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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 binds to distinct genomic regions in cells of different developmental stages.(A) Human embryonic stem cells (ESC), neural progenitor cells (NeuPC), neurons, and IMR90 cells were stained with anti-human NUP98 antibodies (green), mAb414 (red), and Hoechst (blue). (B) Venn diagram of the overlap between NUP98-binding regions in human embryonic stem cells (ESC), neural progenitor cells (NeuPC), and lung fibroblast cells (IMR90). (C) Chromosomal view of NUP98 binding regions on chromosome 1 in ESCs, NeuPCs, and IMR90 cells. Refseq(+) indicates Refseq genes on the (+) strand, and Refseq(−) indicates Refseq genes on the (−) strand. (D) Number of genes bound by NUP98 in ESCs, NeuPCs, and IMR90 cells. (E) Promoter enrichment of NUP98 binding regions in ESCs, NeuPCs, and IMR90 cells. The percentage of NUP98 binding regions that overlap with promoters was normalized against the percentage of promoters in human genome. (F) Intergenic enrichment of NUP98 binding regions in ESCs, NeuPCs, and IMR90 cells. The percentage of NUP98 binding regions that overlap with intergenic regions was normalized against the percentage of intergenic regions in human genome.
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pgen-1003308-g001: NUP98 binds to distinct genomic regions in cells of different developmental stages.(A) Human embryonic stem cells (ESC), neural progenitor cells (NeuPC), neurons, and IMR90 cells were stained with anti-human NUP98 antibodies (green), mAb414 (red), and Hoechst (blue). (B) Venn diagram of the overlap between NUP98-binding regions in human embryonic stem cells (ESC), neural progenitor cells (NeuPC), and lung fibroblast cells (IMR90). (C) Chromosomal view of NUP98 binding regions on chromosome 1 in ESCs, NeuPCs, and IMR90 cells. Refseq(+) indicates Refseq genes on the (+) strand, and Refseq(−) indicates Refseq genes on the (−) strand. (D) Number of genes bound by NUP98 in ESCs, NeuPCs, and IMR90 cells. (E) Promoter enrichment of NUP98 binding regions in ESCs, NeuPCs, and IMR90 cells. The percentage of NUP98 binding regions that overlap with promoters was normalized against the percentage of promoters in human genome. (F) Intergenic enrichment of NUP98 binding regions in ESCs, NeuPCs, and IMR90 cells. The percentage of NUP98 binding regions that overlap with intergenic regions was normalized against the percentage of intergenic regions in human genome.

Mentions: To test whether NUP98 can bind to the mammalian genome during cell differentiation, we performed ChIP-Seq experiments on cultured human embryonic stem cells (ESCs), neural progenitor cells (NeuPCs) that were differentiated in vitro from ESCs, and neurons that were differentiated in vitro from NeuPCs. We also determined the presence of chromatin-bound NUP98 in lung fibroblast IMR90 cells as an example of another differentiated cell type. As expected, in all four cell types, NUP98 was found both on nuclear pores at the nuclear periphery and intranuclear sites, consistent with its reported capacity to move on and off the nuclear pores (Figure 1A) [18], [32]. We first validated the ChIP-Seq method using IMR90 cells with two independent antibodies against human NUP98. As expected, both antibodies stained nuclear pores and a few intranuclear sites in IMR90 cells (Figure S1A). Additionally, both proved efficient and specific in western blot and immunoprecipitation experiments (Figure S1B, S1C). Since Nups were not expected to bind directly to DNA, we employed two cross-linking conditions for the ChIP-Seq experiment, formaldehyde single cross-linking and formaldehyde-disuccinimidyl glutarate double-crosslinking in order to more efficiently crosslink indirect Nup-chromatin contacts. After crosslinking, we immunoprecipitated NUP98 using the two antibodies, purified DNA that was immunoprecipitated, and had DNA amplified and subjected to deep sequencing. Sequencing reads were then mapped to the human genome (Figure S2). The results from the four ChIP-Seq experiments, using two antibodies and two cross-linking conditions, were highly consistent (Figure S1D–S1F), with 73% NUP98-binding regions from pull-down using the first antibody overlapping with 78% NUP98-binding regions using the second antibody. We further validated our results by randomly selecting several NUP98-binding regions called from the ChIP-Seq experiment and confirming the interaction between NUP98 and these regions by ChIP-qPCR (Figure S1G).


Dynamic association of NUP98 with the human genome.

