Cell differentiation is remarkably stable but can be reversed by somatic cell nuclear transfer, cell fusion, and iPS. Nuclear transfer to amphibian oocytes provides a special opportunity to test transcriptional reprogramming without cell division. We show here that, after nuclear transfer to amphibian oocytes, mitotic chromatin is reprogrammed up to 100 times faster than interphase nuclei. We find that, as cells traverse mitosis, their genes pass through a temporary phase of unusually high responsiveness to oocyte reprogramming factors (mitotic advantage). Mitotic advantage is not explained by nuclear penetration, DNA modifications, histone acetylation, phosphorylation, methylation, nor by salt soluble chromosomal proteins. Our results suggest that histone H2A deubiquitination may account, at least in part, for the acquisition of mitotic advantage. They support the general principle that a temporary access of cytoplasmic factors to genes during mitosis may facilitate somatic cell nuclear reprogramming and the acquisition of new cell fates in normal development.

Cells are dividing very actively at a time in development when new gene expression and new cell lineages arise. At mitosis, most transcription factors are temporarily displaced from chromosomes. We show that, after transplantation to oocytes, somatic cell nuclei that have been synchronized in mitosis can be reprogrammed to pluripotency gene expression up to 100 times faster than interphase nuclei. We find that, as cells traverse mitosis, their genes pass through a temporary phase of unusually high responsiveness to oocyte reprogramming factors (mitotic advantage). Many other genes in the genome have also shown a mitotic advantage, which affects the rate rather than the final level of transcriptional enhancement. This is attributable to a chromatin state rather than to more rapid passage of reprogramming factors through the nuclear membrane. Histone H2A deubiquitination at mitosis is required for the acquisition of mitotic advantage. Our results support the general principle that a temporary access of cytoplasmic factors to genes during mitosis facilitates somatic cell nuclear reprogramming and the acquisition of new cell fates in normal development.

Funding: All of the work in this paper was funded by a grant from the Wellcome Trust grant number 088333/Z/09/Z to J.B. Gurdon and by a grant from the Medical Research Council number G1001690, file number 98037. This institute contributes some core facilities, funded by the Wellcome Trust. We are affiliated with the Zoology Department in Cambridge University, but it has not contributed to the funding or to any other aspect of the work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2014 Halley-Stott et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Germinal vesicle (GV) stage oocytes do not replicate or divide. They therefore provide an opportunity to test whether the cell cycle phase of donor nuclei affects the efficiency of nuclear reprogramming as judged by active transcription of previously silenced genes [12] . To our surprise, we found that a mitotic state of donor nuclei dramatically increases the efficiency of activating certain quiescent pluripotency genes in these nuclei. Our results support an idea that a brief period during mitosis facilitates an exchange of gene regulatory factors on chromatin and that this could be an important mechanism to help cells embarking on new cell lineages during normal development.

Normal development, as well as nearly all cases of experimentally induced changes in gene transcription, is accompanied by cell division. It is therefore hard to distinguish those molecular events which prepare cells for, or engage them in, mitosis from those that are required specifically for transcriptional reprogramming. The relationship between the cell cycle and cell fate decisions has for a long time attracted interest [1] . Transition through mitosis is a time when many transcription factors are displaced from chromatin, potentially permitting new transcription factors to occupy chromatin sites on mitotic exit and so direct a postmitotic cell fate change [2] – [5] . Mitotic remodelling has been shown to be of great importance for the efficient replication of erythrocyte nuclei by Xenopus egg extracts [6] , [7] . For new transcription, cell division seems to be needed in some cases [8] , [9] but not in others [10] , [11] . Here we have used nuclear transfer to amphibian oocytes to compare directly the ability of mitotic chromatin or interphase nuclei to be reprogrammed in the absence of cell division.

