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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.

Show MeSH
Oligomer formation between NPM1 and NPM3. (A) Crosslinking experiments. Recombinant His–NPM1 was mixed with increasing amounts of His–NPM3 (1:0 for lanes 1 and 2, 1:0.2 for lanes 3 and 4, 1:1 for lanes 5 and 6, and 1:3 for lanes 7 and 8), denatured in the buffer containing guanidine hydrochloride, and renatured by extensive dialysis. The mixtures were subjected to chemical crosslinking experiment with 0.05% GA. The mixtures treated without (lanes 1, 3, 5 and 7) or with (lanes 2, 4, 6 and 8) GA were separated on 7.5% and 12.5% SDS–PAGE (top and bottom panels, respectively) and visualized with silver staining (left panel) or western blotting with an anti-NPM3 antibody (right panel). Positions of molecular weight markers are indicated at the left, and those of free NPM1 and NPM3 are indicated at the right side of the panel. (B) BN–PAGE analysis of the NPM1–NPM3 complex. NPM1, NPM3 and NPM1–NPM3 complexes as in A were separated on 4–16% BN–PAGE and visualized with silver staining. Lane M is molecular weight markers. Masses of bands shown at the right side of the panel were estimated as in Figure 2B. (C) NPM1/His–NPM3 preparation. His-tagged NPM3 and NPM1 were coexpressed in E. coli and purified as described in ‘Materials and Methods’ section. His–NPM1 and NPM1–His–NPM3 complex were separated on 12.5% SDS–PAGE and visualized with CBB staining. Positions of His–NPM1, non-tagged NPM1 and His–NPM3 are indicated at the right side of the panel. (D) Gel filtration analysis of NPM proteins. His–NPM1 (5 µg), NPM1/His–NPM3 (5 µg NPM1) and His–NPM2 (5 µg) in 20 µl were loaded on Superose 6 PC 3.2/30 column and fractionated. Fractions 12–24 were analyzed by SDS–PAGE and western blotting. Molecular masses were estimated from the elution profile of marker proteins as shown at the top of the panel.
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gks162-F6: Oligomer formation between NPM1 and NPM3. (A) Crosslinking experiments. Recombinant His–NPM1 was mixed with increasing amounts of His–NPM3 (1:0 for lanes 1 and 2, 1:0.2 for lanes 3 and 4, 1:1 for lanes 5 and 6, and 1:3 for lanes 7 and 8), denatured in the buffer containing guanidine hydrochloride, and renatured by extensive dialysis. The mixtures were subjected to chemical crosslinking experiment with 0.05% GA. The mixtures treated without (lanes 1, 3, 5 and 7) or with (lanes 2, 4, 6 and 8) GA were separated on 7.5% and 12.5% SDS–PAGE (top and bottom panels, respectively) and visualized with silver staining (left panel) or western blotting with an anti-NPM3 antibody (right panel). Positions of molecular weight markers are indicated at the left, and those of free NPM1 and NPM3 are indicated at the right side of the panel. (B) BN–PAGE analysis of the NPM1–NPM3 complex. NPM1, NPM3 and NPM1–NPM3 complexes as in A were separated on 4–16% BN–PAGE and visualized with silver staining. Lane M is molecular weight markers. Masses of bands shown at the right side of the panel were estimated as in Figure 2B. (C) NPM1/His–NPM3 preparation. His-tagged NPM3 and NPM1 were coexpressed in E. coli and purified as described in ‘Materials and Methods’ section. His–NPM1 and NPM1–His–NPM3 complex were separated on 12.5% SDS–PAGE and visualized with CBB staining. Positions of His–NPM1, non-tagged NPM1 and His–NPM3 are indicated at the right side of the panel. (D) Gel filtration analysis of NPM proteins. His–NPM1 (5 µg), NPM1/His–NPM3 (5 µg NPM1) and His–NPM2 (5 µg) in 20 µl were loaded on Superose 6 PC 3.2/30 column and fractionated. Fractions 12–24 were analyzed by SDS–PAGE and western blotting. Molecular masses were estimated from the elution profile of marker proteins as shown at the top of the panel.

