The DUF3715 domain has a conserved role in RNA-directed transposon silencing.

RNA-directed transposon silencing operates in the mammalian soma and germline to safeguard genomic integrity. The piRNA pathway and the HUSH complex identify active transposons through recognition of their nascent transcripts, but mechanistic understanding of how these distinct pathways evolved is lacking. TASOR is an essential component of the HUSH complex.

B1 SINE-binding ZFP266 impedes mouse iPSC generation through suppression of chromatin opening mediated by reprogramming factors.

Induced pluripotent stem cell (iPSC) reprogramming is inefficient and understanding the molecular mechanisms underlying this inefficiency holds the key to successfully control cellular identity. Here, we report 24 reprogramming roadblock genes identified by CRISPR/Cas9-mediated genome-wide knockout (KO) screening. Of these, depletion of the predicted KRAB zinc finger protein (KRAB-ZFP) Zfp266 strongly and consistently enhances murine iPSC generation in several reprogramming settings, emerging as the most robust roadblock.

CELLoGeNe - An energy landscape framework for logical networks controlling cell decisions.

Experimental and computational efforts are constantly made to elucidate mechanisms controlling cell fate decisions during development and reprogramming. One powerful computational method is to consider cell commitment and reprogramming as movements in an energy landscape. Here, we develop Computation of Energy Landscapes of Logical Gene Networks (CELLoGeNe), which maps Boolean implementation of gene regulatory networks (GRNs) into energy landscapes.

Conserved regulation of RNA processing in somatic cell reprogramming.

Background: Along with the reorganization of epigenetic and transcriptional networks, somatic cell reprogramming brings about numerous changes at the level of RNA processing. These include the expression of specific transcript isoforms and 3' untranslated regions. A number of studies have uncovered RNA processing factors that modulate the efficiency of the reprogramming process.

Fine-Tuning Mybl2 Is Required for Proper Mesenchymal-to-Epithelial Transition during Somatic Reprogramming.

During somatic reprogramming, Yamanaka's pioneer factors regulate a complex sequence of molecular events leading to the activation of a network of pluripotency factors, ultimately resulting in the acquisition and maintenance of a pluripotent state. Here, we show that, contrary to the pluripotency factors studied so far, overexpression of Mybl2 inhibits somatic reprogramming. Our results demonstrate that Mybl2 levels are crucial to the dynamics of the reprogramming process.

Mapping transcription factor occupancy using minimal numbers of cells in vitro and in vivo.

The identification of transcription factor (TF) binding sites in the genome is critical to understanding gene regulatory networks (GRNs). While ChIP-seq is commonly used to identify TF targets, it requires specific ChIP-grade antibodies and high cell numbers, often limiting its applicability. NA denine ethyltransferase entification (DamID), developed and widely used in , is a distinct technology to investigate protein-DNA interactions.

Constitutively Active SMAD2/3 Are Broad-Scope Potentiators of Transcription-Factor-Mediated Cellular Reprogramming.

Reprogramming of cellular identity using exogenous expression of transcription factors (TFs) is a powerful and exciting tool for tissue engineering, disease modeling, and regenerative medicine. However, generation of desired cell types using this approach is often plagued by inefficiency, slow conversion, and an inability to produce mature functional cells. Here, we show that expression of constitutively active SMAD2/3 significantly improves the efficiency of induced pluripotent stem cell (iPSC) generation by the Yamanaka factors.

Coupling shRNA screens with single-cell RNA-seq identifies a dual role for mTOR in reprogramming-induced senescence.

Expression of the transcription factors OCT4, SOX2, KLF4, and cMYC (OSKM) reprograms somatic cells into induced pluripotent stem cells (iPSCs). Reprogramming is a slow and inefficient process, suggesting the presence of safeguarding mechanisms that counteract cell fate conversion. One such mechanism is senescence.

Mechanisms of iPS cell generation and beyond.

The generation of induced pluripotent stem cells (iPSCs) achieved by overexpression of Oct4, Sox2, Klf4 and c-Myc, transformed our classical views of the cellular epigenetic landscape and delivered a new concept for cell and tissue engineering. In addition to iPSCs, several other cell types have also been generated by master transcription factor (TF)-mediated transdifferentiation. However, the critical molecular mechanisms amongst diverse cellular identity changes are not well understood.

Reprogramming Roadblocks Are System Dependent.

Since the first generation of induced pluripotent stem cells (iPSCs), several reprogramming systems have been used to study its molecular mechanisms. However, the system of choice largely affects the reprogramming efficiency, influencing our view on the mechanisms. Here, we demonstrate that reprogramming triggered by less efficient polycistronic reprogramming cassettes not only highlights mesenchymal-to-epithelial transition (MET) as a roadblock but also faces more severe difficulties to attain a pluripotent state even post-MET.

piggyBac Transposon Mediated Reprogramming and Flow Cytometry Analysis of CD44 and ICAM1 Cell-Surface Marker Changes.

Generation of iPSCs is inefficient and the molecular mechanisms underlying reprogramming are not well understood. While several studies have demonstrated that reprogramming is not entirely a random process and contains predictable stepwise changes, varying degrees of cellular heterogeneity that arise in different reprogramming systems can obscure the process. Among several reprogramming systems available, delivery of polycistronic reprogramming factor expression cassettes with piggyBac transposon into mouse embryonic fibroblasts (MEFs) is one of the simplest and most robust reprogramming approaches that provide a low background of partially reprogrammed cells.

Acute loss of Cited2 impairs Nanog expression and decreases self-renewal of mouse embryonic stem cells.

