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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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Related in: MedlinePlus

The boundary trap assay confirms the links between the NPC and chromatin-bound Prp20p. The ability of Nup2p to bind to the NPC through Nup60p is required for BA. The boundary trap strain, KIY54, and isogenic Δnup2, Δnup60, Δmlp1, Δmlp2, and Δmlp1/Δmlp2 derivatives expressing plasmids encoding the Gal4 DNA binding domain alone, Gbd (pGBC11) or fused to the COOH-terminal portion of the Drosophila BEAF protein, Gbd-BEAF (pGBC11-BEAF-C), a GFP-tagged portion of Cse1p, Gbd-Cse1p (pGBC11-CSE1[474–960]-GFP), full-length Nup2p, Gbd-Nup2p (pGBC12-NUP2[1–720]) or GFP-tagged Prp20p, Gbd-Prp20p-GFP (pGBC12-PRP20-GFP) were serially spotted onto CSM-TRP (T), CSM-TRP+FOA (TF) and CSM-TRP-ADE+FOA (TAF) to assess boundary function. Cells lacking Nup60p were defective in their ability to silence the URA3 reporter, indicated by a reduced viability on media containing 5-FOA. This phenotype was also shared by two independently isolated double-mutant strains lacking both of the Mlp proteins. BA is indicated by growth on media lacking adenine and containing 5-FOA (TAF). A plasmid encoding only Gbd failed to elicit BA and the positive control fusion, Gbd-BEAF, exhibited BA in all genotypes tested. The BA of Gbd-Cse1p was dependent on Nup2p (Ishii et al., 2002), and BA of both Gbd-Cse1p and Gbd-Nup2p was absent in Δnup60 mutants. Double-mutant Δmlp1/Δmlp2 strains exhibited reduced BA. Gdb-Prp20p possesses BA in wild-type and single Mlp mutant strains at frequencies comparable to transport factors (Ishii et al., 2002). Deletion of NUP2 or NUP60 or both MLP1 and MLP2 resulted in dramatically reduced Gbd-Prp20p BA.
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fig2: The boundary trap assay confirms the links between the NPC and chromatin-bound Prp20p. The ability of Nup2p to bind to the NPC through Nup60p is required for BA. The boundary trap strain, KIY54, and isogenic Δnup2, Δnup60, Δmlp1, Δmlp2, and Δmlp1/Δmlp2 derivatives expressing plasmids encoding the Gal4 DNA binding domain alone, Gbd (pGBC11) or fused to the COOH-terminal portion of the Drosophila BEAF protein, Gbd-BEAF (pGBC11-BEAF-C), a GFP-tagged portion of Cse1p, Gbd-Cse1p (pGBC11-CSE1[474–960]-GFP), full-length Nup2p, Gbd-Nup2p (pGBC12-NUP2[1–720]) or GFP-tagged Prp20p, Gbd-Prp20p-GFP (pGBC12-PRP20-GFP) were serially spotted onto CSM-TRP (T), CSM-TRP+FOA (TF) and CSM-TRP-ADE+FOA (TAF) to assess boundary function. Cells lacking Nup60p were defective in their ability to silence the URA3 reporter, indicated by a reduced viability on media containing 5-FOA. This phenotype was also shared by two independently isolated double-mutant strains lacking both of the Mlp proteins. BA is indicated by growth on media lacking adenine and containing 5-FOA (TAF). A plasmid encoding only Gbd failed to elicit BA and the positive control fusion, Gbd-BEAF, exhibited BA in all genotypes tested. The BA of Gbd-Cse1p was dependent on Nup2p (Ishii et al., 2002), and BA of both Gbd-Cse1p and Gbd-Nup2p was absent in Δnup60 mutants. Double-mutant Δmlp1/Δmlp2 strains exhibited reduced BA. Gdb-Prp20p possesses BA in wild-type and single Mlp mutant strains at frequencies comparable to transport factors (Ishii et al., 2002). Deletion of NUP2 or NUP60 or both MLP1 and MLP2 resulted in dramatically reduced Gbd-Prp20p BA.

