Limits...
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.

Show MeSH

Related in: MedlinePlus

DNA regions bound by Prp20p and Nup2p enrich near telomeres and lie in close proximity to ORFs induced in cells lacking Nup2p. (a) Histograms of minimal telomeric distance for the top 5% of significantly enriched intergenic regions bound by Prp20p and Nup2p reveal a telomeric enrichment similar to that observed for ORFs induced in cells lacking Nup2p (see Fig. 3). The shaded histograms represent the distribution of all intergenic regions shown at 1/4 scale on the y-axis. The Prp20p and Nup2p profiles are significantly distinct from the distribution of all intergenic regions (P < 0.000001 and P = 0.000367, respectively). In contrast, the profile of the transcription factor Oaf1p displayed no significant enrichment relative to all intergenic regions (P = 0.368; not depicted). (b) Chromosomal proximity of transcriptionally induced ORFs in Δnup2 cells and ChIP-CHIP enriched intergenic regions. The distance between each enriched intergenic region and the nearest Δnup2-induced ORF was determined for Nup2p, Prp20p, Oaf1p, and 10 randomized datasets. Intergenic regions bound by Nup2p and Prp20p are found much closer to ORFs induced in cells lacking Nup2p, relative to the Oaf1p or randomized datasets, as evidenced by the high number of Nup2p and Prp20p enriched intergenic regions found within 10 kb of Δnup2 ORFs. The Kolmogorov-Smirnov test reveals significant differences between the Oaf1p profile and those obtained with Prp20p and Nup2p (P = 0.0282 and P = 0.00102, respectively).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171315&req=5

fig4: DNA regions bound by Prp20p and Nup2p enrich near telomeres and lie in close proximity to ORFs induced in cells lacking Nup2p. (a) Histograms of minimal telomeric distance for the top 5% of significantly enriched intergenic regions bound by Prp20p and Nup2p reveal a telomeric enrichment similar to that observed for ORFs induced in cells lacking Nup2p (see Fig. 3). The shaded histograms represent the distribution of all intergenic regions shown at 1/4 scale on the y-axis. The Prp20p and Nup2p profiles are significantly distinct from the distribution of all intergenic regions (P < 0.000001 and P = 0.000367, respectively). In contrast, the profile of the transcription factor Oaf1p displayed no significant enrichment relative to all intergenic regions (P = 0.368; not depicted). (b) Chromosomal proximity of transcriptionally induced ORFs in Δnup2 cells and ChIP-CHIP enriched intergenic regions. The distance between each enriched intergenic region and the nearest Δnup2-induced ORF was determined for Nup2p, Prp20p, Oaf1p, and 10 randomized datasets. Intergenic regions bound by Nup2p and Prp20p are found much closer to ORFs induced in cells lacking Nup2p, relative to the Oaf1p or randomized datasets, as evidenced by the high number of Nup2p and Prp20p enriched intergenic regions found within 10 kb of Δnup2 ORFs. The Kolmogorov-Smirnov test reveals significant differences between the Oaf1p profile and those obtained with Prp20p and Nup2p (P = 0.0282 and P = 0.00102, respectively).

Mentions: To further examine the role of Prp20p and Nup2p in endogenous NPC-mediated BA, we performed genome localization studies by chromatin immunopurification microarray (ChIP-CHIP) using myc-epitope tagged Prp20p and Nup2p and microarrays containing 6,081 yeast intergenic regions (Ren et al., 2000). Because the ChIP-CHIP procedure involves cross-linking proteins to DNA followed by PCR amplification of bait-associated DNA fragments, we were able to perform these experiments with myc-tagged Nup2p despite the inability of this protein to stably associate with DNA during standard immunopurification procedures (Nup2p-myc yielded ∼50-fold less DNA than Prp20p-myc as determined by SyBr Green fluorimetry; unpublished data). Strikingly similar to the telomeric bias of induced ORFs observed in Δnup2 cells, the top 5% of Prp20p- and Nup2p-enriched intergenic regions (304 targets) clustered together and enriched near telomeres (compare Fig. 4 a with Fig. 3). This telomerically biased distribution was not observed with numerous transcription factors that we have investigated (unpublished data).


