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. TASOR's DUF3715 domain adopts a pseudo-PARP structure and is required for transposon silencing in a manner independent of complex assembly. TEX15, an essential piRNA pathway factor, also contains the DUF3715 domain. Here, we show that TASOR's and TEX15's DUF3715 domain share extensive structural homology. We found that the DUF3715 domain arose in early eukaryotes and that in vertebrates it is restricted to TEX15, TASOR, and TASORB orthologs. While TASOR-like proteins are found throughout metazoa, TEX15 is vertebrate-specific. The branching of TEX15 and the TASOR-like DUF3715 domain likely occurred in early metazoan evolution. Remarkably, despite this vast evolutionary distance, the DUF3715 domain from divergent TEX15 sequences can functionally substitute the DUF3715 domain of TASOR and mediates transposon silencing. We have thus termed this domain of unknown function as the RNA-directed pseudo-PARP transposon silencing (RDTS) domain. In summary, we show an unexpected functional link between these critical transposon silencing pathways.

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. We show that ZFP266 binds Short Interspersed Nuclear Elements (SINEs) adjacent to binding sites of pioneering factors, OCT4 (POU5F1), SOX2, and KLF4, and impedes chromatin opening. Replacing the KRAB co-suppressor with co-activator domains converts ZFP266 from an inhibitor to a potent facilitator of iPSC reprogramming. We propose that the SINE-KRAB-ZFP interaction is a critical regulator of chromatin accessibility at regulatory elements required for efficient cellular identity changes. In addition, this work serves as a resource to further illuminate molecular mechanisms hindering reprogramming.

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. CELLoGeNe removes inadvertent symmetries in the energy landscapes normally arising from standard Boolean operators. Furthermore, CELLoGeNe provides tools to visualize and stochastically analyze the shapes of multi-dimensional energy landscapes corresponding to epigenetic landscapes for development and reprogramming. We demonstrate CELLoGeNe on two GRNs governing different aspects of induced pluripotent stem cells, identifying experimentally validated attractors and revealing potential reprogramming roadblocks. CELLoGeNe is a general framework that can be applied to various biological systems offering a broad picture of intracellular dynamics otherwise inaccessible with existing methods.

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. However, a comprehensive evaluation of the involvement of RNA processing factors in the reprogramming of somatic mammalian cells is lacking. RESULTS: Here, we used data from a large number of studies carried out in three mammalian species, mouse, chimpanzee and human, to uncover consistent changes in gene expression upon reprogramming of somatic cells. We found that a core set of nine splicing factors have consistent changes across the majority of data sets in all three species. Most striking among these are ESRP1 and ESRP2, which accelerate and enhance the efficiency of somatic cell reprogramming by promoting isoform expression changes associated with mesenchymal-to-epithelial transition. We further identify genes and processes in which splicing changes are observed in both human and mouse. CONCLUSIONS: Our results provide a general resource for gene expression and splicing changes that take place during somatic cell reprogramming. Furthermore, they support the concept that splicing factors with evolutionarily conserved, cell type-specific expression can modulate the efficiency of the process by reinforcing intermediate states resembling the cell types in which these factors are normally expressed.

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. Mybl2 overexpression changes chromatin conformation, affecting the accessibility of pioneer factors to the chromatin and promoting accessibility for early immediate response genes known to be reprogramming blockers. These changes in the chromatin landscape ultimately lead to a deregulation of key genes that are important for the mesenchymal-to-epithelial transition. This work defines Mybl2 level as a gatekeeper for the initiation of reprogramming, providing further insights into the tight regulation and required coordination of molecular events that are necessary for changes in cell fate identity during 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. DNA adenine methyltransferase identification (DamID), developed and widely used in Drosophila, is a distinct technology to investigate protein-DNA interactions. Unlike ChIP-seq, it does not require antibodies, precipitation steps, or chemical protein-DNA crosslinking, but to date it has been seldom used in mammalian cells due to technical limitations. Here we describe an optimized DamID method coupled with next-generation sequencing (DamID-seq) in mouse cells and demonstrate the identification of the binding sites of two TFs, POU5F1 (also known as OCT4) and SOX2, in as few as 1000 embryonic stem cells (ESCs) and neural stem cells (NSCs), respectively. Furthermore, we have applied this technique in vivo for the first time in mammals. POU5F1 DamID-seq in the gastrulating mouse embryo at 7.5 d post coitum (dpc) successfully identified multiple POU5F1 binding sites proximal to genes involved in embryo development, neural tube formation, and mesoderm-cardiac tissue development, consistent with the pivotal role of this TF in post-implantation embryo. This technology paves the way to unprecedented investigation of TF-DNA interactions and GRNs in specific cell types of limited availability in mammals, including in vivo samples.

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. To identify modulators of reprogramming-induced senescence, we performed a genome-wide shRNA screen in primary human fibroblasts expressing OSKM. In the screen, we identified novel mediators of OSKM-induced senescence and validated previously implicated genes such as CDKN1A We developed an innovative approach that integrates single-cell RNA sequencing (scRNA-seq) with the shRNA screen to investigate the mechanism of action of the identified candidates. Our data unveiled regulation of senescence as a novel way by which mechanistic target of rapamycin (mTOR) influences reprogramming. On one hand, mTOR inhibition blunts the induction of cyclin-dependent kinase (CDK) inhibitors (CDKIs), including p16INK4a, p21CIP1, and p15INK4b, preventing OSKM-induced senescence. On the other hand, inhibition of mTOR blunts the senescence-associated secretory phenotype (SASP), which itself favors reprogramming. These contrasting actions contribute to explain the complex effect that mTOR has on reprogramming. Overall, our study highlights the advantage of combining functional screens with scRNA-seq to accelerate the discovery of pathways controlling complex phenotypes.

