http://repub.eur.nl/res/aut/8085/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/34195/ 2011-06-27T00:00:00Z Background: NADPH oxidases play an essential role in reactive oxygen species (ROS)-based signaling in the heart. Previously, we have demonstrated that (peri)nuclear expression of the catalytic NADPH oxidase subunit NOX2 in stressed cardiomyocytes, e.g. under ischemia or high concentrations of homocysteine, is an important step in the induction of apoptosis in these cells. Here this ischemia-induced nuclear targeting and activation of NOX2 was specified in cardiomyocytes. Methods: The effect of ischemia, mimicked by metabolic inhibition, on nuclear localization of NOX2 and the NADPH oxidase subunits p22phoxand p47phox, was analyzed in rat neonatal cardiomyoblasts (H9c2 cells) using Western blot, immuno-electron microscopy and digital-imaging microscopy. Results: NOX2 expression significantly increased in nuclear fractions of ischemic H9c2 cells. In addition, in these cells NOX2 was found to colocalize in the nuclear envelope with nuclear pore complexes, p22phox, p47phoxand nitrotyrosine residues, a marker for the generation of ROS. Inhibition of NADPH oxidase activity, with apocynin and DPI, significantly reduced (peri)nuclear expression of nitrotyrosine. Conclusion: We for the first time show that NOX2, p22phoxand p47phoxare targeted to and produce ROS at the nuclear pore complex in ischemic cardiomyocytes. http://repub.eur.nl/res/pub/32958/ 2010-12-15T00:00:00Z Nuclear pore complexes (NPCs) are multiprotein complexes consisting of nucleoporins and function in transport between the nucleus and the cytoplasm. In yeast, nucleoporins have also been linked to gene expression as well as to chromatin insulating activity. Recently, we identified genomic regions that interact with nucleoporins in Drosophila using DamID technology. We found that nucleoporins in the nucleoplasm interact with active genes and stimulate gene expression. However, genes interacting with nucleoporins at the NPC itself show average gene expression and it remains unclear why they interact with the NPC. Here, we further investigated the function of the genome-NPC interactions. First, to investigate whether a different technique would lead to similar results, we compared our nucleoporin DamID data to recently published nucleoporin chromatin immunoprecipitation (ChIP) data. Then, to further understand the function of interactions between the genome and NPCs, we analyzed the relationship between NPC-interacting genomic regions and chromatin insulators. We found that the insulator protein Su(Hw) was enriched within and near NPC-interacting genomic regions, suggesting a role of this protein in chromatin architecture close to the NPC. This suggests that the NPC may have a function in the structural organization of the genome. http://repub.eur.nl/res/pub/28544/ 2010-04-01T00:00:00Z BICD2 is one of the two mammalian homologues of the Drosophila Bicaudal D, an evolutionarily conserved adaptor between microtubule motors and their cargo that was previously shown to link vesicles and mRNP complexes to the dynein motor. Here, we identified a G2-specific role for BICD2 in the relative positioning of the nucleus and centrosomes in dividing cells. By combining mass spectrometry, biochemical and cell biological approaches, we show that the nuclear pore complex (NPC) component RanBP2 directly binds to BICD2 and recruits it to NPCs specifically in G2 phase of the cell cycle. BICD2, in turn, recruits dynein-dynactin to NPCs and as such is needed to keep centrosomes closely tethered to the nucleus prior to mitotic entry. When dynein function is suppressed by RNA interference-mediated depletion or antibody microinjection, centrosomes and nuclei are actively pushed apart in late G2 and we show that this is due to the action of kinesin-1. Surprisingly, depletion of BICD2 inhibits both dynein and kinesin-1-dependent movements of the nucleus and cytoplasmic NPCs, demonstrating that BICD2 is needed not only for the dynein function at the nuclear pores but also for the antagonistic activity of kinesin-1. Our study demonstrates that the nucleus is subject to opposing activities of dynein and kinesin-1 motors and that BICD2 contributes to nuclear and centrosomal positioning prior to mitotic entry through regulation of both dynein and kinesin-1. http://repub.eur.nl/res/pub/25205/ 2009-04-06T00:00:00Z SRY and other Sox-type transcription factors are important developmental regulators with various implications in human disease. In this study, we identified Exp4 (exportin 4) as an interaction partner of Sox2 in mouse embryonic stem cells and neural progenitors. We show that, besides its established function in nuclear export, Exp4 acts as a bona fide nuclear import receptor for Sox2 and SRY. Thus, Exp4 is an example of a nuclear transport receptor carrying distinct cargoes into different directions. In contrast to a published study, we observed that the import activity of Imp-α (importin-α) isoforms toward Sox2 is negligible. Instead, we found that Imp9 and the Imp-β/7 heterodimer mediate nuclear import of Sox2 in parallel to Exp4. Import signals for the three pathways overlap and include conserved residues in the Sox2 high-mobility group (HMG) box domain that are also critical for DNA binding. This suggests that nuclear import of Sox proteins is