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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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Related in: MedlinePlus

Effects of NPM3 incorporation into the NPM1 pentamer on the NPM1 functions in somatic cells. (A) Purified GST–NPM1/His–NPM3 oligomer. GST–NPM1 was first purified and denatured in the absence or presence of three times molar excess of His–NPM3, the mixtures were dialyzed to refold the proteins and the excess His–NPM3 was removed by purification of the proteins with glutathione sepharose. GST, GST–NPM1 and GST–NPM1/His–NPM3 (lanes 1–3, respectively, 200 ng of GST proteins) were separated on 12.5% SDS–PAGE and visualized with CBB staining. (B) RNA-binding activity of the NPM1–NPM3 complex. Increasing amounts of GST–NPM1 and GST–NPM1/His–NPM3 were mixed with 32P-labeled total RNA purified from HeLa cells. The mixture was filtrated through nitrocellulose membrane. The membrane was extensively washed and the radio-active RNA retained on the membrane was detected by BAS2500 image analyzing system (top panel). Experiments were performed with doublet and the average radioactivity of the retained RNA is graphically shown at the bottom. The radio-active RNA incubated in the absence of NPM proteins (lane 1) retained on the membrane was set as 1.0 and relative amounts of retained RNA were measured. Two independent experiments demonstrated similar results. (C) Depletion of NPM1 from the extracts. NPM1 was immune-depleted from the extracts prepared from HeLa cells stably expressing EF–NPM3 using an anti-NPM1 antibody. Increasing amounts (1, 3 and 10 µg of proteins) of mock- and NPM1-depleted extracts (lanes 1–3 and 4–6, respectively) were separated by SDS–PAGE and analyzed by western blotting with anti-Flag, anti-NPM1 and anti-PCNA antibodies. (D) FRAP analysis of NPM1 and NPM3. HeLa cells stably expressing either EF–NPM1 or EF–NPM3 were grown on the glass-base dishes. FRAP analyses were performed as described in ‘Materials and Methods’ section. Small nucleoli as shown by white circle in the left panels were breached by a 488-nm laser and the fluorescent recovery at the bleached nucleoli was measured every 0.5 s. The fluorescence at the bleached area relative to the initial fluorescence (1.0) was calculated and plotted as a function of time. The data are represented as mean values ± SD from 12 and 10 experiments for NPM1 and NPM3, respectively. The t1/2 of fluorescence recovery (7.10 and 4.29 s for NPM1 and NPM3, respectively) was estimated by curve fitting as described in ‘Materials and Methods’ section. Typical FRAP images of EF–NPM1 and EF–NPM3 before bleaching, and 0 and 6.2 s after bleaching are shown at the bottom.
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gks162-F8: Effects of NPM3 incorporation into the NPM1 pentamer on the NPM1 functions in somatic cells. (A) Purified GST–NPM1/His–NPM3 oligomer. GST–NPM1 was first purified and denatured in the absence or presence of three times molar excess of His–NPM3, the mixtures were dialyzed to refold the proteins and the excess His–NPM3 was removed by purification of the proteins with glutathione sepharose. GST, GST–NPM1 and GST–NPM1/His–NPM3 (lanes 1–3, respectively, 200 ng of GST proteins) were separated on 12.5% SDS–PAGE and visualized with CBB staining. (B) RNA-binding activity of the NPM1–NPM3 complex. Increasing amounts of GST–NPM1 and GST–NPM1/His–NPM3 were mixed with 32P-labeled total RNA purified from HeLa cells. The mixture was filtrated through nitrocellulose membrane. The membrane was extensively washed and the radio-active RNA retained on the membrane was detected by BAS2500 image analyzing system (top panel). Experiments were performed with doublet and the average radioactivity of the retained RNA is graphically shown at the bottom. The radio-active RNA incubated in the absence of NPM proteins (lane 1) retained on the membrane was set as 1.0 and relative amounts of retained RNA were measured. Two independent experiments demonstrated similar results. (C) Depletion of NPM1 from the extracts. NPM1 was immune-depleted from the extracts prepared from HeLa cells stably expressing EF–NPM3 using an anti-NPM1 antibody. Increasing amounts (1, 3 and 10 µg of proteins) of mock- and NPM1-depleted extracts (lanes 1–3 and 4–6, respectively) were separated by SDS–PAGE and analyzed by western blotting with anti-Flag, anti-NPM1 and anti-PCNA antibodies. (D) FRAP analysis of NPM1 and NPM3. HeLa cells stably expressing either EF–NPM1 or EF–NPM3 were grown on the glass-base dishes. FRAP analyses were performed as described in ‘Materials and Methods’ section. Small nucleoli as shown by white circle in the left panels were breached by a 488-nm laser and the fluorescent recovery at the bleached nucleoli was measured every 0.5 s. The fluorescence at the bleached area relative to the initial fluorescence (1.0) was calculated and plotted as a function of time. The data are represented as mean values ± SD from 12 and 10 experiments for NPM1 and NPM3, respectively. The t1/2 of fluorescence recovery (7.10 and 4.29 s for NPM1 and NPM3, respectively) was estimated by curve fitting as described in ‘Materials and Methods’ section. Typical FRAP images of EF–NPM1 and EF–NPM3 before bleaching, and 0 and 6.2 s after bleaching are shown at the bottom.

