Limits...
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.

Show MeSH

Related in: MedlinePlus

Mitotic localization of Nup98 chimeras.HeLa cells were transiently transfected with GFP constructs and fixed and stained after 24 hours for immunofluorescence microscopy. (A) CREST serum, which in particular recognizes CENP-B [36, 55], 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) 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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4816316&req=5

pone.0152321.g002: Mitotic localization of Nup98 chimeras.HeLa cells were transiently transfected with GFP constructs and fixed and stained after 24 hours for immunofluorescence microscopy. (A) CREST serum, which in particular recognizes CENP-B [36, 55], 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) 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.

Mentions: Nup98-HOXA9, Nup98-HOXD10 and Nup98-PMX1 were previously found to associate with kinetochores and chromosomes during mitosis and it was suggested that they localize to the outer kinetochore [36]. To address whether Nup98 chimeras with non-HD proteins would behave similarly, we transiently transfected HeLa cells with GFP-Nup98, GFP-HOXA9, the HD-fusions GFP-Nup98-HOXA9, GFP-Nup98-HHEX, GFP-Nup98-PMX1, as well as with the non-HD fusions GFP-Nup98-JARID1A and GFP-Nup98-RARG, and stained non-synchronized cells with CREST serum staining the inner kinetochore. As shown in Fig 2A, we found that similar to HOXA9, Nup98-HOXA9, Nup98-HHEX and Nup98-PMX1 were concentrated on chromatin during pro-metaphase (and metaphase; data not shown). The association of the fusion proteins with chromatin was dependent on the integrity of the homeodomain: the HD mutant Nup98-HOXA9 N51S no longer bound chromatin, whereas the Nup98-HOXA9 ΔFG mutant was still found on chromatin. Similar results were obtained for the corresponding Nup98-HHEX and Nup98-PMX1 mutants (data not shown). However, no significant co-localization with the inner kinetochore marker CREST was observed for these Nup98-HD fusion proteins. Furthermore, Nup98-HOXA9, Nup98-HHEX and Nup98-PMX1, respectively, also showed no overlay with the outer kinetochore protein Hec1 [38] during pro-metaphase (Fig 2B) or metaphase (data not shown). In contrast to Nup98-HD fusions and similar to Nup98, the Nup98-JARID1A and Nup98-RARG fusions were not found to be concentrated on chromatin. Together our data show that only Nup98-HD fusion proteins co-localize with chromatin, but not with kinetochores.


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)

Mitotic localization of Nup98 chimeras.HeLa cells were transiently transfected with GFP constructs and fixed and stained after 24 hours for immunofluorescence microscopy. (A) CREST serum, which in particular recognizes CENP-B [36, 55], 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) 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4816316&req=5

pone.0152321.g002: Mitotic localization of Nup98 chimeras.HeLa cells were transiently transfected with GFP constructs and fixed and stained after 24 hours for immunofluorescence microscopy. (A) CREST serum, which in particular recognizes CENP-B [36, 55], 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) 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.
Mentions: Nup98-HOXA9, Nup98-HOXD10 and Nup98-PMX1 were previously found to associate with kinetochores and chromosomes during mitosis and it was suggested that they localize to the outer kinetochore [36]. To address whether Nup98 chimeras with non-HD proteins would behave similarly, we transiently transfected HeLa cells with GFP-Nup98, GFP-HOXA9, the HD-fusions GFP-Nup98-HOXA9, GFP-Nup98-HHEX, GFP-Nup98-PMX1, as well as with the non-HD fusions GFP-Nup98-JARID1A and GFP-Nup98-RARG, and stained non-synchronized cells with CREST serum staining the inner kinetochore. As shown in Fig 2A, we found that similar to HOXA9, Nup98-HOXA9, Nup98-HHEX and Nup98-PMX1 were concentrated on chromatin during pro-metaphase (and metaphase; data not shown). The association of the fusion proteins with chromatin was dependent on the integrity of the homeodomain: the HD mutant Nup98-HOXA9 N51S no longer bound chromatin, whereas the Nup98-HOXA9 ΔFG mutant was still found on chromatin. Similar results were obtained for the corresponding Nup98-HHEX and Nup98-PMX1 mutants (data not shown). However, no significant co-localization with the inner kinetochore marker CREST was observed for these Nup98-HD fusion proteins. Furthermore, Nup98-HOXA9, Nup98-HHEX and Nup98-PMX1, respectively, also showed no overlay with the outer kinetochore protein Hec1 [38] during pro-metaphase (Fig 2B) or metaphase (data not shown). In contrast to Nup98-HD fusions and similar to Nup98, the Nup98-JARID1A and Nup98-RARG fusions were not found to be concentrated on chromatin. Together our data show that only Nup98-HD fusion proteins co-localize with chromatin, but not with kinetochores.

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