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Function of homo- and hetero-oligomers of human nucleoplasmin/nucleophosmin family proteins NPM1, NPM2 and NPM3 during sperm chromatin remodeling.

Okuwaki M, Sumi A, Hisaoka M, Saotome-Nakamura A, Akashi S, Nishimura Y, Nagata K - Nucleic Acids Res. (2012)

Bottom Line: Furthermore, the oligomer formation with NPM1 elicited NPM3 nucleosome assembly and sperm chromatin decondensation activity.NPM3 also suppressed the RNA-binding activity of NPM1, which enhanced the nucleoplasm-nucleolus shuttling of NPM1 in somatic cell nuclei.Our results proposed a novel mechanism whereby three NPM proteins cooperatively regulate chromatin disassembly and assembly in the early embryo and in somatic cells.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Medicine and Graduate School of Comprehensive Human Sciences, Initiative for Promotion of Young Scientists' Independent Research, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8577, Japan. mokuwaki@md.tsukuba.ac.jp

ABSTRACT
Sperm chromatin remodeling after oocyte entry is the essential step that initiates embryogenesis. This reaction involves the removal of sperm-specific basic proteins and chromatin assembly with histones. In mammals, three nucleoplasmin/nucleophosmin (NPM) family proteins-NPM1, NPM2 and NPM3-expressed in oocytes are presumed to cooperatively regulate sperm chromatin remodeling. We characterized the sperm chromatin decondensation and nucleosome assembly activities of three human NPM proteins. NPM1 and NPM2 mediated nucleosome assembly independently of other NPM proteins, whereas the function of NPM3 was largely dependent on formation of a complex with NPM1. Maximal sperm chromatin remodeling activity of NPM2 required the inhibition of its non-specific nucleic acid-binding activity by phosphorylation. Furthermore, the oligomer formation with NPM1 elicited NPM3 nucleosome assembly and sperm chromatin decondensation activity. NPM3 also suppressed the RNA-binding activity of NPM1, which enhanced the nucleoplasm-nucleolus shuttling of NPM1 in somatic cell nuclei. Our results proposed a novel mechanism whereby three NPM proteins cooperatively regulate chromatin disassembly and assembly in the early embryo and in somatic cells.

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Oligomer formation of human NPM proteins. (A) Chemical crosslinking experiments. His-tagged NPM1, NPM2 and NPM3 (200 ng each) were treated without (lanes 1, 3 and 5) or with (lanes 2, 4 and 6) 0.05% glutaraldehyde (GA) and the fixed proteins were separated on 7.5% and 12.5% SDS–PAGE (top and bottom panels, respectively). Proteins were analyzed by western blotting with anti-His-tag antibody. Positions of molecular weight markers are indicated at the left side of the panels. (B) BN–PAGE analysis of NPM proteins. Recombinant His-tagged NPM1 (34.5 kDa), NPM2 (26.3 kDa) and NPM3 (21.5 kDa) (500 ng) were separated by 4–16% BN–PAGE and visualized with CBB staining. Lane M indicates molecular weight markers. The mobility of marker proteins was plotted as a function of their molecular weights (Mw) (right panel). The marker proteins were linearly separated and the masses of NPM proteins were estimated as shown at the right side of the gel. (C) Limited proteolysis of human NPM proteins. His-tagged NPM1, NPM2 and NPM3 were treated without or with increasing amounts of trypsin and incubated at 37°C for 5 min. Then the proteins were separated on 15% SDS–PAGE followed by CBB staining (Red) or western blotting with anti-His-tag antibody (Green). Positions of bands corresponding to the NPM core are indicated by arrow heads. CBB-stained gel and western blotting images are merged and shown in the top panel. (D) Alignment of the amino acid sequences of human NPM N-terminal domains. Amino acid sequences of human NPM1, NPM2 and NPM3 were aligned by ClastalW2 software (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and conserved amino acids are highlighted by asterisks at the bottom of sequences. The conserved core domains of NPM proteins are indicated on blue background. The position of acidic ‘A1 tract’ is shown by red line at the top of the sequences.
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gks162-F2: Oligomer formation of human NPM proteins. (A) Chemical crosslinking experiments. His-tagged NPM1, NPM2 and NPM3 (200 ng each) were treated without (lanes 1, 3 and 5) or with (lanes 2, 4 and 6) 0.05% glutaraldehyde (GA) and the fixed proteins were separated on 7.5% and 12.5% SDS–PAGE (top and bottom panels, respectively). Proteins were analyzed by western blotting with anti-His-tag antibody. Positions of molecular weight markers are indicated at the left side of the panels. (B) BN–PAGE analysis of NPM proteins. Recombinant His-tagged NPM1 (34.5 kDa), NPM2 (26.3 kDa) and NPM3 (21.5 kDa) (500 ng) were separated by 4–16% BN–PAGE and visualized with CBB staining. Lane M indicates molecular weight markers. The mobility of marker proteins was plotted as a function of their molecular weights (Mw) (right panel). The marker proteins were linearly separated and the masses of NPM proteins were estimated as shown at the right side of the gel. (C) Limited proteolysis of human NPM proteins. His-tagged NPM1, NPM2 and NPM3 were treated without or with increasing amounts of trypsin and incubated at 37°C for 5 min. Then the proteins were separated on 15% SDS–PAGE followed by CBB staining (Red) or western blotting with anti-His-tag antibody (Green). Positions of bands corresponding to the NPM core are indicated by arrow heads. CBB-stained gel and western blotting images are merged and shown in the top panel. (D) Alignment of the amino acid sequences of human NPM N-terminal domains. Amino acid sequences of human NPM1, NPM2 and NPM3 were aligned by ClastalW2 software (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and conserved amino acids are highlighted by asterisks at the bottom of sequences. The conserved core domains of NPM proteins are indicated on blue background. The position of acidic ‘A1 tract’ is shown by red line at the top of the sequences.

