Book Description

Nuclear pore complexes are best known for their regulation of nucleocytoplasmic transport as integral components of the eukaryotic nuclear envelope. Over the years, their importance in regulation of genome function has become apparent. Many of the 30 individual nuclear pore proteins, Nups, have been found to play distinct roles interacting with and regulating various genomic targets, especially in a cell-type specific manner. The mechanism behind this regulation is often unknown. We have developed a method by which to study the roles of Nups on chromatin using an ectopic-tethering system. Drosophila melanogaster provide a powerful tool with which to combine many genetic elements of interest together in individual organisms quickly and efficiently, and additionally has allowed for powerful high-resolution visualization of chromatin structure perturbations through the imaging of their larval salivary gland polytene chromosomes. Using this system we observed that tethering Nups to chromatin was sufficient to induce chromatin decondensation, visualized by robust and reproducible loss of DNA and histone fluorescene signal associated with Nup binding. Additionally we observed recruitment of chromatin-remodeling complex PBAP, and reliance on PBAP for the observed Nup-induced decondensation, suggesting an important functional relationship between these proteins. We then took our findings and hypotheses generated from this ectopic-tethering imaging system to next conduct functional biochemical analysis of these proteins in Drosophila S2 cell culture. We found that nucleoporin Elys has a robust biochemical interaction with components of PBAP in an endogenous context, supporting the recruitment of these proteins we observed via immunofluorescence. Additionally, MNase experiments determined that Elys was critical for facilitating the formation and/or maintenance of open chromatin, both genome-wide and on a local nucleosomal level at Elys target genes. Together these results demonstrate the importance of nucleoporins in regulation of chromatin structure, and provide one mechanism to explain this phenomenon. These findings are of particular interest in the fields of chromatin biology and the study of nuclear pore protein function, demonstrating a possible explanation for not only associations of NPCs with decondensed chromatin at the nuclear periphery, but also regulation of Nup target gene expression, through regulation of chromatin accessibility.