facilitated by several parallel import pathways. http://repub.eur.nl/res/pub/28741/ 2008-06-01T00:00:00Z The standard model of Wnt signaling specifies that after receipt of a Wnt ligand at the membranous receptor complex, downstream mediators inhibit a cytoplasmic destruction complex, allowing β-catenin to accumulate in the cytosol and nucleus and co-activate Wnt target genes. Unexpectedly, shortly after Wnt treatment, we detected the dephosphorylated form of β-catenin at the plasma membrane, where it displayed a discontinuous punctate labeling. This pool of β-catenin could only be detected in E-cadherin-/-cells, because in E-cadherin+/+cells Wnt-induced, membranous β-catenin was concealed by a constitutive junctional pool. Wnt-signaling-dependent dephosphorylated β-catenin colocalized at the plasma membrane with two members of the destruction complex - APC and axin - and the activated Wnt co-receptor LRP6. β-catenin induced through the Wnt receptor complex was significantly more competent transcriptionally than overexpressed β-catenin, both in cultured cells and in early Xenopus embryos. Our data reveal a new step in the processing of the Wnt signal and suggest regulation of signaling output beyond the level of protein accumulation. http://repub.eur.nl/res/pub/29263/ 2008-05-01T00:00:00Z T-cell acute lymphoblastic leukemia (TALL) is mostly characterized by specific chromosomal abnormalities, some occurring in a mutually exclusive manner that possibly delineate specific T-ALL subgroups. One subgroup, including MLLrearranged, CALM-AFWor inv (7)(p15q34) patients, is characterized by elevated expression of HOXA genes. Using a gene expression-based clustering analysis of 67 T-ALL cases with recurrent molecular genetic abnormalities and 25 samples lacking apparent aberrations, we identified 5 new patients with elevated HOXA levels. Using microarray-based comparative genomic hybridization (array-CGH), a cryptic and recurrent deletion, del (9)(q34.11q34.13), was exclusively identified in 3 of these 5 patients. This deletion results in a conserved SET-NUP214 fusion product, which was also identified in the T-ALL cell line LOUCY. SET-NUP214 binds in the promoter regions of specific HOXA genes, where it interacts with CRM1 and DOT1L, which may transcriptionally activate specific members of the HOXA cluster. Targeted inhibition of SET-NUP214 by siRNA abolished expression of HOXA genes, inhibited proliferation, and induced differentiation in LOUCY but not in other T-ALL lines. We conclude that SETNUP214 may contribute to the pathogenesis of T-ALL by enforcing T-cell differentia. http://repub.eur.nl/res/pub/17815/ 1997-01-08T00:00:00Z The starting point of the work described in tius thesis was a novel gene, CAN, that had been cloned by virtue of its involvement in translocation (6;9) (von Lindern, 1990), a recurrent chromosomal aberration defining a specific subtype of acute myeloid leukemia. Tills gene's predicted amino acid sequence showed no homology to known proteins (chapter 3), and therefore its cellular function was unknown. The protein encoded by CANIs fusion partner in the (6;9) translocation, DEK, also had no significant homology to proteins in the database. Consequently, the role of the chimeric DEK-CAN fusion protein in t(6;9)-associated leukemogenesis was a complete mystery. In addition, illl'itl'o studies to show the oncogenic function ofDEK-CAN consistently proved unsuccessful (M. von Lindem, J. Boer, G. Grosveld, unpublished results). To gain understanding of the possible function ofDEK-CAN, as well as the normal cellular functions ofDEK and CAN, we analyzed their primary amino acid stmctures (chapter 3 and 4), studied their subcellular localization (chapter 3), and looked for interacting proteins (chapter 5 and 6). In these studies, we focused on the CAN protein, since this protein had also been found in another leukemia-related fusion protein, SET-CAN (von Lindem, 1992), which suggests that it could playa more general role in leukemogenesis. In view of the difficulty reproducing the oncogenic effect of the DEK-CAN gene in vitro, and toxic effects caused by overexpression of DEK-CAN, we also set out to create an ill vivo system to mimic translocation (6;9) in transgenic mice using Cre-mediated recombination (chapter 8). http://repub.eur.nl/res/pub/3045/ 1993-01-01T00:00:00Z This article presents the development of a set of new expression vectors for overproduction of proteins in Escherichia coli. The vectors, pETUBI-ES1, 2 and 3, allow in-frame cloning of any sequence with the ubiquitin gene driven by the strong T7f10 promoter. Combination of the T7 expression system with ubiquitin fusion appears to have a synergistic effect on protein overproduction. Large amounts of stable RNA are produced by T7 RNA polymerase, and fusion of ubiquitin to the N-terminus of target proteins seems to confer more efficient translation, better folding or protection against proteolytic degradation. The ubiquitin part can be utilized for purification of the fusion protein, after which it can be easily removed from the fusion product by ubiquitin-specific proteases. The advantage of combining both systems is demonstrated by the synthesis of large quantities (up to 40-50% of the total protein) of the human ERCC1 protein that hitherto was refractory to overproduction in various other E. coli and yeast expression systems.