When transplanted into Xenopus oocytes, the nuclei of mammalian somatic cells are reprogrammed to express stem cell genes such as Oct4, Nanog, and Sox2. We now describe an experimental system in which the pluripotency genes Sox2 and Oct4 are repressed in retinoic acid-treated ES cells but are reprogrammed up to 100% within 24 h by injection of nuclei into the germinal vesicle (GV) of growing Xenopus oocytes. The isolation of GVs in nonaqueous medium allows the reprogramming of individual injected nuclei to be seen in real time. Analysis using fluorescence recovery after photobleaching shows that nuclear transfer is associated with an increase in linker histone mobility. A simultaneous loss of somatic H1 linker histone and incorporation of the oocytespecific linker histone B4 precede transcriptional reprogramming. The loss of H1 is not required for gene reprogramming. We demonstrate both by antibody injection experiments and by dominant negative interference that the incorporation of B4 linker histone is required for pluripotency gene reactivation during nuclear reprogramming. We suggest that the binding of oocyte-specific B4 linker histone to chromatin is a key primary event in the reprogramming of somatic nuclei transplanted to amphibian oocytes. chromatin | nuclear transfer | Xenopus T he transfer of somatic cell nuclei to eggs is an experimental means of reversing the process of cell differentiation in which cells become progressively restricted in the developmental pathways open to them (1-3). By comparison with other procedures (4, 5), nuclear transfer (NT) is relatively efficient (6), and it makes use of natural components of eggs without any accompanying change to the genome. A variant of this technique is NT to Xenopus oocytes (7). Although no new cell types are generated in this type of NT, reactivation of pluripotency genes takes place within a day after NT and in the absence of cell division. The oocyte (M1 prophase I) is the immediate progenitor of an egg (M2 metaphase) and is believed to reprogram transcription in the same way that an egg reprograms the sperm nucleus after fertilization. Therefore the direct and efficient transcriptional reprogramming activity of the Xenopus oocyte makes it a favorable cell in which to analyze an important part of the mechanism of nuclear reprogramming.Our aim is to understand the mechanism of reprogramming by NT in eggs and oocytes. The substrate for this reprogramming activity is the chromatin of transplanted nuclei. The chromatin of eukaryotes contains DNA wrapped around the four core histones arranged as a nucleosome. The linker DNA joining two nucleosomes is also bound by chromatin proteins such as linker histones, high mobility group proteins (8, 9), and poly (ADPribose) polymerase 1 (10). Several linker histone variants are present in somatic cells, and the ratio of the various forms varies from one cell type to another (11). Linker histones initially were thought to have a general function in repressing gene activity. Recent work has demonstrated that linker...

BackgroundNuclear reprogramming is potentially important as a route to cell replacement and drug discovery, but little is known about its mechanism. Nuclear transfer to eggs and oocytes attempts to identify the mechanism of this direct route towards reprogramming by natural components. Here we analyze how the reprogramming of nuclei transplanted to Xenopus oocytes exploits the incorporation of the histone variant H3.3.ResultsAfter nuclear transplantation, oocyte-derived H3.3 but not H3.2, is deposited on several regions of the genome including rDNA, major satellite repeats, and the regulatory regions of Oct4. This major H3.3 deposition occurs in absence of DNA replication, and is HIRA-and transcription-dependent. It is necessary for the shift from a somatic- to an oocyte-type of transcription after nuclear transfer.ConclusionsThis study demonstrates that the incorporation of histone H3.3 is an early and necessary step in the direct reprogramming of somatic cell nuclei by oocyte. It suggests that the incorporation of histone H3.3 is necessary during global changes in transcription that accompany changes in cell fate.

Full-grown Xenopus oocytes in first meiotic prophase contain an immensely enlarged nucleus, the Germinal Vesicle (GV), that can be injected with several hundred somatic cell nuclei. When the nuclei of mammalian somatic cells or cultured cell lines are injected into a GV, a wide range of genes that are not transcribed in the donor cells, including pluripotency genes, start to be transcriptionally activated, and synthesize primary transcripts continuously for several days. Because of the large size and abundance of Xenopus laevis oocytes, this experimental system offers an opportunity to understand the mechanisms by which somatic cell nuclei can be reprogrammed to transcribe genes characteristic of oocytes and early embryos. The use of mammalian nuclei ensures that there is no background of endogenous maternal transcripts of the kind that are induced. The induced gene transcription takes place in the absence of cell division or DNA synthesis and does not require protein synthesis.Here we summarize new as well as established results that characterize this experimental system. In particular, we describe optimal conditions for transplanting somatic nuclei to oocytes and for the efficient activation of transcription by transplanted nuclei. We make a quantitative determination of transcript numbers for pluripotency and housekeeping genes, comparing cultured somatic cell nuclei with those of embryonic stem cells. Surprisingly we find that the transcriptional activation of somatic nuclei differs substantially from one donor cell-type to another and in respect of different pluripotency genes. We also determine the efficiency of an injected mRNA translation into protein.