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Dynamic association of NUP98 with the human genome.

Liang Y, Franks TM, Marchetto MC, Gage FH, Hetzer MW - PLoS Genet. (2013)

Bottom Line: Overexpression of a dominant negative fragment of NUP98 levels decreases expression levels of NUP98-bound genes.Alternatively, genes that are highly induced can interact with NUP98 in the nuclear interior, away from the nuclear pores.This work demonstrates for the first time that NUP98 dynamically associates with the human genome during differentiation, revealing a role of a nuclear pore protein in regulating developmental gene expression programs.

View Article: PubMed Central - PubMed

Affiliation: Salk Institute for Biological Studies, Molecular and Cell Biology Laboratory, La Jolla, California, USA.

ABSTRACT
Faithful execution of developmental gene expression programs occurs at multiple levels and involves many different components such as transcription factors, histone-modification enzymes, and mRNA processing proteins. Recent evidence suggests that nucleoporins, well known components that control nucleo-cytoplasmic trafficking, have wide-ranging functions in developmental gene regulation that potentially extend beyond their role in nuclear transport. Whether the unexpected role of nuclear pore proteins in transcription regulation, which initially has been described in fungi and flies, also applies to human cells is unknown. Here we show at a genome-wide level that the nuclear pore protein NUP98 associates with developmentally regulated genes active during human embryonic stem cell differentiation. Overexpression of a dominant negative fragment of NUP98 levels decreases expression levels of NUP98-bound genes. In addition, we identify two modes of developmental gene regulation by NUP98 that are differentiated by the spatial localization of NUP98 target genes. Genes in the initial stage of developmental induction can associate with NUP98 that is embedded in the nuclear pores at the nuclear periphery. Alternatively, genes that are highly induced can interact with NUP98 in the nuclear interior, away from the nuclear pores. This work demonstrates for the first time that NUP98 dynamically associates with the human genome during differentiation, revealing a role of a nuclear pore protein in regulating developmental gene expression programs.

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NUP98 is functionally relevant for the expression of its binding targets.(A) Fold change in expression levels upon full length NUP98 (-NUP98) overexpression in NeuPCs. Error bars were computed as standard deviation from triplicates. P value was obtained from Student's t-test and comparisons with P value<0.05 indicated with asterisks. (B) Western blot GAPDH and GFP in NeuPCs with overexpression of GFP-NUP98 or untreated condition as negative control. (C) Fold change in expression levels upon NUP98 fragment (-NUP98ΔC) overexpression in NeuPCs. Error bars were computed as standard deviation from triplicates. P value was obtained from Student's t-test and comparisons with P value<0.05 indicated with asterisks. (D) Western blot of GAPDH and GFP in NeuPCs with overexpression of GFP-NUP98 fragment (GFP-NUP98ΔC) or untreated condition as negative control.
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pgen-1003308-g005: NUP98 is functionally relevant for the expression of its binding targets.(A) Fold change in expression levels upon full length NUP98 (-NUP98) overexpression in NeuPCs. Error bars were computed as standard deviation from triplicates. P value was obtained from Student's t-test and comparisons with P value<0.05 indicated with asterisks. (B) Western blot GAPDH and GFP in NeuPCs with overexpression of GFP-NUP98 or untreated condition as negative control. (C) Fold change in expression levels upon NUP98 fragment (-NUP98ΔC) overexpression in NeuPCs. Error bars were computed as standard deviation from triplicates. P value was obtained from Student's t-test and comparisons with P value<0.05 indicated with asterisks. (D) Western blot of GAPDH and GFP in NeuPCs with overexpression of GFP-NUP98 fragment (GFP-NUP98ΔC) or untreated condition as negative control.

