Mitotic localization of Nup98 chimeras.

<p>HeLa cells were transiently transfected with GFP constructs and fixed and stained after 24 hours for immunofluorescence microscopy. (<b>A</b>) CREST serum, which in particular recognizes CENP-B [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152321#pone.0152321.ref036" target="_blank">36</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152321#pone.0152321.ref055" target="_blank">55</a>], 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>B</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.</p

Localization of Nup98 fusion proteins.

<p>HeLa cells were transiently transfected with GFP constructs and visualized after 24 hours by direct fluorescence microscopy. All fusion proteins localize to the nucleus. (<b>A</b>) GFP-Nup98 is found at the nuclear rim and in the nucleoplasm, whereas Nup98 homeodomain fusions exhibit a punctate pattern: (<b>B</b>) GFP-Nup98-HOXA9, (<b>C</b>) GFP-Nup98-HOXA10, (<b>D</b>) GFP-Nup98-HHEX, and (<b>E</b>) GFP-Nup98-PMX1. Nup98 fusions with other chromatin-binding motifs show a different punctate distribution: (<b>F</b>) GFP-Nup98-JARID1A, and (<b>G</b>) GFP-Nup98-PHF23 (<b>H</b>) GFP-Nup98-NSD1, (<b>I</b>) GFP-Nup98-NSD3 and (<b>K</b>) GFP-Nup98-RARG. Nup98 fused to partners that lack chromatin-binding domains localize more dispersed to the nucleoplasm: (<b>L</b>) GFP-Nup98-LEDGF. Disruption of the FG, the HD or the PHD domain disrupts the localization of the Nup98 chimeras: (<b>M</b>) GFP-Nup98-PMX1 N51S, (<b>N</b>) GFP-Nup98-HOXA9 ΔFG, (<b>O</b>) GFP-Nup98-HOXA9 N51S, (<b>P</b>) GFP-Nup98-HHEX ΔFG, (<b>Q</b>) GFP-Nup98-HHEX ΔHD, and (<b>R</b>) GFP-Nup98-JARID1A W1625A. (<b>S</b>) GFP-HOXA9 and (<b>T</b>) GFP-HHEX localize to the nucleoplasm. Shown are representative confocal images. Scale bars, 5 μm.</p

Electron micrographs of HeLa cells expressing Nup98 chimeras.

<p>(<b>A</b>) and (<b>B</b>) Nup98-HOXA9, (<b>C</b>) and (<b>D</b>) Nup98-HHEX, control cells expressing (<b>E</b>) GFP and (<b>F</b>) Nup98. HeLa TRex cells expressing (<b>G</b>) Nup98 and (<b>H</b>) Nup98-HOXA9. Scale bars, 2 μm (A, C, E, F, G, H); 1 μm (B and D).</p

Cell cycle analysis of transiently transfected HeLa cells after double thymidine block.

<p>Cell cycle analysis of transiently transfected HeLa cells after double thymidine block.</p

Nup98-HOXA9 affects lamin A/C and lamin B1 distribution.

<p>HeLa cells were transiently transfected with GFP constructs and fixed and stained after 24 hours for immunofluorescence microscopy. (<b>A</b>) 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>B</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.</p

Expression of Nup98 fusions perturbs the nuclear distribution of lamina-associated polypeptide 2α (LAP2α).

<p>HeLa cells were transiently transfected with GFP constructs and fixed and stained after 24 hours for immunofluorescence microscopy. (<b>A</b>) 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 (<b>A</b>) and Nup98-HHEX (<b>B</b>), LAP2α diminished from the nucleoplasm and aggregates at the nuclear periphery. Disruption of the homeodomain in HOXA9 (<b>A</b>) and HHEX (<b>B</b>) and the FG domain of Nup98 (<b>A</b> and <b>B</b>) prevent the relocation of the lamina proteins. DAPI was used to visualize DNA (merge). (<b>B</b>) Fluorescence intensity of LAP2α staining was determined along the axis shown as line in the fluorescence images and plotted as a graph. (<b>D</b>) Quantification of cells with altered LA/C and LAP2α distribution. About 400 cells were analyzed for each sample. (<b>E</b>) Western blot analysis of the expression levels of LA/C, LAP2α, and LB1.</p

Lamina-associated polypeptide 2α (LAP2α) is altered in Nup98 fusion expressing leukemic cells.

<p>(<b>A</b>) 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>B</b>) Nup98-HHEX and (<b>C</b>) Nup98-HOXA9. Scale bars, 2 μm (C); 1 μm (B). (<b>D</b>) 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.</p

Additional file 1: of MYB deregulation from a EWSR1-MYB fusion at leukemic evolution of a JAK2 V617F positive primary myelofibrosis

Detailed materials/methods and results. (DOC 68 kb