated RNA purified from RPE1-TetR-ChlR1 cells with shRNA expression uninduced or induced at 48 and 72 hours with 2 µg/ml doxycycline. (B) ChlR1 (upper panel) and -tubulin (lower panel) protein levels in RPE1-TetR-EGFP cells or RPE1-TetR-ChlR1 cells with uninduced and induced shRNA for 24 hours with 2 µg/ml doxycycline. (C) RPE1-TetR-ChlR1 cells were grown on coverslips and ChlR1 shRNA was uninduced or induced for 24 hours with 2 µg/ml doxycycline before cells were pre-extracted with 0.1% Triton X-100 and fixed in methanol. Mitotic cells were stained for -tubulin (red) by indirect immunofluorescence and for DNA (blue) using Hoechst no. 33568. More than100 cells were counted per experiment and the mean percentage of mitotic cells for three independent experiements determined and expressed as the mitotic index (± s.d.). (D) More than 50 mitotic cells per experiment were assessed for specific mitotic stage and the percentage of total mitoses for three independent experiments calculated (± s.d.). An increase in pro-metaphase cells with concomitant decrease in other mitotic stages was observed following depletion of ChlR1. Shown is the mean and standard deviation of at least three independent experiments. (E) Representative confocal images of RPE1-TetR-ChlR1 cells with ChlR1 shRNA induced for 24 hours; -tubulin was stained red and DNA stained blue. Bar, 8 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 5. ChlR1 depletion results in mitotic failure. (A) Normal nuclear morphology in uninduced RPE1-TetR-ChlR1 cells (B) RPE1-TetR-ChlR1 cells were induced with 2 µg/ml doxycycline for 96 hours. Representative nuclear abnormalities are indicated (1-4) and enlargements are shown below. -Tubulin was stained red and DNA stained blue. Bar, 40 µm except for enlargements. (C) Quantification of nuclear abnormalities before and after induction of ChlR1 shRNA. Cells within a field of view were counted and the percentage of nuclear abnormalities determined and classified as fragmented (grey) or abnormally large (white). Data are the average of ten fields of view for each sample.

View larger version (77K): [in this window] [in a new window]   Fig. 6. ChlR1 depletion results in mitotic delay and failure. HeLa cells were transfected with siRNA duplexes specific for ChlR1, or GFP as a control. (A) 24 hours following transfection, cells were harvested for western analysis of ChlR1 protein expression using Hel1 antibody. -Tubulin was included as a loading control. Control (B) or ChlR1 siRNA-transfected (C) cells were stained with 1 µM Syto13 to label DNA and filmed by time-lapse imaging using a 20x objective and a TRITC filter set. Numbers in upper left of each panel indicate minutes after start of filming. Bar, 10 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 7. Mammalian cohesin co-immunoprecipitates with ChlR1. HeLa cell lysates were incubated with protein G beads alone or in combination with pre-immune serum or ChlR1 2075 antibody as indicated at the top of the immunoblots. Specific interaction with components of the cohesin complex was detected by western blotting using antibodies to Smc1, Smc3, Scc1 or SA1/2 as indicated on the left. (B) To confirm specificity of the interaction, cells were transfected with 20 nM siRNAs as indicated at the top of the immunoblots. ChlR1 protein levels were efficiently reduced following transfection with specific siRNA but not with GFP control. Scc1 input is shown to be unaltered (middle panel). Depletion of ChlR1 protein levels results in a proportional decrease in the amount of co-immunoprecipitating Scc1 protein compared to controls (lower panel).

View larger version (36K): [in this window] [in a new window]   Fig. 8. Depletion of ChlR1 results in abnormal sister chromatid cohesion. (A) HeLa cells were untransfected (left) or transfected with ChlR1-specific siRNA (right) and metaphase spreads prepared 24 hours following transfection. DNA was stained with propidium iodide and chromosome morphology visualized by confocal microscopy. Bar is 8 µm in top images and 1 µm in enlargements shown underneath. (B) The distance between chromatid pairs was measured using Leica confocal software of at least ten images taken from untransfected (grey) and ChlR1-specific siRNA (black) transfected cells. The graph shows distribution of pair width for each data set. The difference between mock and transfected cells was statistically analyzed using Student's t-test; P<0.0002.

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