Y-complex nucleoporins independently contribute to nuclear pore assembly and gene regulation in neuronal progenitors

From their essential function in building up the nuclear pore complexes, nucleoporins have expanded roles beyond nuclear transport. Hence, their contribution to chromatin organization and gene expression has set them as critical players in development and pathologies. We previously reported that Nup133 and Seh1, two components of the Y-complex subunit of the nuclear pore scaffold, are dispensable in mouse embryonic stem cells but required for their survival during neuroectodermal differentiation. Here, a transcriptomic analysis revealed that Nup133 regulates a subset of genes at early stages of neuroectodermal differentiation, including Lhx1 and Nup210L, encoding a newly validated nucleoporin. These genes were also misregulated in Nup133∆Mid neuronal progenitors, in which NPC basket assembly is impaired, as previously observed in pluripotent cells. However, a four-fold reduction of Nup133, despite affecting basket assembly, is not sufficient to alter Nup210L and Lhx1 regulation. Finally, these two genes are misregulated in Seh1-deficient progenitors that only show a mild decrease in NPC density. Together these data reveal a shared function of Y-complex nucleoporins in gene regulation during neuroectodermal differentiation. We compared the transcriptome of WT and Nup133-/- mESCs at the pluripotent state and after 2 or 3 days of differentiation towards neuroectodermal lineage. We therefore used cell lines from two distinct genetic background, namely the HM1 control cell line (Selfridge et al., 1992, PMID: 1440055) and its CRISPR/Cas9-edited Nup133-/- derivatives (#14 and #19), and the blastocyst-derived control (#1A4) and Nup133-/- (merm, #319) mESC lines (cell lines described in Souquet et al., 2018 - PMID: 30428363).

核孔蛋白（nucleoporins）不仅在构建核孔复合物（nuclear pore complexes）的过程中发挥核心功能，其生物学功能还突破了核质转运的范畴，具备更多样化的作用。因此，核孔蛋白在染色质组织与基因表达调控中的关键作用，使其成为发育进程与病理状态中的核心调控因子。我们此前的研究表明，作为核孔支架Y复合物（Y-complex）亚基的两个组分——Nup133与Seh1，在小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）中是非必需的，但在神经外胚层分化过程中对细胞存活至关重要。本研究通过转录组分析（transcriptomic analysis）发现，Nup133在神经外胚层分化早期调控特定基因子集的表达，其中包括编码新验证核孔蛋白的Lhx1与Nup210L。在NPC篮状结构组装受损的Nup133∆Mid神经元祖细胞中，这些基因同样存在表达失调的情况，这与多能细胞中的观察结果一致。然而，尽管将Nup133的表达量下调四倍会破坏篮状结构的组装，但这并不足以改变Nup210L与Lhx1的表达调控模式。最终，在仅表现出NPC密度轻度下降的Seh1缺陷型祖细胞中，这两个基因同样出现了表达失调。综合以上数据，本研究揭示了Y复合物核孔蛋白在神经外胚层分化过程中参与基因调控的共同功能。
我们对处于多能状态以及向神经外胚层谱系分化2或3天后的野生型（wild type, WT）与Nup133-/-小鼠胚胎干细胞（mESCs）进行了转录组比较分析。本研究使用了两种不同遗传背景的细胞系：一是HM1对照细胞系（Selfridge等，1992，PMID: 1440055）及其经CRISPR/Cas9编辑得到的Nup133-/-衍生细胞系（#14与#19）；二是囊胚来源的对照细胞系（#1A4）与Nup133-/-（merm, #319）小鼠胚胎干细胞系（细胞系信息详见Souquet等，2018，PMID: 30428363）。