Disabling de novo DNA methylation in embryonic stem cells allows an illegitimate fate trajectory.

Genome remethylation is essential for mammalian development but specific reasons are unclear. Here we examined embryonic stem (ES) cell fate in the absence of de novo DNA methyltransferases. We observed that ES cells deficient for both Dnmt3a and Dnmt3b are rapidly eliminated from chimeras. On further investigation we found that in vivo and in vitro the formative pluripotency transition is derailed toward production of trophoblast. This aberrant trajectory is associated with failure to suppress activation of Ascl2Ascl2 encodes a bHLH transcription factor expressed in the placenta. Misexpression of Ascl2 in ES cells provokes transdifferentiation to trophoblast-like cells. Conversely, Ascl2 deletion rescues formative transition of Dnmt3a/b mutants and improves contribution to chimeric epiblast. Thus, de novo DNA methylation safeguards against ectopic activation of Ascl2 However, Dnmt3a/b-deficient cells remain defective in ongoing embryogenesis. We surmise that multiple developmental transitions may be secured by DNA methylation silencing potentially disruptive genes.

A common molecular logic determines embryonic stem cell self-renewal and reprogramming.

During differentiation and reprogramming, new cell identities are generated by reconfiguration of gene regulatory networks. Here, we combined automated formal reasoning with experimentation to expose the logic of network activation during induction of naïve pluripotency. We find that a Boolean network architecture defined for maintenance of naïve state embryonic stem cells (ESC) also explains transcription factor behaviour and potency during resetting from primed pluripotency. Computationally identified gene activation trajectories were experimentally substantiated at single-cell resolution by RT-qPCR Contingency of factor availability explains the counterintuitive observation that Klf2, which is dispensable for ESC maintenance, is required during resetting. We tested 124 predictions formulated by the dynamic network, finding a predictive accuracy of 77.4%. Finally, we show that this network explains and predicts experimental observations of somatic cell reprogramming. We conclude that a common deterministic program of gene regulation is sufficient to govern maintenance and induction of naïve pluripotency. The tools exemplified here could be broadly applied to delineate dynamic networks underlying cell fate transitions.

microRNAs as novel regulators of stem cell pluripotency and somatic cell reprogramming.

Emerging evidence suggests that microRNA (miRNA)-mediated post-transcriptional gene regulation plays an essential role in modulating embryonic stem (ES) cell pluripotency maintenance, differentiation, and reprogramming of somatic cells to an ES cell-like state. Investigations from ES cell-enriched miRNAs, such as mouse miR-290 cluster and human miR-302 cluster, and ES cell-depleted miRNAs such as let-7 family miRNAs, revealed a common theme that miRNAs target diverse cellular processes including cell cycle regulators, signaling pathway effectors, transcription factors, and epigenetic modifiers and shape their protein output. The combinatorial effects downstream of miRNA action allow miRNAs to modulate cell-fate decisions effectively. Furthermore, the transcription and biogenesis of miRNAs are also tightly regulated. Thus, elucidating the interplay between miRNAs and other modes of gene regulation will shed new light on the biology of pluripotent stem cells and somatic cell reprogramming.

Isolation of homozygous mutant mouse embryonic stem cells using a dual selection system.

Obtaining random homozygous mutants in mammalian cells for forward genetic studies has always been problematic due to the diploid genome. With one mutation per cell, only one allele of an autosomal gene can be disrupted, and the resulting heterozygous mutant is unlikely to display a phenotype. In cells with a genetic background deficient for the Bloom's syndrome helicase, such heterozygous mutants segregate homozygous daughter cells at a low frequency due to an elevated rate of crossover following mitotic recombination between homologous chromosomes. We constructed DNA vectors that are selectable based on their copy number and used these to isolate these rare homozygous mutant cells independent of their phenotype. We use the piggyBac transposon to limit the initial mutagenesis to one copy per cell, and select for cells that have increased the transposon copy number to two or more. This yields homozygous mutants with two allelic mutations, but also cells that have duplicated the mutant chromosome and become aneuploid during culture. On average, 26% of the copy number gain events occur by the mitotic recombination pathway. We obtained homozygous cells from 40% of the heterozygous mutants tested. This method can provide homozygous mammalian loss-of-function mutants for forward genetic applications.

A hyperactive piggyBac transposase for mammalian applications.

