Wdr82 is associated with multiple protein complexes. A, T-REx HEK293 stable cell lines that express FLAG-Wdr82 or empty vector were induced with 1 μg/ml doxycycline for 3 days. Nuclear extracts were prepared and subjected to FLAG immunoprecipitation (FLAG-IP), and bound proteins were eluted by FLAG peptide after extensive washing. Proteins were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue. Protein bands were excised and processed for in-gel trypsin digestion. Peptides were analyzed by quadrupole time-of-flight mass spectrometry, and proteins were identified. B, identified proteins are classified into five categories based on known biological activities. C, T-REx HEK293 stable cell lines that express FLAG-Wdr82 or empty vector were induced with 1 μg/ml doxycycline for 3 days. Nuclear extracts were subjected to FLAG immunoprecipitation, and immunoprecipitates were analyzed by Western blotting for representative proteins of each complex class.

Wdr82 forms a complex with PP1 catalytic and regulatory subunits. A, endogenous PNUTS and Tox4 associate with PP1 and Wdr82. Nuclear extracts isolated from HEK293 cells were subjected to immunoprecipitation (IP) using antisera directed against Tox4, PNUTS, Setd1a, or Setd1b. Immunoprecipitates were analyzed by Western blotting using the indicated antisera. B, PNUTS, Tox4, Wdr82, and PP1 catalytic subunits exhibit reciprocal immunoprecipitation. Inducible T-REx HEK293 stable cell lines that express empty vector or the indicated FLAG-tagged protein were induced by 1 μg/ml doxycycline for 3 days. Nuclear extracts were subjected to FLAG immunoprecipitation, and immunoprecipitates were analyzed by Western blotting using the indicated antisera. Asterisks in the FLAG Western blot indicate the expected size of each FLAG-tagged protein. C, PNUTS, Tox4, Wdr82, and PP1 catalytic subunits co-migrate following sucrose gradient fractionation. T-REx HEK293 cells that express FLAG-Wdr82 were induced with 1 μg/ml doxycycline for 3 days. Nuclear extracts were prepared and subjected to FLAG immunoprecipitation, and bound proteins were eluted by FLAG peptide. Wdr82-associated proteins were loaded onto 10–50% sucrose gradients and analyzed by equilibrium centrifugation, as described previously (32). Equal volumes of each fraction were analyzed by Western blotting. Migration of RNA pol II subunits, histone methyltransferase complex components, and PP1-associated proteins such as PNUTS, Tox4, and PP1 catalytic subunits are shown in the gradient. Molecular markers were applied to a parallel gradient. D, endogenous PNUTS, Tox4, and Wdr82 co-migrate following sucrose gradient fractionation analysis. Nuclear extracts from HEK293 cells were loaded onto 10–50% sucrose gradients and analyzed by equilibrium centrifugation. Fractions were collected, and equal volumes of each fraction were analyzed by Western blotting using the indicated antisera.

PNUTS and Tox4 interact nearly exclusively with components of the PTW/PP1 phosphatase complex. A, identification of PNUTS- and Tox4-associated proteins by mass spectrometry. Inducible T-REx HEK293 stable cell lines that express FLAG-PNUTS, FLAG-Tox4, or empty vector were induced with 1 μg/ml doxycycline for 3 days. Nuclear extracts were prepared and subjected to FLAG immunoprecipitation, and bound proteins were eluted by FLAG peptide after extensive washing. Proteins were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue. Specific bands were analyzed by mass spectrometry. The arrows indicate identified proteins. B, summary of the PTW/PP1 phosphatase complex. The domain structure of each component in the PTW/PP1 phosphatase complex is schematically shown. TFS2N, transcription factor II N-terminal domain; RVXF motif, PP1 docking motif; Zinc finger; HMG box, high mobility group box; PP2Ac, catalytic domain of protein phosphatase 2A; WD40, WD40 repeat. The number of amino acids in each component is indicated.

PNUTS mediates PTW/PP1 complex formation by providing a binding platform to each component. A, PNUTS directly interacts with Wdr82 and mediates the association of Wdr82 with PP1α. Recombinant FLAG-Wdr82, His-PNUTS, and His-Tox4 were purified from insect Sf9 cells, and GST-PP1α was purified from E. coli. Purified FLAG-Wdr82 was incubated with various combinations of purified components, and protein interactions were analyzed by in vitro FLAG pull-down assay. Proteins were separated by SDS-PAGE and detected by Coomassie Brilliant Blue staining. The arrows indicate identities of protein bands. B, PNUTS directly interacts with PP1α and mediates the association of PP1α with Wdr82. Purified GST-PP1α was incubated with various combinations of purified components, and protein interactions were analyzed by an in vitro GST pull-down assay. Proteins were analyzed as described above. C, protein-protein interactions within the PTW/PP1 phosphatase complex. A diagram of PNUTS deletion constructs is shown. The numbers indicate amino acid residues of the PNUTS protein. HEK293 cells were transiently transfected with various deletion constructs of PNUTS with an N-terminal FLAG epitope. Nuclear extracts were prepared and subjected to FLAG immunoprecipitation (FLAG-IP), and immunoprecipitates were analyzed by Western blotting using the indicated antisera. D, summary of protein-protein interactions within the PTW/PP1 phosphatase complex.

