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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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Distinct localization of two groups of NUP98-regulated developmental genes.(A) Criteria for counting gene localization as ‘Periphery’ or ‘Non-Periphery’, with LMNB staining in green and FISH signal in red. Genes counted as ‘Periphery’ were localized within 0.5 µm of the nuclear lamina. (B) Percentage of periphery localization of NUP98 binding genes through development, i.e. in ESC (yellow), NeuPC (blue) and Neuron (purple), determined from IF-FISH experiments. Error bars were calculated as standard deviation from triplicates for a total of at least 100 cells using 3D reconstruction of images. (C, D) Representative 3D IF-FISH images showing the localization of (C) group I genes (GRIK1) and (D) group II genes (GPM6B) through development, i.e. in ESC, NeuPC, and Neuron. FISH probes were shown in red, LMNB staining in green, and Hoechst in blue. Each set of images includes the x-y, y-z and x-z planes that cross at the FISH probe signal. (E) Model of two groups of NUP98-gene interaction. Group I genes are at the beginning stage of developmental induction in neural progenitor cells and interact with NUP98 at the nuclear pores in the NE, and subsequently translocate to intranuclear sites upon full induction in neurons. In contrast, group II genes are already greatly activated in NeuPCs and interact with NUP98 at intranuclear sites away from the NE. The percentage of genes observed at the nuclear periphery at each stage was indicated.
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pgen-1003308-g007: Distinct localization of two groups of NUP98-regulated developmental genes.(A) Criteria for counting gene localization as ‘Periphery’ or ‘Non-Periphery’, with LMNB staining in green and FISH signal in red. Genes counted as ‘Periphery’ were localized within 0.5 µm of the nuclear lamina. (B) Percentage of periphery localization of NUP98 binding genes through development, i.e. in ESC (yellow), NeuPC (blue) and Neuron (purple), determined from IF-FISH experiments. Error bars were calculated as standard deviation from triplicates for a total of at least 100 cells using 3D reconstruction of images. (C, D) Representative 3D IF-FISH images showing the localization of (C) group I genes (GRIK1) and (D) group II genes (GPM6B) through development, i.e. in ESC, NeuPC, and Neuron. FISH probes were shown in red, LMNB staining in green, and Hoechst in blue. Each set of images includes the x-y, y-z and x-z planes that cross at the FISH probe signal. (E) Model of two groups of NUP98-gene interaction. Group I genes are at the beginning stage of developmental induction in neural progenitor cells and interact with NUP98 at the nuclear pores in the NE, and subsequently translocate to intranuclear sites upon full induction in neurons. In contrast, group II genes are already greatly activated in NeuPCs and interact with NUP98 at intranuclear sites away from the NE. The percentage of genes observed at the nuclear periphery at each stage was indicated.

Mentions: As a mobile nuclear pore complex component, NUP98 can act both at the nuclear pore complexes and inside the nucleus at sites that are not attached to the nuclear envelope (NE) [8], [17]. Therefore, we wondered if either of the two classes of genes is specifically associated with nuclear pore complexes at the NE. We examined the localization of the group I and group II NUP98 targets by immunofluorescence-fluorescence in situ hybridization (IF-FISH) experiments. We used lamin (LMNB) staining as a marker for the NE, and only counted FISH signals whose center overlaid with the NE (corresponding to <0.5 µm distance from the NE) as ‘periphery’ localization (Figure 7A). We found that the two groups of genes also showed distinct intranuclear localization at the progenitor cell stage. In NeuPCs, group I genes that will become transcriptionally active were localized to the periphery, whereas group II genes that were already expressed at high levels were in the interior of the nucleus (Figure 7B–7D, Figure S11). Upon differentiation into neurons, group I genes moved into the nuclear interior whereas group II genes maintained their interior localization (Figure 7B–7D, Figure S11).


Dynamic association of NUP98 with the human genome.

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

Distinct localization of two groups of NUP98-regulated developmental genes.(A) Criteria for counting gene localization as ‘Periphery’ or ‘Non-Periphery’, with LMNB staining in green and FISH signal in red. Genes counted as ‘Periphery’ were localized within 0.5 µm of the nuclear lamina. (B) Percentage of periphery localization of NUP98 binding genes through development, i.e. in ESC (yellow), NeuPC (blue) and Neuron (purple), determined from IF-FISH experiments. Error bars were calculated as standard deviation from triplicates for a total of at least 100 cells using 3D reconstruction of images. (C, D) Representative 3D IF-FISH images showing the localization of (C) group I genes (GRIK1) and (D) group II genes (GPM6B) through development, i.e. in ESC, NeuPC, and Neuron. FISH probes were shown in red, LMNB staining in green, and Hoechst in blue. Each set of images includes the x-y, y-z and x-z planes that cross at the FISH probe signal. (E) Model of two groups of NUP98-gene interaction. Group I genes are at the beginning stage of developmental induction in neural progenitor cells and interact with NUP98 at the nuclear pores in the NE, and subsequently translocate to intranuclear sites upon full induction in neurons. In contrast, group II genes are already greatly activated in NeuPCs and interact with NUP98 at intranuclear sites away from the NE. The percentage of genes observed at the nuclear periphery at each stage was indicated.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003308-g007: Distinct localization of two groups of NUP98-regulated developmental genes.(A) Criteria for counting gene localization as ‘Periphery’ or ‘Non-Periphery’, with LMNB staining in green and FISH signal in red. Genes counted as ‘Periphery’ were localized within 0.5 µm of the nuclear lamina. (B) Percentage of periphery localization of NUP98 binding genes through development, i.e. in ESC (yellow), NeuPC (blue) and Neuron (purple), determined from IF-FISH experiments. Error bars were calculated as standard deviation from triplicates for a total of at least 100 cells using 3D reconstruction of images. (C, D) Representative 3D IF-FISH images showing the localization of (C) group I genes (GRIK1) and (D) group II genes (GPM6B) through development, i.e. in ESC, NeuPC, and Neuron. FISH probes were shown in red, LMNB staining in green, and Hoechst in blue. Each set of images includes the x-y, y-z and x-z planes that cross at the FISH probe signal. (E) Model of two groups of NUP98-gene interaction. Group I genes are at the beginning stage of developmental induction in neural progenitor cells and interact with NUP98 at the nuclear pores in the NE, and subsequently translocate to intranuclear sites upon full induction in neurons. In contrast, group II genes are already greatly activated in NeuPCs and interact with NUP98 at intranuclear sites away from the NE. The percentage of genes observed at the nuclear periphery at each stage was indicated.
Mentions: As a mobile nuclear pore complex component, NUP98 can act both at the nuclear pore complexes and inside the nucleus at sites that are not attached to the nuclear envelope (NE) [8], [17]. Therefore, we wondered if either of the two classes of genes is specifically associated with nuclear pore complexes at the NE. We examined the localization of the group I and group II NUP98 targets by immunofluorescence-fluorescence in situ hybridization (IF-FISH) experiments. We used lamin (LMNB) staining as a marker for the NE, and only counted FISH signals whose center overlaid with the NE (corresponding to <0.5 µm distance from the NE) as ‘periphery’ localization (Figure 7A). We found that the two groups of genes also showed distinct intranuclear localization at the progenitor cell stage. In NeuPCs, group I genes that will become transcriptionally active were localized to the periphery, whereas group II genes that were already expressed at high levels were in the interior of the nucleus (Figure 7B–7D, Figure S11). Upon differentiation into neurons, group I genes moved into the nuclear interior whereas group II genes maintained their interior localization (Figure 7B–7D, Figure S11).

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