Characterization of genome-nucleoporin interactions in Drosophila links chromatin insulators to the nuclear pore complex 

Abstract

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.

Evolution of a transcriptional regulator from a transmembrane nucleoporin

“…(Fig. 5D), which is similar to what has been shown previously for Nup98 and is indicative of a role in transcriptional initiation (Capelson et al 2010;Kalverda and Fornerod 2010;Liang et al 2013). When we performed DamID experiments with a DamID-Nup98 mutant (DamIDNup98ΔCTD) that has dramatically reduced affinity for the NPC (Griffis et al 2002;Kalverda and Fornerod 2010), we found that its chromatin-binding profile is very similar to sPom121 and wild-type Nup98 (Supplemental Fig.…”

“…Previous studies demonstrated that Nup98 is a potent transcription factor in the nucleoplasm whose dynamics on chromatin are blocked by transcriptional inhibitors (Griffis et al 2002(Griffis et al , 2004Capelson et al 2010;Kalverda and Fornerod 2010;Pascual-Garcia et al 2014). For example, when cells were treated with actinomycin D, the fluorescence recovery of Nup98 by FRAP was much slower as Nup98 becomes immobilized on chromatin and unable to recycle to intranuclear foci (Griffis et al 2002(Griffis et al , 2004.…”

“…Thus, it seems that evolution would favor a more mobile subset of Nups that can regulate gene expression anywhere in the nucleus, even beyond the NPC. Indeed, a subset of peripheral Nups like Nup98 has acquired the ability to move off the NPC in metazoan species such as Drosophila (Capelson et al 2010;Kalverda and Fornerod 2010). Interestingly, however, other than Sec13, no scaffold Nups have been shown to localize in the nucleoplasm in Drosophila cells, implying that gene expression regulation probably does not occur via an NPC-like scaffold in nonmammalian species but instead involves recruitment of chromatin-modifying complexes to chromatin by individual Nups (Capelson et al 2010;Pascual-Garcia et al 2014).…”

“…Nup98 was previously shown to bind to intranuclear gene promoters to regulate transcription (Capelson et al 2010;Kalverda and Fornerod 2010;Liang et al 2013;Light et al 2013). This raised the interesting possibility that sPom121, via its loss of membrane anchoring and NPC targeting, might have acquired the ability to bind to Nup98 target genes independently of localization at the nuclear periphery.…”

“…Supporting this hypothesis, the dynamic movement of a subset of Nups, including Nup50, Nup98, and Nup153, was slowed in the presence of transcriptional inhibitory drugs, suggesting a role for Nup98 and potentially other Nups in the regulation of transcription (Griffis et al 2002(Griffis et al , 2004Buchwalter et al 2014). A breakthrough in our understanding of the function of mobile Nups came from studies in Drosophila and mammalian cells, which determined that Nup98 and several other peripheral Nups such as Nup50, Nup62, and Nup153 can detach from the NPC, bind to intranuclear promoters distal to the NE, and affect regulation of adjacent genes (Capelson et al 2010;Kalverda and Fornerod 2010;Liang et al 2013). Although the mechanism of Nup98-mediated gene regulation in the nucleoplasm is yet to be determined in detail, recent evidence suggests that Nup98 interacts with CBP/p300 and MBD-R2/NSL chromatin-modifying complexes in human and Drosophila cells, respectively, suggesting a possible mechanism by which Nup98 promotes an active chromatin state (Kasper et al 1999;Pascual-Garcia et al 2014).…”