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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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Genetic interactions support links between NUP2, NUP60, PRP20, and HTZ1. Growth rate analysis of Δhtz1, Δnup2, Δnup60, Δnup53, and prp20-7 single-mutant and relevant double-mutant strains at 23, 30, and 37°C. Double-mutant prp20-7 Δhtz1, Δhtz1 Δnup2, Δhtz1 Δnup60, prp20-7 Δnup2, and prp20-7 Δnup60 strains all exhibited more severe growth defects than those detected in their parental strains, whereas double-mutant combinations involving deletion of NUP53 revealed no genetic interactions.
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fig5: Genetic interactions support links between NUP2, NUP60, PRP20, and HTZ1. Growth rate analysis of Δhtz1, Δnup2, Δnup60, Δnup53, and prp20-7 single-mutant and relevant double-mutant strains at 23, 30, and 37°C. Double-mutant prp20-7 Δhtz1, Δhtz1 Δnup2, Δhtz1 Δnup60, prp20-7 Δnup2, and prp20-7 Δnup60 strains all exhibited more severe growth defects than those detected in their parental strains, whereas double-mutant combinations involving deletion of NUP53 revealed no genetic interactions.

Mentions: NUP2 has previously been shown to interact genetically with PRP20 and NUP60 (Booth et al., 1999; Dilworth et al., 2001). Given the requirement of Nup60p for the NPC association of Nup2p and the telomeric biases of aberrantly expressed ORFs in Δnup2 and Δhtz1 cells, we tested for genetic interactions between these genes. The growth rates of strains harboring single- and double-mutant combinations of NUP2, NUP60, PRP20, and HTZ1 at various temperatures were tested. NUP53 (Marelli et al., 1998) was included as a negative control. As shown in Fig. 5, the temperature-sensitive allele of PRP20, prp20-7, conferred increased sensitivity when combined with mutations of NUP2, NUP60, or HTZ1, as these double-mutant strains, but not double-mutants involving NUP53, grew poorly at 30°C. As a confirmation of specificity, we obtained similar results using another temperature-sensitive allele of PRP20, srm1-1 (Clark and Sprague, 1989), with the sole exception that the genetic interaction with HTZ1 was very weak in the srm1-1 background (unpublished data). In addition, we detected HTZ1-NUP2 and HTZ1-NUP60 genetic interactions, which support functional relationships between Nup2p-interacting proteins and the activity of Htz1p. We note, however, that the HTZ1-NUP2 interaction is rather weak as it is readily evident only at 23°C, showing only a modest growth defect at 30°C and none at 37°C.


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)

Genetic interactions support links between NUP2, NUP60, PRP20, and HTZ1. Growth rate analysis of Δhtz1, Δnup2, Δnup60, Δnup53, and prp20-7 single-mutant and relevant double-mutant strains at 23, 30, and 37°C. Double-mutant prp20-7 Δhtz1, Δhtz1 Δnup2, Δhtz1 Δnup60, prp20-7 Δnup2, and prp20-7 Δnup60 strains all exhibited more severe growth defects than those detected in their parental strains, whereas double-mutant combinations involving deletion of NUP53 revealed no genetic interactions.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171315&req=5

fig5: Genetic interactions support links between NUP2, NUP60, PRP20, and HTZ1. Growth rate analysis of Δhtz1, Δnup2, Δnup60, Δnup53, and prp20-7 single-mutant and relevant double-mutant strains at 23, 30, and 37°C. Double-mutant prp20-7 Δhtz1, Δhtz1 Δnup2, Δhtz1 Δnup60, prp20-7 Δnup2, and prp20-7 Δnup60 strains all exhibited more severe growth defects than those detected in their parental strains, whereas double-mutant combinations involving deletion of NUP53 revealed no genetic interactions.
Mentions: NUP2 has previously been shown to interact genetically with PRP20 and NUP60 (Booth et al., 1999; Dilworth et al., 2001). Given the requirement of Nup60p for the NPC association of Nup2p and the telomeric biases of aberrantly expressed ORFs in Δnup2 and Δhtz1 cells, we tested for genetic interactions between these genes. The growth rates of strains harboring single- and double-mutant combinations of NUP2, NUP60, PRP20, and HTZ1 at various temperatures were tested. NUP53 (Marelli et al., 1998) was included as a negative control. As shown in Fig. 5, the temperature-sensitive allele of PRP20, prp20-7, conferred increased sensitivity when combined with mutations of NUP2, NUP60, or HTZ1, as these double-mutant strains, but not double-mutants involving NUP53, grew poorly at 30°C. As a confirmation of specificity, we obtained similar results using another temperature-sensitive allele of PRP20, srm1-1 (Clark and Sprague, 1989), with the sole exception that the genetic interaction with HTZ1 was very weak in the srm1-1 background (unpublished data). In addition, we detected HTZ1-NUP2 and HTZ1-NUP60 genetic interactions, which support functional relationships between Nup2p-interacting proteins and the activity of Htz1p. We note, however, that the HTZ1-NUP2 interaction is rather weak as it is readily evident only at 23°C, showing only a modest growth defect at 30°C and none at 37°C.

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