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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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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.
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pone.0152321.g005: 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.

Mentions: We next asked whether nucleoplasmic lamin-binding proteins, such as the lamin A-binding partner LAP2α, might be affected by Nup98 chimeras [39]. As shown in Fig 5A (top row), LAP2α is a nucleoplasmic protein excluded from the nucleoli, in contrast to the primarily lamina-associated lamin A/C. Expression of GFP-Nup98 (Fig 5A, second row) did not alter the distribution of lamin A/C or LAP2α. In contrast, cells expressing GFP-Nup98-HOXA9 (Fig 5A, third row) or GFP-Nup98-HHEX (Fig 5B, first row) exhibited a dramatically reduced LAP2α staining in the nucleoplasm and a frequent aggregation of LAP2α in small foci at the nuclear periphery in addition to the alterations in lamin A/C. Plotting LAP2α staining intensity profiles across the nuclear diameter confirmed this decrease in LAP2α staining throughout the nucleoplasm (Fig 5C). GFP-HOXA9 (Fig 5A, bottom row) or GFP-HHEX (Fig 5B, bottom row) expression had no effect on the distribution of the two proteins. In contrast, the changes in lamin A/C and LAP2α distribution were also found in HeLa cells expressing GFP-Nup98-PMX1 or the non-HD fusion proteins GFP-Nup98-NSD1 and GFP-Nup98-NSD3 (S5A Fig), but were not seen in cells expressing other AML-related chromosomal translocation products, such as the AML1-ETO fusion protein (S5B Fig).


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4816316&req=5

pone.0152321.g005: 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.
Mentions: We next asked whether nucleoplasmic lamin-binding proteins, such as the lamin A-binding partner LAP2α, might be affected by Nup98 chimeras [39]. As shown in Fig 5A (top row), LAP2α is a nucleoplasmic protein excluded from the nucleoli, in contrast to the primarily lamina-associated lamin A/C. Expression of GFP-Nup98 (Fig 5A, second row) did not alter the distribution of lamin A/C or LAP2α. In contrast, cells expressing GFP-Nup98-HOXA9 (Fig 5A, third row) or GFP-Nup98-HHEX (Fig 5B, first row) exhibited a dramatically reduced LAP2α staining in the nucleoplasm and a frequent aggregation of LAP2α in small foci at the nuclear periphery in addition to the alterations in lamin A/C. Plotting LAP2α staining intensity profiles across the nuclear diameter confirmed this decrease in LAP2α staining throughout the nucleoplasm (Fig 5C). GFP-HOXA9 (Fig 5A, bottom row) or GFP-HHEX (Fig 5B, bottom row) expression had no effect on the distribution of the two proteins. In contrast, the changes in lamin A/C and LAP2α distribution were also found in HeLa cells expressing GFP-Nup98-PMX1 or the non-HD fusion proteins GFP-Nup98-NSD1 and GFP-Nup98-NSD3 (S5A Fig), but were not seen in cells expressing other AML-related chromosomal translocation products, such as the AML1-ETO fusion protein (S5B Fig).

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