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. 2011 May 1;25(9):946-58.
doi: 10.1101/gad.615211.

Nuclear actin polymerization is required for transcriptional reprogramming of Oct4 by oocytes

Kei Miyamoto  1 , Vincent Pasque, Jerome Jullien, John B Gurdon
Affiliations

Nuclear actin polymerization is required for transcriptional reprogramming of Oct4 by oocytes

Kei Miyamoto et al. Genes Dev. .

Abstract

Amphibian oocytes can rapidly and efficiently reprogram the transcription of transplanted somatic nuclei. To explore the factors and mechanisms involved, we focused on nuclear actin, an especially abundant component of the oocyte's nucleus (the germinal vesicle). The existence and significance of nuclear actin has long been debated. Here, we found that nuclear actin polymerization plays an essential part in the transcriptional reactivation of the pluripotency gene Oct4 (also known as Pou5f1). We also found that an actin signaling protein, Toca-1, enhances Oct4 reactivation by regulating nuclear actin polymerization. Toca-1 overexpression has an effect on the chromatin state of transplanted nuclei, including the enhanced binding of nuclear actin to gene regulatory regions. This is the first report showing that naturally stored actin in an oocyte nucleus helps transcriptional reprogramming in a polymerization-dependent manner.

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Figures

Figure 1.
Figure 1.
Visualization of nuclear F-actin. (A) Schematic diagram of the experimental strategy to visualize nuclear F-actin formed in transplanted nuclei. Injected nuclei cannot be observed without dissecting GVs. Dissected GVs can be maintained alive in mineral oil and are transparent enough to observe their interior structure by confocal microscopy. (B) Nuclear F-actin in a Xenopus oocyte nucleus, visualized with GFP-UtrCH probes. A meshwork structure of nuclear F-actin is naturally formed in nucleoplasm. Bar, 5 μm. (C) Nuclear F-actin is observed in injected nuclei. F-actin was labeled with GFP-UtrCH (green) and chromatin of injected nuclei was visualized with Cherry-histone H2B (red). Actin bundles in a transplanted nucleus are marked with white arrows. The image was taken 24 h after NT. Bar, 10 μm.
Figure 2.
Figure 2.
Nuclear actin polymerization is necessary for transcriptional reprogramming of Oct4. (A) Anti-βactin antibody injection (clone AC15) into GVs inhibits the transcriptional reactivation of Oct4 in NT oocytes. IgG was injected as a control. Relative fold increases of gene transcripts to control NT were measured by qPCR. Transcription was analyzed 24 h and 48 h after NT. Data represent mean ± SEM; (**) P = 0.0029; F and T-test against control, antibody injection: n = 8 at 48 h and n = 5 at 24 h; control injection: n = 4 at 48 h and n = 2 at 24 h. (B) Actin mutant expression influences transcriptional reactivation of Oct4 in NT oocytes. Several human actin mutants (G13R, R62D, G15S, and S14C) and human wild-type actin (wt) were expressed in oocytes, and these oocytes were used for NT. Transcript level is relative to control wild type. Data represent mean ± SEM; (*) P = 0.025; F and T-test against wild type, n = 6. (C) The effect of actin-depolymerizing reagent on Oct4 reactivation in NT oocytes. CB was added to culture medium at a concentration of 5 μg/mL during 48 h of culture. Data represent mean ± SEM; CB+: n = 10 at 48 h and n = 4 at 24 h; control: n = 11 at 48 h and n = 4 at 24 h. (D) Oct4 reactivation from transplanted nuclei is inhibited by depolymerizing nuclear actin with CB in oil GVs. Nuclei suspended in CB-containing solution (gray bar, CB+) or DMSO-containing solution (black bar, CB−) were injected into oil GVs, and transcriptional activation was examined by qRT–PCR analysis. Transcript level is relative to control CB−. Data represent mean ± SEM; n = 6. (E) Oct4 reactivation from transplanted nuclei is inhibited by depolymerizing nuclear actin with LA in oil GVs. Nuclei suspended in LA-containing solution (gray bar, LA+) or DMSO-containing solution (black bar, LA−) were injected into oil GVs. Data represent mean ± SEM; (**) P = 0.0016; F and T-test against LA−; n = 3.
Figure 3.
Figure 3.