The PTW/PP1 complex exhibits phosphatase activity in vitro. Wdr82-, PNUTS-, and PP1α-associated proteins were purified from inducible T-REx HEK293 cell lines using FLAG immunoprecipitation. Purified proteins were examined in a phosphatase assay, using GST-CTD phosphorylated by MAPK as a substrate. In vitro phosphorylated GST-CTD was incubated with the immunoprecipitate from vector control, Wdr82, PNUTS, or PP1α in the absence or presence of the PP1-specific inhibitor NIPP1 or inhibitor 2 at 30 °C for 1 h. Reaction products were analyzed by Western blotting using anti-Ser(P)⁵ (Anti-Ser5P) CTD and anti-PP1α antisera. The anti-Ser(P)⁵ CTD antiserum recognizes phosphorylated CTD at Ser⁵, the major product of the MAPK reaction (34). The slightly slower mobility of PP1 in the PP1 immunoprecipitates is due to the presence of the FLAG epitope.

Chromatin association and integrity of the PTW/PP1 complex throughout cell cycle progression. A, chromatin association of PNUTS is regulated throughout cell cycle progression. Inducible T-REx HEK293 cells that express FLAG-PNUTS were cultured on glass bottom collagen-coated dishes and induced by 1 μg/ml doxycycline for 2 days. Cells were stained with anti-FLAG antibody and fluorescein isothiocyanate (FITC)-conjugated secondary antibody. Cells were observed by confocal microscopy after DNA was stained with DAPI. Cell phase was determined based on cell morphology and DAPI stains. B, the PTW/PP1 phosphatase complex is associated with chromatin. HEK293 cells were fractionated by sequential extraction as described previously (42). An equal proportion of each fraction was analyzed by Western blotting. Acetylated histone H3 and NuMA were used as marker proteins for chromatin and matrix fractions, respectively. C, the integrity of the PTW/PP1 phosphatase complex is preserved throughout cell cycle progression. T-REx HEK293 cells expressing FLAG-PNUTS were induced and synchronized by successive thymidine-nocodazole block. Cells were harvested at various time points following release from arrest. A small fraction of cells at each time point was analyzed for cell cycle distribution by PI staining and flow cytometry. Whole cell extracts were prepared and subjected to FLAG immunoprecipitation (FLAG-IP), and immunoprecipitates were analyzed by Western blotting using the indicated antisera.

Dysregulation of the PTW/PP1 phosphatase complex causes cell cycle arrest and cell death. A, the PNUTS (W401A) mutant interacts with Wdr82 and Tox4 but not with PP1. T-REx HEK293 cell lines that express FLAG-PNUTS, FLAG-PNUTS (W401A), or empty vector were induced with 1 μg/ml doxycycline for 3 days. Nuclear extracts were subjected to FLAG immunoprecipitation, and immunoprecipitates were analyzed by Western blotting. B, inducible expression of PNUTS (W401A) causes proliferation defects. T-REx HEK293 cell lines that express empty vector, FLAG-PNUTS, or FLAG-PNUTS (W401A) were cultured in the presence of 1 μg/ml doxycycline for 3 days. Cell morphology was photographed using inverted microscopy. Representative images are shown. C, inducible expression of PNUTS (W401A) leads to cell death. T-REx HEK293 cell lines that express FLAG-PNUTS or FLAG-PNUTS (W401A) were cultured on glass bottom collagen-coated dishes. Cells were induced with 1 μg/ml doxycycline for 4.5 days. Cells were fixed and stained by DAPI and observed by confocal microscopy. Representative images are shown. D, inducible expression of PNUTS (W401A) leads to apoptotic cell death. T-REx HEK293 cell lines that express empty vector or FLAG-PNUTS (W401A) were cultured in the presence of 1 μg/ml doxycycline for 3 days. Apoptotic cells were detected by flow cytometry after Annexin V and PI staining. UR, upper right panel corresponding to dead cells; LL, lower left panel corresponding to healthy cells; LR, lower right panel corresponding to apoptotic cells. Numerical values represent a summary of data from three experiments. *, p < 0.05. E, inducible expression of PNUTS (W401A) causes cell cycle arrest. T-REx HEK293 cell lines that express empty vector, FLAG-PNUTS, or FLAG-PNUTS (W401A) were cultured in the absence or the presence of 1 μg/ml doxycycline for 3 days. Cells were analyzed for cell cycle distribution by PI staining and flow cytometry. The percentage of cells in each phase of the cell cycle was determined.

Depletion of PNUTS results in destabilization of the PTW/PP1 complex leading to cell cycle dysregulation and apoptotic cell death. A, depletion of PNUTS causes destabilization of the PTW/PP1 complex. HEK293 cells were transfected with control or PNUTS siRNA pools according to the manufacturer's instructions. Proteins were analyzed by Western blotting after 3 days of transfection. B, depletion of PNUTS causes abnormal cell cycle distribution. Cell cycle distribution after 3 days of transfection was analyzed by PI staining and flow cytometry. The error bars indicate the mean S.D., and p values were determined by a standard t test. C, depletion of PNUTS causes apoptotic cell death. Apoptotic cells after 3 days of transfection were analyzed by cytometry after Annexin V and PI staining. UR, upper right panel corresponding to dead cells; LL, lower left panel corresponding to healthy cells; LR, lower right panel corresponding to apoptotic cells. Numerical values represent a summary of data from three experiments. *, p < 0.05.

Dysregulation of the PTW/PP1 phosphatase complex causes defects at mitotic exit. T-REx HEK293 cells that express GFP-PNUTS (W401A) were cultured on glass bottom collagen-coated dishes. Cells were cultured in the presence of 1 μg/ml doxycycline for 3 days. Cells were fixed and stained by DAPI and observed by confocal microscopy. The first three columns show representative progression through mitotic stages, and the fourth column shows a representative image of a defective exit from mitosis to interphase.