Mentions: Our analysis of interactions made by Nup2p led us to hypothesize that Nup2p BA is mediated by its interaction on the one hand with Nup60p at the NPC, and on the other hand with Prp20p on chromatin. Using the boundary trap assay (Ishii et al., 2002), we examined if Nup2p-mediated BA was dependent on NPC association, and whether Prp20p possesses its own BA. In this assay, two genes (URA3 and ADE2) are placed within a partially derepressed HML locus with the ADE2 gene flanked by DNA sequences that bind Gal4p (UASg sequence). Boundary activity, such as that exhibited by the control Drosophila protein BEAF, is detected by the ability of proteins fused to the DNA binding domain of Gal4p (Gbd) to allow the expression of the ADE2 marker while maintaining the adjacent URA3 gene in an “OFF” state. This activity is interpreted as a selective insulation of the ADE2 gene from the surrounding silenced chromatin resulting from the binding of the Gbd chimera to the UASg sites flanking ADE2 (Ishii et al., 2002). The BA of Nup2p (and Cse1p) was strictly dependent on its ability to bind to the NPC through Nup60p (Fig. 2). We also found that Prp20p elicited BA at frequencies comparable to those of Gbd-Nup2p and Gbd-Cse1p and, in addition, full activity required Nup2p and Nup60p. It should be noted, however, that a low amount of BA was still detected for Gbd-Prp20p in the absence of Nup2p or Nup60p, which we interpret to be due to the ability of Prp20p to bind to several other components of the nucleocytoplasmic machinery (Floer et al., 1997; Noguchi et al., 1997; Solsbacher et al., 2000; Akhtar et al., 2001), which could offer alternative (albeit less efficient) associations with the NPC. In further support of the pivotal role of Nup2p in BA, neither Nup60p nor Htz1p were active in the boundary trap assay (unpublished data), indicating that the ability to bind to Prp20p is not sufficient to confer BA, and confirming that BA is not a general feature of nups (Ishii et al., 2002).


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)

The boundary trap assay confirms the links between the NPC and chromatin-bound Prp20p. The ability of Nup2p to bind to the NPC through Nup60p is required for BA. The boundary trap strain, KIY54, and isogenic Δnup2, Δnup60, Δmlp1, Δmlp2, and Δmlp1/Δmlp2 derivatives expressing plasmids encoding the Gal4 DNA binding domain alone, Gbd (pGBC11) or fused to the COOH-terminal portion of the Drosophila BEAF protein, Gbd-BEAF (pGBC11-BEAF-C), a GFP-tagged portion of Cse1p, Gbd-Cse1p (pGBC11-CSE1[474–960]-GFP), full-length Nup2p, Gbd-Nup2p (pGBC12-NUP2[1–720]) or GFP-tagged Prp20p, Gbd-Prp20p-GFP (pGBC12-PRP20-GFP) were serially spotted onto CSM-TRP (T), CSM-TRP+FOA (TF) and CSM-TRP-ADE+FOA (TAF) to assess boundary function. Cells lacking Nup60p were defective in their ability to silence the URA3 reporter, indicated by a reduced viability on media containing 5-FOA. This phenotype was also shared by two independently isolated double-mutant strains lacking both of the Mlp proteins. BA is indicated by growth on media lacking adenine and containing 5-FOA (TAF). A plasmid encoding only Gbd failed to elicit BA and the positive control fusion, Gbd-BEAF, exhibited BA in all genotypes tested. The BA of Gbd-Cse1p was dependent on Nup2p (Ishii et al., 2002), and BA of both Gbd-Cse1p and Gbd-Nup2p was absent in Δnup60 mutants. Double-mutant Δmlp1/Δmlp2 strains exhibited reduced BA. Gdb-Prp20p possesses BA in wild-type and single Mlp mutant strains at frequencies comparable to transport factors (Ishii et al., 2002). Deletion of NUP2 or NUP60 or both MLP1 and MLP2 resulted in dramatically reduced Gbd-Prp20p BA.
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Related In: Results  -  Collection