Mentions: NPM1 and NPM3 are ubiquitously expressed, and their expression level is higher in actively growing cells than in quiescent cells. We therefore investigated the biological significance of NPM1 and NPM3 complex formation in growing cells. We previously demonstrated that RNA binding of NPM1 is required for efficient histone chaperone activity in the nucleolus (22). Thus, we tested the RNA-binding activity of the NPM1–NPM3 complex using a filter-binding assay (Figure 8B). The NPM1–NPM3 complex was prepared using GST–NPM1 and His–NPM3 (Figure 8A). GST–NPM1 alone or GST–NPM1 mixed with a 3-fold molar excess of His–NPM3 was denatured, and the proteins were refolded by extensive dialysis. Refolded proteins were further purified with glutathione sepharose to remove free His–NPM3 (lane 3). GST–NPM1 bound to radiolabeled RNA was retained on the membrane in a GST–NPM1 dose-dependent manner (lanes 2–5). The activity of the GST–NPM1/His–NPM3 complex was lower than that of GST–NPM1 alone (lanes 6–9). These results indicated that NPM3 incorporation into the NPM1 pentamer greatly impacted the RNA-binding activity of NPM1, although this incorporation did not completely abolish the RNA-binding activity of NPM1.Figure 8.


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)

Effects of NPM3 incorporation into the NPM1 pentamer on the NPM1 functions in somatic cells. (A) Purified GST–NPM1/His–NPM3 oligomer. GST–NPM1 was first purified and denatured in the absence or presence of three times molar excess of His–NPM3, the mixtures were dialyzed to refold the proteins and the excess His–NPM3 was removed by purification of the proteins with glutathione sepharose. GST, GST–NPM1 and GST–NPM1/His–NPM3 (lanes 1–3, respectively, 200 ng of GST proteins) were separated on 12.5% SDS–PAGE and visualized with CBB staining. (B) RNA-binding activity of the NPM1–NPM3 complex. Increasing amounts of GST–NPM1 and GST–NPM1/His–NPM3 were mixed with 32P-labeled total RNA purified from HeLa cells. The mixture was filtrated through nitrocellulose membrane. The membrane was extensively washed and the radio-active RNA retained on the membrane was detected by BAS2500 image analyzing system (top panel). Experiments were performed with doublet and the average radioactivity of the retained RNA is graphically shown at the bottom. The radio-active RNA incubated in the absence of NPM proteins (lane 1) retained on the membrane was set as 1.0 and relative amounts of retained RNA were measured. Two independent experiments demonstrated similar results. (C) Depletion of NPM1 from the extracts. NPM1 was immune-depleted from the extracts prepared from HeLa cells stably expressing EF–NPM3 using an anti-NPM1 antibody. Increasing amounts (1, 3 and 10 µg of proteins) of mock- and NPM1-depleted extracts (lanes 1–3 and 4–6, respectively) were separated by SDS–PAGE and analyzed by western blotting with anti-Flag, anti-NPM1 and anti-PCNA antibodies. (D) FRAP analysis of NPM1 and NPM3. HeLa cells stably expressing either EF–NPM1 or EF–NPM3 were grown on the glass-base dishes. FRAP analyses were performed as described in ‘Materials and Methods’ section. Small nucleoli as shown by white circle in the left panels were breached by a 488-nm laser and the fluorescent recovery at the bleached nucleoli was measured every 0.5 s. The fluorescence at the bleached area relative to the initial fluorescence (1.0) was calculated and plotted as a function of time. The data are represented as mean values ± SD from 12 and 10 experiments for NPM1 and NPM3, respectively. The t1/2 of fluorescence recovery (7.10 and 4.29 s for NPM1 and NPM3, respectively) was estimated by curve fitting as described in ‘Materials and Methods’ section. Typical FRAP images of EF–NPM1 and EF–NPM3 before bleaching, and 0 and 6.2 s after bleaching are shown at the bottom.