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. Mechanistically, SMAD3 interacts with reprogramming factors and co-activators and co-occupies OCT4 target loci during reprogramming. Unexpectedly, active SMAD2/3 also markedly enhances three other TF-mediated direct reprogramming conversions, from B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons, highlighting broad and general roles for SMAD2/3 as cell-reprogramming potentiators. Our results suggest that co-expression of active SMAD2/3 could enhance multiple types of TF-based cell identity conversion and therefore be a powerful tool for cellular engineering.

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. Through the investigation of reprogramming mechanisms, we recently revealed that over-expression of constitutive active Smad3 boosted not only iPSC generation, but also 3 other master TF-mediated conversions, from B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons. This demonstrated that there were common mechanisms underlying different master TF-mediated cell conversions. To illuminate such mechanisms further, we have recently performed CRISPR/Cas9-mediated genome-wide knockout screening during reprogramming with a lentiviral gRNA library containing 90,000 gRNAs. This screening provided us with ~15 novel reprogramming roadblock genes as well as ~20 candidate genes essential for the reprogramming process but not for ES cell self-renewal. This data set will be a valuable resource to further understand how overexpression of master TFs alters cellular identity, and to achieve more faithful, efficient cell conversions for regenerative medicine.(Presented at the 1934th Meeting, March 17, 2017).

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. Using two novel cell surface markers, ICAM1 and CD44, clear cell population changes undergoing reprogramming can be observed over a time course upon induction of the reprogramming factors. Consequently, this technique allows for easy identification of factors that enhance or delay reprogramming, and can be a useful strategy in elucidating key mechanisms for efficient generation of iPSCs.

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. In contrast, more efficient cassettes can reprogram both wild-type and Nanog(-/-) fibroblasts with comparable efficiencies, routes, and kinetics, unlike the less efficient reprogramming systems. Moreover, we attribute a previously reported variation in the N terminus of KLF4 as a dominant factor underlying these critical differences. Our data establish that some reprogramming roadblocks are system dependent, highlighting the need to pursue mechanistic studies with close attention to the systems to better understand reprogramming.

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. We position Cited2 within the pluripotency gene regulatory network by defining Nanog, Tbx3, and Klf4 as its direct targets. We also demonstrate that the defects caused by Cited2 depletion are, at least in part, rescued by Nanog constitutive expression. Finally, we demonstrate that Cited2 is required for and enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells.

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. Like in vitro differentiation, iPSC generation efficiency and kinetics largely depend on the external environment, as well as the amount and stoichiometry of exogenously expressed reprogramming factors. However, accumulating evidence indicates that when reprogramming succeeds, the process is not random but progresses in an ordered, step-wise manner. In this review, we summarize current knowledge detailing how somatic cells reach a pluripotent state.

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. We also show that reprogramming of epiblast-derived stem cells to naive pluripotency requires NuRD complex function and that increased MBD3/NuRD levels can enhance reprogramming efficiency when coexpressed with the reprogramming factor NANOG. Our results therefore show that the MBD3/NuRD complex plays a key role in reprogramming in certain contexts and that a chromatin complex required for cell differentiation can also promote reversion back to a naive pluripotent cell state.

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. The rapid expansion of minor reprogrammed cells in the heterogeneous population can also obscure investigation of relevant transition processes. Understanding the biological mechanisms essential for successful iPS cell generation requires both accurate capture of cells undergoing the reprogramming process and identification of the associated global gene expression changes. Here we demonstrate that in mouse embryonic fibroblasts, reprogramming follows an orderly sequence of stage transitions, marked by changes in the cell-surface markers CD44 and ICAM1, and a Nanog-enhanced green fluorescent protein (Nanog-eGFP) reporter. RNA-sequencing analysis of these populations demonstrates two waves of pluripotency gene upregulation, and unexpectedly, transient upregulation of several epidermis-related genes, demonstrating that reprogramming is not simply the reversal of the normal developmental processes. This novel high-resolution analysis enables the construction of a detailed reprogramming route map, and the improved understanding of the reprogramming process will lead to new reprogramming strategies.

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. In contrast to previously published work, Erk2-null ES cells exhibited no detectable defect in lineage specification to any of the three germ layers when induced to differentiate in either embryoid bodies or in defined neural induction conditions. However, under self-renewing conditions Erk2-null ES cells express increased levels of the pluripotency-associated transcripts, Nanog and Tbx3, a decrease in Nanog-GFP heterogeneity, and exhibit enhanced self-renewal in colony forming assays. Transgenic add-back of ERK2 is capable of restoring normal pluripotent gene expression and self-renewal capacity. We show that ERK2 contributes to the destabilization of ES cell self-renewal by reducing expression of pluripotency genes, such as Nanog, but is not specifically required for the early stages of germ layer specification.

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. An Oct4 activation domain fusion supported embryonic stem cell self-renewal in vitro at lower concentrations than that required for Oct4 while alleviating the ordinary requirement for the cytokine LIF. At still lower levels of the fusion, LIF dependence was restored. We conclude that the necessary and sufficient function of Oct4 in promoting pluripotency is to activate specific target genes.

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 21.1 years, standard deviation (SD) 0.7 years). Latency data determined with the Motor Control Test of Computerized Dynamic Posturography (CDP) were compared for the three conditions of mastication status, the direction of translation, and the magnitude of translation, using three-way repeated measures ANOVA and lower-order ANOVA with the three conditions separated. Latency was significantly shorter with mastication than with the lower jaw relaxed (P < 0.00001). Mastication alone, however, cannot be considered significant because of the complex interactions involved among the three conditions. Mastication increases not only static balance but also reactive balance in response to unanticipated external disturbances. Gum chewing may therefore reduce falls among elderly persons with impaired balance.