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Nucleus
2011 Jan 01;26:533-9. doi: 10.4161/nucl.2.6.17799.
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Epigenetic stability of repressed states involving the histone variant macroH2A revealed by nuclear transfer to Xenopus oocytes.
Pasque V
,
Halley-Stott RP
,
Gillich A
,
Garrett N
,
Gurdon JB
.
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How various epigenetic mechanisms restrict chromatin plasticity to determine the stability of repressed genes is poorly understood. Nuclear transfer to Xenopus oocytes induces the transcriptional reactivation of previously silenced genes. Recent work suggests that it can be used to analyze the epigenetic stability of repressed states. The notion that the epigenetic state of genes is an important determinant of the efficiency of nuclear reprogramming is supported by the differential reprogramming of given genes from different starting epigenetic configurations. After nuclear transfer, transcription from the inactive X chromosome of post-implantation-derived epiblast stem cells is reactivated. However, the same chromosome is resistant to reactivation when embryonic fibroblasts are used. Here, we discuss different kinds of evidence that link the histone variant macroH2A to the increased stability of repressed states. We focus on developmentally regulated X chromosome inactivation and repression of autosomal pluripotency genes, where macroH2A may help maintain the long-term stability of the differentiated state of somatic cells.
Figure 1. Nuclear transfer to Xenopus oocyte reveals the stability of repressed states. The nuclei of differentiated cells can be transplanted into the germinal vesicle (GV) of first meiotic prophase oocytes. This induces reactivation of previously silenced genes. A repressed transgene on the inactive X (Xi-GFP) is reactivated when EpiSC are used as donors. Reactivation of Xist-induced repression is also seen when ES cells differentiated for 4 d with retinoic acid (ESRA) are used as donors, correlating with the absence of macroH2A on the Xi. However, when TS or MEF nuclei are used as donors, the Xi-GFP fails to reactivate, correlating with the presence of the histone variant macroH2A. Xist RNA is lost from the Xi following nuclear transfer.
Figure 2. Elongating RNA Pol II exclusion from the Xi is maintained in transplanted MEF nuclei, but not in transplanted EpiSC nuclei. The elongating form of RNA Pol II (H5 antibody) remains excluded from the Xi in transplanted female MEF nuclei (arrows), but not in transplanted EpiSC nuclei. Xenopus oocyte GVs containing transplanted nuclei were fixed 48 h post transplantation and immunostained as described.3 The antibodies used were a rabbit IgG anti-H3K27me3 (1/200, Upstate 07–449) and a mouse IgM anti-Serine2 phosphorylated RNA Pol II (1/200, H5 Covance MMS-129R). The secondary antibodies used were: Alexa 488 goat anti-mouse IgG (1/200, Invitrogen), Alexa 647 goat anti-mouse IgM (1/200, Invitrogen). Confocal sections were projected and merged using ImageJ. Number of transplanted nuclei showing RNA Pol II exclusion: MEF 81.15% (n = 16), EpiSC 0% (n = 15). Scale bars = 5 μm.
Figure 3. macroH2A-GFP reveals a major reorganization of chromatin following transplantation of nuclei into Xenopus oocytes. The nuclei of C2C12 cells expressing macroH2A-GFP were transplanted into Xenopus oocyte GVs and imaged immediately (0 h) or 3 d after nuclear transfer (72 h).
Figure 4. Developmental dynamics of X chromosome inactivation and reactivation during female mouse embryogenesis. One active X chromosome (green) is contributed by both egg and sperm to give zygotes in which the two X chromosomes are active (green). Due to maternal imprints, the paternal X chromosome is always inactivated during early mouse development (E2.0, orange). The inactivation of the paternal X is then stably maintained in the extra-embryonic lineage (red), and this is correlated with the incorporation of macroH2A on the inactive X chromosome. In the inner cell mass of the E3.5 blastocyst, induction of pluripotency is linked to the reactivation of the X chromosome (green). Hence, female ES cells, derived from the ICM at E3.5, possess two active X chromosomes. Random X chromosome inactivation is initiated during differentiation of the epiblast from ICM cells (orange). A subset of the epiblast cells is induced to form primordial germ cells, in which X chromosome reactivation is induced (green). Stable maintenance of Xi in the embryo and in the adult is also associated with the incorporation of macroH2A (red). Green: active or reactivation of the X chromosome. Orange: X chromosome inactivation. Red: maintenance of X chromosome inactivation. Adapted with permission, from reference 5 (2011) Macmillan Publishers' Ltd.
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