11 research outputs found
Mitosis Gives a Brief Window of Opportunity for a Change in Gene Transcription
<div><p>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.</p></div
Removal of salt-soluble factors from interphase C2C12 nuclei.
<p>(a) Design of salt depletion procedure. (b) 300 mM salt does not remove the mitotic advantage of mitotic nuclei, nor does it make interphase nuclei behave like mitotic chromatin by loss of DNA-binding factors. Salt-treated samples were injected to oocytes and cultured for 40 h and then analyzed by RT-qPCR. Supporting data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001914#pbio.1001914.s001" target="_blank">Data S1</a>. (c) Two independent experiments show that the majority of chromatin binding factors can be depleted from nuclei by 300 mM salt and Triton when compared to 75 mM salt, which should not remove chromatin-bound factors. The blemish for topoisomerase II (75 mM,M) is not in the position of this protein.</p
Mitotic nuclei are reprogrammed much more efficiently than interphase nuclei.
<p>(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 (<i>n</i> = 3). Supporting data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001914#pbio.1001914.s001" target="_blank">Data S1</a>.</p
Histone ubiquitination can explain the mitotic advantage.
<p>(a) Western blot for histone H2AK119Ub and H2B120Ub from interphase and mitotic donor cells treated with IAA or MG132. (b) ChIP analysis for ubiquitinated histone H2A shows a large difference between mitotic chromatin and interphase nuclei for several genomic regions. (c) IAA largely removes the deubiquitinated state of mitotic chromatin in several gene regions. None of these values are significantly different from one another. Supporting data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001914#pbio.1001914.s001" target="_blank">Data S1</a>. (d) Mitotic advantage is eliminated by IAA treatment. Supporting data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001914#pbio.1001914.s001" target="_blank">Data S1</a>. (e) H2AK119 ubiquitination in interphase nuclei is reduced by MG132. Supporting data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001914#pbio.1001914.s001" target="_blank">Data S1</a>. (f) RT-qPCR of interphase and mitotic nuclei treated with MG132 after nuclear transfer to oocytes; interphase transcription is much enhanced. Supporting data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001914#pbio.1001914.s001" target="_blank">Data S1</a>. (g) Western blot to show <i>in</i> vitro deubiquitination of interphase and mitotic donor nuclei. (h) RT-qPCR transcription analysis of Ubp-M-treated donor nuclei 36 h after nuclear transfer. Supporting data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001914#pbio.1001914.s001" target="_blank">Data S1</a>.</p
Sonication does not eliminate mitotic advantage.
<p>(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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001914#pbio.1001914.s001" target="_blank">Data S1</a>. (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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001914#pbio.1001914.s001" target="_blank">Data S1</a>. (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.</p
Mitotic advantage is independent of nuclear membrane permeability.
<p>(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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001914#pbio.1001914.s001" target="_blank">Data S1</a>.</p
The Expression of TALEN before Fertilization Provides a Rapid Knock-Out Phenotype in <i>Xenopus laevis</i> Founder Embryos
<div><p>Recent advances in genome editing using programmable nucleases have revolutionized gene targeting in various organisms. Successful gene knock-out has been shown in <i>Xenopus</i>, a widely used model organism, although a system enabling less mosaic knock-out in founder embryos (F0) needs to be explored in order to judge phenotypes in the F0 generation. Here, we injected modified highly active transcription activator-like effector nuclease (TALEN) mRNA to oocytes at the germinal vesicle (GV) stage, followed by <i>in vitro</i> maturation and intracytoplasmic sperm injection, to achieve a full knock-out in F0 embryos. Unlike conventional injection methods to fertilized embryos, the injection of TALEN mRNA into GV oocytes allows expression of nucleases before fertilization, enabling them to work from an earlier stage. Using this procedure, most of developed embryos showed full knock-out phenotypes of the pigmentation gene <i>tyrosinase</i> and/or embryonic lethal gene <i>pax6</i> in the founder generation. In addition, our method permitted a large 1 kb deletion. Thus, we describe nearly complete gene knock-out phenotypes in <i>Xenopus laevis</i> F0 embryos. The presented method will help to accelerate the production of knock-out frogs since we can bypass an extra generation of about 1 year in <i>Xenopus laevis</i>. Meantime, our method provides a unique opportunity to rapidly test the developmental effects of disrupting those genes that do not permit growth to an adult able to reproduce. In addition, the protocol shown here is considerably less invasive than the previously used host transfer since our protocol does not require surgery. The experimental scheme presented is potentially applicable to other organisms such as mammals and fish to resolve common issues of mosaicism in founders.</p></div
Phenotypes of embryos that were injected with <i>pax6</i> TALEN mRNAs.
<p>(A) Injection of <i>pax6</i> TALEN mRNAs to GV oocytes, followed by <i>in vitro</i> maturation and ICSI, recapitulated the null mutant phenotype of <i>pax6</i> in F0 embryos. Examples of knock-out tadpoles with different magnifications (a-f; <i>pax6</i> TALEN-expressed tadpoles, g-l; control tadpoles) are shown. The percentages of embryos that showed the knock-out phenotype of <i>pax6</i> are summarized in the graph, as judged by the degree of eye deformation. Embryos without TALEN mRNA injection are used as a control (Non injected). (B) Different phenotypes are observed at the tadpole stage between conventional embryo injection (Fig 2B) and the oocyte injection method (Fig 2A). TALEN mRNAs were injected into fertilized one-cell stage embryos. Examples of <i>pax6</i> TALEN-expressed tadpoles with different magnifications (a-d; <i>pax6</i> TALEN-expressed tadpoles, e-h; control tadpoles) are shown.</p
Injection of TALEN mRNAs into GV oocytes allows efficient <i>tyrosinase</i> gene disruption.
<p>(A) Schematic diagram of the experiment to express TALENs in <i>Xenopus laevis</i> eggs before fertilization. (B) Development of <i>tyrosinase</i> (<i>tyr</i>) TALENs-expressed oocytes. TALEN mRNA-injected GV oocytes, followed by <i>in vitro</i> maturation and ICSI, were able to develop to the swimming tadpole stage although mRNA injection itself seems to decrease the developmental capacity of <i>in vitro</i> matured eggs. Actual numbers of embryos that were injected with sperm and that developed to each developmental stages are indicated next to the corresponding bars. (C-E) Expression of <i>tyr</i> TALENs in GV oocytes allowed almost complete albino phenotypes in F0 embryos and frogs. Enlarged pictures of two TALEN-RL-expressed tadpoles are shown in Fig 1D. Control R represents control embryos and frogs in which only right side TALEN was expressed.</p
Double knock-out of <i>pax6</i> and <i>tyr</i> in <i>Xenopus</i> F0 embryos.
<p>(A) Double knock-out of <i>pax6</i> and <i>tyr</i> was achieved by TALEN mRNA injection to GV oocytes. A double knock-out tadpole is shown. (B) Detection of <i>pax6</i> and <i>tyr</i> mutants by RFLP analysis. Wild type embryos are cut by restriction enzymes (Ui: uninjected embryo), while mutant embryos are not digested. Digested PCR products appear at lower bands (marked by open arrowheads) and the undigested is marked by closed arrowheads. M represents 100 bp ladder marker. (C) Sequencing of three different embryos (#3, #6, #9; the numbers correspond to those in Fig 3B) revealed the 100% mutation rate in <i>pax6</i> and <i>tyr</i> double knock-out embryos.</p