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. Read more (in Dutch): Het Nut van Junk. NRC Handelsblad, 12-13 augustus 2006.

Searching for a holy grail: 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? Read more: Terminating Wnt signals: a novel nuclear export mechanism targets activated β-Catenin. Journal of Cell Biology, 2005

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. Read more: The needs of the pore. Journal of Cell Biology, 2002