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)

DNA regions bound by Prp20p and Nup2p enrich near telomeres and lie in close proximity to ORFs induced in cells lacking Nup2p. (a) Histograms of minimal telomeric distance for the top 5% of significantly enriched intergenic regions bound by Prp20p and Nup2p reveal a telomeric enrichment similar to that observed for ORFs induced in cells lacking Nup2p (see Fig. 3). The shaded histograms represent the distribution of all intergenic regions shown at 1/4 scale on the y-axis. The Prp20p and Nup2p profiles are significantly distinct from the distribution of all intergenic regions (P < 0.000001 and P = 0.000367, respectively). In contrast, the profile of the transcription factor Oaf1p displayed no significant enrichment relative to all intergenic regions (P = 0.368; not depicted). (b) Chromosomal proximity of transcriptionally induced ORFs in Δnup2 cells and ChIP-CHIP enriched intergenic regions. The distance between each enriched intergenic region and the nearest Δnup2-induced ORF was determined for Nup2p, Prp20p, Oaf1p, and 10 randomized datasets. Intergenic regions bound by Nup2p and Prp20p are found much closer to ORFs induced in cells lacking Nup2p, relative to the Oaf1p or randomized datasets, as evidenced by the high number of Nup2p and Prp20p enriched intergenic regions found within 10 kb of Δnup2 ORFs. The Kolmogorov-Smirnov test reveals significant differences between the Oaf1p profile and those obtained with Prp20p and Nup2p (P = 0.0282 and P = 0.00102, respectively).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171315&req=5

fig4: DNA regions bound by Prp20p and Nup2p enrich near telomeres and lie in close proximity to ORFs induced in cells lacking Nup2p. (a) Histograms of minimal telomeric distance for the top 5% of significantly enriched intergenic regions bound by Prp20p and Nup2p reveal a telomeric enrichment similar to that observed for ORFs induced in cells lacking Nup2p (see Fig. 3). The shaded histograms represent the distribution of all intergenic regions shown at 1/4 scale on the y-axis. The Prp20p and Nup2p profiles are significantly distinct from the distribution of all intergenic regions (P < 0.000001 and P = 0.000367, respectively). In contrast, the profile of the transcription factor Oaf1p displayed no significant enrichment relative to all intergenic regions (P = 0.368; not depicted). (b) Chromosomal proximity of transcriptionally induced ORFs in Δnup2 cells and ChIP-CHIP enriched intergenic regions. The distance between each enriched intergenic region and the nearest Δnup2-induced ORF was determined for Nup2p, Prp20p, Oaf1p, and 10 randomized datasets. Intergenic regions bound by Nup2p and Prp20p are found much closer to ORFs induced in cells lacking Nup2p, relative to the Oaf1p or randomized datasets, as evidenced by the high number of Nup2p and Prp20p enriched intergenic regions found within 10 kb of Δnup2 ORFs. The Kolmogorov-Smirnov test reveals significant differences between the Oaf1p profile and those obtained with Prp20p and Nup2p (P = 0.0282 and P = 0.00102, respectively).
Mentions: To further examine the role of Prp20p and Nup2p in endogenous NPC-mediated BA, we performed genome localization studies by chromatin immunopurification microarray (ChIP-CHIP) using myc-epitope tagged Prp20p and Nup2p and microarrays containing 6,081 yeast intergenic regions (Ren et al., 2000). Because the ChIP-CHIP procedure involves cross-linking proteins to DNA followed by PCR amplification of bait-associated DNA fragments, we were able to perform these experiments with myc-tagged Nup2p despite the inability of this protein to stably associate with DNA during standard immunopurification procedures (Nup2p-myc yielded ∼50-fold less DNA than Prp20p-myc as determined by SyBr Green fluorimetry; unpublished data). Strikingly similar to the telomeric bias of induced ORFs observed in Δnup2 cells, the top 5% of Prp20p- and Nup2p-enriched intergenic regions (304 targets) clustered together and enriched near telomeres (compare Fig. 4 a with Fig. 3). This telomerically biased distribution was not observed with numerous transcription factors that we have investigated (unpublished data).

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