Table 1.
Nup98 fusion proteins employed in this study.
Fig 1.
Localization of Nup98 fusion proteins.
HeLa cells were transiently transfected with GFP constructs and visualized after 24 hours by direct fluorescence microscopy. All fusion proteins localize to the nucleus. (A) GFP-Nup98 is found at the nuclear rim and in the nucleoplasm, whereas Nup98 homeodomain fusions exhibit a punctate pattern: (B) GFP-Nup98-HOXA9, (C) GFP-Nup98-HOXA10, (D) GFP-Nup98-HHEX, and (E) GFP-Nup98-PMX1. Nup98 fusions with other chromatin-binding motifs show a different punctate distribution: (F) GFP-Nup98-JARID1A, and (G) GFP-Nup98-PHF23 (H) GFP-Nup98-NSD1, (I) GFP-Nup98-NSD3 and (K) GFP-Nup98-RARG. Nup98 fused to partners that lack chromatin-binding domains localize more dispersed to the nucleoplasm: (L) GFP-Nup98-LEDGF. Disruption of the FG, the HD or the PHD domain disrupts the localization of the Nup98 chimeras: (M) GFP-Nup98-PMX1 N51S, (N) GFP-Nup98-HOXA9 ΔFG, (O) GFP-Nup98-HOXA9 N51S, (P) GFP-Nup98-HHEX ΔFG, (Q) GFP-Nup98-HHEX ΔHD, and (R) GFP-Nup98-JARID1A W1625A. (S) GFP-HOXA9 and (T) GFP-HHEX localize to the nucleoplasm. Shown are representative confocal images. Scale bars, 5 μm.
Fig 2.
Mitotic localization of Nup98 chimeras.
HeLa cells were transiently transfected with GFP constructs and fixed and stained after 24 hours for immunofluorescence microscopy. (A) CREST serum, which in particular recognizes CENP-B [36, 55], was used to detect the inner kinetochore and DAPI to visualize DNA. Nup98-HD fusion proteins (Nup98-HOXA9, Nup98-HHEX, Nup98-PMX1; green) associate with chromatin (blue), but not with the inner kinetochore (red) during prometaphase. No association with chromatin was found for Nup98 or Nup98 fused to non-HD partners (i.e. Nup98-JARID1A and Nup98-RARG). Disruption of the HD domain of Nup98-HOXA9 (Nup98-HOXA9 N51S), but not of the FG domain (Nup98-HOXA9 ΔFG) affects chromatin association of the fusion protein. Shown are single confocal sections. Scale bars, 5 μm. (B) Anti-Hec1 antibodies were used to detect the outer kinetochore (red), but no co-localization of Nup98-HOXA9, Nup98-HHEX, and Nup98-PMX1, respectively (green) was observed in prometaphase cells. The fusion proteins exclusively associated with chromatin (blue). Shown are single confocal sections. Scale bars, 5 μm.
Fig 3.
Nup98-HOXA9 affects lamin A/C and lamin B1 distribution.
HeLa cells were transiently transfected with GFP constructs and fixed and stained after 24 hours for immunofluorescence microscopy. (A) Lamin A/C (LA/C, red) and lamin B1 (LB1, magenta) concentrate at the nuclear envelope (NE) in HeLa cells expressing Nup98 (green), but relocate to the nucleoplasm in cells expressing Nup98-HOXA9. White arrowheads point to some lobules decorating the NE. Disruption of the homeodomain of HOXA9 (Nup98-HOXA9 N51S) and the FG domain of Nup98 (Nup98-HOXA9 ΔFG) prevent the relocation of the lamina proteins. Scale bars, 5 μm. (B) Fluorescence intensity of LA/C (left) and LB1 (right) staining was determined along the axis shown as line in the fluorescence images and plotted as a graph.
Fig 4.
Electron micrographs of HeLa cells expressing Nup98 chimeras.
