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

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

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

Identification of methylated deoxyadenosines in vertebrates reveals diversity in DNA modifications.

Methylation of cytosine deoxynucleotides generates 5-methylcytosine (m(5)dC), a well-established epigenetic mark. However, in higher eukaryotes much less is known about modifications affecting other deoxynucleotides. Here, we report the detection of N(6)-methyldeoxyadenosine (m(6)dA) in vertebrate DNA, specifically in Xenopus laevis but also in other species including mouse and human. Our methylome analysis reveals that m(6)dA is widely distributed across the eukaryotic genome and is present in different cell types but is commonly depleted from gene exons. Thus, direct DNA modifications might be more widespread than previously thought.M.J.K. was supported by the Long-Term Human Frontiers Fellowship (LT000149/2010-L), the Medical Research Council grant (G1001690), and by the Isaac Newton Trust Fellowship (R G76588). The work was sponsored by the Biotechnology and Biological Sciences Research Council grant BB/M022994/1 (J.B.G. and M.J.K.). The Gurdon laboratory is funded by the grant 101050/Z/13/Z (J.B.G.) from the Wellcome Trust, and is supported by the Gurdon Institute core grants, namely by the Wellcome Trust Core Grant (092096/Z/10/Z) and by the Cancer Research UK Grant (C6946/A14492). C.R.B. and G.E.A. are funded by the Wellcome Trust Core Grant. We are grateful to D. Simpson and R. Jones-Green for preparing X. laevis eggs and oocytes, F. Miller for providing us with M. musculus tissue, T. Dyl for X. laevis eggs and D. rerio samples, and to Gurdon laboratory members for their critical comments. We thank U. Ruether for providing us with M. musculus kidney DNA (Entwicklungs- und Molekularbiologie der Tiere, Heinrich Heine Universitaet Duesseldorf, Germany). We also thank J. Ahringer, S. Jackson, A. Bannister and T. Kouzarides for critical input and advice, M. Sciacovelli and E. Gaude for suggestions.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nsmb.314

Epigenetic memory in induced pluripotent stem cells

Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics, these two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an ‘epigenetic memory’ of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment.National Institutes of Health (U.S.) (NIH grant RO1-DK70055)National Institutes of Health (U.S.) (NIH Grant RO1-DK59279)National Institutes of Health (U.S.) (American Recovery and Reinvestment Act (RC2-HL102815))National Institutes of Health (U.S.) (NIH (K99HL093212-01))Cooley’s Anemia FoundationNational Institutes of Health (U.S.) (NIH LLS (3567-07))National Institutes of Health (U.S.) (NIH grant R37CA054358)National Institutes of Health (U.S.) (NIH grant P50HG003233)National Institutes of Health (U.S.) (NIH grant R01AI047457)National Institutes of Health (U.S.) (NIH Grant R01AI047458)National Institutes of Health (U.S.) (CA86065)National Institutes of Health (U.S.) (HL099999)Thomas and Stacey Siebel FoundationCalifornia Institute for Regenerative Medicine (Fellowship T1-00001

A strategy to discover new organizers identifies a putative heart organizer

Organizers are regions of the embryo that can both induce new fates and impart pattern on other regions. So far, surprisingly few organizers have been discovered, considering the number of patterned tissue types generated during development. This may be because their discovery has relied on transplantation and ablation experiments. Here we describe a new approach, using chick embryos, to discover organizers based on a common gene expression signature, and use it to uncover the anterior intestinal portal (AIP) endoderm as a putative heart organizer. We show that the AIP can induce cardiac identity from non-cardiac mesoderm and that it can pattern this by specifying ventricular and suppressing atrial regional identity. We also uncover some of the signals responsible. The method holds promise as a tool to discover other novel organizers acting during development

DNA repair, genome stability and cancer: a historical perspective

The multistep process of cancer progresses over many years. The prevention of mutations by DNA repair pathways led to an early appreciation of a role for repair in cancer avoidance. However, the broader role of the DNA damage response (DDR) emerged more slowly. In this Timeline article, we reflect on how our understanding of the steps leading to cancer developed, focusing on the role of the DDR. We also consider how our current knowledge can be exploited for cancer therapy

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

Calcium Dependent CAMTA1 in Adult Stem Cell Commitment to a Myocardial Lineage

The phenotype of somatic cells has recently been found to be reversible. Direct reprogramming of one cell type into another has been achieved with transduction and over expression of exogenous defined transcription factors emphasizing their role in specifying cell fate. To discover early and novel endogenous transcription factors that may have a role in adult-derived stem cell acquisition of a cardiomyocyte phenotype, mesenchymal stem cells from human and mouse bone marrow and rat liver were co-cultured with neonatal cardiomyocytes as an in vitro cardiogenic microenvironment. Cell-cell communications develop between the two cell types as early as 24 hrs in co-culture and are required for elaboration of a myocardial phenotype in the stem cells 8-16 days later. These intercellular communications are associated with novel Ca(2+) oscillations in the stem cells that are synchronous with the Ca(2+) transients in adjacent cardiomyocytes and are detected in the stem cells as early as 24-48 hrs in co-culture. Early and significant up-regulation of Ca(2+)-dependent effectors, CAMTA1 and RCAN1 ensues before a myocardial program is activated. CAMTA1 loss-of-function minimizes the activation of the cardiac gene program in the stem cells. While the expression of RCAN1 suggests involvement of the well-characterized calcineurin-NFAT pathway as a response to a Ca(2+) signal, the CAMTA1 up-regulated expression as a response to such a signal in the stem cells was unknown. Cell-cell communications between the stem cells and adjacent cardiomyocytes induce Ca(2+) signals that activate a myocardial gene program in the stem cells via a novel and early Ca(2+)-dependent intermediate, up-regulation of CAMTA1