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 for mouse embryonic stem cell viability 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 also misregulated in Seh1-deficient neural 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, which seem independent of nuclear pore basket assembly.


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As channels embedded in the nuclear envelope, the nuclear pore complexes (NPCs) 43 constitute the only gateway for selective transport of macromolecules between the 44 cytoplasm and the nucleus. These impressive structures are composed of proteins called 45 nucleoporins (Nups) that assemble in a highly organized and modular manner (reviewed in 46 Dultz et al., (2022)). The Y-complex -also named Nup107-160 complex -that comprises in 47 vertebrates nine distinct proteins, is a key structural subunit of the NPC scaffold. 16 copies of 48 this complex assemble on the nuclear and cytoplasmic sides of the NPC to build up its outer 49 rings, to which cytoplasmic filaments and the nuclear basket are anchored. 50 In addition to their canonical nuclear transport function, many Nups are also known to have Earlier studies had found that the vertebrate Y-complex is, as an entity, critically required for 73 NPC assembly both at the end of mitosis and during interphase (Doucet and Hetzer, 2010;74 Harel et al., Vollmer et al., 2015;Walther et al., 2003). The viability of Nup133 -/-, Seh1 -75 /and Nup43 -/-mESCs however indicated that the corresponding Y-complex Nups were 76 individually largely dispensable for nuclear pore assembly in these pluripotent cells. 77 Consistently, we previously showed that mutations of Nups that form the short arm of the Y-78 complex, namely Nup43, Nup85 and Seh1, only lead to a mild decrease in NPC density in 79 pluripotent mESCs (Gonzalez-Estevez, Verrico et al., 2021). In contrast, pluripotent Nup133 -/-80 mESCs feature a normal NPC density, but show specific nuclear basket defects, with half of 81 NPCs lacking Tpr while Nup153 dynamics was increased (Souquet et al., 2018). How Y-82 complex Nups contribute to NPC assembly in differentiating mESCs was unknown. The impaired neuroectodermal differentiation of Nup133 -/-mESCs initially described in Lupu 98 et al. (2008), was also observed in HM1-derived Nup133 -/-mESCs as revealed by their altered 99 growth and increased cell death (Figure 1A, B). To assess the potential effect of Nup133 100 deficiency in gene regulation upon neuroectodermal differentiation, we first determined the 101 mRNA levels of genes expressed in pluripotent cells (Oct4 and Nanog) and in early neuronal 102 progenitors (Sox1 and Pax6) that are considered markers for the respective states. RT-qPCR 103 analyses showed that these genes were properly repressed and activated, respectively, in 104 Nup133 -/cells stimulated to differentiate towards neuroectoderm ( Figure 1C). This 105 indicated that despite their impaired viability at early stages of differentiation ( Figure 1A, B) 106 the surviving Nup133 -/cells are able to exit pluripotency and to commit towards the 107 neuronal lineage, without overt defects in the expression of these markers.

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To more broadly explore the impact of Nup133 on gene expression, we compared the 109 transcriptome of WT and Nup133 -/-mESCs at the pluripotent state and after 2 or 3 days of 110 differentiation towards neuroectodermal lineage. We therefore used cell lines from two 111 distinct genetic backgrounds, namely the HM1 control cell line and its CRISPR/Cas9-edited 112 Nup133 -/derivatives (#14 and #19), and the blastocyst-derived control (#1A4) and Nup133 -/-113 (merm, #319) mESC lines (Souquet et al., 2018 and Table S1). This analysis revealed that the 114 transcriptomes of pluripotent WT and Nup133 -/-mESCs were overall similar, whereas an 115 increasing number of genes were misregulated at day 3 of differentiation (Figures 1D and 116 S1A). 117 We assayed by RT-qPCR the altered expression of a subset of these genes, filtered by criteria 118 of differential expression (logFC>2 or <-2), significance (adjusted p-value<0.05) and lastly by 119 average expression level (average number of reads with a log2(CPM)>1, to ensure proper 120 detection by RT-qPCR) (Figure S1B-E). In addition to WT (HM1) and Nup133 -/-(#14) used for 121 the initial RNA-seq experiment, these analyses were also conducted on samples from 122 Nup133 "Rescue" cell lines generated by inserting the GFP-Nup133 transgene in Nup133 -/-123 (#14) mESCs at the permissive Tigre locus (Zeng et al., 2008). As an additional control, we 124 used an HM1-derived cell line that carries a transgene (OsTIR) similarly inserted in the Tigre 125 locus ( Figure S2A and Table S1). In contrast to the impaired viability of Nup133 -/-mESCs 126 upon neuroectodermal differentiation, the survival of the Rescue and WT (OsTIR) cell lines 127 were similar, confirming the functionality of the GFP-Nup133 transgene (Figure 2A-C). 128

