Investigation of transcriptional response upon loss of individual Y-complex nucleoporins
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE134097 | GEO | 2020/07/10
REPOSITORIES: GEO
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Project description:Investigation of transcriptional response upon loss of individual Y-complex nucleoporins
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Project description:The origin recognition complex (ORC) binds throughout the genome to initiate DNA replication. In metazoans, it is still unclear how ORC is targeted to specific loci to facilitate helicase loading and replication initiation. Here, we performed immunoprecipitations coupled with mass spectrometry for ORC2 in Drosophila embryos. Surprisingly, we found that ORC2 associates with several subunits of the Nup107-160 subcomplex of the nuclear pore. Bioinformatic analysis revealed that, relative to all modENCODE factors, nucleoporins are among the most enriched factors at ORC2 binding sites. Critically, depletion of the nucleoporin Elys, a member of the Nup107-160 complex, results in decrease ORC2 loading onto chromatin. Depleting Elys also sensitized cells to replication fork stalling, which could reflect a defect in establishing dormant replication origins. Our work reveals a new connection between ORC, replication initiation and nucleoporins, highlighting a previously unrecognized function of nucleoporins in metazoan replication initiation.
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Project description:The origin recognition complex (ORC) binds throughout the genome to initiate DNA replication. In metazoans, it is still unclear how ORC is targeted to specific loci to facilitate helicase loading and replication initiation. Here, we performed immunoprecipitations coupled with mass spectrometry for ORC2 in Drosophila embryos. Surprisingly, we found that ORC2 associates with multiple subunits of the Nup107-160 subcomplex of the nuclear pore. Bioinformatic analysis revealed that, relative to all modENCODE factors, nucleoporins are among the most enriched factors at ORC2 binding sites. Critically, depletion of the nucleoporin Elys, a member of the Nup107-160 complex, results in decreased ORC2 loading onto chromatin. Depleting Elys also sensitized cells to replication fork stalling, which could reflect a defect in establishing dormant replication origins. Our work reveals a new connection between ORC, replication initiation and nucleoporins, highlighting a previously unrecognized function of nucleoporins in metazoan replication initiation.
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Project description: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.
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Project description:We generated NUP133 mutant podocytes to model human hereditary SRNS in vitro. A subtype of SRNS is caused by monogenetic mutations of the nuclear pore complex including NUP133. Human immortalized podocytes were genome edited applying the CRISPR/Cas9 technique to create NUP133 KO cells (“KO-1”). cDNA of NUP133-WT or causative variants NUP133 c.2922T>G (“Mutation-1”) or NUP133 c.3335-11T>A (“Mutation-2”) were stable expressed into this KO background by lentiviral transduction. Transcriptome profiling (RNA-Sequencing) and differential gene expression analysis was performed in triplicate for each cell line. NUP133 mutant podocytes showed no overt transcriptional changes compared to NUP133-WT cells.
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Project description:We generated NUP133 WT and KO podocytes to model human hereditary SRNS in vitro. A subtype of SRNS is caused by monogenetic mutations of the nuclear pore complex including NUP133. Immortalized human podocytes were genome edited applying the CRISPR/Cas9 technique and two independent NUP133 sgRNAs. Two monoclonal cell backgrounds were used to create two matching sets of NUP133 WT and KO cells (WT-1 versus KO-1 and WT-2 versus KO-2). Transcriptome profiling (RNA-Sequencing) and differential gene expression analysis was performed in duplicate for each cell line. NUP133 KO podocytes exhibit vast transcriptional changes compared to WT cells.
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Project description:We recently identified a missense mutation in Nucleoporin107 (Nup107; D447N) underlying XX-ovarian-dysgenesis (XX-OD), a disorder characterized by underdeveloped and dysfunctional ovaries. Specific knockdown of Nup107 in the somatic gonadal cells and moreover, modelling of the human mutation in Drosophila result in ovarian-dysgenesis-like phenotypes in female flies. The aberrant phenotypes in larval and adult ovaries compromised for Nup107 are associated with hyperactivation of BMP signalling. Transcriptomic analysis identified the somatic sex-determination gene Doublesex (dsx) and the extracellular metalloprotease AdamTS-A as targets of Nup107. Either loss or gain of Dsx in the gonadal soma is sufficient to respectively mimic or rescue the phenotypes induced by Nup107 loss. Furthermore, adamTS-A is transcriptionally regulated by Dsx, and its knockdown in the somatic gonad hyperactivates BMP signaling and to a large extent recapitulates loss of Nup107 phenotypes. Thus, Dsx acts downstream of Nup107 to impact female germline stem cells via sex-specific modulation of the BMP pathway.
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Project description:To study the function of zebrafish nuclear pores during early embryogenesis, we generated maternal zygotic double mutant of nup85;nup133 (MZnup85;nup133) using CRISPR/Cas9 and report the transcriptome-wide changes in comparison to wild-type (WT) embryos. Our analysis reveals a dramatic delay of maternal mRNA degradation and zygotic genome activation in MZnup85;nup133 embryos during maternal-to-zygotic transition.
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Project description:Investigation of transcriptional response upon loss of RanBP3
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