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

NUP98 binds to distinct genomic regions in cells of different developmental stages.(A) Human embryonic stem cells (ESC), neural progenitor cells (NeuPC), neurons, and IMR90 cells were stained with anti-human NUP98 antibodies (green), mAb414 (red), and Hoechst (blue). (B) Venn diagram of the overlap between NUP98-binding regions in human embryonic stem cells (ESC), neural progenitor cells (NeuPC), and lung fibroblast cells (IMR90). (C) Chromosomal view of NUP98 binding regions on chromosome 1 in ESCs, NeuPCs, and IMR90 cells. Refseq(+) indicates Refseq genes on the (+) strand, and Refseq(−) indicates Refseq genes on the (−) strand. (D) Number of genes bound by NUP98 in ESCs, NeuPCs, and IMR90 cells. (E) Promoter enrichment of NUP98 binding regions in ESCs, NeuPCs, and IMR90 cells. The percentage of NUP98 binding regions that overlap with promoters was normalized against the percentage of promoters in human genome. (F) Intergenic enrichment of NUP98 binding regions in ESCs, NeuPCs, and IMR90 cells. The percentage of NUP98 binding regions that overlap with intergenic regions was normalized against the percentage of intergenic regions in human genome.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585015&req=5

pgen-1003308-g001: NUP98 binds to distinct genomic regions in cells of different developmental stages.(A) Human embryonic stem cells (ESC), neural progenitor cells (NeuPC), neurons, and IMR90 cells were stained with anti-human NUP98 antibodies (green), mAb414 (red), and Hoechst (blue). (B) Venn diagram of the overlap between NUP98-binding regions in human embryonic stem cells (ESC), neural progenitor cells (NeuPC), and lung fibroblast cells (IMR90). (C) Chromosomal view of NUP98 binding regions on chromosome 1 in ESCs, NeuPCs, and IMR90 cells. Refseq(+) indicates Refseq genes on the (+) strand, and Refseq(−) indicates Refseq genes on the (−) strand. (D) Number of genes bound by NUP98 in ESCs, NeuPCs, and IMR90 cells. (E) Promoter enrichment of NUP98 binding regions in ESCs, NeuPCs, and IMR90 cells. The percentage of NUP98 binding regions that overlap with promoters was normalized against the percentage of promoters in human genome. (F) Intergenic enrichment of NUP98 binding regions in ESCs, NeuPCs, and IMR90 cells. The percentage of NUP98 binding regions that overlap with intergenic regions was normalized against the percentage of intergenic regions in human genome.
Mentions: To test whether NUP98 can bind to the mammalian genome during cell differentiation, we performed ChIP-Seq experiments on cultured human embryonic stem cells (ESCs), neural progenitor cells (NeuPCs) that were differentiated in vitro from ESCs, and neurons that were differentiated in vitro from NeuPCs. We also determined the presence of chromatin-bound NUP98 in lung fibroblast IMR90 cells as an example of another differentiated cell type. As expected, in all four cell types, NUP98 was found both on nuclear pores at the nuclear periphery and intranuclear sites, consistent with its reported capacity to move on and off the nuclear pores (Figure 1A) [18], [32]. We first validated the ChIP-Seq method using IMR90 cells with two independent antibodies against human NUP98. As expected, both antibodies stained nuclear pores and a few intranuclear sites in IMR90 cells (Figure S1A). Additionally, both proved efficient and specific in western blot and immunoprecipitation experiments (Figure S1B, S1C). Since Nups were not expected to bind directly to DNA, we employed two cross-linking conditions for the ChIP-Seq experiment, formaldehyde single cross-linking and formaldehyde-disuccinimidyl glutarate double-crosslinking in order to more efficiently crosslink indirect Nup-chromatin contacts. After crosslinking, we immunoprecipitated NUP98 using the two antibodies, purified DNA that was immunoprecipitated, and had DNA amplified and subjected to deep sequencing. Sequencing reads were then mapped to the human genome (Figure S2). The results from the four ChIP-Seq experiments, using two antibodies and two cross-linking conditions, were highly consistent (Figure S1D–S1F), with 73% NUP98-binding regions from pull-down using the first antibody overlapping with 78% NUP98-binding regions using the second antibody. We further validated our results by randomly selecting several NUP98-binding regions called from the ChIP-Seq experiment and confirming the interaction between NUP98 and these regions by ChIP-qPCR (Figure S1G).

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