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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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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.
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pone.0152321.g003: 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.

Mentions: We next examined the effects of Nup98 fusion protein expression on several components of the NE, i.e. the major constituents of the nuclear lamina, lamin A/C (LA/C) and lamin B1 (LB1) as well as NPCs. NPCs were detected using a monoclonal antibody that recognizes several FG-repeats containing nucleoporins (mAb414). While the organization of the NPC appeared normal (S1 Fig), expression of Nup98 fusion proteins had a strong effect on the nuclear lamina. As shown in Fig 3A, lamin A/C and lamin B1 were found concentrated in the lamina with lower levels in the nucleoplasm of HeLa cells transiently expressing GFP-Nup98 (second row), as well as in untransfected control cells (first row). In contrast, expression of GFP-tagged Nup98 fusion proteins (i.e. Nup98-HOXA9, Nup98-HHEX, Nup98-PMX1, Nup98-NSD1, Nup98-LEDGF) clearly altered the appearance of the lamina: the accumulation of both lamin A/C and lamin B1 in the lamina was reduced, the fraction in the nucleoplasm increased and the NE appeared somehow lobulated (Fig 3A, rows 3–6; S2 Fig). Consistent with this partial displacement of lamins from the lamina other lamina components, such as Sun1, Sun2, and emerin, were partially diminished from the INM, whereas components of the ONM, such as Nesprin-2, remained unaffected (S3 Fig). Intensity profiles across the horizontal axis through nuclei revealed a decrease in the peripheral lamins A/C and B1 along with an increase in the nucleoplasmic lamins A/C and B1 in cells expressing GFP-Nup98-HOXA9 as compared to GFP-Nup98 (Fig 3B). The aberrant NE phenotype was dependent on the integrity of both the HD and the FG domain in Nup98: HeLa cells expressing the HD mutant Nup98-HOXA9 N51S or the Nup98-HOXA9 ΔFG mutant showed lamina staining indistinguishable from cells expressing Nup98 (Fig 3A). Similar results were obtained for the corresponding Nup98-HHEX mutants (data not shown).


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)

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.
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Related In: Results  -  Collection

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pone.0152321.g003: 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.
Mentions: We next examined the effects of Nup98 fusion protein expression on several components of the NE, i.e. the major constituents of the nuclear lamina, lamin A/C (LA/C) and lamin B1 (LB1) as well as NPCs. NPCs were detected using a monoclonal antibody that recognizes several FG-repeats containing nucleoporins (mAb414). While the organization of the NPC appeared normal (S1 Fig), expression of Nup98 fusion proteins had a strong effect on the nuclear lamina. As shown in Fig 3A, lamin A/C and lamin B1 were found concentrated in the lamina with lower levels in the nucleoplasm of HeLa cells transiently expressing GFP-Nup98 (second row), as well as in untransfected control cells (first row). In contrast, expression of GFP-tagged Nup98 fusion proteins (i.e. Nup98-HOXA9, Nup98-HHEX, Nup98-PMX1, Nup98-NSD1, Nup98-LEDGF) clearly altered the appearance of the lamina: the accumulation of both lamin A/C and lamin B1 in the lamina was reduced, the fraction in the nucleoplasm increased and the NE appeared somehow lobulated (Fig 3A, rows 3–6; S2 Fig). Consistent with this partial displacement of lamins from the lamina other lamina components, such as Sun1, Sun2, and emerin, were partially diminished from the INM, whereas components of the ONM, such as Nesprin-2, remained unaffected (S3 Fig). Intensity profiles across the horizontal axis through nuclei revealed a decrease in the peripheral lamins A/C and B1 along with an increase in the nucleoplasmic lamins A/C and B1 in cells expressing GFP-Nup98-HOXA9 as compared to GFP-Nup98 (Fig 3B). The aberrant NE phenotype was dependent on the integrity of both the HD and the FG domain in Nup98: HeLa cells expressing the HD mutant Nup98-HOXA9 N51S or the Nup98-HOXA9 ΔFG mutant showed lamina staining indistinguishable from cells expressing Nup98 (Fig 3A). Similar results were obtained for the corresponding Nup98-HHEX mutants (data not shown).

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