DNA transposons have been widely used for transgenesis and insertional mutagenesis in various organisms. Among the transposons active in mammalian cells, the moth-derived transposon piggyBac is most promising with its highly efficient transposition, large cargo capacity, and precise repair of the donor site. Here we report the generation of a hyperactive piggyBac transposase. The active transposition of piggyBac in multiple organisms allowed us to screen a transposase mutant library in yeast for hyperactive mutants and then to test candidates in mouse ES cells. We isolated 18 hyperactive mutants in yeast, among which five were also hyperactive in mammalian cells. By combining all mutations, a total of 7 aa substitutions, into a single reading frame, we generated a unique hyperactive piggyBac transposase with 17-fold and ninefold increases in excision and integration, respectively. We showed its applicability by demonstrating an increased efficiency of generation of transgene-free mouse induced pluripotent stem cells. We also analyzed whether this hyperactive piggyBac transposase affects the genomic integrity of the host cells. The frequency of footprints left by the hyperactive piggyBac transposase was as low as WT transposase (~1%) and we found no evidence that the expression of the transposase affects genomic integrity. This hyperactive piggyBac transposase expands the utility of the piggyBac transposon for applications in mammalian genetics and gene therapy.

Mobilization of giant piggyBac transposons in the mouse genome.

The development of technologies that allow the stable delivery of large genomic DNA fragments in mammalian systems is important for genetic studies as well as for applications in gene therapy. DNA transposons have emerged as flexible and efficient molecular vehicles to mediate stable cargo transfer. However, the ability to carry DNA fragments >10 kb is limited in most DNA transposons. Here, we show that the DNA transposon piggyBac can mobilize 100-kb DNA fragments in mouse embryonic stem (ES) cells, making it the only known transposon with such a large cargo capacity. The integrity of the cargo is maintained during transposition, the copy number can be controlled and the inserted giant transposons express the genomic cargo. Furthermore, these 100-kb transposons can also be excised from the genome without leaving a footprint. The development of piggyBac as a large cargo vector will facilitate a wider range of genetic and genomic applications.

Transposon-mediated genome manipulation in vertebrates.

Transposable elements are DNA segments with the unique ability to move about in the genome. This inherent feature can be exploited to harness these elements as gene vectors for genome manipulation. Transposon-based genetic strategies have been established in vertebrate species over the last decade, and current progress in this field suggests that transposable elements will serve as indispensable tools. In particular, transposons can be applied as vectors for somatic and germline transgenesis, and as insertional mutagens in both loss-of-function and gain-of-function forward mutagenesis screens. In addition, transposons will gain importance in future cell-based clinical applications, including nonviral gene transfer into stem cells and the rapidly developing field of induced pluripotent stem cells. Here we provide an overview of transposon-based methods used in vertebrate model organisms with an emphasis on the mouse system and highlight the most important considerations concerning genetic applications of the transposon systems.

Human naive epiblast cells possess unrestricted lineage potential.

Classic embryological experiments have established that the early mouse embryo develops via sequential lineage bifurcations. The first segregated lineage is the trophectoderm, essential for blastocyst formation. Mouse naive epiblast and derivative embryonic stem cells are restricted accordingly from producing trophectoderm. Here we show, in contrast, that human naive embryonic stem cells readily make blastocyst trophectoderm and descendant trophoblast cell types. Trophectoderm was induced rapidly and efficiently by inhibition of ERK/mitogen-activated protein kinase (MAPK) and Nodal signaling. Transcriptome comparison with the human embryo substantiated direct formation of trophectoderm with subsequent differentiation into syncytiotrophoblast, cytotrophoblast, and downstream trophoblast stem cells. During pluripotency progression lineage potential switches from trophectoderm to amnion. Live-cell tracking revealed that epiblast cells in the human blastocyst are also able to produce trophectoderm. Thus, the paradigm of developmental specification coupled to lineage restriction does not apply to humans. Instead, epiblast plasticity and the potential for blastocyst regeneration are retained until implantation.

A lncRNA fine tunes the dynamics of a cell state transition involving Lin28, let-7 and de novo DNA methylation.

Execution of pluripotency requires progression from the naïve status represented by mouse embryonic stem cells (ESCs) to a state capacitated for lineage specification. This transition is coordinated at multiple levels. Non-coding RNAs may contribute to this regulatory orchestra. We identified a rodent-specific long non-coding RNA (lncRNA) linc1281, hereafter Ephemeron (Eprn), that modulates the dynamics of exit from naïve pluripotency. Eprn deletion delays the extinction of ESC identity, an effect associated with perduring Nanog expression. In the absence of Eprn, Lin28a expression is reduced which results in persistence of let-7 microRNAs, and the up-regulation of de novo methyltransferases Dnmt3a/b is delayed. Dnmt3a/b deletion retards ES cell transition, correlating with delayed Nanog promoter methylation and phenocopying loss of Eprn or Lin28a. The connection from lncRNA to miRNA and DNA methylation facilitates the acute extinction of naïve pluripotency, a pre-requisite for rapid progression from preimplantation epiblast to gastrulation in rodents. Eprn illustrates how lncRNAs may introduce species-specific network modulations.

