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The mobile nucleoporin Nup2p and chromatin-bound Prp20p function in endogenous NPC-mediated transcriptional control.

Dilworth DJ, Tackett AJ, Rogers RS, Yi EC, Christmas RH, Smith JJ, Siegel AF, Chait BT, Wozniak RW, Aitchison JD - J. Cell Biol. (2005)

Bottom Line: Nuclear pore complexes (NPCs) govern macromolecular transport between the nucleus and cytoplasm and serve as key positional markers within the nucleus.Among these, Nup2p is unique as it transiently associates with NPCs and, when artificially tethered to DNA, can prevent the spread of transcriptional activation or repression between flanking genes, a function termed boundary activity.These data combined with functional assays of boundary activity and epigenetic variegation suggest that Nup2p and the Ran guanylyl-nucleotide exchange factor, Prp20p, interact at specific chromatin regions and enable the NPC to play an active role in chromatin organization by facilitating the transition of chromatin between activity states.

View Article: PubMed Central - PubMed

Affiliation: Institute for Systems Biology, Seattle, WA 98103, USA.

ABSTRACT
Nuclear pore complexes (NPCs) govern macromolecular transport between the nucleus and cytoplasm and serve as key positional markers within the nucleus. Several protein components of yeast NPCs have been implicated in the epigenetic control of gene expression. Among these, Nup2p is unique as it transiently associates with NPCs and, when artificially tethered to DNA, can prevent the spread of transcriptional activation or repression between flanking genes, a function termed boundary activity. To understand this function of Nup2p, we investigated the interactions of Nup2p with other proteins and with DNA using immunopurifications coupled with mass spectrometry and microarray analyses. These data combined with functional assays of boundary activity and epigenetic variegation suggest that Nup2p and the Ran guanylyl-nucleotide exchange factor, Prp20p, interact at specific chromatin regions and enable the NPC to play an active role in chromatin organization by facilitating the transition of chromatin between activity states.

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Loss of Nup2p or Gsp2p results in subtelomeric gene silencing defects that are distinct from those observed for strains lacking Nup60p or Htz1p. (a) The expression status of a telomerically encoded α2 reporter gene was assayed in single cells by monitoring the response of cells to the α-factor. Cells that do not express α2 (OFF) respond to α-factor arrest in G1 and shmoo; cells expressing α2 (ON) continue to bud and divide. The initial OFF proportion was determined by scoring cells after a 4-h treatment with α-factor. (b) Determination of OFF maintenance ratio. α2 OFF maintenance was assayed by monitoring α-factor arrested cells over time. Cells that have switched to the ON state give rise to microcolonies, whereas stably arrested cells do not divide. (c) The wild-type normalized initial OFF ratios (y-axis) and OFF maintenance ratios (x-axis) were plotted for each strain (error bars indicate the SD for three independent experiments). Strains lacking Nup2p or Gsp2p exhibited very similar phenotypes (marginally increased initial OFF ratios [y ≥ 1], and poor OFF state maintenance [x < 1]). Cells lacking Nup60p exhibited a steady-state defect in the establishment of the OFF state and an inability to maintain the OFF state (x and y < 1), whereas cells lacking Htz1p showed the opposite phenotype (x and y > 1).
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fig6: Loss of Nup2p or Gsp2p results in subtelomeric gene silencing defects that are distinct from those observed for strains lacking Nup60p or Htz1p. (a) The expression status of a telomerically encoded α2 reporter gene was assayed in single cells by monitoring the response of cells to the α-factor. Cells that do not express α2 (OFF) respond to α-factor arrest in G1 and shmoo; cells expressing α2 (ON) continue to bud and divide. The initial OFF proportion was determined by scoring cells after a 4-h treatment with α-factor. (b) Determination of OFF maintenance ratio. α2 OFF maintenance was assayed by monitoring α-factor arrested cells over time. Cells that have switched to the ON state give rise to microcolonies, whereas stably arrested cells do not divide. (c) The wild-type normalized initial OFF ratios (y-axis) and OFF maintenance ratios (x-axis) were plotted for each strain (error bars indicate the SD for three independent experiments). Strains lacking Nup2p or Gsp2p exhibited very similar phenotypes (marginally increased initial OFF ratios [y ≥ 1], and poor OFF state maintenance [x < 1]). Cells lacking Nup60p exhibited a steady-state defect in the establishment of the OFF state and an inability to maintain the OFF state (x and y < 1), whereas cells lacking Htz1p showed the opposite phenotype (x and y > 1).