Results

Mitotic Chromatin Is Reprogrammed Much More Rapidly Than Interphase Nuclei Permeabilized mouse C2C12 cells, a cultured myoblast cell line which we have used extensively in our oocyte nuclear transfer experiments, were arrested at specific stages of the cell cycle (Figure S1a) and were injected into the GV of oocytes (Figure 1a). The DNA content of these donor cell populations (Figure 1b) confirmed cell cycle arrest in each of the cell cycle stages. The transcriptional reactivation of three silent genes quiescent in C2C12 cells (Nanog, Oct4, and Sox2) was assessed by RT-qPCR 38 h after nuclear transplantation (Figure 1c). Nuclei at a late stage of the cell cycle (M) show greatly enhanced transcription of each of the genes when compared to unsynchronized nuclei (predominantly G1 and S), whereas an already active gene (c-jun) shows little increase in transcript level. Particularly impressive is the 100-fold enhancement in Sox 2 expression from mitotic donor nuclei when compared to interphase donor nuclei (Figure 1c). In over 50 experiments, donor cells arrested in mitosis or in late G2 always generated more Sox2 transcripts from reactivated genes at 25–48 h after injection to oocytes than unsynchronized donor cells. This difference ranged from a few fold to over 100-fold and is much affected by the exact duration of nocodazole treatment. Sox2 is a gene that is more widely expressed than most others, notably in early embryos, in most stem cells, and in the nervous system [13]. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Mitotic nuclei are reprogrammed much more efficiently than interphase nuclei. (a) Nuclear transplantation procedure used in this and the following experiments. (b) DNA content analysis of donor cells used for nuclear transplantation to oocytes confirms enrichment of specific cell cycles stages. (c) Donor nuclei in the later stages of the cell cycle reprogram better than those from earlier stages. Nuclei from C2C12 cells arrested at each stage of the cell cycle or growing in the absence of inhibitor were used as donor material for NT to oocyte GVs. The figure shows the relative expression for each of the indicated genes at 38 h after transplantation compared to unarrested donor cells (n = 3). Supporting data can be found in Data S1. https://doi.org/10.1371/journal.pbio.1001914.g001 To test whether this result is a peculiarity of this donor cell type (C2C12 myoblasts) or is a nonspecific effect of nocodazole, we repeated these experiments with 10T1/2 donor nuclei (Figure S1b) or prepared mitotic C2C12 donor cells without any inhibitors by a shake-off procedure (Figure S1c). In both cases, enhanced transcription from mitotic donors was observed, although the magnitude of mitotic advantage was lower (particularly in the case of the shake-off samples, many cells of which appeared to be apoptotic by visual inspection). Mitotic donor nuclei were also prepared using another cell synchronization agent (Taxol), and the mitotic advantage was again seen (Figure S1d). When G1/G0 cells were exposed to nocodazole for the same period of time as used to prepare mitotic cells, no enhancement of transcription of the genes was observed (Figure S1e). These results indicate that the observed mitotic advantage is not due to a nonspecific activity of nocodazole nor to a peculiarity of one line of cells (C2C12). To ask if this mitotic advantage applies more widely in the genome than to the pluripotency genes so far tested, we compared by RNAseq the genes transcribed in injected oocytes by interphase nuclei or mitotic chromatin. We focussed our analysis on genes that were found to be consistently expressed by interphase nuclei after nuclear transfer. One experiment indicated that 617 genes were transcribed in oocytes at least 2-fold more in mitotic nuclei compared to interphase nuclei. Of these mitotically up-regulated genes, Sox2 was 4-fold more transcribed than in interphase nuclear transfers, and over half of the 617 genes were more strongly transcribed than Sox2. The list of these genes is in Table S1.

