Initiation of epigenetic reprogramming of the X chromosome in somatic nuclei transplanted to a mouse oocyte

Abstract

The active and inactive X chromosomes have distinct epigenetic marks in somatic nuclei, which undergo reprogramming after transplantation into oocytes. We show that, despite the disappearance of Xist RNA coating in 30 min, the epigenetic memory of the inactive X persists with the precocious appearance of histone H3 trimethylation of lysine 27 (H3-3meK27), without the expected colocalization with Eed/Ezh2. Subsequently, Xist re-appears on the original inactive X, and the silent Xist on the active X undergoes re-activation, resulting in unusual biallelic Xist RNA domains. Despite this abnormal Xist expression pattern, colocalization of H3-3meK27 and Eed is thereafter confined to a single Xist domain, which is presumably on the original inactive X. These epigenetic events differ markedly from the kinetics of preferential paternal X inactivation in normal embryos. All the epigenetic marks on the X are apparently erased in the epiblast, suggesting that the oocyte and epiblast may have distinct properties for stepwise programming of the genome.

Figure 1

Progressive loss of Xist RNA coating from somatic nuclei in SCNT embryos. Shown are RNA fluorescence in situ hybridization for Xist RNA coating (green) and DNA staining with TOTO3 (blue). (A) An Xist RNA domain was seen in somatic donor nucleus before fusion with enucleated oocyte (Xist RNA domain/total, 29/29). (B) At 30 min after SCNT, the Xist domain disappeared from a significant number of cells (Xist RNA domain/total, 4/14). (C) After 2 h, there was a further reduction in the number of cells with Xist RNA domains in SCNT embryos (Xist RNA domain/total, 1/18). (D) The coating of Xist RNA was completely lost at the two-cell stage (Xist RNA domain/total, 0/24).

Figure 2

Detection of H3-3meK27 and Eed in one- to two-cell SCNT embryos. Embryos were immunostained at 0, 2, 8 and 24 h after SCNT with H3-3meK27 (green) and Eed (red) antibodies. The white arrowheads indicate a domain of H3-3meK27. (A–C) A weak signal for H3-3meK27 was detected without Eed in somatic nucleus before transfer to oocytes, and also at 8 h after nuclear transfer. (D) At 24 h after SCNT (two-cell stage), both H3-3meK27 and Eed accumulated to a single domain in each blastomere. (E) There was no similar detectable signal or colocalization of H3-3meK27 and Eed in the two-cell stage control embryo.

Figure 3

Xist RNA coating in relation to H3-3meK27 from four-cell stages to morula stages. (A–C) SCNT embryos and (D–F) control embryos were immunostained with H3-3meK27 (red) combined with Xist RNA fluorescence in situ hybridization (green). DNA was stained with TOTO3 (shown in blue). White arrowheads indicate the blastomeres that showed two domains of Xist RNA coating. The magnified field is indicated in the figure with asterisks and white arrows indicate colocalization of Xist RNA and H3-3meK27. (A) Xist RNA was not detectable in most of the SCNT embryos at the four-cell stage. The light-blue arrowhead indicates a domain of H3-3meK27 on the X chromosome. (B) Two domains of Xist RNA coating were observed from six- to eight-cell embryos, but only one of the two domains colocalized with H3-3meK27 in more than half the embryos examined. (C) The two domains of Xist RNA coating continued in morula, and in early blastocyst (not shown). (D) Control in vitro-fertilized embryos show a single domain of Xist RNA coating at the four-cell stages, which continued to (E) eight-cell stages and (F) morula stages, which do not colocalize with H3-3meK27 except in some blastomeres of morula (white arrow).

Figure 4

Verification of allelic expression of Xist transcript in SCNT embryos. (A) Complementary DNA from individual SCNT embryo was PCR amplified using Xist transcriptspecific primer sets, and the PCR products were digested with HindIII. Tail fibroblast cDNAs from C57BL/6 (B6), Mus spretus (Spr) and (B6 × Spr) F1 and cDNA from (B6 × Spr) F1 in vitro-fertilized (IVF) embryos were used as positive controls. (B) Summary of allelic expression of Xist transcript. In the control IVF embryos ((B6 × Spr) F1), monoallelic Xist expression (blue bar) was detected from the four-cell stage onwards. In contrast, Xist expression in the SCNT embryo was delayed and mainly detected from the eight-cell stage onwards (68% of total analysed embryos), and the proportion of cells with Xist increased progressively at the morula (71%) and the blastocyst stage (100%). Among the cells showing Xist RNA domains, 91% at the eight-cell stage, 60% at the morula stage and 40% at the blastocyst stage showed biallelic Xist RNA domains in SCNT embryos (denoted by red bars).

Figure 5

Comparative kinetics of epigenetic modifications of X chromosome in SCNT (top) and control embryos (bottom). There is rapid disappearance of Xist RNA coating from Xi in SCNT embryos commencing within 30 min after activation of the reconstructed embryo. Xist RNA re-appeared in these embryos from the four- to eight-cell stages, and colocalized with the H3-3meK27 and Eed/Ezh2 protein complex. Colocalization of G9a histone methyltransferase with H3-3meK27 and H3-2meK9 was also observed 30 min after activation, but disappeared at the two-cell stage. H3-3meK27 appeared precociously and initially in the absence of the Eed/Ezh2 complex at 8 h in SCNT embryos. Biallelic expression of Xist RNA was observed from the four- to eight-cell stages onwards in SCNT embryos, but eventually declined in blastocysts. The H3-3meK27 was associated with and persisted on only one of the two Xist domains associated with the original Xi. Compare the kinetics of the epigenetic events in SCNT with control embryos, which differ markedly. See text for detailed explanations.