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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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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.
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pone.0152321.g001: 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.

Mentions: Nup98 is known to localize to NPCs and the nucleoplasm [5, 7, 35], while Nup98-HD fusion proteins were previously found in a distinctive punctate pattern throughout the nuclear interior with exclusion from the nucleolus [23, 32, 36]. We first asked whether Nup98 chimeras in which Nup98 is fused to non-HD partners might adapt a similar speckled pattern in the nucleus as the Nup98-HD chimeras. To determine the localization, GFP-tagged fusion proteins (Table 1) were visualized in transiently transfected HeLa cells. GFP-Nup98 (Fig 1A) localized to the nuclear rim in a pattern typical for nucleoporins, to the nucleoplasm, and to nuclear foci, also referred as “GLFG bodies”, as previously described [5, 23, 36]. The fusion proteins GFP-Nup98-HOXA9 (Fig 1B), GFP-Nup98-HOXA10 (Fig 1C), GFP-Nup98-HHEX (Fig 1D) and GFP-Nup98-PMX1 (Fig 1E) were present in a characteristic speckled pattern in the nucleus, as previously seen for GFP-Nup98-HOXA9, GFP-Nup98-HHEX, and GFP-Nup98-PMX1 [23, 32, 36]. In contrast to the Nup98-HD fusion proteins, Nup98 chimeras with PHD finger, zinc-finger or SET domain proteins, such as GFP-Nup98-JARID1A (Fig 1F), GFP-Nup98-PHF23 (Fig 1G), GFP-Nup98-NSD1 (Fig 1H), or GFP-Nup98-NSD3 (Fig 1I), exhibited a nuclear localization with a different, finer punctate pattern very similar as recently described for the GFP-Nup98-RARG fusion (Fig 1K) [37]. These chimeras also showed a higher tendency to form aggregates, similar to GFP-Nup98. Fusions of Nup98 to partners lacking any of these DNA-binding domains, such as GFP-Nup98-LEDGF (Fig 1L) showed no particular distribution within the nucleoplasm. Mutational disruption of the HD (GFP-Nup98-PMX1 N51S, Fig 1M; GFP-Nup98-HOXA9 N51S, Fig 1O; GFP-Nup98-HHEX ΔHD, Fig 1Q), the PHD domain (GFP-Nup98-JARID1A W1625A Fig 1R) or the GLFG domain (GFP-Nup98-HOXA9 ΔFG, Fig 1N; GFP-Nup98-HHEX ΔFG Fig 1P) displaced the Nup98 chimeras from their specific locations. GFP-HOXA9 (Fig 1S) and GFP-HHEX (Fig 1T) were both found diffusely distributed in the nucleoplasm. Together our data indicate that Nup98 chimeras generally localize to the nuclear interior, but their specific intra-nuclear distribution is varying and dependent on the fusion partner, the integrity of the GLFG domain in Nup98 and the integrity of the DNA-binding domains of the respective fusion partner.


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4816316&req=5

pone.0152321.g001: 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.
Mentions: Nup98 is known to localize to NPCs and the nucleoplasm [5, 7, 35], while Nup98-HD fusion proteins were previously found in a distinctive punctate pattern throughout the nuclear interior with exclusion from the nucleolus [23, 32, 36]. We first asked whether Nup98 chimeras in which Nup98 is fused to non-HD partners might adapt a similar speckled pattern in the nucleus as the Nup98-HD chimeras. To determine the localization, GFP-tagged fusion proteins (Table 1) were visualized in transiently transfected HeLa cells. GFP-Nup98 (Fig 1A) localized to the nuclear rim in a pattern typical for nucleoporins, to the nucleoplasm, and to nuclear foci, also referred as “GLFG bodies”, as previously described [5, 23, 36]. The fusion proteins GFP-Nup98-HOXA9 (Fig 1B), GFP-Nup98-HOXA10 (Fig 1C), GFP-Nup98-HHEX (Fig 1D) and GFP-Nup98-PMX1 (Fig 1E) were present in a characteristic speckled pattern in the nucleus, as previously seen for GFP-Nup98-HOXA9, GFP-Nup98-HHEX, and GFP-Nup98-PMX1 [23, 32, 36]. In contrast to the Nup98-HD fusion proteins, Nup98 chimeras with PHD finger, zinc-finger or SET domain proteins, such as GFP-Nup98-JARID1A (Fig 1F), GFP-Nup98-PHF23 (Fig 1G), GFP-Nup98-NSD1 (Fig 1H), or GFP-Nup98-NSD3 (Fig 1I), exhibited a nuclear localization with a different, finer punctate pattern very similar as recently described for the GFP-Nup98-RARG fusion (Fig 1K) [37]. These chimeras also showed a higher tendency to form aggregates, similar to GFP-Nup98. Fusions of Nup98 to partners lacking any of these DNA-binding domains, such as GFP-Nup98-LEDGF (Fig 1L) showed no particular distribution within the nucleoplasm. Mutational disruption of the HD (GFP-Nup98-PMX1 N51S, Fig 1M; GFP-Nup98-HOXA9 N51S, Fig 1O; GFP-Nup98-HHEX ΔHD, Fig 1Q), the PHD domain (GFP-Nup98-JARID1A W1625A Fig 1R) or the GLFG domain (GFP-Nup98-HOXA9 ΔFG, Fig 1N; GFP-Nup98-HHEX ΔFG Fig 1P) displaced the Nup98 chimeras from their specific locations. GFP-HOXA9 (Fig 1S) and GFP-HHEX (Fig 1T) were both found diffusely distributed in the nucleoplasm. Together our data indicate that Nup98 chimeras generally localize to the nuclear interior, but their specific intra-nuclear distribution is varying and dependent on the fusion partner, the integrity of the GLFG domain in Nup98 and the integrity of the DNA-binding domains of the respective fusion partner.

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