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

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.

This dataset contains processed aligned reads (.bam files) from four main questions we addressed in our paper:
1) Where do Crm1, Nups, and RNA Pol II bind in the genome? [ChEC-seq2]
2) What is the relationship between Crm1 and Nup2? Are they interdependent or does the binding of one factor depend on the other? [ChEC-seq2]
3) Does loss of Crm1 or Nups (Nup1/Nup2) affect nascent transcription? If so, how? [SLAM-seq]
4) How does Leptomycin B (LMB, inhibitor of Crm1) affect binding of Crm1 and transcription factor Gcn4 to the genome? [ChEC-seq2]

ChEC-seq2 data was obtained and processed as described in VanBelzen et al. 2024, with reads trimmed with Trimmomatic and mapped to the Saccharomyces cerevisiae genome (sacCer3) using Bowtie2. For peak calling analyses, we processed the bam files by trimming them to the first base pair and called peaks using DoubleChEC (VanBelzen et al. 2024). SLAM-seq was performed as described in Herzog et al. 2017 and Alalam et al., 2022, using 0.2 mM 4-thiouracil (4sU) for 6 min at 30°C prior to harvesting, and processed using SLAM-DUNK (Herzog et al. 2017), which generated the read counts (.tsv files). Differential expression was analyzed using DESeq2 (Love et al., 2014). All sample information (BioProject, Biosample, SRA, GEO) is included in the Sample information spreadsheet.

核孔蛋白（Nuclear pore proteins，Nups）可与数百个染色体位点发生物理相互作用，进而影响转录过程。在酵母中，转录因子介导核孔蛋白与增强子、启动子之间的相互作用。为阐明这一机制的分子基础，我们利用了Gcn4转录因子中的功能分离突变，该突变可阻断其与核孔复合体（NPC）的相互作用。该突变会减弱Gcn4与高度保守的核输出因子Crm1/Xpo1之间的相互作用。Crm1与核孔蛋白共同占据增强子区域，且Crm1抑制可阻断核孔蛋白Nup2与基因组的结合。在体内环境中，Crm1可与核孔复合体稳定结合；在体外实验中，Crm1可直接结合Gcn4与Nup2。值得注意的是，Crm1与Gcn4之间的相互作用既不需要Ran-GTP，也不需要核输出序列结合位点。最后，Crm1与Ran-GTP可促进Gcn4的DNA结合能力，这表明Crm1-Ran-GTP结合与DNA结合之间的别构偶联，可促进结合转录因子的增强子在核孔复合体处的停靠。

本数据集包含本研究论文中针对四个核心问题所产生的经处理比对读数（.bam格式文件）：
1) Crm1、核孔蛋白Nups与RNA聚合酶II（RNA Pol II）在基因组中的结合位点分布如何？[ChEC-seq2]
2) Crm1与Nup2之间存在何种关联？二者是相互依赖的关系，还是其中一种因子的结合依赖于另一种？[ChEC-seq2]
3) 敲除Crm1或核孔蛋白Nups（Nup1/Nup2）是否会影响新生转录？若会，具体机制是什么？[SLAM-seq]
4) 莱普霉素B（Leptomycin B，LMB，Crm1抑制剂）对Crm1与转录因子Gcn4在基因组中的结合存在何种影响？[ChEC-seq2]

ChEC-seq2数据的获取与处理参照VanBelzen等（2024）的研究方法，使用Trimmomatic对测序读数进行质量修剪，并通过Bowtie2将修剪后的读数比对至酿酒酵母（Saccharomyces cerevisiae）参考基因组（sacCer3）。在峰调用分析中，我们将BAM文件修剪至首个碱基对，并使用DoubleChEC（VanBelzen等，2024）进行峰调用。SLAM-seq实验参照Herzog等（2017）及Alalam等（2022）的方法开展：在样本收获前，于30℃下使用0.2 mM 4-硫尿嘧啶（4sU）处理6分钟，随后通过SLAM-DUNK（Herzog等，2017）进行数据处理，最终生成读数计数文件（.tsv格式）。差异表达分析采用DESeq2（Love等，2014）完成。所有样本相关信息（BioProject、Biosample、SRA、GEO）均已收录于样本信息电子表格中。