Mentions: Since NUP98 associated with neural development genes during neural differentiation, we asked if this nuclear pore complex component plays a role in their expression. We randomly selected 24 genes from the 54 genes in the ‘nervous system development’ gene ontology category that showed specific enrichment in NeuPCs (Figure 2B) together with GAPDH as well as additional genes that did not bind NUP98 as negative controls, and examined how their expression levels were affected by NUP98 overexpression in neural progenitor cells using qRT-PCR (Figure 5A, 5B, Figure S8A). To do this, NeuPCs were transfected with NUP98 and the overexpressed NUP98 localized to both nuclear pores and nucleoplasm (Figure S9). Strikingly, we found that 12 NUP98-associated neural developmental genes showed statistically significant increase in expression levels upon NUP98 overexpression, indicating that NUP98 regulates the transcription of these genes. Since not all genes responded to NUP98 overexpression, we suspect that NUP98 might not be rate-limiting in all its target genes. We then overexpressed a fragment of NUP98 (amino acid 1–504) in NeuPCs, which lacks a C-terminal domain of NUP98 that is no longer capable of binding to the nuclear pore complex (Figure S9). We were interested in this region of NUP98 because this is the same fragment as appeared in multiple NUP98-involved leukemic fusions and this fragment has been found to interfere with the differentiation of haematopoietic progenitor cells [39]. Given reported evidences for a role of NUP98 in gene regulation [8], [17] and our observation of the association between NUP98 and developmental genes at the progenitor cell stage, we hypothesized that this NUP98 fragment might interfere with the expression of NUP98 targets required for neural differentiation. We found that overexpression of this fragment of NUP98 had a dominant negative effect on the expression of NUP98-binding neural developmental genes, and 20 of the 24 genes exhibited statistically significant decrease in expression levels (Figure 5C, 5D). No significant effects on gene expression have been observed for GAPDH as well as additional genes that did not bind NUP98 (Figure 5C, 5D, Figure S8B). This suggests that the C-terminal domain of NUP98 is required for the expression of NUP98 target genes because the fragment lacking this domain could not stimulate expression of target genes as the full length NUP98 protein did. As an additional negative control, we overexpressed NUP35 using the same vector and found no effects on the expression of NUP98-binding genes (Figure S10). We did not examine the effect of NUP98 knockdown on gene expression because NUP98 is encoded on the same mRNA with a core component of the nuclear pore, NUP96, which is essential to nuclear pore biogenesis [32]. Knockdown of NUP98 causes simultaneous knockdown of NUP96 and a failure in nuclear pore formation and cell death (data not shown). Therefore, it was not possible to specifically analyze the gene regulatory function of NUP98 from such knockdown experiments.


Dynamic association of NUP98 with the human genome.

Liang Y, Franks TM, Marchetto MC, Gage FH, Hetzer MW - PLoS Genet. (2013)

NUP98 is functionally relevant for the expression of its binding targets.(A) Fold change in expression levels upon full length NUP98 (-NUP98) overexpression in NeuPCs. Error bars were computed as standard deviation from triplicates. P value was obtained from Student's t-test and comparisons with P value<0.05 indicated with asterisks. (B) Western blot GAPDH and GFP in NeuPCs with overexpression of GFP-NUP98 or untreated condition as negative control. (C) Fold change in expression levels upon NUP98 fragment (-NUP98ΔC) overexpression in NeuPCs. Error bars were computed as standard deviation from triplicates. P value was obtained from Student's t-test and comparisons with P value<0.05 indicated with asterisks. (D) Western blot of GAPDH and GFP in NeuPCs with overexpression of GFP-NUP98 fragment (GFP-NUP98ΔC) or untreated condition as negative control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585015&req=5

