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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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Prp20p and Nup2p interact with chromatin remodeling factors. (a) Prp20p is nucleosome associated. (Left) Prp20p-prA was immunopurified from yeast whole-cell lysates and abundant copurifying proteins were identified by MS analysis of gel slices. Components of the histone octamer, H2A, H2B, H3, and H4, were present as well as the linker histone, H1 and Ran/Gsp1p. The presence of Mgm101p is not specific (see text). (Right) Prp20p-prA eluates contain DNA. Eluates were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. Prp20p-prA associated DNA is ∼600 bp in length due to chromatin shearing during the cell lysis procedures. (b) The interaction between Nup2p and the Prp20p–nucleosome complex can be reconstituted in vitro. (Top) Bacterially expressed and purified Prp20p was incubated with glutathione resin coated with GST or GST-Nup2p. Unbound and bound proteins were resolved by SDS-PAGE and visualized with Coomassie blue. (Middle) The Prp20p–nucleosome complex was bound to IgG-coated magnetic beads and then incubated with bacterially expressed and purified GST-Nup2p-CFP, GST-CFP, or GST-Nup2p. Immunoblotting for GST revealed that although GST alone could not bind to the Prp20p–nucleosome complex, GST chimeras containing Nup2p bound efficiently (asterisks). Arrows indicate nonspecific immunoreactive proteins. (Bottom) Glutathione resin coated with GST-Nup2p, but not GST alone, was able to capture Prp20p-prA from yeast extracts. (c) Interactions between the NPC and chromatin. Proteins present at Coomassie blue–detectable levels in Nup2p, Nup60p, and/or Prp20p immunopurifications are connected by solid black lines. Other known physical and yeast two-hybrid interactions are shown by dotted and dashed lines, respectively (Dingwall et al., 1995; Rexach and Blobel, 1995; Denning et al., 2001; Feuerbach et al., 2002). High coverage tandem MS (MS/MS) of immunopurification eluates from Nup2p, Prp20p, and Nup60p,as well as Kap95p and Nup49p (as controls) was also performed. The inset list (Eluate MS/MS) shows proteins present exclusively in Nup2p eluates (top) or Prp20p eluates (bottom) and those present in both eluates (middle). (d) Nucleosomes associated with Prp20p and Htz1p possess unique acetylation patterns suggestive of boundary chromatin. The acetylation levels of residues K5, K8, K12, and K16 of histone H4 were quantified by mass spectrometry for global (white), Prp20p-associated (dark gray), and Htz1p-associated (light gray) nucleosomes.
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fig1: Prp20p and Nup2p interact with chromatin remodeling factors. (a) Prp20p is nucleosome associated. (Left) Prp20p-prA was immunopurified from yeast whole-cell lysates and abundant copurifying proteins were identified by MS analysis of gel slices. Components of the histone octamer, H2A, H2B, H3, and H4, were present as well as the linker histone, H1 and Ran/Gsp1p. The presence of Mgm101p is not specific (see text). (Right) Prp20p-prA eluates contain DNA. Eluates were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. Prp20p-prA associated DNA is ∼600 bp in length due to chromatin shearing during the cell lysis procedures. (b) The interaction between Nup2p and the Prp20p–nucleosome complex can be reconstituted in vitro. (Top) Bacterially expressed and purified Prp20p was incubated with glutathione resin coated with GST or GST-Nup2p. Unbound and bound proteins were resolved by SDS-PAGE and visualized with Coomassie blue. (Middle) The Prp20p–nucleosome complex was bound to IgG-coated magnetic beads and then incubated with bacterially expressed and purified GST-Nup2p-CFP, GST-CFP, or GST-Nup2p. Immunoblotting for GST revealed that although GST alone could not bind to the Prp20p–nucleosome complex, GST chimeras containing Nup2p bound efficiently (asterisks). Arrows indicate nonspecific immunoreactive proteins. (Bottom) Glutathione resin coated with GST-Nup2p, but not GST alone, was able to capture Prp20p-prA from yeast extracts. (c) Interactions between the NPC and chromatin. Proteins present at Coomassie blue–detectable levels in Nup2p, Nup60p, and/or Prp20p immunopurifications are connected by solid black lines. Other known physical and yeast two-hybrid interactions are shown by dotted and dashed lines, respectively (Dingwall et al., 1995; Rexach and Blobel, 1995; Denning et al., 2001; Feuerbach et al., 2002). High coverage tandem MS (MS/MS) of immunopurification eluates from Nup2p, Prp20p, and Nup60p,as well as Kap95p and Nup49p (as controls) was also performed. The inset list (Eluate MS/MS) shows proteins present exclusively in Nup2p eluates (top) or Prp20p eluates (bottom) and those present in both eluates (middle). (d) Nucleosomes associated with Prp20p and Htz1p possess unique acetylation patterns suggestive of boundary chromatin. The acetylation levels of residues K5, K8, K12, and K16 of histone H4 were quantified by mass spectrometry for global (white), Prp20p-associated (dark gray), and Htz1p-associated (light gray) nucleosomes.

