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The nuclear envelope arguably is the most important border within the eukaryotic cell. Borders in general are stable, but can reflect the dynamics of an entire organization. This makes them an attractive platform from which to study a complex system -- such as the eukaryotic cell.
Chromatin meets the Nuclear Pore Complex
Apart from forming a physical barrier between the nucleus and the cytoplasm, allowing selective molecular exchange through nuclear pore complexes, the nuclear envelope plays an important role in chromatin organization. In particular the nuclear lamina, which is a thin layer of microfilaments coating the inner nuclear membrane, had been implicated in the spacial organization of the eukaryotic genome. Less is known about the nature of chromatin interacting with the NPC.
In yeast, nuclear pore complexes also interact with active genes, attracting or retaining them at the nuclear periphery. In higher eukaryotes, some NPC components (nucleoporins) are also found in the nucleoplasm, with so far unknown function. We have functionally characterized nucleoporin-chromatin interactions specifically at the NPC or within the nucleoplasm in Drosophila. We analyzed genomic interactions of full-length nucleoporins Nup98, Nup50 and Nup62 and nucleoplasmic and NPC-tethered forms of Nup98. We found that nucleoporins predominantly interacted with transcriptionally active genes inside the nucleoplasm. A smaller set of non-active genes interacted with the NPC. Genes strongly interacting with nucleoplasmic Nup98 were downregulated upon Nup98 depletion and activated on nucleoplasmic Nup98 overexpression. Thus, nucleoporins stimulate gene expression away from the NPC by interacting with these genes inside the nucleoplasm.
Nuclear pore complex components and cancer
Genes that interact with and respond to nucleoplasmic pools of Nup98 or Nup50 are highly enriched in developmental genes, suggesting an important function of the nucleoplasmic pool of nucleoporins on fly development. In adition, genes that interact with and respond to nucleoplasmic Nup98 are enriched in genes that are directly linked to the cell cycle. These include for example Cyclin B, Bub1 and Mad2. Interestingly, several of the nucleporin-regulated cell cycle genes have also been implicated in human cancer. For example, overexpression of Mad2 leads to tumors in transgenic mice and the human homologues of several of the group are included in "death-from-cancer" gene signatures: high expression of this signature set of genes correlates with an unfavourable outcome in several types of cancer.
Nup98 plays a causative yet incompletely understood role in human leukemia. A large number of different chromosome translocations in mainly acute myeloid leukemia (AML) result in chimaeric proteins containing the FG repeat part of Nup98 and a wide set of proteins, including homeobox transcription factors such as HoxA9. Common to all oncogenic Nup98 fusion proteins is that their localization is inside the nucleoplasm, not at the NPC.
Future research will focus on the role of nucleoplasmic nucleoporins expressed as a consequence of leukemia-associated chromosome translocations, and whether they contribute to oncogenesis by promoting expression of cell cycle genes.
Nuclear envelope chromatin interactions: From the inside looking in
Many observations suggest that the nuclear envelope plays a key role in the spatial and functional organization of chromatin. In close collaboration with the lab of Bas van Steensel (Dept. of Molecular Genetics), we are analyzing the behaviour of genes in respect to various nuclear envelope components.
The nuclear lamina (NL) has long been thought to be an anchoring site of chromatin and to participate in the regulation of gene expression, but genomic sequences that interact with the NL in vivo have remained unknown. We used a genome-wide approach to identify nearly 500 Drosophila genes that interact in vivo with the NL component Lam, a B-type lamin.
These genes are transcriptionally silent, late replicating, lack active histone marks, and are widely spaced. These factors collectively predict Lamin binding behavior, indicating the NL integrates variant and invariant chromatin features. Consistently, NE proximity is partly conserved between cell types and induction of gene expression or active histone marks reduces Lam binding. Lam target genes cluster in the genome, and these clusters are coordinately expressed during development. This genome-wide analysis gives clear insight into the nature and dynamic behavior of the genome at the NL and implies that intergenic or "junk" DNA functions in the global organization of chromatin in the nucleus.
Leucine-rich nuclear export signals (NESs) mediate rapid nuclear export of proteins via interaction with CRM1. Although this interaction is stimulated by RanGTP, it remains of a relatively low affinity. For this reason NESs are difficult to detect biochemically. NESs are difficult to predict as well. as the consensus sequence is very loose. In other words: NESs are a pain. But why?
To answer this, we screened a 15-mer random peptide library for CRM1 binding in order to identify strong signals. In this way we identified NESs with a very high affinity for CRM1 in vitro. In fact, the affinity is so high that they stably bind without the requirement of RanGTP. We have dubbed these signals "supraphysiological" NESs, or superNESs for short.
SuperNESs behave peculiarly in vivo: they are inefficient in mediating nuclear export and instead accumulate together with CRM1 at nucleoporin Nup358. From this we conclude that NESs have evolved to maintain low affinity for CRM1; allowing efficient export complex disassembly and release from Nup358. These results stress the importance of weak, almost invisible interactions.
While supraNESs are inefficient to promote regular nuclear export, viruses have employed them for different purposes. The small mouse parvovirus MVM uses a supraNES to promote nuclear export of virus particles, while the new world alphavirus VEEV uses a supraNES in combination with a regular NLS to obstruct the nuclear pore complex.
Nuclear export of β-Catenin
β-Catenin is the nuclear effector of the Wnt signaling cascade. The mechanism by which nuclear activity of β-Catenin is regulated is not very well defined. We identified a cofactor of CRM1-mediated nuclear export, Ran-binding protein 3 (RanBP3) as a novel β-Catenin interacting protein that binds directly to β-Catenin in a RanGTP stimulated manner.
In vivo, RanBP3 inhibits β-Catenin-mediated transcriptional activation both in Wnt1 or β-Catenin stimulated human cells. In Xenopus embryos, RanBP3 interferes with β-Catenin-induced dorsoventral axis formation. Furthermore, RanBP3 depletion stimulates the Wnt pathway, both in human cells and Drosophila embryos. In human cells, this is accompanied by an increase of active β-Catenin in the nucleus. Conversely, overexpression of RanBP3 leads to a shift of active β-Catenin towards the cytoplasm.
Interestingly, RanBP3 only acts on a very small dephosphorylated nuclear fraction of total β-Catenin, that correlates with transcriptional activity. We can visualize this fraction only in certain colon carcinoma cells that contain "massive" amounts of this active β-Catenin; still only a very small fraction of total β-Catenin. We find that modulation of β-Catenin activity and localization by RanBP3 is independent of APC and CRM1.
We conclude that RanBP3 is a direct export enhancer for β-Catenin, independent of its role as a CRM1 associated nuclear export cofactor, and ask -- what is active β-Catenin?
The closer you look the less you see: structure-function of the nuclear pore complex
Thanks to the genius and hard work of numerous labs over the last decade, we know almost everything about the structure and function of the nuclear pore complex -- or do we? In any case we've learned quite a bit of what it's not.
Our lab has contributed to the "negative knowledge" by providing evidence that the cytoplasmic filaments of the NPC are not required for selective nuclear import, which had been hypothesized previously. Instead, the lab has provided evidence that the cytoplasmic filaments play a supportive role in disassembly of nuclear export complexes.
In addition we have identified Nup358 as the main consituent of the cytoplasmic filaments of the NPC, which is anchored to the "core NPC by Nup88.
The current focus includes the question which nucleoporins are involved in nuclear export, and whether different types of cargo have different requirements.
Dept of Gene Regulation / B4
Netherlands Cancer Institute
Plesmanlaan 121 - 1066 CX Amsterdam, The Netherlands
Tel: +31-(0)20-512 2024