Exportin-1 functions as an adaptor for transcription factor-mediated docking of chromatin at the nuclear pore complex

Abstract

Nuclear pore proteins (Nups) in yeast, flies and mammals physically interact with hundreds or thousands of chromosomal sites, which impacts transcriptional regulation. In budding yeast, transcription factors mediate interaction of Nups with enhancers of highly active genes. 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) without altering its DNA binding or activation domains. SILAC mass spectrometry revealed that this mutation reduces the interaction of Gcn4 with the highly conserved nuclear export factor Crm1/Xpo1. Crm1 both interacts with the same sites as Nups genome-wide and is required for Nup2 to interact with the yeast genome. In vivo, Crm1 undergoes extensive and stable interactions with the NPC. In vitro, Crm1 binds to Gcn4 and these proteins form a complex with the nuclear pore protein Nup2. Importantly, the interaction between Crm1 and Gcn4 does not require Ran-GTP, suggesting that it is not through the nuclear export sequence binding site. Finally, Crm1 stimulates DNA binding by Gcn4, supporting a model in which allosteric coupling between Crm1 binding and DNA binding permits docking of transcription factor-bound enhancers at the NPC.

Chromatin endogenous cleavage provides a global view of RNA polymerase II transcription kinetics

“…This suggests that the PD GCN4 either enhances the transfer of RNAPII from the UAS to the promoter or stabilizes the association of RNAPII with the promoter. Genetic interactions between nuclear pore proteins and Mediator suggest that these two components function at the same step in transcription (Ge et al, 2024). Together with the smRNA FISH result, this suggests that nuclear pore proteins stimulate enhancer function by stabilizing RNAPII association with the PIC.…”

“…Single molecule RNA FISH (smRNA FISH) in strains bearing mutations that blocked the interaction of the GAL1-10 promoter with the NPC showed a decrease in the fraction of cells that exhibit transcription (Brickner et al, 2016). A mutation in the Gcn4 ssTF that blocks its ability to mediate peripheral localization and interaction with the NPC leads to a defect in expression of Gcn4 target genes (gcn4-pd; Figure 7; Brickner et al, 2019) and inactivation of nuclear pore proteins essential for chromatin interaction leads to a global transcriptional defect (Ge et al, 2024). Applying RNAPII ChEC-seq2, we have explored the phenotype of the gcn4-pd mutant.…”

“…The kin28is mutations V21C and L83G (Rodríguez-Molina et al, 2016) were introduced by two subsequent rounds of CRISPR-Cas9 mediated mutagenesis as described (Anand et al, 2017). The GCN4-sm and gcn4-pd mutations were introduced by CRISPR-Cas9 mediated mutagenesis and are described (Ge et al, 2024).…”

“…Sequencing was performed on a HiSeq 4000 (Illumina) in the single-end, 50 bp format at the Northwestern University NUseq core facility. In the case of SLAMseq performed with JBY555 (gcn4-pd-GFP) and JBY556 (GCN4sm-GFP) (Ge et al, 2024), cells were shifted into SDC-uracil with 2 mM 4-thiouracil for 6 min.…”

“…The cleavage sites can be identified by next generation sequencing (ChEC-seq2;VanBelzen et al, 2024;Zentner et al, 2015). For ssTFs and nuclear pore proteins, ChEC-seq2 gives results very similar to ChIP-seq or ChIP-exo (Ge et al, 2024;VanBelzen et al, 2024). Likewise, ChEC-seq2 with co-activators and Mediator resembles ChIP (Bruzzone et al, 2018;Grünberg et al, 2016;Saleh et al, 2022).…”