Mentions: Genes within subtelomeric regions are capable of epigenetically switching between transcriptionally active (“ON”) and silent (“OFF”) states (Gottschling et al., 1990). Our transcriptomic data suggest that Nup2p plays a role in the maintenance of subtelomeric gene expression states. To test this, we used a single-cell telomeric silencing assay that detects expression variegation in individual cells based on their response to mating pheromone (Iida and Araki, 2004). When grown in the presence of α-factor, the vast majority of yeast a-type cells (MATa) arrest in G1 before START and form pseudopod-like projections, termed shmoos. Diploid a/α cells (MATa/MATα) and α cells (MATα) are insensitive to α-factor and therefore continue to divide in its presence. However, if haploid MATa cells express the normally silenced α2 gene, they are insensitive to α-factor. Exploiting this phenomenon, Iida and Araki (2004) incorporated a subtelomeric copy of the α2 gene in a MATa background, allowing the variegated expression of the α2 gene to be determined in single cells by their response to α-factor—cells in the ON state are insensitive and continue to divide, whereas cells in the OFF state arrest (Fig. 6 a). Consequently, by monitoring the ability of individual cells to maintain the arrested or insensitive phenotype over a time course, the OFF to ON switching rate can be determined (Fig. 6 b).


The mobile nucleoporin Nup2p and chromatin-bound Prp20p function in endogenous NPC-mediated transcriptional control.

Dilworth DJ, Tackett AJ, Rogers RS, Yi EC, Christmas RH, Smith JJ, Siegel AF, Chait BT, Wozniak RW, Aitchison JD - J. Cell Biol. (2005)

Loss of Nup2p or Gsp2p results in subtelomeric gene silencing defects that are distinct from those observed for strains lacking Nup60p or Htz1p. (a) The expression status of a telomerically encoded α2 reporter gene was assayed in single cells by monitoring the response of cells to the α-factor. Cells that do not express α2 (OFF) respond to α-factor arrest in G1 and shmoo; cells expressing α2 (ON) continue to bud and divide. The initial OFF proportion was determined by scoring cells after a 4-h treatment with α-factor. (b) Determination of OFF maintenance ratio. α2 OFF maintenance was assayed by monitoring α-factor arrested cells over time. Cells that have switched to the ON state give rise to microcolonies, whereas stably arrested cells do not divide. (c) The wild-type normalized initial OFF ratios (y-axis) and OFF maintenance ratios (x-axis) were plotted for each strain (error bars indicate the SD for three independent experiments). Strains lacking Nup2p or Gsp2p exhibited very similar phenotypes (marginally increased initial OFF ratios [y ≥ 1], and poor OFF state maintenance [x < 1]). Cells lacking Nup60p exhibited a steady-state defect in the establishment of the OFF state and an inability to maintain the OFF state (x and y < 1), whereas cells lacking Htz1p showed the opposite phenotype (x and y > 1).
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Related In: Results  -  Collection