An actin signaling protein, Toca-1, enhances Oct4 reactivation. (A) Schematic diagram of the experimental strategy to assess the effect of actin nucleators and actin signaling proteins on transcriptional reprogramming from transplanted C2C12 nuclei. (B) Four factors tagged with HA are expressed as proteins in both the GV and cytoplasm. These include Xenopus tropicalis JMY (xtJMY), X. tropicalis TOCA-1 (xtTOCA-1), mouse N-WASP (mN-WASP), and X. laevis RAC1 (xlRAC1). The proteins were detected by Western blot analysis using anti-HA antibody 24 h after mRNA injection. Actin was used as a loading control. (C) The effect of overexpression of the actin polymerization regulator on transcriptional reprogramming. Relative fold increases of gene transcripts to control water-injected samples are shown, measured by qPCR. Data represent mean ± SEM; (*) P < 0.05; F and T-test against the control water injection; n = 4. (D) A mutant form of Toca-1, Toca-1(W518K), does not enhance Oct4 reactivation, while wild-type Toca-1 enhances it. Toca-1(W518K) was similarly overexpressed in oocytes by mRNA injection. Relative fold increases of gene transcripts to control water-injected samples are shown, measured by qPCR. Data represent mean ± SEM; (*) P = 0.018; F and T-test against the control; Toca1: n = 3; others: n = 4). (E) Reactivation of Oct4, Gdf3, and Eif4e1b genes was enhanced by the overexpression of TOCA-1. Gdf3 is a pluripotency gene and Eif4e1b is an oocyte-specific gene. Data represent mean ± SEM; (**) P < 0.02; (*) P < 0.05; F and T-test against the control; n = 5–6).
Figure 4.
Figure 4.
Toca-1 exists in a transplanted nucleus and enhances nuclear actin polymerization. (A) TOCA1-CHERRY is present in injected nuclei. Toca1-Cherry and GFP-UtrCH mRNA were injected before NT. The injected nuclei were observed 48 h after NT by confocal microscopy using the oil GV method. A nucleus in which TOCA1-CHERRY is present shows nuclear F-actin formation (white arrow). In contrast, nuclear F-actin is not detected in a nucleus without TOCA1-CHERRY (blue arrow). Bar, 20 μm. (B) TOCA1 overexpression increases nuclear F-actin formation. Different amounts of TOCA1 were expressed in oocytes by mRNA injection with different concentrations. These injected oocytes were used for NT. The oocytes were subjected to immunofluorescence analysis and phalloidin staining 48 h after NT. Images were obtained from randomly selected areas. Nuclear F-actin amounts were quantified using the ImageJ software (Materials and Methods). Statistical differences were measured by F and T-tests. Data represent mean ± SEM; Toca-1 1.5 ng: n = 45; Toca-1 4.6 ng and 13.8 ng: n = 55. (C) Actin fractionation experiment indicates that TOCA-1 overexpression increases the F-actin population in GVs. TOCA-1 was expressed in oocytes by mRNA injection. GVs were collected from these injected oocytes or noninjected oocytes, and total actin was fractionated to monomeric and polymeric actin (G- and F-actin, respectively). The amounts of G- and F-actin were measured by Western blot analysis using anti-βactin antibody. CB was added to culture medium at a 1 μg/mL concentration to check that enhanced actin polymerization was diminished with this concentration. F-actin proportion in total actin was compared among three samples, and fold differences relative to the control noninjected oocytes (Toca− and CB−) are shown. Data represent mean ± SEM; n = 2. (D) Actin fractionation experiment indicates that overexpression of a TOCA-1 mutant, W518K, does not increase the F-actin population in GVs. F-actin ratio in total actin is relative to control water-injected oocytes. Data represent mean ± SEM; n = 2.
Figure 5.
Figure 5.
Nuclear actin polymerization is important for both ongoing transcription and transcriptional reprogramming of Oct4. (A) Anti-βactin antibody injection into GVs inhibits Oct4 transcription from transplanted ES nuclei. IgG was injected as a control. Relative fold increases of gene transcripts to control NT were measured by qPCR. Transcription was analyzed 48 h after NT. Data represent mean ± SEM; (**) P = 0.0076; F and T-test against the control; n = 5. (B) TOCA-1 overexpression does not enhance Oct4 transcription from transplanted ES nuclei, while it enhances Oct4 transcription from C2C12 nuclei. Transcript level is relative to control water-injected samples. Data represent mean ± SEM; ES nuclei: n = 4; C2C12 nuclei: n = 3.
Figure 6.
Figure 6.
Association of nuclear actin with chromatin proteins and chromatin of transplanted nuclei. (A) Coimmunoprecipitation analysis in GV lysates to examine binding of nuclear actin to Pol II and the BAF complex. GV proteins were immunoprecipitated by IgG and antibodies against BAF57, BRG1, BAF53A, and Pol II, and were analyzed by Western blots by probing for β-actin and actin. Five percent input was loaded. (B) TOCA-1 overexpression enhances nuclear β-actin binding to the Oct4 gene. ChIP analysis using anti-βactin antibody was performed with TOCA-1-overexpressed NT oocytes (Toca1+) and normal NT oocytes (Toca1−) 48 h after NT using RA-ES cells. The Oct4 gene regulatory region was expressed as distal enhancer (DE), proximal enhancer (PE), and proximal promoter (PP). As a control, ChIP analysis using nonspecific IgG was carried out with normal NT oocytes. The relative abundance of precipitated DNA against total input DNA was measured by qPCR. Data represent mean ± SEM; n = 5.
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