Mentions: First, it was established that Prp20p binds avidly to nucleosomal DNA as detected by ethidium bromide staining of eluates immunopurified from whole-cell lysates and separated by agarose gel electrophoresis (Fig. 1 a). This was in contrast to Nup2p, and the majority of yeast proteins that, using similar methods, do not yield readily detectable DNA. To address the interaction between Nup2p and Prp20p (Fig. 1 b), we first performed solution binding assays using recombinant proteins to confirm that these proteins do indeed interact directly in vitro. Second, we used whole-cell lysates to immobilize the Prp20p–nucleosome complex on beads and then assessed the ability of this complex to bind to bacterially expressed and purified Nup2p. The presence of Nup2p in the bound fractions suggests that Nup2p can interact with Prp20p in this context, which is analogous to chromatin-bound Prp20p. With the third experimental approach, we showed that beads coated with GST-Nup2p, but not GST alone, were able to capture Prp20p from yeast whole-cell extracts. Importantly, none of these experiments exclude the possibility that, rather than associate with nucleosome-bound Prp20p, Nup2p binds instead to a small pool of free Prp20p. To address this uncertainty, we resolved bound and unbound protein mixtures over 10–70% glycerol gradients. Nup2p was detected in the highest glycerol densities only when the Prp20p–nucleosome complex was present (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200509061/DC1). Together with the observation that all detectable Prp20p was found in the high density fractions, the data support the existence of a large DNA–protein complex with which Nup2p can associate. When these data are combined with previously published physical and yeast two-hybrid interactions involving Nup2p, Nup60p, and Prp20p (Dingwall et al., 1995; Rexach and Blobel, 1995; Denning et al., 2001; Feuerbach et al., 2002), they suggest that the Nup2p–Prp20p interaction provides a link between the NPC and chromatin (Fig. 1 c). As a final test of an in vivo interaction between these proteins, we immunopurified Nup2p-prA from formaldehyde cross-linked cells that coexpressed myc-tagged Prp20p. In this case, we were able to detect the myc epitope in Nup2p eluates by immunoblotting using a monoclonal myc antibody and by mass spectrometry (Fig. S2). Together, these data support an in vivo interaction between Nup2p and Prp20p.


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)

Prp20p and Nup2p interact with chromatin remodeling factors. (a) Prp20p is nucleosome associated. (Left) Prp20p-prA was immunopurified from yeast whole-cell lysates and abundant copurifying proteins were identified by MS analysis of gel slices. Components of the histone octamer, H2A, H2B, H3, and H4, were present as well as the linker histone, H1 and Ran/Gsp1p. The presence of Mgm101p is not specific (see text). (Right) Prp20p-prA eluates contain DNA. Eluates were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. Prp20p-prA associated DNA is ∼600 bp in length due to chromatin shearing during the cell lysis procedures. (b) The interaction between Nup2p and the Prp20p–nucleosome complex can be reconstituted in vitro. (Top) Bacterially expressed and purified Prp20p was incubated with glutathione resin coated with GST or GST-Nup2p. Unbound and bound proteins were resolved by SDS-PAGE and visualized with Coomassie blue. (Middle) The Prp20p–nucleosome complex was bound to IgG-coated magnetic beads and then incubated with bacterially expressed and purified GST-Nup2p-CFP, GST-CFP, or GST-Nup2p. Immunoblotting for GST revealed that although GST alone could not bind to the Prp20p–nucleosome complex, GST chimeras containing Nup2p bound efficiently (asterisks). Arrows indicate nonspecific immunoreactive proteins. (Bottom) Glutathione resin coated with GST-Nup2p, but not GST alone, was able to capture Prp20p-prA from yeast extracts. (c) Interactions between the NPC and chromatin. Proteins present at Coomassie blue–detectable levels in Nup2p, Nup60p, and/or Prp20p immunopurifications are connected by solid black lines. Other known physical and yeast two-hybrid interactions are shown by dotted and dashed lines, respectively (Dingwall et al., 1995; Rexach and Blobel, 1995; Denning et al., 2001; Feuerbach et al., 2002). High coverage tandem MS (MS/MS) of immunopurification eluates from Nup2p, Prp20p, and Nup60p,as well as Kap95p and Nup49p (as controls) was also performed. The inset list (Eluate MS/MS) shows proteins present exclusively in Nup2p eluates (top) or Prp20p eluates (bottom) and those present in both eluates (middle). (d) Nucleosomes associated with Prp20p and Htz1p possess unique acetylation patterns suggestive of boundary chromatin. The acetylation levels of residues K5, K8, K12, and K16 of histone H4 were quantified by mass spectrometry for global (white), Prp20p-associated (dark gray), and Htz1p-associated (light gray) nucleosomes.