Identifying novel players of the pluripotency gene regulatory network centered on Oct4, Sox2, and Nanog as well as delineating the interactions within the complex network is key to understanding self-renewal and early cell fate commitment of embryonic stem cells (ESC). While overexpression of the transcriptional regulator Cited2 sustains ESC pluripotency, its role in ESC functions remains unclear. Here, we show that Cited2 is important for proliferation, survival, and self-renewal of mouse ESC.

Routes to induced pluripotent stem cells.

The generation of induced pluripotent stem cells (iPSCs) with Oct4, Sox2, Klf4, c-Myc has been described as 'direct' reprogramming in contrast to reprogramming via nuclear transfer. Interestingly, recent studies have suggested that the conversion process itself includes transient up-regulation and down-regulation of hundreds of genes, making unique intermediate populations. In a sense, the process of 4 factor reprogramming is indirect.

MBD3/NuRD facilitates induction of pluripotency in a context-dependent manner.

The Nucleosome Remodeling and Deacetylase (NuRD) complex is essential for embryonic development and pluripotent stem cell differentiation. In this study, we investigated whether NuRD is also involved in the reverse biological process of induction of pluripotency in neural stem cells. By knocking out MBD3, an essential scaffold subunit of the NuRD complex, at different time points in reprogramming, we found that efficient formation of reprogramming intermediates and induced pluripotent stem cells from neural stem cells requires NuRD activity.

High-resolution analysis with novel cell-surface markers identifies routes to iPS cells.

The generation of induced pluripotent stem (iPS) cells presents a challenge to normal developmental processes. The low efficiency and heterogeneity of most methods have hindered understanding of the precise molecular mechanisms promoting, and roadblocks preventing, efficient reprogramming. Although several intermediate populations have been described, it has proved difficult to characterize the rare, asynchronous transition from these intermediate stages to iPS cells.

ERK2 suppresses self-renewal capacity of embryonic stem cells, but is not required for multi-lineage commitment.

Activation of the FGF-ERK pathway is necessary for naïve mouse embryonic stem (ES) cells to exit self-renewal and commit to early differentiated lineages. Here we show that genetic ablation of Erk2, the predominant ERK isozyme expressed in ES cells, results in hyper-phosphorylation of ERK1, but an overall decrease in total ERK activity as judged by substrate phosphorylation and immediate-early gene (IEG) induction. Normal induction of this subset of canonical ERK targets, as well as p90RSK phosphorylation, was rescued by transgenic expression of either ERK1 or ERK2 indicating a degree of functional redundancy.

Transcriptional activation by Oct4 is sufficient for the maintenance and induction of pluripotency.

Oct4 is an essential regulator of pluripotency in vivo and in vitro in embryonic stem cells, as well as a key mediator of the reprogramming of somatic cells into induced pluripotent stem cells. It is not known whether activation and/or repression of specific genes by Oct4 is relevant to these functions. Here, we show that fusion proteins containing the coding sequence of Oct4 or Xlpou91 (the Xenopus homolog of Oct4) fused to activating regions, but not those fused to repressing regions, behave as Oct4, suppressing differentiation and promoting maintenance of undifferentiated phenotypes in vivo and in vitro.

The effect of mastication on reaction latency to unanticipated external disturbances in the standing position.

Previous research has shown that mastication reduces shifts in the center of gravity of persons standing still. The present research was conducted to determine whether mastication improves reactive balance in the standing position in response to unanticipated external disturbances. The subjects were 32 healthy male adults (mean age 21.

New strategies to generate induced pluripotent stem cells.

Direct reprogramming of somatic cells to a pluripotent state, substantiated only three years prior, is one of the most rapidly developing areas of stem cell research. The generation of patient-derived pluripotent cells applicable to disease modelling, drug screening, toxicology tests and, ultimately, autologous cell-based therapies, has the potential to revolutionize medicine. Since 2006, when Takahashi and Yamanaka first reported the generation of induced pluripotent stem cells from murine fibroblasts via retroviral transduction of a defined set of transcription factors, various new methods have been developed to refine and improve reprogramming technology.

piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells.

Transgenic expression of just four defined transcription factors (c-Myc, Klf4, Oct4 and Sox2) is sufficient to reprogram somatic cells to a pluripotent state. The resulting induced pluripotent stem (iPS) cells resemble embryonic stem cells in their properties and potential to differentiate into a spectrum of adult cell types. Current reprogramming strategies involve retroviral, lentiviral, adenoviral and plasmid transfection to deliver reprogramming factor transgenes.

Virus-free induction of pluripotency and subsequent excision of reprogramming factors.

Reprogramming of somatic cells to pluripotency, thereby creating induced pluripotent stem (iPS) cells, promises to transform regenerative medicine. Most instances of direct reprogramming have been achieved by forced expression of defined factors using multiple viral vectors. However, such iPS cells contain a large number of viral vector integrations, any one of which could cause unpredictable genetic dysfunction.

Mbd3, a component of the NuRD co-repressor complex, is required for development of pluripotent cells.

Mbd3 is a core component of the NuRD (Nucleosome Remodeling and Histone Deacetylation) co-repressor complex, and NuRD-mediated silencing has been implicated in cell fate decisions in a number of contexts. Mbd3-deficient embryonic stem (ES) cells made by gene targeting are viable but fail to form a stable NuRD complex, are severely compromised in the ability to differentiate, and show LIF-independent self-renewal. Mbd3 is known to be essential for postimplantation embryogenesis in mice, but the function of Mbd3 in vivo has not previously been addressed.

The NuRD component Mbd3 is required for pluripotency of embryonic stem cells.

Cells of early mammalian embryos have the potential to develop into any adult cell type, and are thus said to be pluripotent. Pluripotency is lost during embryogenesis as cells commit to specific developmental pathways. Although restriction of developmental potential is often associated with repression of inappropriate genetic programmes, the role of epigenetic silencing during early lineage commitment remains undefined.