Mentions: We next examined the oligomer formation ability of human NPM proteins. Bacterially expressed human NPM proteins were purified, and their oligomerization ability was tested using a chemical crosslinking assay (Figure 2A). NPM1 was subjected to SDS–PAGE and bands with 40 kDa and >200 kDa were detected even in the absence of chemical crosslinking (lane 1), indicating that NPM1 formed a stable oligomer. After glutaraldehyde (GA) treatment, ∼200- and 160-kDa protein bands were observed for NPM1 and NPM2, respectively (lanes 2 and 4). In addition, bands >200 kDa were also detected. This result suggested that human NPM1 and NPM2 both formed stable pentamers and that some of the pentamers formed decamers in solution. Conversely, the band corresponding to a pentamer was not detected when NPM3 was subjected to GA treatment (lanes 5 and 6). Instead, a band similar to that of the NPM3 monomer and a minor population of a 55–60 kDa bands were detected. To further confirm the oligomerization status of NPM proteins, the proteins were analyzed by BN–PAGE (Figure 2B). BN–PAGE was originally developed for the separation of mitochondrial membrane proteins and has been used to determine the native mass and oligomeric status of proteins (28). By comparison with the molecular standards separated on the same gel (Figure 2B, right panel), the native masses of NPM1, NPM2 and NPM3 were estimated to be 358, 231 and 42 kDa, respectively. In parallel, gel filtration analysis revealed that His–NPM1 and His–NPM2 were fractionated at the positions corresponding to approximately 370 and 270 kDa, respectively (Figure 6D). In addition, we examined the mass of NPM3 by gel filtration, but it distributed broadly and we could not estimate native mass (data not shown). From these results and previous structural analyses (9–11,13), we concluded that both human NPM1 (34.7 kDa including the His-tag) and NPM2 (26.3 kDa including the His-tag) formed stable pentamers in solution and that two pentamers are assembled into decamers. On the other hand, NPM3 (21.5 kDa including the His-tag) is suggested to from dimers in solution. Consistent with this data, preliminary results from electrospray ionization mass spectrometry analyses suggested that NPM3 formed a dimer at low salt concentrations (data not shown). NPM1 and NPM2 treated with GA and separated by SDS–PAGE were mainly detected at around pentamer positions, while these proteins were suggested to form decamers by BN–PAGE and gel filtration assays. This could be due to the assumption that the pentamer–pentamer interface is not efficiently crosslinked by GA, and two pentamers are dissociated under SDS–PAGE conditions.Figure 2.


