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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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Association between human NPM proteins. (A) Localization of EGFP-tagged NPM proteins. EF, EF–NPM1, EF–NPM2 and EF–NPM3 were transiently expressed in HeLa cells and the localization of EGFP proteins was examined by fluorescent microscopy. Bottom panels are phase contrast images. Bar at the bottom indicates 10 µm. (B) Localization of endogenous NPM1 and NPM3 in HeLa cells. HeLa cells were subjected to immunofluorescence analysis using anti-NPM1 and anti-NPM3 antibodies. DNA was stained with TO-PRO-3. Images were observed under confocal microscopy. Bar at the bottom indicates 10 µm. (C) Glycerol density gradient of HeLa cell nuclear extracts. Nuclear extracts from HeLa cells were fractionated by 15–35% glycerol density gradient. Fractions collected from the top were analyzed by western blotting with anti-nucleolin (NCL), anti-B23 and anti-NPM3 antibodies. (D) Immunoprecipitation with anti-NPM proteins. HeLa cell extracts were subjected to immunoprecipitation with control Ig (lanes 6 and 9), anti-NPM1 (lanes 7 and 10), and anti-NPM3 (lanes 8 and 11) antibodies. Bound proteins were eluted with SDS-sample buffer (30 µl) and 3 (lanes 6–8) or 9 (lanes 9–11) µl of samples were separated on SDS–PAGE and analyzed by western blotting with anti-NPM1 and anti-NPM3 antibodies (top and bottom panels, respectively). As standard, recombinant His-NPM1 (50, 25, 12 ng for lanes 1–3) and His–NPM3 (5, 2.5, 1.2 ng for lanes 1–3), and increasing amounts of input extracts (lanes 4–5) were also loaded. (E) Immunoprecipitation of transiently expressed NPM proteins. EF, EF–NPM1, -NPM2 and -NPM3 were transiently expressed in HEK293T cells and cell extracts were prepared. Immunoprecipitation with anti-Flag antibody beads was performed. Input (lanes 1–4) and immunoprecipitated (lanes 5–8) proteins were analyzed by western blotting with anti-NPM1, -NPM3 and -Flag antibodies. Positions of NPM proteins are indicated at the right side of the panel. Blank arrow head shows the protein likely to be corresponded to NPM3 degradation product. (F) Expression level of NPM proteins in mouse MII oocytes. MII oocytes (30 and 60 cells for lanes 7 and 8, respectively) and cumulus cells (3 × 103 and 10 × 103 cells for lanes 9 and 10) were prepared from mice and analyzed by western blotting with anti-NPM1, -NPM3 and -NPM2 antibodies. Recombinant human NPM1 and mouse NPM3 were loaded on the same gel as standards (lanes 1–6). Positions of NPM proteins are indicated by arrow heads.
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gks162-F1: Association between human NPM proteins. (A) Localization of EGFP-tagged NPM proteins. EF, EF–NPM1, EF–NPM2 and EF–NPM3 were transiently expressed in HeLa cells and the localization of EGFP proteins was examined by fluorescent microscopy. Bottom panels are phase contrast images. Bar at the bottom indicates 10 µm. (B) Localization of endogenous NPM1 and NPM3 in HeLa cells. HeLa cells were subjected to immunofluorescence analysis using anti-NPM1 and anti-NPM3 antibodies. DNA was stained with TO-PRO-3. Images were observed under confocal microscopy. Bar at the bottom indicates 10 µm. (C) Glycerol density gradient of HeLa cell nuclear extracts. Nuclear extracts from HeLa cells were fractionated by 15–35% glycerol density gradient. Fractions collected from the top were analyzed by western blotting with anti-nucleolin (NCL), anti-B23 and anti-NPM3 antibodies. (D) Immunoprecipitation with anti-NPM proteins. HeLa cell extracts were subjected to immunoprecipitation with control Ig (lanes 6 and 9), anti-NPM1 (lanes 7 and 10), and anti-NPM3 (lanes 8 and 11) antibodies. Bound proteins were eluted with SDS-sample buffer (30 µl) and 3 (lanes 6–8) or 9 (lanes 9–11) µl of samples were separated on SDS–PAGE and analyzed by western blotting with anti-NPM1 and anti-NPM3 antibodies (top and bottom panels, respectively). As standard, recombinant His-NPM1 (50, 25, 12 ng for lanes 1–3) and His–NPM3 (5, 2.5, 1.2 ng for lanes 1–3), and increasing amounts of input extracts (lanes 4–5) were also loaded. (E) Immunoprecipitation of transiently expressed NPM proteins. EF, EF–NPM1, -NPM2 and -NPM3 were transiently expressed in HEK293T cells and cell extracts were prepared. Immunoprecipitation with anti-Flag antibody beads was performed. Input (lanes 1–4) and immunoprecipitated (lanes 5–8) proteins were analyzed by western blotting with anti-NPM1, -NPM3 and -Flag antibodies. Positions of NPM proteins are indicated at the right side of the panel. Blank arrow head shows the protein likely to be corresponded to NPM3 degradation product. (F) Expression level of NPM proteins in mouse MII oocytes. MII oocytes (30 and 60 cells for lanes 7 and 8, respectively) and cumulus cells (3 × 103 and 10 × 103 cells for lanes 9 and 10) were prepared from mice and analyzed by western blotting with anti-NPM1, -NPM3 and -NPM2 antibodies. Recombinant human NPM1 and mouse NPM3 were loaded on the same gel as standards (lanes 1–6). Positions of NPM proteins are indicated by arrow heads.

