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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 orthologues. While TASOR-like proteins are found throughout metazoa, TEX15 is vertebrate specific. The branching of TEX15 and 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 for the same domain in 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.

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 (1-7), 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., Several reports have suggested that reprogramming progresses in an ordered manner (3,5,6,8-10). To identify markers whose expression changed concurrent with pluripotency gene expression, we performed time course microarray analysis using [...]

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 (1-7). However, such iPS cells contain a large number of viral vector integrations (1,8), any one of which could cause unpredictable genetic dysfunction. Whereas c-Myc is dispensable for reprogramming (9,10), complete elimination of the other exogenous factors is also desired because ectopic expression of either Oct4 (also known as Pou5f1) or Klf4 can induce dysplasia (11,12). Two transient transfection-reprogramming methods have been published to address this issue (13,14). However, the efficiency of both approaches is extremely low, and neither has been applied successfully to human cells so far. Here we show that non-viral transfection of a single multiprotein expression vector, which comprises the coding sequences of c-Myc, Klf4, Oct4 and Sox2 linked with 2A peptides, can reprogram both mouse and human fibroblasts. Moreover, the transgene can be removed once reprogramming has been achieved. iPS cells produced with this nonviral vector show robust expression of pluripotency markers, indicating a reprogrammed state confirmed functionally by in vitro differentiation assays and formation of adult chimaeric mice. When the single-vector reprogramming system was combined with a piggyBac transposon (15,16), we succeeded in establishing reprogrammed human cell lines from embryonic fibroblasts with robust expression of pluripotency markers. This system minimizes genome modification in iPS cells and enables complete elimination of exogenous reprogramming factors, efficiently providing iPS cells that are applicable to regenerative medicine, drug screening and the establishment of disease models., Efficient multiprotein expression has been reported in a variety of cell types, including human embryonic stem (ES) cells, using the 2A peptide sequence of foot and mouth disease virus (F2A) [...]

Transgenic expression of just four defined transcription factors (c-Myc, Klf4, Oct4 and Sox2) is sufficient to reprogram somatic cells to a pluripotent state (1-4). 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 (1), lentivirals (5), adenoviral (6) and plasmid (7) transfection to deliver reprogramming factor transgenes. Although the latter two methods are transient and minimize the potential for insertion mutagenesis, they are currently limited by diminished reprogramming efficiencies. piggyBac (PB) transposition is host-factor independent, and has recently been demonstrated to be functional in various human and mouse cell lines (8-11). The PB transposon/transposase system requires only the inverted terminal repeats flanking a transgene and transient expression of the transposase enzyme to catalyse insertion or excision events (12). Here we demonstrate successful and efficient reprogramming of murine and human embryonic fibroblasts using doxycycline-inducible transcription factors delivered by PB transposition (13). Stable iPS cells thus generated express characteristic pluripotency markers and succeed in a series of rigorous differentiation assays. By taking advantage of the natural propensity of the PB system for seamless excision (12), we show that the individual PB insertions can be removed from established iPS cell lines, providing an invaluable tool for discovery. In addition, we have demonstrated the traceless removal of reprogramming factors joined with viral 2A sequences (14) delivered by a single transposon from murine iPS lines. We anticipate that the unique properties of this virus-independent simplification of iPS cell production will accelerate this field further towards full exploration of the reprogramming process and future cell-based therapies., To explore the utility of PB as a vector for somatic cell reprogramming, it was necessary to achieve temporal expression control and transgene silencing, as PB transposons are not purposefully [...]

CD9 is a membrane protein belonging to the tetraspanin family. Despite CD9's broad tissue distribution, the only abnormality observed in CD9-deficient mice was infertility of females, which was responsible for a defect in the sperm-egg fusion process. However, the function of CD9 in sperm-egg fusion is not clear at all because the technique to analyze the activity of molecules in sperm-egg fusion has not been established. We demonstrated that the exogenous mouse CD9, expressed by polyadenylated mRNA injection at the germinal-vesicle stage oocytes, was precisely localized to the egg plasma membrane, and the expression reversed the infertility of [CD9.sup.-/-] eggs. Then, two other tetraspanins, human CD9 and mouse CD81, overexpressed with this technique on [CD9.sup.-/-] eggs restored the fertilization rate up to ~90 and ~50% against that of wild type eggs, respectively. Moreover, in the presence of an anti-mouse CD9 mAb, which blocks sperm-egg fusion, expression of human CD9 or mouse CD81 on eggs also rescued the fusibility. These results suggested that human CD9 plays a crucial role in human fertilization, and mouse CD81 has the potential to compensate for CD9 function in sperm-egg fusion. In addition, the polyadenylated mRNA injection is effective for molecular analysis of sperm-egg fusion. Key Words: fertilization; sperm-egg fusion; polyadenylated mRNA injection; CD9; CD81.

This paper presents the first neutron scattering measurement of 1,2-1,4-polybutadienes with varying microstructure. Incoherent inelastic scattering experiments have been performed in the range of moderate (ΔE≊300 μeV) and high (ΔE≊20 μeV) energy resolution. For the moderate resolution experiments the vibration–relaxation model has been used as a guideline for interpretation. The most striking influence of the microstructure variation can be found in the position of the boson peak. Its position at low temperature as well as its temperature dependence is significantly different among the measured samples. The high resolution data have been Fourier transformed into the time domain yielding the intermediate incoherent scattering function in a time range of 5,...,150 ps. There, the basic properties found for 1,4-polybutadiene earlier could be reproduced (separation of α and βslow regimes of the slow relaxation, Kohlrausch–Williams–Watts function with β≊0.4). On the basis of the new data, no microstructure induced differences can be detected except for the changes of temperature dependence due to the strongly different glass-transition temperatures and different fragility. © 1996 American Institute of Physics. [ABSTRACT FROM AUTHOR]

Glassy 1-butene (CH2=CHCH2CH3) was studied by incoherent inelastic neutron scattering below 10 meV in the temperature range 18≤T≤80 K covering the glass transition temperature (Tg=60 K). A broad excitation peak due to the low-energy excitation was found at 3–4 meV. The spectra corrected for the Bose factor could be scaled to a unique curve for temperatures below Tg. The absolute density of vibrational states was derived from the spectrum at 18 K and the previously measured heat capacity data. The number of vibrational states associated with the low energy excitation was 1.4 per molecule. This is much larger than those for SiO2 and other amorphous materials studied so far. The density of vibrational states was well reproduced by the soft potential model originally conceived for network glasses. © 1996 American Institute of Physics. [ABSTRACT FROM AUTHOR]

Dynamics of amorphous cis-1,4-polybutadiene was studied by incoherent inelastic and quasielastic neutron scattering below 10 meV in a wide temperature range covering the glass transition temperature Tg. The spectra at low temperatures below Tg show a broad excitation peak at about 2–3 meV, which is the so-called low-energy excitation and believed to be an origin of anomalous thermal properties of amorphous materials at low temperatures. It was found that the temperature dependence of the low-energy excitation intensity can be normalized by the Bose factor at temperatures low enough below Tg. With increasing temperature, a very fast motion appears in the energy range below ∼4 meV at around the Vogel–Fulcher temperature T0, which is ∼50 K below Tg. This fast motion is often interpreted as the β process predicted by the mode coupling theory. In this paper, both the low-energy excitation and the fast motion were analyzed in terms of a common microscopic picture, i.e., an asymmetric double well potential. The natures of the two motions are discussed focusing on the relation between the two motions as well as the glass transition dynamics. [ABSTRACT FROM AUTHOR]