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fig6: Loss of Nup2p or Gsp2p results in subtelomeric gene silencing defects that are distinct from those observed for strains lacking Nup60p or Htz1p. (a) The expression status of a telomerically encoded α2 reporter gene was assayed in single cells by monitoring the response of cells to the α-factor. Cells that do not express α2 (OFF) respond to α-factor arrest in G1 and shmoo; cells expressing α2 (ON) continue to bud and divide. The initial OFF proportion was determined by scoring cells after a 4-h treatment with α-factor. (b) Determination of OFF maintenance ratio. α2 OFF maintenance was assayed by monitoring α-factor arrested cells over time. Cells that have switched to the ON state give rise to microcolonies, whereas stably arrested cells do not divide. (c) The wild-type normalized initial OFF ratios (y-axis) and OFF maintenance ratios (x-axis) were plotted for each strain (error bars indicate the SD for three independent experiments). Strains lacking Nup2p or Gsp2p exhibited very similar phenotypes (marginally increased initial OFF ratios [y ≥ 1], and poor OFF state maintenance [x < 1]). Cells lacking Nup60p exhibited a steady-state defect in the establishment of the OFF state and an inability to maintain the OFF state (x and y < 1), whereas cells lacking Htz1p showed the opposite phenotype (x and y > 1).
Mentions: Genes within subtelomeric regions are capable of epigenetically switching between transcriptionally active (“ON”) and silent (“OFF”) states (Gottschling et al., 1990). Our transcriptomic data suggest that Nup2p plays a role in the maintenance of subtelomeric gene expression states. To test this, we used a single-cell telomeric silencing assay that detects expression variegation in individual cells based on their response to mating pheromone (Iida and Araki, 2004). When grown in the presence of α-factor, the vast majority of yeast a-type cells (MATa) arrest in G1 before START and form pseudopod-like projections, termed shmoos. Diploid a/α cells (MATa/MATα) and α cells (MATα) are insensitive to α-factor and therefore continue to divide in its presence. However, if haploid MATa cells express the normally silenced α2 gene, they are insensitive to α-factor. Exploiting this phenomenon, Iida and Araki (2004) incorporated a subtelomeric copy of the α2 gene in a MATa background, allowing the variegated expression of the α2 gene to be determined in single cells by their response to α-factor—cells in the ON state are insensitive and continue to divide, whereas cells in the OFF state arrest (Fig. 6 a). Consequently, by monitoring the ability of individual cells to maintain the arrested or insensitive phenotype over a time course, the OFF to ON switching rate can be determined (Fig. 6 b).

Bottom Line: Nuclear pore complexes (NPCs) govern macromolecular transport between the nucleus and cytoplasm and serve as key positional markers within the nucleus.Among these, Nup2p is unique as it transiently associates with NPCs and, when artificially tethered to DNA, can prevent the spread of transcriptional activation or repression between flanking genes, a function termed boundary activity.These data combined with functional assays of boundary activity and epigenetic variegation suggest that Nup2p and the Ran guanylyl-nucleotide exchange factor, Prp20p, interact at specific chromatin regions and enable the NPC to play an active role in chromatin organization by facilitating the transition of chromatin between activity states.

View Article: PubMed Central - PubMed

Affiliation: Institute for Systems Biology, Seattle, WA 98103, USA.

ABSTRACT
Nuclear pore complexes (NPCs) govern macromolecular transport between the nucleus and cytoplasm and serve as key positional markers within the nucleus. Several protein components of yeast NPCs have been implicated in the epigenetic control of gene expression. Among these, Nup2p is unique as it transiently associates with NPCs and, when artificially tethered to DNA, can prevent the spread of transcriptional activation or repression between flanking genes, a function termed boundary activity. To understand this function of Nup2p, we investigated the interactions of Nup2p with other proteins and with DNA using immunopurifications coupled with mass spectrometry and microarray analyses. These data combined with functional assays of boundary activity and epigenetic variegation suggest that Nup2p and the Ran guanylyl-nucleotide exchange factor, Prp20p, interact at specific chromatin regions and enable the NPC to play an active role in chromatin organization by facilitating the transition of chromatin between activity states.

Show MeSH
Related in: MedlinePlus