H3.3 but not H3.2 is accumulated in transplanted nuclei. (A) H3.2 vs. H3.3 content in oocyte GVs, eggs, and st28 embryos. Soluble pool of H3.2 and H3.3 were separated by Triton Acid Urea (TAU) gel electrophoresis and analyzed by western blot with an anti-H3 antibody. We loaded the equivalent of 20 and 10 GV, and about two eggs or st28 embryos. (B) Schematic outline of injection procedure, where mRNAs coding for tagged Xenopus histones are injected into oocyte cytoplasm 1 day before injection of donor nuclei. Oocytes containing transplanted nuclei are then incubated for 1 to 2 days and submitted to either live imaging, ChIP, or qRT-PCR analysis. (C) H3.2-cherry labelled ESC nuclei were transplanted to the germinal vesicle of H3.2-GFP or H3.3-GFP containing oocytes. Forty-eight hours after transplantation germinal vesicles were isolated and analyzed by confocal microscopy. Images are projection of Z-stack. (D) Quantification of H3.3-Cherry or H3.2-Cherry loss from transplanted nuclei. Donor ESC nuclei expressing H3.3-Cherry or H3.2-Cherry were transplanted to GVs isolated in oil and were imaged immediately (0 h) or 12 h after transplantation. The histogram shows fluorescently labeled histone signal averaged from 40 nuclei in each condition (Error bars indicate s.e.m). ★ indicate P value < 0.05, (T-TEST). (E) FRAP analysis of oocyte H3.3 and HP1alpha associated with transplanted nuclei. ESC nuclei were transplanted to H3.3-Cherry and GFP-HP1-alpha expressing oocytes. Forty-eight hours after transplantation, GVs were isolated and H3.3-cherry and GFP-HP1alpha accumulated on transplanted chromatin was subjected to FRAP analysis. Images show a clump of labeled nuclei at various time points during the FRAP procedure. The graph indicates fluorescence intensity changes over time for the tested chromatin region as well as in the surrounding GVplasm. Fluorescence intensity of H3.3 Cherry on transplanted chromatin at the beginning of the experiment is set to 1. Note that H3.3 Cherry fluorescence intensity in the GVplasm amounts to about 40% of H3.3-Cherry accumulated on chromatin, and remains constant during the course of the FRAP experiment.