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fig1: Prp20p and Nup2p interact with chromatin remodeling factors. (a) Prp20p is nucleosome associated. (Left) Prp20p-prA was immunopurified from yeast whole-cell lysates and abundant copurifying proteins were identified by MS analysis of gel slices. Components of the histone octamer, H2A, H2B, H3, and H4, were present as well as the linker histone, H1 and Ran/Gsp1p. The presence of Mgm101p is not specific (see text). (Right) Prp20p-prA eluates contain DNA. Eluates were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. Prp20p-prA associated DNA is ∼600 bp in length due to chromatin shearing during the cell lysis procedures. (b) The interaction between Nup2p and the Prp20p–nucleosome complex can be reconstituted in vitro. (Top) Bacterially expressed and purified Prp20p was incubated with glutathione resin coated with GST or GST-Nup2p. Unbound and bound proteins were resolved by SDS-PAGE and visualized with Coomassie blue. (Middle) The Prp20p–nucleosome complex was bound to IgG-coated magnetic beads and then incubated with bacterially expressed and purified GST-Nup2p-CFP, GST-CFP, or GST-Nup2p. Immunoblotting for GST revealed that although GST alone could not bind to the Prp20p–nucleosome complex, GST chimeras containing Nup2p bound efficiently (asterisks). Arrows indicate nonspecific immunoreactive proteins. (Bottom) Glutathione resin coated with GST-Nup2p, but not GST alone, was able to capture Prp20p-prA from yeast extracts. (c) Interactions between the NPC and chromatin. Proteins present at Coomassie blue–detectable levels in Nup2p, Nup60p, and/or Prp20p immunopurifications are connected by solid black lines. Other known physical and yeast two-hybrid interactions are shown by dotted and dashed lines, respectively (Dingwall et al., 1995; Rexach and Blobel, 1995; Denning et al., 2001; Feuerbach et al., 2002). High coverage tandem MS (MS/MS) of immunopurification eluates from Nup2p, Prp20p, and Nup60p,as well as Kap95p and Nup49p (as controls) was also performed. The inset list (Eluate MS/MS) shows proteins present exclusively in Nup2p eluates (top) or Prp20p eluates (bottom) and those present in both eluates (middle). (d) Nucleosomes associated with Prp20p and Htz1p possess unique acetylation patterns suggestive of boundary chromatin. The acetylation levels of residues K5, K8, K12, and K16 of histone H4 were quantified by mass spectrometry for global (white), Prp20p-associated (dark gray), and Htz1p-associated (light gray) nucleosomes.
Mentions: First, it was established that Prp20p binds avidly to nucleosomal DNA as detected by ethidium bromide staining of eluates immunopurified from whole-cell lysates and separated by agarose gel electrophoresis (Fig. 1 a). This was in contrast to Nup2p, and the majority of yeast proteins that, using similar methods, do not yield readily detectable DNA. To address the interaction between Nup2p and Prp20p (Fig. 1 b), we first performed solution binding assays using recombinant proteins to confirm that these proteins do indeed interact directly in vitro. Second, we used whole-cell lysates to immobilize the Prp20p–nucleosome complex on beads and then assessed the ability of this complex to bind to bacterially expressed and purified Nup2p. The presence of Nup2p in the bound fractions suggests that Nup2p can interact with Prp20p in this context, which is analogous to chromatin-bound Prp20p. With the third experimental approach, we showed that beads coated with GST-Nup2p, but not GST alone, were able to capture Prp20p from yeast whole-cell extracts. Importantly, none of these experiments exclude the possibility that, rather than associate with nucleosome-bound Prp20p, Nup2p binds instead to a small pool of free Prp20p. To address this uncertainty, we resolved bound and unbound protein mixtures over 10–70% glycerol gradients. Nup2p was detected in the highest glycerol densities only when the Prp20p–nucleosome complex was present (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200509061/DC1). Together with the observation that all detectable Prp20p was found in the high density fractions, the data support the existence of a large DNA–protein complex with which Nup2p can associate. When these data are combined with previously published physical and yeast two-hybrid interactions involving Nup2p, Nup60p, and Prp20p (Dingwall et al., 1995; Rexach and Blobel, 1995; Denning et al., 2001; Feuerbach et al., 2002), they suggest that the Nup2p–Prp20p interaction provides a link between the NPC and chromatin (Fig. 1 c). As a final test of an in vivo interaction between these proteins, we immunopurified Nup2p-prA from formaldehyde cross-linked cells that coexpressed myc-tagged Prp20p. In this case, we were able to detect the myc epitope in Nup2p eluates by immunoblotting using a monoclonal myc antibody and by mass spectrometry (Fig. S2). Together, these data support an in vivo interaction between Nup2p and Prp20p.

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