(A) and (B) Nup98-HOXA9, (C) and (D) Nup98-HHEX, control cells expressing (E) GFP and (F) Nup98. HeLa TRex cells expressing (G) Nup98 and (H) Nup98-HOXA9. Scale bars, 2 μm (A, C, E, F, G, H); 1 μm (B and D).
Fig 5.
Expression of Nup98 fusions perturbs the nuclear distribution of lamina-associated polypeptide 2α (LAP2α).
HeLa cells were transiently transfected with GFP constructs and fixed and stained after 24 hours for immunofluorescence microscopy. (A) Lamin A/C (LA/C, red) concentrates at the nuclear envelope in HeLa control cells and in Nup98 expressing cells (green), while LAP2α (magenta) is found throughout the nucleoplasm. In HeLa cells expressing Nup98-HOXA9 (A) and Nup98-HHEX (B), LAP2α diminished from the nucleoplasm and aggregates at the nuclear periphery. Disruption of the homeodomain in HOXA9 (A) and HHEX (B) and the FG domain of Nup98 (A and B) prevent the relocation of the lamina proteins. DAPI was used to visualize DNA (merge). (B) Fluorescence intensity of LAP2α staining was determined along the axis shown as line in the fluorescence images and plotted as a graph. (D) Quantification of cells with altered LA/C and LAP2α distribution. About 400 cells were analyzed for each sample. (E) Western blot analysis of the expression levels of LA/C, LAP2α, and LB1.
Fig 6.
Lamina-associated polypeptide 2α (LAP2α) is altered in Nup98 fusion expressing leukemic cells.
(A) Mouse bone marrow cells were transduced with retroviral particles to express untagged Nup98-HOXA9 and Nup98-HHEX and stained for immunofluorescence microscopy. Expression of these fusion proteins induced lobulations in the NE as evident from the lamin B1 staining (LB1, green). Whereas lamin A/C is not expressed in mouse BM cells (LA/C, magenta), LAP2α (red) is evenly distributed in nuclei from total bone marrow (TBM) cells and lineage minus precursor cells (Lin-), it is diminished from the nucleoplasm and aggregates at the nuclear periphery of mouse BM cells expressing Nup98-HHEX and Nup98-HOXA9, respectively. Electron micrographs of mouse bone marrow cells transduced with (B) Nup98-HHEX and (C) Nup98-HOXA9. Scale bars, 2 μm (C); 1 μm (B). (D) Lamin B1 staining of patient-derived bone marrow cells revealed irregular NE contour. Lamin A/C is not consistently expressed in patient cells and LAP2α is diminished from the nucleoplasm and aggregates at the nuclear periphery. Patient 1 harbored a Nup98-RAP1GDS1, patient 2 a Nup98-NSD1, and patient 3 a Nup98-DDX10 fusion, respectively. Scale bars, 10 μm.
Fig 7.
Expression of Nup98 fusion proteins deregulates cell cycle progression.
HeLa cells were transiently transfected with GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 and analyzed by live cell imaging and flow cytometry after release from a double-thymidine block. (A) Schematic presentation of the time course for live cell imaging and flow cytometric analysis. (B) GFP and differential interference contrast time-lapse images of HeLa cells after release from double-thymidine block are presented. Cells expressing Nup98-HOXA9 and Nup98-PMX1, respectively, show a delay in the re-entry into the cell cycle. Time is indicated in minutes. (C) Boxplot display of the quantitative analysis of the time between double-thymidine release and onset of mitosis. (D) Quantitative analysis of the relative time of mitosis onset. The differences between GFP, GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 expressing cells were statistically highly relevant (**P>0.01; ***P>0.001). (E) DNA flow cytometry of control, GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 expressing cells nine hours after release a double thymidine block. The differences between GFP, GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 expressing cells were statistically highly relevant (**P>0.01; ***P>0.001; ****P>0.0001).
Table 2.
Cell cycle analysis of transiently transfected HeLa cells after double thymidine block.