Mentions: We first examined the localization of human NPM proteins in HeLa cells. Enhanced green fluorescent protein (EGFP)-tagged NPM proteins were expressed in HeLa cells, and their localization was examined using fluorescent microscopy (Figure 1A). As previously reported (23,25,26), EF-tagged NPM1 and NPM3 preferentially localized in the nucleoli, whereas EF–NPM2 was distributed throughout the nucleus. The level of the EF–NPM3 signal was higher than that of NPM1 in both the nucleoplasm and cytoplasm. Furthermore, the expression pattern of endogenous NPM1 and NPM3 in HeLa cells was confirmed by immunofluorescence using specific antibodies (Figure 1B). Endogenous NPM1 and NPM3 were localized in the nucleolus: the staining patterns of NPM1 and NPM3 suggested that both are mainly found in the granular component of the nucleolus.Figure 1.


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)

Association between human NPM proteins. (A) Localization of EGFP-tagged NPM proteins. EF, EF–NPM1, EF–NPM2 and EF–NPM3 were transiently expressed in HeLa cells and the localization of EGFP proteins was examined by fluorescent microscopy. Bottom panels are phase contrast images. Bar at the bottom indicates 10 µm. (B) Localization of endogenous NPM1 and NPM3 in HeLa cells. HeLa cells were subjected to immunofluorescence analysis using anti-NPM1 and anti-NPM3 antibodies. DNA was stained with TO-PRO-3. Images were observed under confocal microscopy. Bar at the bottom indicates 10 µm. (C) Glycerol density gradient of HeLa cell nuclear extracts. Nuclear extracts from HeLa cells were fractionated by 15–35% glycerol density gradient. Fractions collected from the top were analyzed by western blotting with anti-nucleolin (NCL), anti-B23 and anti-NPM3 antibodies. (D) Immunoprecipitation with anti-NPM proteins. HeLa cell extracts were subjected to immunoprecipitation with control Ig (lanes 6 and 9), anti-NPM1 (lanes 7 and 10), and anti-NPM3 (lanes 8 and 11) antibodies. Bound proteins were eluted with SDS-sample buffer (30 µl) and 3 (lanes 6–8) or 9 (lanes 9–11) µl of samples were separated on SDS–PAGE and analyzed by western blotting with anti-NPM1 and anti-NPM3 antibodies (top and bottom panels, respectively). As standard, recombinant His-NPM1 (50, 25, 12 ng for lanes 1–3) and His–NPM3 (5, 2.5, 1.2 ng for lanes 1–3), and increasing amounts of input extracts (lanes 4–5) were also loaded. (E) Immunoprecipitation of transiently expressed NPM proteins. EF, EF–NPM1, -NPM2 and -NPM3 were transiently expressed in HEK293T cells and cell extracts were prepared. Immunoprecipitation with anti-Flag antibody beads was performed. Input (lanes 1–4) and immunoprecipitated (lanes 5–8) proteins were analyzed by western blotting with anti-NPM1, -NPM3 and -Flag antibodies. Positions of NPM proteins are indicated at the right side of the panel. Blank arrow head shows the protein likely to be corresponded to NPM3 degradation product. (F) Expression level of NPM proteins in mouse MII oocytes. MII oocytes (30 and 60 cells for lanes 7 and 8, respectively) and cumulus cells (3 × 103 and 10 × 103 cells for lanes 9 and 10) were prepared from mice and analyzed by western blotting with anti-NPM1, -NPM3 and -NPM2 antibodies. Recombinant human NPM1 and mouse NPM3 were loaded on the same gel as standards (lanes 1–6). Positions of NPM proteins are indicated by arrow heads.
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Related In: Results  -  Collection