Function of homo- and hetero-oligomers of human nucleoplasmin/nucleophosmin family proteins NPM1, NPM2 and NPM3 during sperm chromatin remodeling.

Okuwaki M, Sumi A, Hisaoka M, Saotome-Nakamura A, Akashi S, Nishimura Y, Nagata K - Nucleic Acids Res. (2012)

Oligomer formation of human NPM proteins. (A) Chemical crosslinking experiments. His-tagged NPM1, NPM2 and NPM3 (200 ng each) were treated without (lanes 1, 3 and 5) or with (lanes 2, 4 and 6) 0.05% glutaraldehyde (GA) and the fixed proteins were separated on 7.5% and 12.5% SDS–PAGE (top and bottom panels, respectively). Proteins were analyzed by western blotting with anti-His-tag antibody. Positions of molecular weight markers are indicated at the left side of the panels. (B) BN–PAGE analysis of NPM proteins. Recombinant His-tagged NPM1 (34.5 kDa), NPM2 (26.3 kDa) and NPM3 (21.5 kDa) (500 ng) were separated by 4–16% BN–PAGE and visualized with CBB staining. Lane M indicates molecular weight markers. The mobility of marker proteins was plotted as a function of their molecular weights (Mw) (right panel). The marker proteins were linearly separated and the masses of NPM proteins were estimated as shown at the right side of the gel. (C) Limited proteolysis of human NPM proteins. His-tagged NPM1, NPM2 and NPM3 were treated without or with increasing amounts of trypsin and incubated at 37°C for 5 min. Then the proteins were separated on 15% SDS–PAGE followed by CBB staining (Red) or western blotting with anti-His-tag antibody (Green). Positions of bands corresponding to the NPM core are indicated by arrow heads. CBB-stained gel and western blotting images are merged and shown in the top panel. (D) Alignment of the amino acid sequences of human NPM N-terminal domains. Amino acid sequences of human NPM1, NPM2 and NPM3 were aligned by ClastalW2 software (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and conserved amino acids are highlighted by asterisks at the bottom of sequences. The conserved core domains of NPM proteins are indicated on blue background. The position of acidic ‘A1 tract’ is shown by red line at the top of the sequences.
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Related In: Results  -  Collection