pgen-1003308-g005: NUP98 is functionally relevant for the expression of its binding targets.(A) Fold change in expression levels upon full length NUP98 (-NUP98) overexpression in NeuPCs. Error bars were computed as standard deviation from triplicates. P value was obtained from Student's t-test and comparisons with P value<0.05 indicated with asterisks. (B) Western blot GAPDH and GFP in NeuPCs with overexpression of GFP-NUP98 or untreated condition as negative control. (C) Fold change in expression levels upon NUP98 fragment (-NUP98ΔC) overexpression in NeuPCs. Error bars were computed as standard deviation from triplicates. P value was obtained from Student's t-test and comparisons with P value<0.05 indicated with asterisks. (D) Western blot of GAPDH and GFP in NeuPCs with overexpression of GFP-NUP98 fragment (GFP-NUP98ΔC) or untreated condition as negative control.
Mentions: Since NUP98 associated with neural development genes during neural differentiation, we asked if this nuclear pore complex component plays a role in their expression. We randomly selected 24 genes from the 54 genes in the ‘nervous system development’ gene ontology category that showed specific enrichment in NeuPCs (Figure 2B) together with GAPDH as well as additional genes that did not bind NUP98 as negative controls, and examined how their expression levels were affected by NUP98 overexpression in neural progenitor cells using qRT-PCR (Figure 5A, 5B, Figure S8A). To do this, NeuPCs were transfected with NUP98 and the overexpressed NUP98 localized to both nuclear pores and nucleoplasm (Figure S9). Strikingly, we found that 12 NUP98-associated neural developmental genes showed statistically significant increase in expression levels upon NUP98 overexpression, indicating that NUP98 regulates the transcription of these genes. Since not all genes responded to NUP98 overexpression, we suspect that NUP98 might not be rate-limiting in all its target genes. We then overexpressed a fragment of NUP98 (amino acid 1–504) in NeuPCs, which lacks a C-terminal domain of NUP98 that is no longer capable of binding to the nuclear pore complex (Figure S9). We were interested in this region of NUP98 because this is the same fragment as appeared in multiple NUP98-involved leukemic fusions and this fragment has been found to interfere with the differentiation of haematopoietic progenitor cells [39]. Given reported evidences for a role of NUP98 in gene regulation [8], [17] and our observation of the association between NUP98 and developmental genes at the progenitor cell stage, we hypothesized that this NUP98 fragment might interfere with the expression of NUP98 targets required for neural differentiation. We found that overexpression of this fragment of NUP98 had a dominant negative effect on the expression of NUP98-binding neural developmental genes, and 20 of the 24 genes exhibited statistically significant decrease in expression levels (Figure 5C, 5D). No significant effects on gene expression have been observed for GAPDH as well as additional genes that did not bind NUP98 (Figure 5C, 5D, Figure S8B). This suggests that the C-terminal domain of NUP98 is required for the expression of NUP98 target genes because the fragment lacking this domain could not stimulate expression of target genes as the full length NUP98 protein did. As an additional negative control, we overexpressed NUP35 using the same vector and found no effects on the expression of NUP98-binding genes (Figure S10). We did not examine the effect of NUP98 knockdown on gene expression because NUP98 is encoded on the same mRNA with a core component of the nuclear pore, NUP96, which is essential to nuclear pore biogenesis [32]. Knockdown of NUP98 causes simultaneous knockdown of NUP96 and a failure in nuclear pore formation and cell death (data not shown). Therefore, it was not possible to specifically analyze the gene regulatory function of NUP98 from such knockdown experiments.

Bottom Line: Overexpression of a dominant negative fragment of NUP98 levels decreases expression levels of NUP98-bound genes.Alternatively, genes that are highly induced can interact with NUP98 in the nuclear interior, away from the nuclear pores.This work demonstrates for the first time that NUP98 dynamically associates with the human genome during differentiation, revealing a role of a nuclear pore protein in regulating developmental gene expression programs.

View Article: PubMed Central - PubMed

Affiliation: Salk Institute for Biological Studies, Molecular and Cell Biology Laboratory, La Jolla, California, USA.

ABSTRACT
Faithful execution of developmental gene expression programs occurs at multiple levels and involves many different components such as transcription factors, histone-modification enzymes, and mRNA processing proteins. Recent evidence suggests that nucleoporins, well known components that control nucleo-cytoplasmic trafficking, have wide-ranging functions in developmental gene regulation that potentially extend beyond their role in nuclear transport. Whether the unexpected role of nuclear pore proteins in transcription regulation, which initially has been described in fungi and flies, also applies to human cells is unknown. Here we show at a genome-wide level that the nuclear pore protein NUP98 associates with developmentally regulated genes active during human embryonic stem cell differentiation. Overexpression of a dominant negative fragment of NUP98 levels decreases expression levels of NUP98-bound genes. In addition, we identify two modes of developmental gene regulation by NUP98 that are differentiated by the spatial localization of NUP98 target genes. Genes in the initial stage of developmental induction can associate with NUP98 that is embedded in the nuclear pores at the nuclear periphery. Alternatively, genes that are highly induced can interact with NUP98 in the nuclear interior, away from the nuclear pores. This work demonstrates for the first time that NUP98 dynamically associates with the human genome during differentiation, revealing a role of a nuclear pore protein in regulating developmental gene expression programs.

Show MeSH
Related in: MedlinePlus