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Show All Figures
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gks162-F1: Association between human NPM proteins. (A) Localization of EGFP-tagged NPM proteins. EF, EF–NPM1, EF–NPM2 and EF–NPM3 were transiently expressed in HeLa cells and the localization of EGFP proteins was examined by fluorescent microscopy. Bottom panels are phase contrast images. Bar at the bottom indicates 10 µm. (B) Localization of endogenous NPM1 and NPM3 in HeLa cells. HeLa cells were subjected to immunofluorescence analysis using anti-NPM1 and anti-NPM3 antibodies. DNA was stained with TO-PRO-3. Images were observed under confocal microscopy. Bar at the bottom indicates 10 µm. (C) Glycerol density gradient of HeLa cell nuclear extracts. Nuclear extracts from HeLa cells were fractionated by 15–35% glycerol density gradient. Fractions collected from the top were analyzed by western blotting with anti-nucleolin (NCL), anti-B23 and anti-NPM3 antibodies. (D) Immunoprecipitation with anti-NPM proteins. HeLa cell extracts were subjected to immunoprecipitation with control Ig (lanes 6 and 9), anti-NPM1 (lanes 7 and 10), and anti-NPM3 (lanes 8 and 11) antibodies. Bound proteins were eluted with SDS-sample buffer (30 µl) and 3 (lanes 6–8) or 9 (lanes 9–11) µl of samples were separated on SDS–PAGE and analyzed by western blotting with anti-NPM1 and anti-NPM3 antibodies (top and bottom panels, respectively). As standard, recombinant His-NPM1 (50, 25, 12 ng for lanes 1–3) and His–NPM3 (5, 2.5, 1.2 ng for lanes 1–3), and increasing amounts of input extracts (lanes 4–5) were also loaded. (E) Immunoprecipitation of transiently expressed NPM proteins. EF, EF–NPM1, -NPM2 and -NPM3 were transiently expressed in HEK293T cells and cell extracts were prepared. Immunoprecipitation with anti-Flag antibody beads was performed. Input (lanes 1–4) and immunoprecipitated (lanes 5–8) proteins were analyzed by western blotting with anti-NPM1, -NPM3 and -Flag antibodies. Positions of NPM proteins are indicated at the right side of the panel. Blank arrow head shows the protein likely to be corresponded to NPM3 degradation product. (F) Expression level of NPM proteins in mouse MII oocytes. MII oocytes (30 and 60 cells for lanes 7 and 8, respectively) and cumulus cells (3 × 103 and 10 × 103 cells for lanes 9 and 10) were prepared from mice and analyzed by western blotting with anti-NPM1, -NPM3 and -NPM2 antibodies. Recombinant human NPM1 and mouse NPM3 were loaded on the same gel as standards (lanes 1–6). Positions of NPM proteins are indicated by arrow heads.
Mentions: We first examined the localization of human NPM proteins in HeLa cells. Enhanced green fluorescent protein (EGFP)-tagged NPM proteins were expressed in HeLa cells, and their localization was examined using fluorescent microscopy (Figure 1A). As previously reported (23,25,26), EF-tagged NPM1 and NPM3 preferentially localized in the nucleoli, whereas EF–NPM2 was distributed throughout the nucleus. The level of the EF–NPM3 signal was higher than that of NPM1 in both the nucleoplasm and cytoplasm. Furthermore, the expression pattern of endogenous NPM1 and NPM3 in HeLa cells was confirmed by immunofluorescence using specific antibodies (Figure 1B). Endogenous NPM1 and NPM3 were localized in the nucleolus: the staining patterns of NPM1 and NPM3 suggested that both are mainly found in the granular component of the nucleolus.Figure 1.

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