Mitotic Advantage Is Due to an Increased Rate of Reprogramming The enhanced reprogramming from mitotic donor material could be due to an increased rate or to a greater eventual level of reprogramming. To distinguish these ideas and to measure the rate of reprogramming, we measured the incorporation of GFP-tagged histone B4 (an early marker of oocyte reprogramming) [14] and the association of Cherry-labelled histone H2B by live imaging of mixed populations of mitotic and interphase donor cells after injection into oocytes (see Figure S2a for design). Mitotic donor material becomes very rapidly marked with both histone B4 and histone H2B, whereas interphase donor nuclei show a lag in the association of both and particularly of H2B (Figure 2a). In support of a difference in the rate of reprogramming, we find that oocyte-derived TBP2 marks the transplanted mitotic cells more strongly than interphase donor cells (Figure S2b; compare white mitotic with yellow interphase arrows). We then asked if there is a more rapid association and activation of RNA polymerase II with mitotic chromatin. We used immunostaining for the elongating form of RNA polymerase II on a mixed population of mitotic and interphase nuclei injected into oocyte GVs. Mitotic donor material is clearly marked with elongating Pol II before interphase donor material (Figure 2b, compare panels ii and iv for pol II). In view of this difference between the two nuclear types in the onset of global pol II transcription after nuclear transfer, we asked whether reprogrammed genes are activated at a different rate in mitotic donor cells compared to interphase cells or if the magnitude of activation is greater. A time course of reprogramming from oocytes injected with either interphase or mitotic donor cells was assessed by RT-qPCR and revealed that genes from mitotic donor cells are activated more rapidly than the same genes from interphase cells (Figure 2c); the accumulation of transcripts reached by 63 h is similar. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. Mitotic advantage is due to an increased rate of reprogramming. (a) Live imaging of mixed mitotic and interphase nuclei injected into the GVs of oocytes expressing fluorescently labelled histone B4 or histone H2B. Mitotic chromatin becomes decorated with oocyte-derived factors to a far greater degree than interphase nuclei 2 h after nuclear transplantation. The arrows indicate interphase (I) or mitotic (M) nuclei. (b) Mitotic donor nuclei (M) display actively transcribing RNA Pol II far more rapidly than interphase nuclei (I) when these nuclei are co-injected into oocyte GVs. Immunofluorescent staining against a mixture of interphase and mitotic donor nuclei injected into the oocyte GV and fixed at 3 min or between 1 and 2 h after transplantation using antibodies against active poI II (magenta). (c) A time course of expression by mitotic and interphase donor nuclei shows that the difference between these nuclei decreases with time, suggesting that the eventual amount of reprogramming is similar in the two nuclear types but that initiation of transcription is much more rapid in mitotic nuclei. Supporting data can be found in Data S1. https://doi.org/10.1371/journal.pbio.1001914.g002 We conclude that the difference in reprogramming between interphase and mitotic donor material giving this mitotic advantage reflects the rate of reprogramming rather than the eventual magnitude of transcript generation from these two types of nucleus.

Mitotic Advantage Is Independent of Nuclear Membrane Permeability The most obvious explanation for this mitotic advantage is the absence of a nuclear envelope in the mitotic karyoplasts. We have quantitated this difference in membrane permeability by time course imaging a mixture of injected interphase nuclei and mitotic karyoplasts. We carried out a “double permeabilization,” in which both the cell and nuclear membranes, of interphase or mitotic donor cells, were permeabilized as illustrated in the scheme in Figure 3a. We then compared the rate of oocyte factor uptake with the rate of reprogramming by RT-qPCR. A difference in the amount of B4 and H2B uptake is indeed seen after plasma permeabilization with digitonin (Figure 3b) but is no longer seen after double permeabilization of the nuclear envelope with Triton (Figure 3c). Nevertheless, the mitotic difference between interphase and mitotic chromatin does persist in respect of the transcriptional reprogramming of silenced genes (Figure 3d). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 3. Mitotic advantage is independent of nuclear membrane permeability. (a) Design of permeability assay. (b) Under normal conditions of plasma membrane permeabilization by digitonin with no nuclear permeabilization, mitotic chromatin (M arrows) takes up histones B4 and H2B faster than interphase nuclei (I arrows). (c) When double permeabilized by Digitonin and Triton X, interphase nuclei and mitotic chromatin take up these histones at a similar rate. (d) After double permeabilization, the mitotic advantage of mitotic nuclei is still very large, as judged by RT-qPCR. Supporting data can be found in Data S1. https://doi.org/10.1371/journal.pbio.1001914.g003 We have confirmed this conclusion using permeabilization by different reagents. Streptolysin 0 (SLO) permeabilizes the plasma membrane but not the nuclear membrane; SLO and Lysolecithin (LL) together permeabilize the plasma membrane and nuclear membrane [15]. Permeabilization was tested using different sizes of dextran (Figure S3a). We then compared transcription from transplanted nuclei, comparing those treated with SLO alone and those treated with SLO and LL. The transcription ratio following these two procedures shows no advantage when the nuclear envelope is permeabilized (Figure S3b). We conclude that the presence of an intact interphase nuclear envelope does not explain the mitotic advantage.