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For the candidates genes localized on the short arm of the Y chromosome (Ddx3y and 130 Eif2s3y, also located in close proximity to the loci of Uty, Uba1y, Kdm5d and Zfy (Subrini and 131 Turner, 2021)) we observed clone-dependent expression variations ( Figure S1B). This 132 suggests that their apparent shared misregulations, also reported in Tet1 and Tet2 mutant  In contrast, we could validate the increased mRNA levels in Nup133 -/compared to WT of 136 Nuggc at day 0, and of Nup210L and Lhx1 at day 3 of differentiation ( Figure S1C). We also 137 confirmed the reduced mRNA levels in Nup133 -/compared to WT for Magohb and Wfikkn1 138 (but not Acta2) at day 3 of differentiation ( Figure S1D). Finally, reduced mRNA levels of the 139 assayed candidate genes at the pluripotent state (day 0) were not significant due to high 140 variability among replicates or between control cell lines (HM1 and OsTIR) ( Figure S1E). 141 Importantly, among the validated candidate genes, Lhx1, Nup210L, Nuggc and Magohb were 142 all efficiently restored to wild-type levels by the GFP-Nup133 transgene.  Figure 2D). 152 The other candidate gene we further characterized, Nup210L, is the differentially expressed 153 gene (DEG) with the most significant p-value at day 3 of differentiation ( Figure 1D). It is also 154 one of the rare DEGs whose expression already increased at day 2 compared to WT cells 155 ( Figure S1A). In mice, Nup210L mRNA is mainly detected in the testis and to a lesser extent 156 in the embryonic brain (https://www.ncbi.nlm.nih.gov/gene/77595); in humans, besides testis, Nup210L expression was also detected in the prefrontal cortex neurons of rare 158 individuals (Gusev et al., 2019). Analyses at later stages (day 5 and 7) of differentiation 159 towards neuroectoderm showed that Nup210L was still more expressed in Nup133 -/-160 compared to WT and Rescue cells, although a progressive increase of its expression was also 161 observed in the latter cell lines (Figure 2E). 162 As its name implies, Nup210L encodes a potential homologue of the transmembrane 163 nucleoporin Nup210/gp210. However, its putative NPC localization had never been   Figure 2C). 184 In contrast, RT-qPCR analysis showed that Nup210L and Lhx1 were similarly misregulated 185 upon neuronal differentiation in Nup133∆mid and in Nup133 -/cells (Figure 2D and E). 186 The improved survival upon differentiation of Nup133∆mid compared to Nup133 -/cells 187 enabled us to perform immunofluorescence analyses to determine whether the NPC basket 188 assembly defects, previously observed in pluripotent mESCs lacking Nup133 or its middle 189 domain, also occurred at the differentiated stage. Quantitative immunofluorescence 190 analyses, performed after 5 days of differentiation, showed that the intensity of Tpr at the 191 nuclear envelope was comparable between the WT and Rescue cell lines. In contrast, a two-192 fold decrease was observed in Nup133∆mid neuronal progenitors ( Figure 3A, B), a defect 193 comparable to the one previously observed at the pluripotent state (Souquet et al., 2018). In 194 addition, we also measured an increased Nup153 intensity at the nuclear envelope in   Figure S2B). 212 The resulting Nup133-degron cell lines maintained normal Nup133 mRNA expression during 213 differentiation ( Figure S3A), but actual Nup133 protein levels (without Auxin 214 treatment) were only ~25% of that found in WT cells (Figure 4A and S3B). This could be due  (Figures 4D and S4A). 242 Overall, these results thus demonstrated that a correct Nup133 stoichiometry is critical for 243 nuclear basket assembly, yet is not required for cell viability or gene regulation upon 244 neuroectodermal differentiation. Taken together these data also reveal that a properly 245 assembled nuclear basket at all NPCs is not required to regulate the expression of Nup133-246 target genes.

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Nup210L mRNA levels rapidly increase in response to Nup133 or Seh1 depletion. 249 We next aimed to determine if the altered expression of Nup210L and Lhx1 was specific for Nup153 levels were not altered in auxin-treated Seh1-degron cells, suggesting, as also 277 observed in Nup133∆mid cells, an increased stoichiometry of Nup153 per NPC (Figure S4F). 278 We also note that a 24h auxin treatment, applied to Seh1-mAID-GFP cells at day 2 of 279 neuronal differentiation was sufficient to cause an important increase in Nup210L mRNA 280 levels ( Figure 5E). Likewise, a 24h auxin treatment of Nup133-degron cells induced Nup210L 281 expression ( Figure 5E). In contrast, 24h of auxin treatment did not lead to an altered 282 regulation of Lhx1 in Seh1-degron or Nup133-degron cells at day 3 ( Figure 5F). Together,  Hence, Y-complex dependent gene regulation may take place either at NPCs or "off-pore".  Cell lines used in this study are listed in Table S1 and were grown as previously described   Table S1). Library preparation and Illumina sequencing were 364 performed at the Ecole normale superieure genomics core facility (Paris, France). Messenger 365 (polyA+) RNAs were purified from 1 µg of total RNA using oligo(dT). Libraries were prepared 366 using the strand specific RNA-Seq library preparation TruSeq Stranded mRNA kit (Illumina).   Figures 1D and S1A were generated in R using ggplot2 (v.3.3.5).

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The RNASeq gene expression data and raw fastq files are available on the GEO repository  Figure S2). Plasmids are listed in Table S2, 406 gRNAs designed using the Benchling website (https://benchling.com) are listed in Table S3, 407 and PCR primers used to generate homology-directed repair templates are listed in Table S4.  Table S1.  Immunofluorescence and quantification of nucleoporin intensity at the nuclear envelope.