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fig2: The boundary trap assay confirms the links between the NPC and chromatin-bound Prp20p. The ability of Nup2p to bind to the NPC through Nup60p is required for BA. The boundary trap strain, KIY54, and isogenic Δnup2, Δnup60, Δmlp1, Δmlp2, and Δmlp1/Δmlp2 derivatives expressing plasmids encoding the Gal4 DNA binding domain alone, Gbd (pGBC11) or fused to the COOH-terminal portion of the Drosophila BEAF protein, Gbd-BEAF (pGBC11-BEAF-C), a GFP-tagged portion of Cse1p, Gbd-Cse1p (pGBC11-CSE1[474–960]-GFP), full-length Nup2p, Gbd-Nup2p (pGBC12-NUP2[1–720]) or GFP-tagged Prp20p, Gbd-Prp20p-GFP (pGBC12-PRP20-GFP) were serially spotted onto CSM-TRP (T), CSM-TRP+FOA (TF) and CSM-TRP-ADE+FOA (TAF) to assess boundary function. Cells lacking Nup60p were defective in their ability to silence the URA3 reporter, indicated by a reduced viability on media containing 5-FOA. This phenotype was also shared by two independently isolated double-mutant strains lacking both of the Mlp proteins. BA is indicated by growth on media lacking adenine and containing 5-FOA (TAF). A plasmid encoding only Gbd failed to elicit BA and the positive control fusion, Gbd-BEAF, exhibited BA in all genotypes tested. The BA of Gbd-Cse1p was dependent on Nup2p (Ishii et al., 2002), and BA of both Gbd-Cse1p and Gbd-Nup2p was absent in Δnup60 mutants. Double-mutant Δmlp1/Δmlp2 strains exhibited reduced BA. Gdb-Prp20p possesses BA in wild-type and single Mlp mutant strains at frequencies comparable to transport factors (Ishii et al., 2002). Deletion of NUP2 or NUP60 or both MLP1 and MLP2 resulted in dramatically reduced Gbd-Prp20p BA.
Mentions: Our analysis of interactions made by Nup2p led us to hypothesize that Nup2p BA is mediated by its interaction on the one hand with Nup60p at the NPC, and on the other hand with Prp20p on chromatin. Using the boundary trap assay (Ishii et al., 2002), we examined if Nup2p-mediated BA was dependent on NPC association, and whether Prp20p possesses its own BA. In this assay, two genes (URA3 and ADE2) are placed within a partially derepressed HML locus with the ADE2 gene flanked by DNA sequences that bind Gal4p (UASg sequence). Boundary activity, such as that exhibited by the control Drosophila protein BEAF, is detected by the ability of proteins fused to the DNA binding domain of Gal4p (Gbd) to allow the expression of the ADE2 marker while maintaining the adjacent URA3 gene in an “OFF” state. This activity is interpreted as a selective insulation of the ADE2 gene from the surrounding silenced chromatin resulting from the binding of the Gbd chimera to the UASg sites flanking ADE2 (Ishii et al., 2002). The BA of Nup2p (and Cse1p) was strictly dependent on its ability to bind to the NPC through Nup60p (Fig. 2). We also found that Prp20p elicited BA at frequencies comparable to those of Gbd-Nup2p and Gbd-Cse1p and, in addition, full activity required Nup2p and Nup60p. It should be noted, however, that a low amount of BA was still detected for Gbd-Prp20p in the absence of Nup2p or Nup60p, which we interpret to be due to the ability of Prp20p to bind to several other components of the nucleocytoplasmic machinery (Floer et al., 1997; Noguchi et al., 1997; Solsbacher et al., 2000; Akhtar et al., 2001), which could offer alternative (albeit less efficient) associations with the NPC. In further support of the pivotal role of Nup2p in BA, neither Nup60p nor Htz1p were active in the boundary trap assay (unpublished data), indicating that the ability to bind to Prp20p is not sufficient to confer BA, and confirming that BA is not a general feature of nups (Ishii et al., 2002).

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