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 between NPM1 and NPM3. (A) Crosslinking experiments. Recombinant His–NPM1 was mixed with increasing amounts of His–NPM3 (1:0 for lanes 1 and 2, 1:0.2 for lanes 3 and 4, 1:1 for lanes 5 and 6, and 1:3 for lanes 7 and 8), denatured in the buffer containing guanidine hydrochloride, and renatured by extensive dialysis. The mixtures were subjected to chemical crosslinking experiment with 0.05% GA. The mixtures treated without (lanes 1, 3, 5 and 7) or with (lanes 2, 4, 6 and 8) GA were separated on 7.5% and 12.5% SDS–PAGE (top and bottom panels, respectively) and visualized with silver staining (left panel) or western blotting with an anti-NPM3 antibody (right panel). Positions of molecular weight markers are indicated at the left, and those of free NPM1 and NPM3 are indicated at the right side of the panel. (B) BN–PAGE analysis of the NPM1–NPM3 complex. NPM1, NPM3 and NPM1–NPM3 complexes as in A were separated on 4–16% BN–PAGE and visualized with silver staining. Lane M is molecular weight markers. Masses of bands shown at the right side of the panel were estimated as in Figure 2B. (C) NPM1/His–NPM3 preparation. His-tagged NPM3 and NPM1 were coexpressed in E. coli and purified as described in ‘Materials and Methods’ section. His–NPM1 and NPM1–His–NPM3 complex were separated on 12.5% SDS–PAGE and visualized with CBB staining. Positions of His–NPM1, non-tagged NPM1 and His–NPM3 are indicated at the right side of the panel. (D) Gel filtration analysis of NPM proteins. His–NPM1 (5 µg), NPM1/His–NPM3 (5 µg NPM1) and His–NPM2 (5 µg) in 20 µl were loaded on Superose 6 PC 3.2/30 column and fractionated. Fractions 12–24 were analyzed by SDS–PAGE and western blotting. Molecular masses were estimated from the elution profile of marker proteins as shown at the top of the panel.
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Related In: Results  -  Collection

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Show All Figures
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gks162-F6: Oligomer formation between NPM1 and NPM3. (A) Crosslinking experiments. Recombinant His–NPM1 was mixed with increasing amounts of His–NPM3 (1:0 for lanes 1 and 2, 1:0.2 for lanes 3 and 4, 1:1 for lanes 5 and 6, and 1:3 for lanes 7 and 8), denatured in the buffer containing guanidine hydrochloride, and renatured by extensive dialysis. The mixtures were subjected to chemical crosslinking experiment with 0.05% GA. The mixtures treated without (lanes 1, 3, 5 and 7) or with (lanes 2, 4, 6 and 8) GA were separated on 7.5% and 12.5% SDS–PAGE (top and bottom panels, respectively) and visualized with silver staining (left panel) or western blotting with an anti-NPM3 antibody (right panel). Positions of molecular weight markers are indicated at the left, and those of free NPM1 and NPM3 are indicated at the right side of the panel. (B) BN–PAGE analysis of the NPM1–NPM3 complex. NPM1, NPM3 and NPM1–NPM3 complexes as in A were separated on 4–16% BN–PAGE and visualized with silver staining. Lane M is molecular weight markers. Masses of bands shown at the right side of the panel were estimated as in Figure 2B. (C) NPM1/His–NPM3 preparation. His-tagged NPM3 and NPM1 were coexpressed in E. coli and purified as described in ‘Materials and Methods’ section. His–NPM1 and NPM1–His–NPM3 complex were separated on 12.5% SDS–PAGE and visualized with CBB staining. Positions of His–NPM1, non-tagged NPM1 and His–NPM3 are indicated at the right side of the panel. (D) Gel filtration analysis of NPM proteins. His–NPM1 (5 µg), NPM1/His–NPM3 (5 µg NPM1) and His–NPM2 (5 µg) in 20 µl were loaded on Superose 6 PC 3.2/30 column and fractionated. Fractions 12–24 were analyzed by SDS–PAGE and western blotting. Molecular masses were estimated from the elution profile of marker proteins as shown at the top of the panel.
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