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Show All Figures
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gks162-F2: Oligomer formation of human NPM proteins. (A) Chemical crosslinking experiments. His-tagged NPM1, NPM2 and NPM3 (200 ng each) were treated without (lanes 1, 3 and 5) or with (lanes 2, 4 and 6) 0.05% glutaraldehyde (GA) and the fixed proteins were separated on 7.5% and 12.5% SDS–PAGE (top and bottom panels, respectively). Proteins were analyzed by western blotting with anti-His-tag antibody. Positions of molecular weight markers are indicated at the left side of the panels. (B) BN–PAGE analysis of NPM proteins. Recombinant His-tagged NPM1 (34.5 kDa), NPM2 (26.3 kDa) and NPM3 (21.5 kDa) (500 ng) were separated by 4–16% BN–PAGE and visualized with CBB staining. Lane M indicates molecular weight markers. The mobility of marker proteins was plotted as a function of their molecular weights (Mw) (right panel). The marker proteins were linearly separated and the masses of NPM proteins were estimated as shown at the right side of the gel. (C) Limited proteolysis of human NPM proteins. His-tagged NPM1, NPM2 and NPM3 were treated without or with increasing amounts of trypsin and incubated at 37°C for 5 min. Then the proteins were separated on 15% SDS–PAGE followed by CBB staining (Red) or western blotting with anti-His-tag antibody (Green). Positions of bands corresponding to the NPM core are indicated by arrow heads. CBB-stained gel and western blotting images are merged and shown in the top panel. (D) Alignment of the amino acid sequences of human NPM N-terminal domains. Amino acid sequences of human NPM1, NPM2 and NPM3 were aligned by ClastalW2 software (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and conserved amino acids are highlighted by asterisks at the bottom of sequences. The conserved core domains of NPM proteins are indicated on blue background. The position of acidic ‘A1 tract’ is shown by red line at the top of the sequences.
Mentions: We next examined the oligomer formation ability of human NPM proteins. Bacterially expressed human NPM proteins were purified, and their oligomerization ability was tested using a chemical crosslinking assay (Figure 2A). NPM1 was subjected to SDS–PAGE and bands with 40 kDa and >200 kDa were detected even in the absence of chemical crosslinking (lane 1), indicating that NPM1 formed a stable oligomer. After glutaraldehyde (GA) treatment, ∼200- and 160-kDa protein bands were observed for NPM1 and NPM2, respectively (lanes 2 and 4). In addition, bands >200 kDa were also detected. This result suggested that human NPM1 and NPM2 both formed stable pentamers and that some of the pentamers formed decamers in solution. Conversely, the band corresponding to a pentamer was not detected when NPM3 was subjected to GA treatment (lanes 5 and 6). Instead, a band similar to that of the NPM3 monomer and a minor population of a 55–60 kDa bands were detected. To further confirm the oligomerization status of NPM proteins, the proteins were analyzed by BN–PAGE (Figure 2B). BN–PAGE was originally developed for the separation of mitochondrial membrane proteins and has been used to determine the native mass and oligomeric status of proteins (28). By comparison with the molecular standards separated on the same gel (Figure 2B, right panel), the native masses of NPM1, NPM2 and NPM3 were estimated to be 358, 231 and 42 kDa, respectively. In parallel, gel filtration analysis revealed that His–NPM1 and His–NPM2 were fractionated at the positions corresponding to approximately 370 and 270 kDa, respectively (Figure 6D). In addition, we examined the mass of NPM3 by gel filtration, but it distributed broadly and we could not estimate native mass (data not shown). From these results and previous structural analyses (9–11,13), we concluded that both human NPM1 (34.7 kDa including the His-tag) and NPM2 (26.3 kDa including the His-tag) formed stable pentamers in solution and that two pentamers are assembled into decamers. On the other hand, NPM3 (21.5 kDa including the His-tag) is suggested to from dimers in solution. Consistent with this data, preliminary results from electrospray ionization mass spectrometry analyses suggested that NPM3 formed a dimer at low salt concentrations (data not shown). NPM1 and NPM2 treated with GA and separated by SDS–PAGE were mainly detected at around pentamer positions, while these proteins were suggested to form decamers by BN–PAGE and gel filtration assays. This could be due to the assumption that the pentamer–pentamer interface is not efficiently crosslinked by GA, and two pentamers are dissociated under SDS–PAGE conditions.Figure 2.

Bottom Line: Furthermore, the oligomer formation with NPM1 elicited NPM3 nucleosome assembly and sperm chromatin decondensation activity.NPM3 also suppressed the RNA-binding activity of NPM1, which enhanced the nucleoplasm-nucleolus shuttling of NPM1 in somatic cell nuclei.Our results proposed a novel mechanism whereby three NPM proteins cooperatively regulate chromatin disassembly and assembly in the early embryo and in somatic cells.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Medicine and Graduate School of Comprehensive Human Sciences, Initiative for Promotion of Young Scientists' Independent Research, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8577, Japan. mokuwaki@md.tsukuba.ac.jp

ABSTRACT
Sperm chromatin remodeling after oocyte entry is the essential step that initiates embryogenesis. This reaction involves the removal of sperm-specific basic proteins and chromatin assembly with histones. In mammals, three nucleoplasmin/nucleophosmin (NPM) family proteins-NPM1, NPM2 and NPM3-expressed in oocytes are presumed to cooperatively regulate sperm chromatin remodeling. We characterized the sperm chromatin decondensation and nucleosome assembly activities of three human NPM proteins. NPM1 and NPM2 mediated nucleosome assembly independently of other NPM proteins, whereas the function of NPM3 was largely dependent on formation of a complex with NPM1. Maximal sperm chromatin remodeling activity of NPM2 required the inhibition of its non-specific nucleic acid-binding activity by phosphorylation. Furthermore, the oligomer formation with NPM1 elicited NPM3 nucleosome assembly and sperm chromatin decondensation activity. NPM3 also suppressed the RNA-binding activity of NPM1, which enhanced the nucleoplasm-nucleolus shuttling of NPM1 in somatic cell nuclei. Our results proposed a novel mechanism whereby three NPM proteins cooperatively regulate chromatin disassembly and assembly in the early embryo and in somatic cells.

Show MeSH