A single-copy Sleeping Beauty transposon mutagenesis screen identifies new PTEN-cooperating tumor suppressor genes.

The overwhelming number of genetic alterations identified through cancer genome sequencing requires complementary approaches to interpret their significance and interactions. Here we developed a novel whole-body insertional mutagenesis screen in mice, which was designed for the discovery of Pten-cooperating tumor suppressors. Toward this aim, we coupled mobilization of a single-copy inactivating Sleeping Beauty transposon to Pten disruption within the same genome. The analysis of 278 transposition-induced prostate, breast and skin tumors detected tissue-specific and shared data sets of known and candidate genes involved in cancer. We validated ZBTB20, CELF2, PARD3, AKAP13 and WAC, which were identified by our screens in multiple cancer types, as new tumor suppressor genes in prostate cancer. We demonstrated their synergy with PTEN in preventing invasion in vitro and confirmed their clinical relevance. Further characterization of Wac in vivo showed obligate haploinsufficiency for this gene (which encodes an autophagy-regulating factor) in a Pten-deficient context. Our study identified complex PTEN-cooperating tumor suppressor networks in different cancer types, with potential clinical implications.

The piggyBac transposon displays local and distant reintegration preferences and can cause mutations at noncanonical integration sites.

The DNA transposon piggyBac is widely used as a tool in mammalian experimental systems for transgenesis, mutagenesis, and genome engineering. We have characterized genome-wide insertion site preferences of piggyBac by sequencing a large set of integration sites arising from transposition from two separate genomic loci and a plasmid donor in mouse embryonic stem cells. We found that piggyBac preferentially integrates locally to the excision site when mobilized from a chromosomal location and identified other nonlocal regions of the genome with elevated insertion frequencies. piggyBac insertions were associated with expressed genes and markers of open chromatin structure and were excluded from heterochromatin. At the nucleotide level, piggyBac prefers to insert into TA-rich regions within a broader GC-rich context. We also found that piggyBac can insert into sites other than its known TTAA insertion site at a low frequency (2%). Such insertions introduce mismatches that are repaired with signatures of host cell repair pathways. Transposons could be mobilized from plasmids with the observed noncanonical flanking regions, indicating that piggyBac could generate point mutations in the genome.

Genome-wide forward genetic screens in mouse ES cells.

Mouse embryonic stem (ES) cells are an attractive model system for investigating mammalian biology. Their relatively stable genome and high amenability to genome modification enables the generation of large-scale mutant libraries, which can be subsequently used for phenotype-driven genetic screens. While retroviral vectors have traditionally been used to generate insertional mutations in ES cells, their severe distribution-bias in the mammalian genome substantially limits genome-wide mutagenesis. The recent development of the DNA transposon piggyBac offers an efficient and highly versatile alternative for achieving more unbiased mutagenesis. Furthermore, heterozygous mutations created by insertional mutagens can be converted in parallel to homozygosity by using Blm-deficient ES cells, allowing genome-wide loss-of-function screens to be conducted. In this chapter, we describe the principles underpinning genetic screens in mouse ES cells with examples of previously successful screens. Protocols are provided for piggyBac transposon-mediated mutagenesis, production of the corresponding homozygous mutants in a Blm-deficient genetic background, and methods for mapping and validation of mutations recovered from screens of such libraries.

PiggyBac transposon mutagenesis: a tool for cancer gene discovery in mice.

Transposons are mobile DNA segments that can disrupt gene function by inserting in or near genes. Here, we show that insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery in mice. PiggyBac transposition in genetically engineered transposon-transposase mice induced cancers whose type (hematopoietic versus solid) and latency were dependent on the regulatory elements introduced into transposons. Analysis of 63 hematopoietic tumors revealed that PiggyBac is capable of genome-wide mutagenesis. The PiggyBac screen uncovered many cancer genes not identified in previous retroviral or Sleeping Beauty transposon screens, including Spic, which encodes a PU.1-related transcription factor, and Hdac7, a histone deacetylase gene. PiggyBac and Sleeping Beauty have different integration preferences. To maximize the utility of the tool, we engineered 21 mouse lines to be compatible with both transposon systems in constitutive, tissue- or temporal-specific mutagenesis. Mice with different transposon types, copy numbers, and chromosomal locations support wide applicability.