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Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype.

Fahrenkrog B, Martinelli V, Nilles N, Fruhmann G, Chatel G, Juge S, Sauder U, Di Giacomo D, Mecucci C, Schwaller J - PLoS ONE (2016)

Bottom Line: Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML).In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors.Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Medicine, Université Libre de Bruxelles, Charleroi, Belgium.

ABSTRACT
Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

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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).
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pone.0152321.g004: 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).

Mentions: Electron microscopic imaging of HeLa cells expressing GFP-Nup98-HOXA9 (Fig 4A and 4B) and GFP-Nup98-HHEX (Fig 4C and 4D) clearly revealed nuclear deformation and NE lobulations, which were not observed in GFP (Fig 4E) or GFP-Nup98 (Fig 4F) expressing cells. To further strengthen the specific effect of Nup98-HOXA9 expression on NE organization and nuclear shape, we generated stable HeLa T-Rex cell lines expressing GFP-Nup98-HOXA9 in a tetracycline-inducible manner. Expression levels in clonal cell lines were analyzed by Western blot analysis and protein localization was determined by direct fluorescence microscopy (S4 Fig). At the ultrastructural level, we observed the same alterations in the nuclear shape as well as NE lobulation in HeLa T-Rex cells conditionally expressing GFP-Nup98-HOXA9 (Fig 4H) as in transiently transfected HeLa cells. In the absence of tetracycline and GFP-Nup98-HOXA9 expression, these cells had normal nuclear shape and NE characteristics (Fig 4G).


Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype.

Fahrenkrog B, Martinelli V, Nilles N, Fruhmann G, Chatel G, Juge S, Sauder U, Di Giacomo D, Mecucci C, Schwaller J - PLoS ONE (2016)

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).
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pone.0152321.g004: 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).
Mentions: Electron microscopic imaging of HeLa cells expressing GFP-Nup98-HOXA9 (Fig 4A and 4B) and GFP-Nup98-HHEX (Fig 4C and 4D) clearly revealed nuclear deformation and NE lobulations, which were not observed in GFP (Fig 4E) or GFP-Nup98 (Fig 4F) expressing cells. To further strengthen the specific effect of Nup98-HOXA9 expression on NE organization and nuclear shape, we generated stable HeLa T-Rex cell lines expressing GFP-Nup98-HOXA9 in a tetracycline-inducible manner. Expression levels in clonal cell lines were analyzed by Western blot analysis and protein localization was determined by direct fluorescence microscopy (S4 Fig). At the ultrastructural level, we observed the same alterations in the nuclear shape as well as NE lobulation in HeLa T-Rex cells conditionally expressing GFP-Nup98-HOXA9 (Fig 4H) as in transiently transfected HeLa cells. In the absence of tetracycline and GFP-Nup98-HOXA9 expression, these cells had normal nuclear shape and NE characteristics (Fig 4G).

Bottom Line: Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML).In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors.Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Medicine, Université Libre de Bruxelles, Charleroi, Belgium.

ABSTRACT
Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

Show MeSH
Related in: MedlinePlus