ChEC-seq2 profiling of Crm1, Nucleoporins (nups), and RNA Pol II from budding yeast grown in SDC

Nuclear pore proteins (Nups) physically interact with hundreds of chromosomal sites, impacting transcription. In yeast, transcription factors mediate interactions between Nups and enhancers and promoters. To define the molecular basis of this mechanism, we exploited a separation-of-function mutation in the Gcn4 transcription factor that blocks its interaction with the nuclear pore complex (NPC). This mutation reduces the interaction of Gcn4 with the highly conserved nuclear export factor Crm1/Xpo1. Crm1 and Nups co-occupy enhancers and Crm1 inhibition blocks interaction of the nuclear pore protein Nup2 with the genome. In vivo, Crm1 interacts stably with the NPC. In vitro, Crm1 binds both Gcn4 and Nup2 directly. Importantly, the interaction between Crm1 and Gcn4 requires neither Ran-GTP nor the nuclear export sequence binding site. Finally, Crm1 and Ran-GTP stimulate DNA binding by Gcn4, suggesting that allosteric coupling between Crm1-Ran-GTP binding and DNA binding facilitates docking of transcription factor-bound enhancers at the NPC. ChEC-seq2 was used to assess Crm1, Nups, and Pol II localization in synthetic complete medium containing glucose (SDC) in budding yeast.