Long-term association of a transcription factor with its chromatin binding site can stabilize gene expression and cell fate commitment.

Some lineage-determining transcription factors are overwhelmingly important in directing embryonic cells to a particular differentiation pathway, such as Ascl1 for nerve. They also have an exceptionally strong ability to force cells to change from an unrelated pathway to one preferred by their action. Transcription factors are believed to have a very short residence time of only a few seconds on their specific DNA or chromatin-binding sites. We have developed a procedure in which DNA containing one copy of the binding site for the neural-inducing factor Ascl1 is injected directly into a Xenopus oocyte nucleus which has been preloaded with a limiting amount of the Ascl1 transcription factor protein. This is followed by a further injection of DNA as a competitor, either in a plasmid or in chromosomal DNA, containing the same binding site but with a different reporter. Importantly, expression of the reporter provides a measure of the function of the transcription factor in addition to its residence time. The same long residence time and resistance to competition are seen with the estrogen receptor and its DNA response elements. We find that in this nondividing oocyte, the nerve-inducing factor Ascl1 can remain bound to a specific chromatin site for hours or days and thereby help to stabilize gene expression. This stability of transcription factor binding to chromatin is a necessary part of its action because removal of this factor causes discontinuation of its effect on gene expression. Stable transcription factor binding may be a characteristic of nondividing cells

DNA-induced spatial entrapment of general transcription machinery can stabilize gene expression in a nondividing cell.

Funder: Wellcome TrustAn important characteristic of cell differentiation is its stability. Only rarely do cells or their stem cell progenitors change their differentiation pathway. If they do, it is often accompanied by a malfunction such as cancer. A mechanistic understanding of the stability of differentiated states would allow better prospects of alleviating the malfunctioning. However, such complete information is yet elusive. Earlier experiments performed in Xenopus oocytes to address this question suggest that a cell may maintain its gene expression by prolonged binding of cell type-specific transcription factors. Here, using DNA competition experiments, we show that the stability of gene expression in a nondividing cell could be caused by the local entrapment of part of the general transcription machinery in transcriptionally active regions. Strikingly, we found that transcriptionally active and silent forms of the same DNA template can stably coexist within the same nucleus. Both DNA templates are associated with the gene-specific transcription factor Ascl1, the core factor TBP2, and the polymerase II (Pol-II) ser5 C-terminal domain (CTD) phosphorylated form, while Pol-II ser2 CTD phosphorylation is restricted to the transcriptionally dominant template. We discover that the active and silent DNA forms are physically separated in the oocyte nucleus through partition into liquid-liquid phase-separated condensates. Altogether, our study proposes a mechanism of transcriptional regulation involving a spatial entrapment of general transcription machinery components to stabilize the active form of a gene in a nondividing cell

The Expression of TALEN before Fertilization Provides a Rapid Knock-Out Phenotype in Xenopus laevis Founder Embryos.

Recent advances in genome editing using programmable nucleases have revolutionized gene targeting in various organisms. Successful gene knock-out has been shown in Xenopus, 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 in vitro 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 tyrosinase and/or embryonic lethal gene pax6 in the founder generation. In addition, our method permitted a large 1 kb deletion. Thus, we describe nearly complete gene knock-out phenotypes in Xenopus laevis 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 Xenopus laevis. 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.K.M. was a Research Fellow at Wolfson College and was supported by the Herchel Smith Postdoctoral Fellowship.This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pone.014294

Initiation and maintenance of pluripotency gene expression in the absence of cohesin

Cohesin is implicated in establishing and maintaining pluripotency. Whether this is because of essential cohesin functions in the cell cycle or in gene regulation is unknown. Here we tested cohesin’s contribution to reprogramming in systems that reactivate the expression of pluripotency genes in the absence of proliferation (embryonic stem [ES] cell heterokaryons) or DNA replication (nuclear transfer). Contrary to expectations, cohesin depletion enhanced the ability of ES cells to initiate somatic cell reprogramming in heterokaryons. This was explained by increased c-Myc (Myc) expression in cohesin-depleted ES cells, which promoted DNA replication-dependent reprogramming of somatic fusion partners. In contrast, cohesin-depleted somatic cells were poorly reprogrammed in heterokaryons, due in part to defective DNA replication. Pluripotency gene induction was rescued by Myc, which restored DNA replication, and by nuclear transfer, where reprogramming does not require DNA replication. These results redefine cohesin’s role in pluripotency and reveal a novel function for Myc in promoting the replication-dependent reprogramming of somatic nuclei

Tsukushi Modulates Xnr2, FGF and BMP Signaling: Regulation of Xenopus Germ Layer Formation

Cell-cell communication is essential in tissue patterning. In early amphibian development, mesoderm is formed in the blastula-stage embryo through inductive interactions in which vegetal cells act on overlying equatorial cells. Members of the TGF-beta family such as activin B, Vg1, derrière and Xenopus nodal-related proteins (Xnrs) are candidate mesoderm inducing factors, with further activity to induce endoderm of the vegetal region. TGF-beta-like ligands, including BMP, are also responsible for patterning of germ layers. In addition, FGF signaling is essential for mesoderm formation whereas FGF signal inhibition has been implicated in endoderm induction. Clearly, several signaling pathways are coordinated to produce an appropriate developmental output; although intracellular crosstalk is known to integrate multiple pathways, relatively little is known about extracellular coordination

Laws of biology: why so few?