The Mitotic Advantage Is Due to Chromatin Composition Because the difference in reprogramming rate between interphase and mitotic donor cells is maintained after extensive permeabilization of the interphase nuclear membrane, we asked if the source of the difference lies in the chromatin of the two donor cell preparations. To answer this, we mildly sonicated both interphase and mitotic donor cell preparations to give fragments of chromatin of similar sizes (Figure 4a and b), injected these preparations in parallel with a permeabilized cell preparation into oocyte GVs, and assessed gene reactivation by RT-qPCR (Figure 4c). It is clear that the difference in the rate of gene reactivation from interphase and mitotic nuclei is maintained when the injected material is sonicated chromatin as opposed to whole nuclei. This suggests that the “mitotic advantage” is present in the chromatin of mitotic cells. This result also confirms that the difference between interphase and mitotic donor cells is not due to the interphase nuclear membrane, nor to any other aspect of nuclear organization that is eliminated by sonication. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 4. Sonication does not eliminate mitotic advantage. (a) Interphase and mitotic donor nuclei were mildly sonicated to fragment the chromatin as shown by DAPI staining of the four kinds of donor nuclei. (b) The major proportion of DNA in both sonicated samples is above the size exclusion limit of the gel, confirming mild sonication. (c) Interphase and mitotic nuclei or corresponding sonicated chromatin preparations were transplanted into oocyte GVs and gene reactivation analyzed by RT-qPCR after 42 h. The mitotic advantage is retained on fragments of chromatin. Supporting data can be found in Data S1. (d) Genomic DNA prepared from interphase and mitotic cells was injected into oocyte GVs and gene transcription assessed by RT-qPCR. There is no significant difference between interphase and mitotic DNA with respect to gene activation in the oocyte at either of the indicated time points. Supporting data can be found in Data S1. (e) There is no observable difference in DNA methylation between interphase and mitotic cells as determined by pyrosequencing of bisulphite-converted genomic DNA (horizontal lines represent the indicated DNA sequences, with balls representing individual CpG dinucleotides; black filling represents the percentage of methylation for each site). Solid black bars represent the positions of known transcription factor binding sites, such as SP1/HRE. OS is Oct-Sox, PD is Pou-Domain, and SRR is the Sox2 Regulatory Region, and genomic distances are presented below each map, set relative to the transcriptional start site of each gene. https://doi.org/10.1371/journal.pbio.1001914.g004 The difference between interphase and mitotic reprogramming is, however, abolished when genomic DNA prepared from donor nuclei is injected into oocyte GVs (Figure 4d); this excludes differences at the DNA level (sequence and DNA methylation for example) as possible sources of the difference in reprogramming between interphase and mitotic samples. The possibility of DNA methylation accounting for the mitotic effect was further excluded by bisulphite analysis of specific loci on mitotic and interphase DNAs, as this revealed no mitosis-specific differences (Figure 4e). These two results indicate that whatever accounts for the difference between mitotic and interphase donor cells is not present at the level of genomic DNA itself but is in non-DNA components of chromatin.