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gks162-F8: Effects of NPM3 incorporation into the NPM1 pentamer on the NPM1 functions in somatic cells. (A) Purified GST–NPM1/His–NPM3 oligomer. GST–NPM1 was first purified and denatured in the absence or presence of three times molar excess of His–NPM3, the mixtures were dialyzed to refold the proteins and the excess His–NPM3 was removed by purification of the proteins with glutathione sepharose. GST, GST–NPM1 and GST–NPM1/His–NPM3 (lanes 1–3, respectively, 200 ng of GST proteins) were separated on 12.5% SDS–PAGE and visualized with CBB staining. (B) RNA-binding activity of the NPM1–NPM3 complex. Increasing amounts of GST–NPM1 and GST–NPM1/His–NPM3 were mixed with 32P-labeled total RNA purified from HeLa cells. The mixture was filtrated through nitrocellulose membrane. The membrane was extensively washed and the radio-active RNA retained on the membrane was detected by BAS2500 image analyzing system (top panel). Experiments were performed with doublet and the average radioactivity of the retained RNA is graphically shown at the bottom. The radio-active RNA incubated in the absence of NPM proteins (lane 1) retained on the membrane was set as 1.0 and relative amounts of retained RNA were measured. Two independent experiments demonstrated similar results. (C) Depletion of NPM1 from the extracts. NPM1 was immune-depleted from the extracts prepared from HeLa cells stably expressing EF–NPM3 using an anti-NPM1 antibody. Increasing amounts (1, 3 and 10 µg of proteins) of mock- and NPM1-depleted extracts (lanes 1–3 and 4–6, respectively) were separated by SDS–PAGE and analyzed by western blotting with anti-Flag, anti-NPM1 and anti-PCNA antibodies. (D) FRAP analysis of NPM1 and NPM3. HeLa cells stably expressing either EF–NPM1 or EF–NPM3 were grown on the glass-base dishes. FRAP analyses were performed as described in ‘Materials and Methods’ section. Small nucleoli as shown by white circle in the left panels were breached by a 488-nm laser and the fluorescent recovery at the bleached nucleoli was measured every 0.5 s. The fluorescence at the bleached area relative to the initial fluorescence (1.0) was calculated and plotted as a function of time. The data are represented as mean values ± SD from 12 and 10 experiments for NPM1 and NPM3, respectively. The t1/2 of fluorescence recovery (7.10 and 4.29 s for NPM1 and NPM3, respectively) was estimated by curve fitting as described in ‘Materials and Methods’ section. Typical FRAP images of EF–NPM1 and EF–NPM3 before bleaching, and 0 and 6.2 s after bleaching are shown at the bottom.
Mentions: NPM1 and NPM3 are ubiquitously expressed, and their expression level is higher in actively growing cells than in quiescent cells. We therefore investigated the biological significance of NPM1 and NPM3 complex formation in growing cells. We previously demonstrated that RNA binding of NPM1 is required for efficient histone chaperone activity in the nucleolus (22). Thus, we tested the RNA-binding activity of the NPM1–NPM3 complex using a filter-binding assay (Figure 8B). The NPM1–NPM3 complex was prepared using GST–NPM1 and His–NPM3 (Figure 8A). GST–NPM1 alone or GST–NPM1 mixed with a 3-fold molar excess of His–NPM3 was denatured, and the proteins were refolded by extensive dialysis. Refolded proteins were further purified with glutathione sepharose to remove free His–NPM3 (lane 3). GST–NPM1 bound to radiolabeled RNA was retained on the membrane in a GST–NPM1 dose-dependent manner (lanes 2–5). The activity of the GST–NPM1/His–NPM3 complex was lower than that of GST–NPM1 alone (lanes 6–9). These results indicated that NPM3 incorporation into the NPM1 pentamer greatly impacted the RNA-binding activity of NPM1, although this incorporation did not completely abolish the RNA-binding activity of NPM1.Figure 8.

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
Related in: MedlinePlus