Finding fundamental organizing principles is the current intellectual front end of systems biology. From a hydrogen atom to the whole cell level, organisms manage massively parallel and massively interactive processes over several orders of magnitude of size. To manage this scale of informational complexity it is natural to expect organizing principles that determine higher order behavior. Currently, there are only hints of such organizing principles but no absolute evidences. Here, we present an approach as old as Mendel that could help uncover fundamental organizing principles in biology. Our approach essentially consists of identifying constants at various levels and weaving them into a hierarchical chassis. As we identify and organize constants, from pair-wise interactions to networks, our understanding of the fundamental principles in biology will improve, leading to a theory in biology

Enhanced Generation of Induced Pluripotent Stem Cells from a Subpopulation of Human Fibroblasts

BACKGROUND: The derivation of induced pluripotent stem cells (iPSCs) provides new possibilities for basic research and novel cell-based therapies. Limitations, however, include our current lack of understanding regarding the underlying mechanisms and the inefficiency of reprogramming. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report identification and isolation of a subpopulation of human dermal fibroblasts that express the pluripotency marker stage specific embryonic antigen 3 (SSEA3). Fibroblasts that expressed SSEA3 demonstrated an enhanced iPSC generation efficiency, while no iPSC derivation was obtained from the fibroblasts that did not express SSEA3. Transcriptional analysis revealed NANOG expression was significantly increased in the SSEA3 expressing fibroblasts, suggesting a possible mechanistic explanation for the differential reprogramming. CONCLUSIONS/SIGNIFICANCE: To our knowledge, this study is the first to identify a pluripotency marker in a heterogeneous population of human dermal fibroblasts, to isolate a subpopulation of cells that have a significantly increased propensity to reprogram to pluripotency and to identify a possible mechanism to explain this differential reprogramming. This discovery provides a method to significantly increase the efficiency of reprogramming, enhancing the feasibility of the potential applications based on this technology, and a tool for basic research studies to understand the underlying reprogramming mechanisms

Reprogramming towards totipotency is greatly facilitated by synergistic effects of small molecules

Animal cloning has been achieved in many species by transplanting differentiated cell nuclei to unfertilized oocytes. However, the low efficiencies of cloning have remained an unresolved issue. Here we find that the combination of two small molecules, trichostatin A (TSA) and vitamin C (VC), under culture condition with bovine serum albumin deionized by ion-exchange resins, dramatically improves the cloning efficiency in mice and 15% of cloned embryos develop to term by means of somatic cell nuclear transfer (SCNT). The improvement was not observed by adding the non-treated, rather than deionized, bovine serum. RNA-seq analyses of SCNT embryos at the two-cell stage revealed that the treatment with TSA and VC resulted in the upregulated expression of previously identified reprogramming-resistant genes. Moreover, the expression of early-embryo-specific retroelements was upregulated by the TSA and VC treatment. The enhanced gene expression was relevant to the VC-mediated reduction of histone H3 lysine 9 methylation in SCNT embryos. Our study thus shows a simply applicable method to greatly improve mouse cloning efficiency, and furthers our understanding of how somatic nuclei acquire totipotency.This research was supported by Japan Society for the Promotion of Science KAKENHI numbers JP15H06753, JP16H01321, JP16H01222 to K. Miyamoto, JP25712035 to K. Yamagata, JP23658292 to K. Matsumoto, and 23580390 to M.Y.; by the Sumitomo Foundation Grant for Basic Science Research Projects (150810 to K. Miyamoto); by a Kindai University Research Grant (15-I-2 to K. Miyamoto and M.A.); by the grant from Ministry of Education, Science and Culture (grants 24380172 and 26292168 to H.I.); by a Japan Science and Technology Agency grant for Exploratory Research from A-STEP (AS221Z0334E to M.Y.); and by a grant from the Wellcome Trust (101050/Z/13/Z to J.B.G.). K. Miyamoto was supported by the Herchel Smith Postdoctoral Fellowship from the University of Cambridge

Reprogramming of Embryonic Human Fibroblasts into Fetal Hematopoietic Progenitors by Fusion with Human Fetal Liver CD34+ Cells

Experiments with somatic cell nuclear transfer, inter-cellular hybrid formation_ENREF_3, and ectopic expression of transcription factors have clearly demonstrated that cell fate can be dramatically altered by changing the epigenetic state of cell nuclei. Here we demonstrate, using chemical fusion, direct reprogramming of the genome of human embryonic fibroblasts (HEF) into the state of human fetal liver hFL CD34+ (hFL) hematopoietic progenitors capable of proliferating and differentiating into multiple hematopoietic lineages. We show that hybrid cells retain their ploidy and can differentiate into several hematopoietic lineages. Hybrid cells follow transcription program of differentiating hFL cells as shown by genome-wide transcription profiling. Using whole-genome single nucleotide polymorphism (SNP) profiling of both donor genomes we demonstrate reprogramming of HEF genome into the state of hFL hematopoietic progenitors. Our results prove that it is possible to convert the fetal somatic cell genome into the state of fetal hematopoietic progenitors by fusion. This suggests a possibility of direct reprogramming of human somatic cells into tissue specific progenitors/stem cells without going all the way back to the embryonic state. Direct reprogramming of terminally differentiated cells into the tissue specific progenitors will likely prove useful for the development of novel cell therapies